The composition and emission of volatile organic compounds in Eucalyptus spp. leaves.
Anthony J. Winters
Submitted in fulfillment of the requirements of the degree Doctor of Philosophy
March, 2010
School of Biological, Earth and Environmental Sciences University of New South Wales PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet
Surname or Family name: WINTERS
First name: Anthony Other name/s: Jon
Abbreviation for degree as given in the University calendar:
School: Biological, Earth and Environmental Sciences Faculty: Science
Title: The composition and emission of volatile organic compounds in Eucalyptus spp. leaves.
Abstract 350 words maximum: (PLEASE TYPE)
The emission of volatile organic compounds (VOCs) from plants has important effects on atmospheric chemistry and the carbon dynamics on plants. Eucalypts are amongst the highest emitters of VOCs, yet there are relatively few studies of emissions from this genus. The few studies published have examined emissions rates in relation to uncontrolled environmental variables. Only one study has examined these effects under controlled conditions. This thesis examined the factors that regulate VOC emissions from Eucalyptus globulus, one of the most widely cultivated eucalypt species.
The emissions of isoprene, monoterpenes and the short-chained carbonyls formaldehyde, acetaldehyde and acetone were determined from four eucalypt species. Carbonyl emissions were generally comparable with rates reported for other species. There was large variation in diurnal isoprene and monoterpene emissions between species, but under standard conditions, isoprene emissions were much lower than previous reports. Monoterpenes were as much as six times greater than previous reports for some species. Emission of each carbonyl was correlated with its ambient concentration across different species.
The effects of temperature and light on isoprene and monoterpene emissions from E. globulus were studied under laboratory conditions. Isoprene emissions exhibited the well described enzymatic response to increasing temperature, with a peak in emission at around 45 ƱC. Monoterpenes exhibited a large transient burst in response to incremental increases in leaf temperature. Analysis of the decay rate suggested a single pool contributed to the transient burst. I argue that emissions occur directly from oil glands, regulated only by a thin layer of cap cells above the gland. Light-dependent emission of cis-ocimene was also observed.
Fumigation of leaves identified four compounds incorporated 13 C; isoprene (C5), cis-ocimene (C10), trans-caryophyllene (C15) and the tentatively identified C5 compound iso-valeraldehyde. Emissions were only detected in emitted compounds and no labelled uptake was identified in either oil glands or the remaining leaf tissue. The fraction of final 13C incorporation in the three terpenoids appeared to match the established paths of biosynthesis.
Together, these data suggest that isoprene emissions from eucalypts may be a factor of two or more lower than previously estimated, while the case for monoterpene emissions remains unclear.
Declaration relating to disposition of project thesis/dissertation
I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation.
I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only).
…………………………………………………………… ……………………………………..……………… ……….……………………...…….… Signature Witness Date
The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research.
FOR OFFICE USE ONLY Date of completion of requirements for Award:
THIS SHEET IS TO BE GLUED TO THE INSIDE FRONT COVER OF THE THESIS Abstract
The emission of volatile organic compounds (VOCs) from plants has important effects on atmospheric chemistry and the carbon dynamics on plants. They also constitute a fascinating evolutionary response to the many challenges faced by plants. Eucalypts are amongst the highest emitters of VOCs, yet there are relatively few studies of emissions from this genus. While known to emit isoprene and monoterpenes, the few published studies have examined emissions rates and composition in relation to uncontrolled environmental variables. Only one study has examined these effects under controlled conditions, but examined only three terpenoid compounds. This thesis examined the factors that regulate VOC emissions from Eucalyptus globulus, one of the most widely cultivated eucalypt species.
The first major section of research examined emissions of isoprene, monoterpenes and the short-chained carbonyls formaldehyde, acetaldehyde and acetone from four species
(Eucalyptus camaldulensis, Eucalyptus globulus, Eucalyptus grandis, and Eucalytpus viminalis) in south-western Australia. A smaller comparative study of carbonyl emissions was conducted on E. camaldulensis in south-eastern Australia. Carbonyl emissions were generally comparable with rates reported for other species, with diurnal emissions peaking at about 4, 75 and 34 nmol m-2 min-1 for acetone, formaldehyde and acetaldehyde respectively. There was considerable variation in diurnal isoprene and monoterpene emissions between species, but under standard conditions, isoprene emissions were between c. 440 and 1400 ug C m-2 h-1 across all species. In E. globulus, the best studied species to date, these emission rates are two to eight times lower than previous reports. Conversely, standard emission rates of monoterpenes ranged between
360 – 3300 ug C m-2 h-1 which, where data exists, is as much as six times greater than
i previous reports for some species. Emission of each carbonyl was correlated with its ambient concentration across different species, but more weakly related to temperature.
Acetaldehyde emission in particular was significantly correlated with transpiration, but not with sap flow or with ethanol concentrations in xylem sap. This suggests fermentation within the leaf and stomatal conductance are primary controlling processes, since differences in acetaldehyde exchange velocities between sites suggest stomata may indeed exert long term emission regulation, in contrast to compounds for which no biological sink exists.
The second major section examined the effects of temperature and light on isoprene and monoterpene emissions from E. globulus. Isoprene emissions exhibited the well described enzymatic response to increasing temperature, with a peak in emission at around 45 °C. Even at high temperatures, the loss of carbon as a result of isoprene emission did not exceed 3 %. Monoterpenes, on the other hand, exhibited a large transient burst in response to incremental increases in leaf temperature that was c. 2 –
16 times the steady-state emission rate. These bursts took one hour or more to decay to a steady state. Analysis of the decay rate suggested a single pool contributed to the transient burst. Using evidence from this study and previous published reports, I argue that emissions occur directly from oil glands, regulated only by a thin layer of cap cells above the gland. Evidence is also presented for the light-dependent emission of cis- ocimene. These data suggest emission estimates of eucalypts using conventional models may be in serious error.
The final piece of research sought to identify the source of monoterpenes from within leaves of E. globulus. Fumigation of leaves identified four compounds incorporated 13
C; isoprene (C5), cis-ocimene (C10), trans-caryophyllene (C15) and the tentatively identified C5 compound iso-valeraldehyde. Importantly, emissions were only detected
ii in emitted compounds and no labelled uptake was identified in either oil glands or the remaining leaf tissue. The fraction of final 13C incorporation in the three terpenoids appeared to match the established paths of biosynthesis. The uptake of 13C incorporation agreed with monoterpene synthase activity studied in leaves of the same age. Confirmation and further elucidation of iso-valeraldehyde emission is required.
The identification of 13C uptake by a single monoterpene and sesquiterpene is discussed against the dynamics of emission over the age of a eucalypt leaf.
Together, these data suggest that isoprene emissions from eucalypts may be a factor of two or more lower than previously estimated, while the case for monoterpene emissions remains unclear. Estimates based on temperature-correcting models may be adequate under temperatures between 35 and 40 °C, but subject to more uncertainty below and above these temperatures. Elucidation of those mechanisms responsible for the novel behaviour reported here, and further model development, will go some way to resolving this uncertainty.
iii ORIGINALITY STATEMENT
‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree or diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis. I also declare that the intellectual content of this thesis is the product of my own work, except to the extent that assistance from others in the project's design and conception or in style, presentation and linguistic expression is acknowledged.’
Signed ……………………………………………......
Date ……………………………………………......
iv COPYRIGHT STATEMENT
‘I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.'
Signed ……………………………………………......
Date ……………………………………………......
AUTHENTICITY STATEMENT
‘I certify that the Library deposit digital copy is a direct equivalent of the final officially approved version of my thesis. No emendation of content has occurred and if there are any minor variations in formatting, they are the result of the conversion to digital format.’
Signed ……………………………………………......
Date ……………………………………………......
v Preface
Chapter 4 of this thesis has been published as:
Winters, A.J., Adams, M.A., Bleby, T.M., Rennenberg, H., Steigner, D., Steinbrecher,
R., Kreuzwieser, J., 2009. Emissions of isoprene, monoterpene and short-
chained carbonyl compounds from Eucalyptus spp. in southern Australia.
Atmospheric Environment 43: 3035-3043.
The work involved in writing Chapter 4 and the publication above was conducted by
me. Experimental set up and sampling involved the co-authors above, plus assistance
from Vera Brands, Frances Müller, Dagmar Weiss and Sebastian Pfautsch. D.
Steigner and R. Steinbrecher prepared and analysed adsorbent cartridges as part of the
sampling program. Carbonyl compounds were analysed by J. Kreuzwieser.
The work involved in carrying out the experiments and writing the remainder of the
thesis was conducted by me.
Assistance with the enzyme assays in Chapter 6 was provided by Ina Zimmer and Jogi
Schnitzler of the Research Centre Karlsruhe, Institute for Meteorology and Climate
Research, Germany. They continued to work on these assays after the end of my stay
with them in order to improve the results.
vi Ackowledgements
My profound appreciation is extended to many people.
To Mark Adams for taking me on and seeing through a rather difficult project. To
Heinz Rennenberg and Jürgen Kruezwieser, for their hospitality over a number of stays in Freiburg and their valued input throughout the project.
Charles Hocart of the RSBS Mass Spectrometry facility deserves special mention. This work has been infinitely improved by Charles’ selfless assistance at all stages and I am deeply grateful for his help and advice.
Marilyn Ball hosted me within the Functional Ecology lab at RSBS after the CRC for
Greenhouse Accounting wound up. Marilyn provided office and lab space and buckets of hospitality. Stephen Roxburgh provided support throughout much of this project. The sadly defunct CRC for Greenhouse Accounting provided substantial financial support for this work. Substantial contributions were also generously provided by the Bushfire
CRC. Most of this work was conducted at the Research School of Biological Sciences at the Australian National University. In the RSBS workshops, Brian McNamara taught, tested, built, repaired and advised on the many electronic aspects of this project. Istvan
‘Ishty’ Zaveczky rushed through the many parts I needed machining and provided much advice on component design. Bill Speed and Shane Green also went above and beyond in providing assistance. Steve Dempsey and Sue Lyons kept the plants happy and healthy. Chin Wong, Hilary Stuart-Williams and Peter Groeneveld from Environmental
Biology, sadly disbanded, provided advice and assistance throughout. David Kelly of the Research School of Physics and Engineering at the ANU really got me on my feet with LabVIEW programming.
vii Ina Zimmer and Jogi Schnitzler were extraordinarily accommodating and helpful during the terpene synthase work conducted in Garmisch-Partenkirchen.
Finally, to my wife and daughter. my profound gratitude, für alles.
viii Table of Contents
Chapter 1
Introduction...... 1
1.1 General introduction...... 1
1.2 Atmospheric chemistry ...... 3
1.3 Ecological and physiological function of volatile organic compounds ...... 4
1.4 Biosynthesis of terpenoid and carbonyl VOCs ...... 6
1.5 Mechanisms and rates of VOC emissions from plants ...... 8
1.6 VOC emissions from eucalypts...... 10
1.7 Project aims...... 11
Chapter 2
Method development ...... 13
2.1 Introduction...... 13
2.2 Design and construction of an automated GC sampling system...... 15
2.2.1 Introduction...... 15
2.2.2 Initial configuration of the GC...... 16
2.2.3 Initial modification and proof of concept tests ...... 17
2.2.4 Final configuration...... 18
2.2.5 Programmed control of the automated sampling system...... 23
ix 2.2.6 Software...... 25
2.2.7 Sampling sequence ...... 26
2.2.8 Trap performance...... 29
2.2.9 Gas chromatography...... 30
2.2.10 GC calibration and detection limits ...... 31
2.3 Proton transfer reaction – mass spectrometry ...... 33
2.3.1 Introduction...... 33
2.3.2 Instrument operation...... 34
2.3.3 Calibration, sensitivity, detection limits and transmission efficiency ...... 36
2.3.4 Compound fragmentation ...... 40
2.4 Gas exchange system ...... 44
2.4.1 Cuvette and plumbing layout...... 44
2.4.2 Gas exchange calculations...... 45
2.4.3 Calibration of mass flow controllers...... 46
2.5 Error estimation...... 48
2.6 Other instrumental analyses ...... 49
Chapter 3
Characterisation of terpenoid oils in juvenile leaves of Eucalyptus globulus ...... 50
3.1 Introduction...... 50
3.2 Methods and Materials...... 53
x 3.2.1 Plant material ...... 53
3.2.2 Determination of optimum solvent extraction time...... 53
3.2.3 Extraction of terpenoid oils from individual oil glands...... 54
3.2.4 Effect of light on the composition of oil in glands and whole leaves...... 55
3.2.5 Analysis of terpenoid oils by GC-MS...... 56
3.2.6 Statistical analyses ...... 57
3.3 Results and Discussion...... 58
3.3.1 Optimum solvent extraction time ...... 58
3.3.2 Spatial variation in gland composition ...... 59
3.3.3 The effect of light and location on oil composition...... 67
3.4 Concluding remarks ...... 68
Chapter 4
Emissions of isoprene, monoterpene and short-chained carbonyl compounds from
Eucalyptus spp. in southern Australia...... 69
4.1 Introduction...... 69
4.2 Material and Methods ...... 72
4.2.1 Field sites and plant material ...... 72
4.2.2 Gas exchange systems and VOC sampling...... 73
4.2.3 VOC analysis ...... 74
4.2.4 Sap sampling and analysis ...... 75
xi 4.2.5 Error, data and statistical analyses...... 76
4.3 Results and Discussion...... 78
4.3.1 Isoprenoid emissions...... 78
4.3.2 Carbonyl emissions...... 85
4.4 Concluding remarks ...... 92
Chapter 5
Temperature and lights effects on terpenoid emissions from juvenile leaves of
Eucalyptus globulus...... 93
5.1 Introduction...... 93
5.2 Methods and materials ...... 96
5.2.1 Plant material ...... 96
5.2.2 Gas exchange system ...... 96
5.2.3 Sampling of volatile organic compounds ...... 97
5.2.4 Verification of results and cuvette characterisation...... 98
5.2.5 Data analysis and error determination ...... 99
5.3 Results...... 100
5.3.1 Temperature effects on total monoterpene emission patterns...... 100
5.3.2 Characterisation of cuvette sorption effects...... 108
5.3.3 Temperature effects on the emission of individual monoterpenes ...... 111
5.3.4 Response of monoterpene emissions to changes in light level...... 114
xii 5.3.5 Temperature effects on isoprene emission...... 116
5.3.6 Light effects on isoprene emission ...... 121
5.4 Discussion ...... 122
5.4.1 Monoterpene burst-decay emission patterns...... 122
5.4.2 Implications of monoterpene emission bursts for emission modelling .. 130
5.4.3 Evidence for light- and temperature-dependent monoterpene emission 134
5.4.4 Isoprene emissions from eucalypts ...... 134
5.5 Concluding remarks ...... 136
Chapter 6
The origin of terpenoid emissions from juvenile leaves of Eucalyptus globulus revealed
13 by CO2 fumigation ...... 137
6.1 Introduction...... 137
6.2 Methods and materials ...... 140
6.2.1 Plant material ...... 140
6.2.2 Gas exchange system ...... 140
6.2.3 Sampling of volatile organic compounds ...... 140
6.2.4 Charcoal adsorption cartridges ...... 141
6.2.5 Identification of 13C-labelled terpenoids within leaf tissue ...... 142
6.2.6 GC-MS analysis of individual terpenes...... 142
6.2.7 Extraction and temperature assay of monoterpene synthase ...... 142
xiii 6.2.8 Data analysis and error determination ...... 144
6.3 Results...... 145
6.3.1 Origin of 13C-labelled terpenoid compounds...... 145
6.3.2 Identification and kinetics of 13C-labelled compounds...... 145
6.3.3 Monoterpene synthase activity ...... 153
6.4 Discussion ...... 155
6.4.1 The origin of labelled terpenes ...... 155
6.4.2 Identification of 13C labelling ...... 157
6.4.3 The putative importance of de novo synthesis...... 159
6.5 Concluding remarks ...... 161
Chapter 7
Conclusions...... 162
References...... 168
Appendix 1...... 194
Appendix 2...... 196
xiv List of tables
Chapter 1
Table 1.1 Isoprenoid classification, examples and putative sites of synthesis in higher plants...... 7
Chapter 3
Table 3.1. The relative composition of compounds in the oil glands and whole leaf of E. globulus under light and dark conditions...... 64
Chapter 4
Table 4.1. Comparison of Eucalyptus isoprene and monoterpene emission data from this study with that from the literature...... 81
Table 4.2. Exchange velocities from E. camaldulensis of formaldehyde, actealdehyde and acetone at the Barmah Forest Site and the Albany Tree Farm...... 89
xv List of figures
Chapter 2
Figure 2.1. The original configuration of the SRI gas chromatograph (GC)...... 17
Figure 2.2. The first attempt at a temperature-controlled trap...... 20
Figure 2.3. Elevation view of a partly assembled adsorbent VOC trap...... 22
Figure 2.4. Layout of the water trap ...... 24
Figure 2.5. Coordination of temperature programs for the GC oven, and VOC and water traps...... 27
Figure 2.6. Schematic of the GC system with water and adsorbent VOC trap ...... 28
Figure 2.7. Determination of the trap breakthrough volume for representative mixing ratios of three gas standards...... 30
Figure 2.8. Layout of the major components of the proton transfer reaction mass spectrometer...... 36
Figure 2.9 Representative transmission curve determined from a calibration gas ...... 39
Figure 2.10. Comparative fragmentation spectra of isoprene at acceptable and excessive concentrations...... 43
Figure 2.11. Plumbing diagram of the sampling train used in the laboratory experiments
...... 44
Figure 2.12. Representative calibration curve of an Aalborg mass flow controller ...... 47
xvi Chapter 3
Figure 3.1. Juvenile leaf of E. globulus showing the approximate positions of the 8 regions sampled to identify differences in oil gland composition...... 56
Figure 3.2. Effect of extraction time on yield of terpenoids from leaves of E. globulus...... 59
Figure 3.3. Comparison of extraction time and yield for the sum of limonene and eucalyptol...... 59
Figure 3.4 Typical chromatogram from the analysis of leaf oils of E. globulus ...... 63
Figure 3.5. Treatment effects of light or dark conditions and position on the relative composition of cis-ocimene in leaves of E. globulus ...... 65
Figure 3.6. The mean relative composition of a given compound in glands compared to whole leaves in the light or the dark...... 65
Chapter 4
Figure 4.1. Diurnal course of isoprene and monoterpene emissions of formaldehyde, acetaldehyde and acetone emissions from E. globulus...... 79
Figure 4.2. Composition of monoterpene emissions from Eucalytpus viminalis, E. grandis, E. camaldulensis and E. globulus...... 84
Figure 4.3. Correlation of emission rate with leaf temperature for formaldehyde, acetaldehyde and actone from Eucalyptus grandis and E. globulus...... 86
Figure 4.4. Tempearture influence on carbonyl emission from E. camaldulensis at the
Barmah natural forest site and the water- and nutrient-rich Albany plantation...... 87
xvii Figure 4.5. The effect of ambient carbonyl concentrations on the exchange of formaldehyde, acetaldehyde and acetone from leaves of E. camaldulensis...... 90
Figure 4.6. Relationships between actealdehyde emisison and trunk xylem sap flow, xylem ethanol concentration and leaf transpiration rates in E. camaldulensis and E. globulus at the Albany plantation site...... 90
Chapter 5
Figure 5.1. Emission of monoterpenes in response to stepwise increases in leaf temperature ...... 101
Figure 5.2. The proportional change in steady-state emission rate resulting from an increase in leaf temperature ...... 101
Figure 5.3. Two typical monoterpene emission patterns from leaves in which leaf temperature was increased before monoterpene emissions stabilised...... 103
Figure 5.4. The rate of increase in monoterpene emissions at each increment of temperature change...... 104
Figure 5.5. Single-exponential models fitted to the decay phase of monoterpene emissions at each temperature step-increase ...... 106
Figure 5.6. Half-life times of the time-dependent monoterpene emission decay for each temperature increase ...... 107
Figure 5.7. Sorption isotherm produced from the step-increase and -decrease of eucalyptol gas standard into the cuvette air stream ...... 109
Figure 5.8. The contribution of monoterpenes desorbed from the cuvette wall to the monoterpene signal following an increase in wall temperature of the cuvette...... 110
xviii Figure 5.9. Changes in the concentration of monoterpenes within the mixed air volume of the cuvette in response to increasing leaf temperature...... 110
)LJXUH3DQHO$(PLVVLRQRIĮ-pinene, FDPSKHQHȕ-pinene, myrcene, p-cymene, cis-ocimene from leaves of E. globulus as a function of leaf temperature...... 112
)LJXUH 3DQHO % (PLVVLRQ RI HXFDO\SWRO OLPRQHQH Ȗ-terpinene and terpinolene from leaves of E. globulus as a function of leaf temperature ...... 113
Figure 5.11. The relative contribution of each monoterpene to the total flux of monoterpenes from leaves of E. globulus at each temperature step...... 113
Figure 5.12. The response of cis-ocimene to different levels of PAR...... 115
Figure 5.13. Representative comparison of isoprene emissions quantified using a GC and PTR-MS ...... 116
Figure 5.14. Isoprene emission rates determined by GC from leaves of E. globulus as a function of temperature...... 117
Figure 5.15. Isoprene emission in response to increasing leaf temperature measured using PTR-MS ...... 118
Figure 5.16. Representative patterns of gas exchange observed in leaves of E. globulus subjected to increasing temperature...... 119
Figure 5.17. The ratio of carbon assimilated through photosynthesis to that lost through isoprene emission over the temperature range 20 – 50 °C ...... 120
Figure 5.18. Isoprene emission from leaves of E. globulus in response to increasing levels of irradiance following overnight darkness...... 121
Figure 5.19. Comparison of the Guenther algorithm with the data and model developed in this experiemnt...... 133
xix Chapter 6
Figure 6.1 Time course of 13C incorporation and wash-out into isoprene emitted from leaves of E. globulus...... 146
Figure 6.2. Time course of 13C labelling for the m/z range 85 – 92. Ions at m/z 87 and
13 m/z 85 declined in response to fumigation with CO2 ...... 146
Figure 6.3. Fragmentation spectra generated by PTR-MS ...... 149
Figure 6.4. Mass spectra of A, cis-ocimene and B, trans-caryophyllene, emitted from leaves of E. globulus showing the shift in fragmentation resulting from incorporation
13C into the molecule ...... 151
Figure 6.5. Time course of 13C incorporation into cis-ocimene and trans-caryophyllene emitted from leaves of E. globulus ...... 152
Figure 6.6. Production of cis-ocimene in response to incrementally higher incubation temperatures during the monoterpene synthase assay for leaves of E. globulus and E. viminalis...... 154
xx Chapter 1 Introduction
1. Introduction
1.1 General introduction
As stationary organisms, plants need means other than movement with which to interact and respond to their environment. The production and emission of secondary compounds for this purpose provides such effective mechanisms that thousands of compounds are produced in the plant kingdom. Traditionally termed ‘secondary metabolites’, they had been considered unintended consequences of ‘biosynthetic prodigality’ (Goodwin and Mercer 1972). While use of the term ‘secondary’ persists, our growing understanding of the many functions of these compounds has led some to prefer the term specialised compounds, signifying their adaptive significance
(Pichersky, 2009).
Specialised compounds are broadly defined as those that are not involved in primary metabolism – the photosynthetic and respiratory processes – which loosely include amino acids, carbohydrates and chlorophylls (Seigler, 1998; Heldt, 2005). The distinction is a pragmatic one but nonetheless captures many thousands of compounds, from which only a fraction is known to have a definable function (Seigler, 1998). More
1 Chapter 1 Introduction formal classification within this group proceeds according to their physicochemical, structural or biochemical characteristics. These classes are extensive (see Seigler, 1998) and include alkaloids, flavonoids, tannins, phenylpropanoids, quinones, polyketides, waxes, fatty acids and terpenoids among many others.
The quantitatively most important specialised compounds emitted by plants are the isoprenoid and short-chained oxygenated compounds (Guenther et al., 1995,
Kesselmeier and Staudt, 1999; Seco et al., 2007), with global annual emissions of around 1250 Tg C (Guenther et al., 1995). When under water stress, emissions of volatile secondary compounds derived from recently fixed carbon can represent greater than 10% of this fixed carbon (isoprene in particular). These compounds, once considered non-essential to plant metabolism, have revealed functions that impart adaptive advantages at an amazingly wide range of spatial and temporal scales; a breadth of function that in some cases, a single compound may impart from the molecular to the meteorological scale.
It is the volatile terpenoids and to a lesser extent, short-chained oxygenated compounds, emitted by eucalypts that form the focus of this thesis and to which discussion will be restricted. This thesis will examine some of the factors that regulate emissions of isoprenoid (in particular) and short-chained oxygenated organic compounds from eucalypts. However many factors contribute to these processes and these will be discussed more generally here.
2 Chapter 1 Introduction
1.2 Atmospheric chemistry
Globally, biogenic sources are emitted at an estimated 1250 Tg C yr-1 (Guenther et al.,
1995; 2006), which is five to ten times greater than VOC emissions from anthropogenic sources (Müller, 1992; Guenther et al., 1995). The possession of at least one double bond in this structurally complex group makes them highly reactive with O3, HO·, NO3· and Cl· (Finlayson-Pitts and Pitts, 2000). As such they typically have short atmospheric lifetimes according to the species and concentration of reactants, and the time of day.
For example, at typical diurnal concentrations of OH (day), NO3 (night) and O3 (day), the hemiterpene isoprene persists for 1.4 h, 50 min and 1.3 days respectively (Atkinson,
2000). The reaction products have long been of interest to atmospheric chemists and
Fehsenfeld et al. (1992) summarised five major impacts biogenic VOCs have on the atmosphere: (1), production of ozone in atmospheres of high [NOx]; (2), preferential reaction with OH suppresses OH radical concentrations, enhancing [HO2] which is further involved in the conversion of NO2 to ozone; (3), photooxidation of biogenic
VOCs can be an important source of CO, accounting for perhaps 10 – 20 % of global
[CO] (e.g. Röckmann et al., 1998); (4), generation of organic nitrates which participate in ozone formation; (5), the formation of secondary organic aerosols following oxidation (e.g. Hatakeyama et al., 1989; Kavouras et al., 1998) and sesquiterpenes
(Hoffmann et al., 1997; Jaoui et al., 2003); and (6), the increased lifetime of other greenhouse gases, such as methane, as a result of the reactions described in point 2 above. While secondary organic aerosols contribute to the visible effects of haze, it their putative roles as cloud condensation nuclei (Cruz and Pandis, 1997) and influence on the reflection and scattering of solar radiation (Beerling et al., 2007), potentially influencing forest primary productivity (Roderick et al., 2001), that is attracting much attention today. 3 Chapter 1 Introduction
1.3 Ecological and physiological function of volatile organic compounds
Specialised compounds play fascinating ecological and physiological roles in plants.
The long-since debunked waste product hypothesis (Seigler, 1998) has been steadily supplanted by a range of demonstrated functions including endogenous and exogenous signalling, feeding defence, wound healing, protection against temperature extremes and oxidising components of the atmosphere. Each of these roles are fulfilled predominantly, but not exclusively, by low molecular mass & FRPSRXQGV7ZR( of the perhaps most recognised roles for plant volatiles are the production of synomones for attracting pollinating insect and animal species, and allomones, compounds that confer protection to the plant. Both classes may be stored or produced during certain periods of plant development. However, herbivory can also induce a number of defence responses in plants. For example, the response to herbivory can elicit the production of jasmonic acid (and its derivatives), signalling compounds that stimulate a number of chemical defences. For example, insect attack can release the fatty acid linolenic acid, the precursor for jasmonic acid, from membrane lipids (Turner et al.,
2002). Through a set of species-specific biosynthetic steps, jasmonic acid induces the formation of traumatic resin ducts, seen in Picea abies (Martin et al., 2002).
Conversely, application of methyl jasmonate in eucalypts did not alter differences in the composition of constitutive leaf defence compounds or feeding preference by specialist herbivores, concurring with evidence that most perennial evergreen species lack foliar- induced defence chemicals, relying instead on constitutive compounds (Henery et al.,
2008; Rapley et al., 2008). However, in Eucalyptus globulus, wounding induced traumatic oil gland formation in the woody tissue around the wound site in the two years following injury (Eyles et al., 2004). Allelopathic inhibition of competing species is another well documented function, often noticeable in the understorey of eucalypt
4 Chapter 1 Introduction forests (del Moral et al., 1978; del Moral and Muller, 1970; May and Ash, 1990). Less conclusive, but not without empirical bases, are the hypotheses that VOCs confer tolerance to oxidative stress and high temperatures. Numerous lines of evidence suggest isoprene scavenges reactive oxygen species, such as H2O2, produced as ozone enters the leaf, mitigating its accumulation in plant cells (Vickers et al., 2009) and oxidation of membrane lipids (Loreto and Velikova, 2001; Loreto et al., 2001). Isoprene has also been shown to impart a greater tolerance in plants to high temperatures and while the phenomenon has been well documented (Singsaas et al., 1997; Logan and Monson,
1999; Sharkey et al., 2001; Peñuelas et al., 2005), empirical demonstration of the purported mechanism of membrane stabilisation (Siwko et al., 2007) remains elusive.
Many oil-bearing species occur in Mediterranean climates, such as the eucalypts of southern Australia, but also the gymnosperms of North America and the Mediterranean, and their propensity to burn has not escaped notice. The relationship between leaf flammability and terpene content has only recently received attention (Alessio et al.,
2008a,b; de Lillis et al., 2009), but it appears moisture content is as important as terpene content. There is ample ecological evidence that some oil-bearing species of eucalypt, such as E. regnans, require fire for seed germination, yet adults of this same species are sensitive to fire (e.g. Ashton and Martin, 1996). The adaptive implications of leaf flammability and fire adaptation, or indeed dependence, are yet to be fully examined.
5 Chapter 1 Introduction
1.4 Biosynthesis of terpenoid and carbonyl VOCs
The diversity of terpenoids is achieved by myriad branches of two highly localised biosynthetic pathways that each produces the universal isoprenoid precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The first to be identified was the mevalonic acid (MVA) pathway, which was long believed to take place within the cytosol but is now known to be more localised, proceeding within peroxisomes and the endoplasmic reticulum (Sapir-Mir et al., 2008). The more recently discovered methyl-erythritol phosphate (MEP) pathway is similarly localised, being restricted to chloroplasts in higher plants. While the precursors IPP and DMAPP are common to both pathways, it is only in the plastidic MEP pathway that both compounds are produced simultaneously (Bouvier et al., 2005), in a ratio of approximately 5:1
IPP:DMAPP (Altincicek et al., 2002). In the mevalonate pathway, DMAPP is produced solely from IPP.
DMAPP is the substrate from which isoprene and other hemiterpenoids are formed via their respective synthases. Higher terpenoids are also formed from IPP and DMAPP, but this occurs in step-wise condensations catalysed via prenyl transferases specific to each class of compound (Table 1.1). For example, monoterpenes have geranyl-PP
(GPP) as their common precursor, formed when the prenyl transferase GPP synthase binds one molecule of DMAPP, catalysing the cleavage of the pyrophosphate group and adding a molecule of IPP. Similarly, the sesquiterpene precursor farnesyl-PP (FPP) is formed when FPP synthase binds DMAPP and, in two steps, incorporates two molecules of IPP to form FPP. An analogous process occurs for the diterpene precursor geranylgeranyl pyrophosphate.
6 Chapter 1 Introduction
Table 1.1 Isoprenoid classification, examples and putative sites of synthesis in higher plants (adapted from Heldt, 2005).
Precursor Compound Example Known or putative class site of synthesis C5: Dimethylallyl pyrophosphate Hemiterpenes Isoprene Plastid C5: Isopentenyl-PP Cytokinin side chain C10: Geranyl-PP Monoterpenes a-pinene, myrcene, linalool Plastid
C15: Farnesyl-PP Sesquiterpenes Caryophellene Cytosol/ER C20: Geranylgeranyl-PP Diterpenes Gibberellin Plastid C30: 2 FPP Triterpenes Stigmasterol Cytosol C40: 2 GGPP Tetraterpenes Carotenoids Plastid
Isotopic labelling and pathway inhibition studies have been the primary tools used to identify the intracellular location of compound synthesis. Production of isoprene and 2- methyl-3-buten-2-ol (MBO), products that branch directly from DMAPP, are produced from the MEP pathway in all species studied (e.g. Zeidler et al., 1998). A chloroplastic origin has also been identified for IPP incorporated into monoterpenes and sesquiterpenes, although the latter are synthesised in the cytosol (McCaskill and
Croteau, 1995).
Carbonyl compounds, in particular, acetaldehyde, formaldehyde and acetone, are emitted in important quantities (e.g. up to 100 nmol m-2 min-1, Cojocariu et al., 2004) by many species (Kesselmeier, 2001). Comparatively less is known about the biosynthesis of these VOCs, with the exception of acetaldehyde. Alcoholic fermentation in the root system produced by anoxia produces ethanol, which is transported to the leaves via the transpiration stream. Here it is oxidised via alcohol dehydrogenase to produce acetaldehyde, a fraction of which is emitted to the atmosphere (Kreuzwieser et al.,
1999; 2001). Acetone formation may occur in cyanogenic plants via disruption to the
7 Chapter 1 Introduction cell membrane, where the amino acid valine is converted over a number of stages to acetone and hydrogen cyanide (Fall, 2003). Decarboxylation of acetoacetate has also been suggested as a possible source of acetone (MacDonald & Fall, 1993).
Formaldehyde is thought to originate from the degradation of pectin during plant developmental processes such as cell wall expansion and degradation (Kreuzwieser et al 2002). In this pathway, pectin methyl esterase acts on pectin to produce methanol, which undergoes enzymatic conversion to formaldehyde (Giese et al., 1994).
1.5 Mechanisms and rates of VOC emissions from plants
The influence of VOC emissions on atmospheric chemistry and their role in the plant’s carbon balance have driven much of the work to identify the rates, composition and regulating factors of VOC emissions. The constitutive composition of terpenoid VOCs is under genetic control (e.g. Vernet et al., 1986; Dudai et al., 2001; Shelton et al.,
2002), regulated by developmental and environmental factors. Emission rates and regulation, at short time scales in particular, are influenced more by environmental variables (Kesselmeier and Staudt, 1999). Of the many VOCs emitted by plants, the isoprenoids are the best studied. Even within this class of compounds, tremendous variability exists between plant species, and the extent to which the environment controls emissions can only be resolved by examining individual species. Despite this, fundamental principles drive emissions. Isoprene (C5), monoterpenes (C10) and sesquiterpenes (C15), emitted by many plant species, are governed by synthesis rates and diffusional resistances from source to atmosphere. Both processes are influenced to varying degrees by light and temperature, although other factors are involved.
Temperature dependence was identified in the very early days and shown to regulate
8 Chapter 1 Introduction monoterpene emissions from storage pools in an exponential manner (Tyson et al.,
1974). Light dependence was identified in isoprene (e.g. Tingey et al., 1979), but this has since extended to include monoterpenes and sesquiterpenes de novo synthesised in plants (Shao et al., 2001; Dindorf et al., 2005). Isoprene emission is directly coupled to its synthesis which takes place predominantly in the light, although as carbon is derived from more than one source, low night time emissions are commonly observed. In both isoprene and recently synthesised terpenes, light and temperature regulate the activity of enzymes responsible for their production. In addition, light and temperature also regulate their flux through the leaf. Temperature influences both the partial pressure and hence the solubility of a given compound, while light can affect stomatal aperture, regulating the emission of compounds with low partial pressures in the leaf internal air volume (Niinemets et al., 2002b). Monoterpene-emitting plants without storage organs possess small transient pools that can be seen as a slower stage of emission decay when synthesis ceases (Niinemets and Reichstein, 2002, 2003). Absent in highly volatile compounds, stomatal regulation was nonetheless suspected in a number of plant species
(Valentini et al., 1997). Niinemets and Reichstein (2003) postulated the theoretical basis for this control and identified data that supported this role (see references therein).
Other factors such as humidity (Yokouchi and Ambe, 1984; Guenther et al., 1991),
[CO2] (Guenther et al., 1991; Rosenstiel et al., 2003), nutrient availability (Funk et al.,
2006), seasonality (Monson et al., 1994; Pier and McDuffie, 1997), and physical injury
(Sharkey, 1996) have been shown to influence emissions. Water stress in particular has produced varied results on VOC emissions from plants and it is perhaps for this reason that this factor is poorly represented in emissions models. A reduction in emissions has been found in some species (Bertin and Staudt, 1996; Staudt, 1996), while others have produced unequivocal results (Fang et al., 1996; Blanch et al., 2007) or enhanced
9 Chapter 1 Introduction emissions (Staudt et al., 2008). Lavoir et al. (2009) pointed out that many of these studies have been conducted in pots and sought to redress this with a water stress field study in Quercus ilex. These plants did indeed reduce emissions as a result of water stress, and attributed this to deficit in the availability of carbon substrate rather than monoterpene synthase activity.
A number of studies have also identified circadian control of isoprene (Wilkinson et al.,
2006), and monoterpene emissions (Dudareva et al., 2005; Wang et al., 2007) and isoprene synthase activity (Loivamaki et al., 2007) in taxonomically distinct species. In each study there was no direct relationship to light and temperature.
To date, light and temperature remain the basis by which empirically-developed
(Tingey et al., 1991; Guenther et al., 1993) and processed-based (Zimmer et al., 2000;
Niinemets et al., 2002c) models function.
1.6 VOC emissions from eucalypts
With over 800 species, eucalypts are the dominant tree in the Australian landscape, and are found in all but the driest environments. Colloquially know as eucalypts, they consist of three very closely related genera (Eucalyptus, Corymbia and Angophora). In addition to their extensive natural distribution, they are planted widely in Australia and overseas, primarily for pulp and fibre production, or structural timber. Eucalypt leaf oils have for many years been the subject of intense study, primarily for utilitarian purposes. But these studies have documented strong compositional differences between species and ecotypes (e.g. Li et al., 1994, 1995, 1996), environmental conditions (King et al., 2006) and seasons (Simmons and Parsons, 1987). A consistent thread of study has
10 Chapter 1 Introduction examined the ecological and evolutionary basis for essential oil production in eucalypts, and with improvements in analytical techniques, ideas in this area continue to be tested.
Within the plant-based VOC literature, eucalypts have been rarely studied. Eucalyptus globulus was used to develop the widely applied isoprene and monoterpene emission model of Guenther et al. (1991), but beyond this they have featured in only a few field
(Street et al., 1997) and laboratory (He et al., 2000a; Velikova et al., 2007) studies.
Fundamental details such as field-based emission estimates, changes in composition with changing environmental conditions, the source of compounds within leaves and the influence of basic regulating factors such as light and temperature, are yet to be identified. Filling in these gaps will provide far more robust data for regional emissions estimates and provide insight into the mechanisms regulating emissions in this genus.
1.7 Project aims
This thesis examines some of the fundamental aspects of VOC emission from
Eucalyptus globulus Labill. subsp. globulus. The natural range of this species is restricted to small patches of the south-eastern corner of the Australian mainland, areas of Tasmania and adjacent islands (Brooker and Kleinig, 1999), but it is planted widely around the world due to its high growth rate and suitability for pulp (Turnbull and
Booth, 2002). Important as an emissions source in this regard, it may also represent
VOC production and emission processes some of the other many hundreds of species of eucalypt that possess oil glands.
11 Chapter 1 Introduction
This thesis therefore sought to address three questions regarding VOC emissions from eucalypts:
1. What range of compounds and at what rates are terpenoids and carbonyls
emitted from field-grown eucalypts cultivated under conditions of non-limiting
nutrient and soil water availability, and how do environmental factors influence
these emissions?
2. How do temperature and light influence terpenoid emissions from E. globulus,
given there has been no overlap between detailed laboratory-based studies of a
few compounds with field-based studies covering a greater spectrum of
compounds?
3. Do de novo synthesised terpenes make any contribution to emissions from E.
globulus?
To address these questions, an analytical system was built and characterised (Chapter 2) for VOC sampling in later experiments. The composition of oils within leaves (Chapter
3) was characterised as a basis for identifying compounds emitted from leaves.
Emissions from field-grown, adult plants were examined to identify the range and composition of emissions and to ascertain the influence of environmental effects
(Chapter 4). Temperature and light effects in particular were examined in more detail in laboratory-based studies of juvenile leaves (Chapter 5). The presence of de novo synthesised terpenes was determined in a fumigation study in Chapter 6 and finally, the results of this work are drawn together in Chapter 7.
12 Chapter 2 Method development
2. Method development
2.1 Introduction
Biogenic VOCs comprise thousands of compounds that vary widely in their vapour pressure, boiling point, polarity and solubility in water. Once synthesised by plants, their physio-chemical properties influence both the route and rate at which they travel through leaf tissues and into the atmosphere. To a large extent, these properties also dictate the methods used for identification and quantification, that fall into two broad categories.
The first is continuous, online monitoring methods such as Fourier transform infrared spectroscopy (FT-IR) and proton transfer reaction mass spectrometry (PTR-MS). The second is chromatographic methods, mostly gas chromatography (GC), where samples must be collected and concentrated before analysis. Many VOCs can be analysed by more than one technique. In many cases, a second technique is required to confirm results, particularly identification when VOCs may vary by as little as one atomic unit of mass or in their structure. For example, GC is a highly sensitive method, capable of separating and detecting a wide range of compounds depending on column chemistry.
13 Chapter 2 Method development
Column chemistry and detector type also determine the detection and quantification limits for the method. Conversely, GC provides no structural information when used without either a mass selective or infra red detector, and has comparatively long run times of up to 30 – 40 min, providing limited time resolution. PTR-MS on the other hand provides very high temporal resolution and low detection limits, in addition to structural information. However, unless a particular ion has been confirmed to be free of contaminants, the signal from a particular ion may include signals from isobaric species, fragments and adducts of other compounds within the matrix gas. FTIR can also provide highly specific structural information about functional groups, but is limited in time resolution and detection limits. GC has also been coupled to PTR-MS to separate analytes (e.g. de Gouw et al., 2003), but this is at the expense of temporal resolution.
In this chapter, both PTR-MS and GC were used to identify and quantify biogenic VOC emissions from leaves of E. globulus. The rationale for their use in this task, as well as the development and application of the specific techniques, is described here. Also described here is the combined sampling system, incorporating all instruments used in the laboratory-based experiments described in Chapters 5 and 6.
14 Chapter 2 Method development
2.2 Design and construction of an automated GC sampling system
2.2.1 Introduction
The impetus for the design and construction of an automated sampling system coupled to a GC came about following the field work described in Chapter 4. In that series of field campaigns, cartridges were used to sample terpenoid and carbonyl compounds from eucalypts at a number of sites in southern Australia. Following sampling, cartridges were shipped overseas for analysis. The major benefit of this approach was that limited resources were needed and deployed in the field, allowing sampling at a larger number of sites. However, and as is often the case, we had a limited number of cartridges that could be analysed at the Research Centre Karlsruhe, Garmisch-
Partenkirchen. This constrained both temporal and spatial sampling. These limitations and the loss of samples due to contamination and breakage, left considerable gaps in the data and compromised output. Similar problems have been previously identified and storage and transport can diminish sample integrity through sample loss, degradation or contamination (Ballesta et al., 2001; Apel et al., 2003).
A major problem with this style of research is that deficiencies in the data set cannot be identified until well after the field-work has been completed. Hence there was a need to design and build an analytical system that could routinely condition, desorb and analyse cartridges either in the field or laboratory. This section describes the system I built to do this.
Sampling and analysis of biogenic hydrocarbons by GC involves three major steps: collection, separation and detection. Samples are commonly collected on adsorbent cartridges or in evacuated canisters. In the laboratory, VOCs adsorbed onto the
15 Chapter 2 Method development cartridges are desorbed and refocussed on a cryogenically-cooled intermediate trap.
Ballistic heating of the intermediate trap then releases a pulse of analytes onto the head of the GC column. Analysing samples on-site can avoid some of the collection, storage and transport problems previously identified. One common field technique involves coupling an automated sampling system to a portable GC. All applications of this technique rely on a solid adsorbent trap cooled to sub-ambient temperatures during sampling, followed by rapid heating to desorb analytes onto the column (see Helmig,
1999; Tanner et al., 2006, for reviews). Cryogenic cooling offers superior trap and oven temperature control, providing greater flexibility in column selection, but has high operating and maintenance costs (Tanner et al., 2006). Liquid CO2 and electrical cooling provide popular alternatives, but electrical cooling requires the least maintenance. Cooling using Peltier thermoelectric modules has been used to sample methane and C2 – C5 compounds (e.g. Harrison et al., 2000; Tanner et al., 2006), as well as biogenic terpenoids (Harrison et al., 2001a,b).
2.2.2 Initial configuration of the GC
An SRI 8610C gas chromatograph obtained from SRI GC (Torrance, CA, USA) included a flame ionization detector (FID), a heated, on-column injector, a 4-channel data acquisition system and 10-port heated injection valve (Figure 2.1). Hydrogen was supplied by an H2-50 hydrogen generator (SRI GC), at a maximum rate of 50 mL H2 min-1. This was enough to supply both the carrier gas and FID combustion gas. Air for the FID was delivered by a built-in air compressor at approximately 250 mL min-1. The
16 Chapter 2 Method development
5 1 2
3 4
Figure 2.1. The original configuration of the SRI gas chromatograph (GC). 1, GC chassis; 2, GC oven; 3, a combined FID and electrocatalytic detector (ECD, not included in our system); 4, heated 10-port valve.
5, housing for the TO-14 VOC sampling traps. The air sampling pump and H2 generator are not shown. Source: SRI GC.
system included the SRI TO-14 Air Concentrator sampling system, consisting of two sorbent traps arranged in series, each housed in an aluminium heater block fitted with
100 W cartridge heaters. An external vacuum pump, operated by the GC control software PeakSimple, drew air through the sampling traps. The single-bed traps (3.175 mm O.D. x 150 mm long) were pre-packed with approximately 120 mg of adsorbent material. The factory-configured CG and sampling system was controlled by the
PeakSimple 3.44 software package.
2.2.3 Initial modification and proof of concept tests
The capacity for an adsorbent material to remove a given compound from a gas stream is dependent on its affinity for that compound, the sorbent’s effective surface area and the pressure and temperature of the gas (Shaw, 1992). The mass of a given compound
17 Chapter 2 Method development that can be stripped from an air stream depends, in addition to the factors above, on its concentration in the matrix and the mass of adsorbent material used. Temperature plays a fundamental role in all physical and chemical processes involved in adsorption (Shaw,
1992).
In the original, manufacturer-supplied GC configuration, the trapping system suffered from flaws that resulted in poor repeatability and chromatography. The first was the inability to control trap temperature during sampling. During initial testing, fluctuations in ambient temperature during sampling or between samples had a clear influence on the reproducibility of sampling. A second problem was the slow rate (3-4 minutes) at which the trap heated to desorption temperatures of 250 – 280 °C. This resulted in poor peak resolution, particularly for low boiling point compounds. To rectify this, and keep the sampling system in its original configuration, an intermediate trap was required at the head of the column. An alternative approach was to redesign the trap to improve temperature stability during sampling while increasing trap heating rate during desorption. Automation of these processes could also improve reproducibility.
The initial approach relied as much as possible, on the original configuration. One of the two traps was removed leaving a single adsorbent trap containing Tenax GR. A new heater block was built to house a 100 W cartridge heater that could increase the rate of heating. With two heaters operating, the trap desorption temperature could be reached in around 50 s.
An electrically-powered cooling system was designed and built to reduce and stabilise the temperature of the VOC trap during sampling. After a number of trials, the cooling system was fixed to one side of the heater. The cooling system consisted of two Peltier modules, side by side, that transferred heat from the trap and heater block assembly to a
18 Chapter 2 Method development fan-cooled heat sink. When tested in isolation the trap temperature could be held at -20
°C. Unfortunately, when the cooling and heating components were assembled, the heat sink drew too much heat from the trap that consequently failed to reach the desorption temperature set point. The assembled system is shown in Figure 2.2. A new design, described below, was built to meet system requirements for heating and cooling.
2.2.4 Final configuration
A second and final configuration was based in part on that developed by Tanner et al.
(2006), who modified a similar GC for automated sampling of C2 to C6 VOCs. A single trap was again used (Figure 2.3), consisting of a smaller stainless steel (SS) tube,
1.83 mm internal diameter (I.D.) and 2.11 mm outer diameter (O.D.), (HTX-14TW,
Small Parts, Florida, USA). The trap was packed with 60 mg of Tenax TA and 30 mg of
Carbopack X (Sigma Aldrich, Sydney, Australia). These materials have been widely used in combination for trapping biogenic C5 to C10 VOCs emitted by vegetation
(Steinbrecher et al, 1994). Small wads of silanised glass wool (Alltech Associates,
Sydney, Australia) held the adsorbent in place. The trap was connected to the GC with a straight 1/8” SS union at one end and a 1/8” SS tee-union (all fittings from Swagelok
Pty Ltd, Sydney, Australia). Custom made single-piece ploytetrafluoroethylene (PTFE) ferrules were used to seal the smaller diameter trap tube to the fittings. Temperature feedback from the trap was monitored with a sheathed, electrically isolated Type-K thermocouple (443-7967, RS Components, Sydney, Australia) embedded within the adsorbent material. This thermocouple provided temperature feedback for the heating and cooling phases of the trap and was fed into the trap through one arm of a tee-union
19 Chapter 2 Method development
12345678
Figure 2.2. The first attempt at a temperature-controlled trap. The system was mounted onto the side of the GC, using the space occupied by the initial system. The fans (1) and heat sinks (2) were on the outside of the system, coupled to a large copper plate (3). The Peltier modules obscured from view by foam insulation (4) drew heat from the aluminium block (5) surrounding the trap. Cartridge heaters (6) supplied heat to the trap and the system was insulated and enclosed by a lid. A fan (7) mounted in the lid (8) help remove heat from the insulated area in the transition between heating and cooling.
20 Chapter 2 Method development
(the remaining arm being for gas flow) and sealed airtight with a graphite ferrule. As the trap was resistively heated using electrical current, it had to be electrically isolated from the rest of the GC chassis. The trap itself was isolated by a coating of PTFE (170-
6282, RS Components, Sydney, Australia), that functioned effectively and safely at up to 300 °C. Short lengths of PTFE tubing placed between the trap and the GC gas lines were also used to electrically isolate the trap from the GC chassis.
The final configuration retained the Peltier modules and fan-cooled heat sinks for cooling the VOC trap. Two copper plates, 3 mm thick x 100 mm long x 40 mm wide, each with a groove running the length of the plate to sandwich the trap, were made and formed the cold plates from which the Peltier modules drew heat. On each side of these two plates were positioned high temperature Peltier thermoelectric modules (HT-8-12-
F2-4040, Econocool Aust Pty Ltd, Cleveland, Australia). During operation, heat was drawn from the cold side of Peltier modules and transferred to larger copper plates that in turn transferred the heat into fan-cooled heat sinks (Swiftech MCXC-370, CoolPC,
Brisbane, Australia). The ‘cold’ components of the system, the trap and cold plates, were mounted in a sealed, insulated box to limit the influx of heat and the volume of air that had to be cooled.
A water trap, of a similar design to the adsorbent trap, was placed upstream to prevent water freezing in the adsorbent trap during sampling. The water trap (Figure 2.4) used two 6.35 mm O.D., 100 mm lengths of SS tube. A single piece of 6.35 mm I.D. PTFE tubing was threaded through both SS tubes, allowing the tubing to be configured into a
‘U’ shape with the SS tubing positioned on each arm of the ‘U’. The SS tubes were sandwiched between copper plates 4 mm thick x 75 mm x 45 mm. Silanised glass wool was loosely packed into the second end of the ‘U’ to provide a greater surface area for trapping vapour. A Type-T thermocouple made from 30 AWG thermocouple wire
21 Chapter 2 Method development
W
Figure 2.3. Elevation view of a partly assembled adsorbent VOC trap. 1, heat sink assembly including fan (fan has been removed from bottom heat sink); 2, copper heat sink; 3, copper spacer; 4, Peltier module; 5, stainless steel tubing containing adsorbent material; 6, copper cold plates sandwiching the SS tubing; 7, insulating foil-lined foam. The white paste (W) visible between the copper cold plates and the Peltier modules is thermal conducting paste. Electrically isolating PTFE heat shrink is just visible where the SS tube exits the copper plates at each end. Fittings and cables passing current from the car battery through the trap have been removed, but connect to the trap immediately after the PTFE heat shrink. Four insulated panels are fitted to the assembled trap enclosing the volume between the copper plates (2), limiting the influx of heat via convection and conduction during the cooling phase. Fittings connecting the trap tubing to the GC plumbing and the thermocouple fitting are removed for clarity.
22 Chapter 2 Method development
(Onetemp Pty Ltd, Sydney, Australia) was sandwiched with the tubing between the copper plates to provide feedback for temperature control. Water vapour was removed via condensation from the sample stream by holding the trap at -20 °C during sampling.
2.2.5 Programmed control of the automated sampling system
The VOC and water traps were controlled with a National Instruments Compact
FieldPoint (cFP-1804) system that consisted of a digital output module (cFP-DO-401), a thermocouple input module (cFP-TC-120) and pulse width modulated digital output module (cFP-PWM-250; National Instruments, Sydney, Australia). The electrical operation of these devices is described below, followed by a description of the software and sampling sequence.
The VOC trap was resistively heated to desorption temperature via current from a 12 V,
35 A h deep cycle battery passed through the stainless steel tube of the trap.
Temperature was controlled by varying the amount of current passing through the trap.
Current was controlled by varying or modulating the width of a digital pulse generated by rapid switching of the current supply. One of the eight channels of the cFP-PWM-
520 module was used to rapidly switch a solid-state relay (SY-4086, Jaycar Electronics,
Canberra, Australia; used for all solid state relays) according to temperature feedback from the Type-K thermocouple embedded in the trap. Pulse width modulation is highly effective in controlling processes where a rapid response to an output stimulus is needed. Early testing of the completed system showed the trap could be heated to
>1440 °C (the limit of the thermocouple) in less than 1 s.
23 Chapter 2 Method development
5
6 3 7 4
1 2
Figure 2.4. Layout of the water trap. 1, entry; 2, exit; 3, cold plates; 4, Peltier modules; 5, U-shaped section; 6, trap insulation; 7, heat sink assembly. Water vapour from the sampled air stream passed twice through the cold section of a trap, the second arm of which was packed with silanised glass wool. After sampling, the trap is heated to 70 °C to by reversing the polarity on the Peltier modules. trapped water is flushed from the air lines by reversing the polarity on the modules, allowing the trap to be heated to 70 °C. In this image the trap is assembled, showing the prominent fans and heat sinks used to dissipate heat during trap cooling.
24 Chapter 2 Method development
Power to the Peltier modules for cooling used the same module and relay configuration and power was supplied from a 12 V, 30 Amp regulated power supply. A second 12 V,
30 Amp regulated power supply provided power for the Peltier modules used to cool the water trap and the control system was configured identically to the cooling system of the adsorbent VOC trap. Water that had accumulated in the trap during the sampling phase was removed by heating. The polarity of the Peltier modules was reversed using a double pole-double throw (DPDT) mechanical relay and activated by one channel of the cFP-DO-401 digital output module. This reversed heat exchange (trap rose to 70 °C), when combined with the continuous air-flow, removed all condensed water.
2.2.6 Software
A program was written using LabVIEW 8.5 (National Instruments, Sydney, Australia) to automate the sampling and analysis system. PeakSimple retained control of the GC, but the cyclic operation of the entire system was controlled and initiated by the
LabVIEW program. The cooling and heating of both traps was coordinated with the operation of the GC according to timing and temperature settings entered by the operator. Temperature set points throughout the sequence were reached and maintained using proportional-integrative-differential (PID) algorithms provided within the software. Variations of these algorithms are used in almost all process control applications; the three-component algorithm being useful where rapid and large system responses were needed. The algorithm was tuned for the system in a specific state or operating environment. Heating of the VOC trap was the most time- and temperature- critical process in this series of operations. The design of the system was such that temperature was consistently held within 0.07% of the set point.
25 Chapter 2 Method development
2.2.7 Sampling sequence
Sequence timing and temperature set points (Figure 2.5) were assigned by the operator prior to commencement of a sampling sequence, and correspond to the system components in Figure 2.6. A typical sampling sequence would begin with the VOC trap in standby at 70 °C; continuously purged with carrier gas but isolated from the sample air stream. At this stage, the GC oven was at or cooling to 40 °C, while the water trap was at its standby temperature of 25 °C. Upon commencing the sampling sequence, the
VOC and water traps cooled to their sampling temperatures (-10 and -20 °C respectively). Approximately five minutes was allowed for the traps to reach their sampling temperature, after which a heated 10-port valve on the GC was switched, redirecting sample air flow onto the adsorbent trap at 75 mL min-1. After the sampling period was complete (15 min), the 10-port valve was used to redirect carrier gas through the trap (in the opposite direction to sampling flow) in order to flush it free of oxygen. Trap cooling then ceased and it was instead rapidly heated to 280 °C over 12 s and held there for 60 s, sweeping analytes directly onto the GC column. While faster rates of heating were possible, Tanner et al. (2006) found optimum recovery of analytes when the trap was heated to its desorption temperature over 10 - 20 s. The trap was then allowed to cool to 75 °C, where it was held until near the end of the GC run, when both the GC column and trap were simultaneously baked to remove any remaining traces of analytes. The baking cycle involved holding the trap at 290 °C for 60 s. After the 15 minute sampling period, the water trap temperature was increased to 70 °C and held there for 23 min before returning to its standby temperature of 25 °C.
26 Chapter 2 Method development
Figure 2.5. Coordination of temperature programs for the GC oven, and VOC and water traps. The system was designed to operate continuously and the first 3 – 4 min show the GC oven completing its baking period before returning to the cycle-start temperature of 40 C. Although the programmed temperature drops suddenly, the oven required 10 min to return to the designated start temperature. During oven cool-down, the VOC and water traps approach return to their programmed set points before sampling starts. A heated 10-port valve on the GC is used to direct flow onto the VOC trap. As sampling nears completion, the GC oven is ready for another run. Following rapid desorption of analytes from the trap, both the water and VOC traps spend most of the GC run time at ~ 70 °C to remove analytes from their adsorptive surfaces. The VOC trap is baked a second time while the GC oven is at 240 °C.
27 Chapter 2 Method development
Figure 2.6. Schematic of the GC system with water and adsorbent VOC trap. P1, pump drawing air from sample stream and operating continuously; P2, pump drawing air through VOC trap and activated during sampling; MFC1 and MFC2, mass flow controllers. All tubing within the GC is stainless steel with the exception of short lengths between the VOC trap and the 10-port valve, which along with the remaining tubing is made of PTFE. The valve is shown in the ‘LOAD’ position with carrier gas flowing through the trap and onto the column.
28 Chapter 2 Method development
2.2.8 Trap performance
The safe sampling volume of the trap was determined to ensure analytes would not be lost due to saturation. The adsorbents selected for the trap have been used previously for sampling biogenic VOC (e.g. Steinbrecher et al., 1994). The likely concentration of terpenoid compounds were estimated during preliminary work and a gas standard containing propane, isoprene and camphene was used to determine the safe sampling volume. At typical concentrations, isoprene and camphene were quantitatively retained at volumes in excess of 2.5 L (Figure 2.7). ‘Breakthrough volume’ is usually defined as that when a compound at a given concentration is no longer retained on the trap. For example, breakthrough volume for propane was > 1.75 L. Sampling volumes were thus conservatively set at 1.2 L. This ensured avoidance of breakthrough volume of the most volatile calibration gas, and remained well within the safe range for all known compounds of interest. Breakthrough was further tested by collecting exhaust gases from the trap into a Tedlar bag. In this arrangement, the bag was placed into an air tight
Pelican™ case and the bag inlet connected to the trap exhaust. A pump and flow meter were used to evacuate the case, drawing sample air through trap and into the second bag at the same flow rate used previously. Reanalysis of the bag contents found no detectable terpenoid compounds.
29 Chapter 2 Method development
600
500
400
300 Peak area Peak 200
100
0 00.511.522.53 Sampling volume (L)
Figure 2.7. Determination of the trap breakthrough volume for representative mixing ratios of 3 gas standards of propane (Ƈ LVRSUHQH Ÿ) and camphene (Ŷ
2.2.9 Gas chromatography
After the sampling volume was reached (determined by time and flow rate), the trap was rapidly heated and compounds flushed onto the head of the GC column. The SRI
8610C GC was fitted with a fused silica capillary column (60 m x 0.32 I.D.) with a 100
% polydimethylsiloxane stationary phase (1μm film thickness, BP-1, SGE Scientific,
Melbourne, Australia). The column was temperature programmed from 40 °C (held for
-1 -1 5 min) to 150 °C at 7.85 °C min and then at 18 °C min to 240 °C (held for 7 min. H2 from the H2 generator (~ 83 kPa, 12 p.s.i.) was passed sequentially through a charcoal filter to remove hydrocarbons and an oxygen scavenger before flowing into the column and FID. Air flow to the FID was 250 mL min-1.
30 Chapter 2 Method development
2.2.10 GC calibration and detection limits
Compounds were identified by comparing retention times with authentic liquid standards. These were introduced onto the trap in concentrations similar to those found during collection of plant-emitted VOCs by adding 1 – 2 μL of liquid standard to a 1 L
Tedlar® bag (Sigma-Aldrich, Sydney, Australia) filled with ~ 1 L of dry ultra high purity nitrogen (BOC Gases, Sydney, Australia).
Quantitative calibration was performed with an eight component gas standard containing acetaldehyde, methanol, acetonitrile, acetone, isoprene, 2-butanone, benzene and camphene, each at 5 ppm ± 5% in N2 (Apel-Reimer Environmental, Colorado).
However, isoprene and camphene were the only terpenoids in the standard, and other terpenes emitted from E. globulus were calibrated against the camphene standard and corrected using effective carbon number. Relative response factors for compounds were determined from effective carbon number (Henrich, 1995). The response factor of a compound in the FID is the ratio of the signal generated per unit mass of carbon.
Aliphatic and aromatic carbons have an effective carbon number (ECN) of 1 while heteroatoms (O, S, halogens, etc.) influence the response in a predictable manner
(Henrich, 1995 and references therein). Terpenoid emissions from E. globulus consist mainly of aliphatic, aromatic and olefinic compounds, with heteroatoms present in only a few compounds, such as the ether bond in eucalyptol. Instrument drift was monitored with frequent two-point calibration using a propane standard (2 ppm ± 0.2 % propane in nitrogen, BOC Gases).
Mass flow controllers (Section 2.4.3) were used to mix the propane standard with dry nitrogen to generate propane concentrations in the range 6 – 190 ppb, over linear range
31 Chapter 2 Method development of detector response. The instrument limit of detection (IDL) was 0.326 ± 0.016 ppb
(Equation 2.1),
ܼ. ݏ ܫܦܮ = ܵ
(2.1)
where z is the signal-to-noise ratio set to 3, sblank is the standard deviation of seven blank runs and s is the sensitivity of the signal response to changes in analyte concentration.
32 Chapter 2 Method development
2.3 Proton transfer reaction – mass spectrometry
2.3.1 Introduction
Proton transfer reaction – mass spectrometry (PTR-MS) has been widely applied in the last 15 years to the analysis of VOCs, including plant biogenic VOCs (e.g. see Tholl et al., 2006 for its application to plant analysis). In contrast to chromatographic methods,
PTR-MS allows on-line monitoring of VOCs within complex gas mixtures. This is
+ made possible by chemically ionising the analytes with hydronium (H3O ) reagent ions.
PTR-MS has two important advantages. The first is limited fragmentation of analytes and thus simpler and more easily interpreted mass spectra. Secondly, only compounds that have a proton affinity greater than that of water (691 kJ mol-1) react. Hence, major constituents of atmospheric air, N2, O2, Ar and CO2 are not ionised and do not interfere with the analysis of constituent VOCs (Lindinger et al., 1998). In addition, quantification is possible from first principles, provided conditions within the reaction chamber are well defined. With some generally well understood limitations, the technique can be used to monitor trace concentrations (ppb-ppt) of volatile organic compounds with a very high time resolution and sensitivity. This feature is particularly useful for investigating physical and chemical processes over relatively short time scales. This is beyond the resolution of most chromatographic methods. The instrument used in this thesis is the high sensitivity version of the PTR-MS produced by Ionicon
Analytic GmbH, Innsbruck, fitted with a third turbomolecular pump in the mass filter/detector region for greater vacuum (10-7 torr).
33 Chapter 2 Method development
2.3.2 Instrument operation
The PTR-MS consists of four major components: a hollow discharge ion source, drift
+ tube, quadrupole analyser and ion detector (Figure 2.8). The ion source produces H3O from water vapour by bombarding water molecules with electrons generated from the hollow cathode discharge in the following reaction sequence:
- + - H2O + e H2O + 2e (2.2)
+ + H2O + H2OH3O + OH (2.3)
+ The ion source generates the reagent ions (H3O ) from water vapour delivered from a reservoir of distilled water at 4 – 10 mL min-1. A proton is transferred from the reagent ion to those compounds that have a proton affinity greater than water (Reaction 2.1):
+ + H3O + VOC VOCH + H2O (2.4)
When the reactive components of air are present in trace quantities (< 5%) and the
+ + + number density of H3O , [H3O ], greatly exceeds that of the product ions [VOCH ], the density of the product ions can be determined by
+ –k[R]t + [VOCH+] = [H3O ]0(1 – e ) §[H3O ]0[R]kt (2.5)
+ where [H3O ]0 is the number density of the reagent ion in the absence of reactant
+ molecules R, k is the compound specific reaction rate constant for protonation by H3O and t is the average time ions spend in the drift tube. The proton transfer reaction rate k is unknown for many compounds. In this thesis, a default value of 2.0 x 10-9 cm3 s-1 is assumed. A number of other products are produced during or subsequent to ionisation but under normal operating conditions most can be ignored as they quickly form hydronium ions. Dimolecular oxygen can provide a serious source of error, resulting in
34 Chapter 2 Method development
+ + a charge transfer reaction from H2O to O2, producing O2 . This in turn undergoes charge transfer reactions with neutral species and contaminates the spectrum.
Maintaining the oxygen signal at 0.5 – 2 % of the primary ion signal limits the effect of this process.
The proton transfer reaction is mild compared to electron ionisation. Many molecules can be identified by their protonated molecular mass [VOC + H]+. While largely a non- dissociative process, there is some fragmentation depending on the compound and the conditions within the drift tube. The degree of fragmentation can be influenced by settings applied to the drift tube; namely the field strength, pressure and temperature.
These settings, particularly the ratio of field strength E (usually in the range 50 - 60 V cm-1) to number density of the buffer gas N, influence the formation of cluster ions
+ H3O Ɣ(H2O)n which also act as reagent ions, resulting in a spectrum that is difficult to interpret. Standard operating conditions typically involve an E/N ratio of 120 – 140 Td
(Hewitt et al., 2003). 126 Td was used in the work presented here. Higher field strengths effectively suppress the formation of clusters but in turn reduce the reaction time and sensitivity of the instrument. Conversely, lower E/N ratios increase cluster formation but also increase sensitivity (Hewitt et al., 2003, Hansel et al., 1995).
35 Chapter 2 Method development
Figure 2.8 Layout of the major components of the proton transfer reaction mass spectrometer. TMP, turbomolecular pump; Q, quadrupole; SEM, secondary electron multiplier. Source: High sensitivity PTR- MS manual, Ionicon Analytic GmbH.
2.3.3 Calibration, sensitivity, detection limits and transmission efficiency
A brief description of the PTR-MS approach used in this thesis follows. Two factors, transmission curves and fragmentation patterns, are discussed in particular as they are key steps in the quantification process.
Quantifying VOCs using PTR-MS follows the same principles of trace analysis used in other analytical methods. Calibration against a known gas standard is the preferred method. However, unique to the PTR-MS is a semi-quantitative method based on the
36 Chapter 2 Method development first-order reaction kinetics in the drift tube (Taipale et al. 2008, and references therein;
Ammann et al. 2004; Steinbacher et al. 2004). The PTR-MS was initially touted as not needing external calibration, as ion-molecule interactions were so well understood that volume mixing ratios (VMRs) could be determined from theory alone (Lindinger et al.,
1998). It is now known that this is not the case and that calibration with an external standard is essential. Nevertheless, theoretical determination of VMRs remains an essential step as calibration gases, including isotopologues, are unavailable for many compounds.
Calibration with a standard gas accounts for all processes from the entry of the analyte into the instrument to the response generated at the detector. The error associated with this is the sum of errors associated with gas standard and flow measurement. In the absence of a calibration gas, the VMR of a compound can be determined from theoretical principles. The same eight component mixed used to calibrate the GC was also used for the PTR-MS. Calculations used to determine VMRs for all compounds, including sensitivity, detection limits and calibration curves were calculated according to the manufacturer’s guidelines.
The detection limit for PTR-MS is influenced by the background signal for a particular mass. These signals can be determined by passing sample gas through a catalytic converter to destroy hydrocarbons, or through selectively bypassing components of the sampling system that might be a source of VOCs (for example, using ultra pure cylinder air). Both techniques were used in this work. Pt-coated alumina pellets (Sigma-Aldrich,
Sydney, Australia) were held in a stainless steel tube heated to > 450 °C through which the air stream was periodically directed. There was no detectible change in signal when zero air was passed through the converter or run directly into the PTR-MS.
37 Chapter 2 Method development
Transmission efficiency is an important consideration in many linear mass spectrometers. It refers to the fraction of ions that are transmitted from the ion source, through the mass filter, to the ion detector (Boyd et al., 2008). Linear quadrupole mass filters generally show lower transmission efficiency for heavier ions as they spend more time in the fringing fields of the filter and are thus subjected to a greater degree of dispersion (Steinbacher et al., 2004). Transmission efficiency of PTR-MSs incorporates those processes that result in compound or signal loss between the drift tube and the detector. Clearly, efficiency depends on conditions that may vary according to experimental design and condition of the instrument. Detector age; temperature, humidity, and pressure are all variable, and all contribute to efficiency. It is important that transmission be determined at frequent intervals (e.g. Taipale et al., 2008).
Transmission efficiency must be considered when determining VMRs from first principles (theoretical VMR). Although the processes that contribute to mass discrimination are understood, we still lack a theoretical basis for quantification of mass discrimination (Taipale et al., 2008, Ammann et al., 2004), and transmission curves must be determined experimentally using non-fragmenting compounds. Two methods are recommended: the use of a calibration standard, against which the calculated concentration is compared; and the use of pure liquid standards, where the increase in a compound’s signal relative to a standardised reduction in the primary ion signal indicates transmission efficiency. The former is operationally simpler and incorporates information about the proton transfer reaction rate, which offsets instrumental error.
The gas calibration mix used in these studies did not cover a wide enough range of masses and included compounds susceptible to fragmentation. Liquid standards were used instead, applying the Tedlar bag method described for GC calibration above and as applied to PTR-MS transmission curve determination by Ammann et al. (2004).
38 Chapter 2 Method development
Five compounds were selected to determine transmission efficiency (with protonated masses): acetonitrile (m/z 42), toluene (m/z 93), 1,2,4-trimethyl benzene (m/z 121), p- cymene (m/z 135) and camphor (m/z 153). A representative transmission curve is shown in Figure 2.9, along with the original transmission curve provided by the instrument manufacturer. The geometry of the curves is somewhat different, but as there is no theoretical basis for their geometry, the reasons for this are uncertain. Transmission curves tend to carry a considerable degree of temporal uncertainty (Ammann et al.,
2004; Steinbacher et al., 2004) and VMR determined by calculation typically produce errors in the range of 10 – 30% (Hewitt et al., 2003; Ammann et al., 2004; Steinbacher et al., 2004).
1.6
1.4
1.2
1
0.8
0.6
0.4
Normalised transmission Normalised transmission factor 0.2
0 0 20 40 60 80 100 120 140 160 180 m/z
Figure 2.9 Representative transmission curve (black line) determined from a calibration gas mix covering the range of mass to charges m/z ratios (essentially the ionised compound mass) examined in this study (black diamonds, mean ± SD, n = 3). For comparison, the original transmission curve for the same mass range provided by the instrument manufacturer (broken grey line).
39 Chapter 2 Method development
2.3.4 Compound fragmentation
As the PTR-MS includes no chromatographic step, the signal observed is the sum of all of the compounds contributing to a particular mass. This includes the molecular ion and fragments from other compounds present in the sample stream. In the linear quadrupole version of the instrument, this effect, unless well characterised, can make interpretation of the mass spectrum or selected ion signal rather difficult. The degree of fragmentation depends on the dipole moment and polarisation of a given compound, as well as the operating conditions within the drift tube, including humidity of the buffer gas, the E/N ratio and the presence of oxygen. The latter can be controlled to some extent to enhance the signal of a particular compound class, but as mentioned above, it is a trade off between competing effects.
In determining the mass discrimination effect, the fragmentation patterns of various compounds were examined to identify suitable candidates. In addition, fragmentation patterns of representative compounds emitted by leaves of E. globulus were also examined. In these series of studies, drift tube temperature was 40 °C, drift tube pressure was 2.11 mbar and E/N was 126 Td and conditions were held constant for all experiments.
Mass spectra of representative compounds are presented in Appendix 1. Methanol is often used to determine transmission as it is believed not to fragment (e.g. Taipale et al.,
2008). In scan mode the scan range has an effective lower limit at m/z 21 as the primary ion signal is found at m/z 19; scanning at this mass would cause the detector to rapidly age. However, monitoring in selected ion mode revealed methanol does indeed have a
+ signal from the CH3 fragment (m/z 15), but this amounted to only 0.5 % of the primary
40 Chapter 2 Method development methanol signal and ignoring this fragment would have little effect on determining the signal response for methanol.
The fragmentation of the terpenoids has attracted some attention given the common occurrence of these compounds in plants and the difficulty in identifying individual compounds within complex mixtures with the PTR-MS. Papers examining the fragmentation of monoterpenoids include those by Holzinger et al. (2000), Maleknia et al. (2007), Ruuskanen et al. (2006), Steeghs et al. (2007) and Tani et al. (2003, 2004).
However, the spectra of a range of compounds reported by Maleknia et al. (2007) vary considerably from the literature and those reported here. This is of considerable interest as the same instrument was used to perform the experiments described in this thesis.
Maleknia et al. (2007) report the formation of dimers and novel fragments hitherto unreported despite the considerable study these compounds have received in the literature. Examples include the finding that the most abundant ion for isoprene was the fragment at m/z 39 and significant formation of hydrated dimers (i.e. greater in mass than the parent ion) in methanol ethanol, acetylacetone, acetone and isoprene. The degree of fragmentation of the lower alcohols, as well as isoprene and monoterpenes is strongly affected by the ratio E/N. The authors did not explicitly report the E/N, but a reduction of the drift tube voltage, which is in part a proxy for the E/N ratio, increased the primary ion signal for many terpenes. This phenomenon is well known and has been studied in detail elsewhere (e.g. Tani et al., 2003), as has the importance of vapour pressure on monoterpene fragment ion formation (Tani et al., 2004). In the work reported here, numerous attempts were made to produce VOC concentrations within the linear range of the instrument (for the purposes of determining mass discrimination and calibration) using a variety of methods. Comparing the spectra produced from these early attempts with those obtained at appropriate concentrations (ppt – several hundred
41 Chapter 2 Method development ppb) indicated that the anomalous results of Maleknia et al. (2007) result from standards used at too high a concentration. Examination of the methods used to introduce standards into the system confirms this. For example, dimer formation in isoprene was induced by increasing the concentration of ~1 μL of neat liquid standard into a 1 L bag to a 4 μL injection (Figure 2.10). This also increased the formation of unusual fragment ions at m/z 41, which became the most abundant ion, in addition to the formation of the
(2M + H)+ dimer and the loss of fragments from the dimer. These patterns were repeated in other compounds over-concentrated in the sample bags. The advice of
Maleknia et al. (2007) to reduce the drift voltage (E/N ratio) to enhance the molecular ion signal is indeed correct, but results in an unnecessary loss of sensitivity when in fact the exceedingly high concentrations used were the problem. Different instruments, standards and conditions will inevitably produce differences in the fragmentation spectrum of these compounds between studies. Despite this, the spectra of the compounds studied here are essentially comparable with other reports in both ion fragments and their intensity.
42 Chapter 2 Method development
Figure 2.10. Comparative fragmentation spectra of isoprene (inset) without (A) and with (B) dimer formation, evident at m/z 137. The PTR-MS was operated at 40 °C with an E/N of 126 Td.
43 Chapter 2 Method development
2.4 Gas exchange system
2.4.1 Cuvette and plumbing layout
Experiments to determine the exchange of VOCs, water vapour and CO2 from leaves of
E. globulus were conducted using dynamic, flow through cuvettes. The field study described in Chapter 4 used a different system to that used in the following laboratory- based studies (Chapters 5 and 6) and this is elaborated in Chapter 4.
In the laboratory-based studies, the cuvette system (kindly provided by Jogi Schnitzler of the Research Centre Karlsruhe GmbH) consisted of an aluminium frame with
Plexiglass covers. The top layer comprised a Plexiglass bilayer to allow water from a temperature-controlled bath to flush through the lid and control leaf temperature.
Aluminium surfaces were coated with Tedlar film to limit adsorption effects and two 12
V fans mixed the air within the cuvette.
PTFE tubing was used throughout with stainless steel valves, unions and fittings. Mass flow controllers and other components that provided potentially large surface areas for compound adsorption were placed downstream of the sampling point to limit this effect.
Air supplied to the cuvette comprised zero grade air (21% O2, 79% N2, BOC Gases,
Sydney, Australia) mixed with medical grade CO2 using mass flow controllers to deliver into the cuvette with a CO2 mixing ratio of 400 ppb. Air was humidified to 30 %
RH by passing the mixed air through a bubbler (containing water filtered by a MilliQ™ water filter) held on ice at 3 °C. The general layout of the plumbing system is shown in
Figure 2.11. Other details specific to each experiment are given in the relevant chapter.
44 Chapter 2 Method development
Figure 2.11. Plumbing diagram of the sampling train used in the laboratory experiments. Not all components were used in all studies and these are indicated in the relevant section of the thesis.
2.4.2 Gas exchange calculations
In laboratory experiments using custom made cuvettes, a CIRAS II infra red gas analyser (IRGA, PP Systems, Hertfordshire, UK) was used to determine the volume mixing ratios of water and carbon dioxide. The concentration of H2O and CO2 were determined by the IRGA and plant gas exchange of these gases calculated according to the equations laid out for this instrument (PP Systems, 2002). Calculations for the exchange of all gases were corrected to standard temperature and pressure.
45 Chapter 2 Method development
2.4.3 Calibration of mass flow controllers
Accurate flow rates were a critical part of the sampling train requiring accurate calibration. Aalborg (Orangeburg, New York, USA) mass flow controllers (MFC) of 0
– 500 mL min-1, 0 – 2 L min-1 and 0 – 15 L min-1, each with 1.5 % full scale errors were used to monitor and control gas flow through this series of laboratory experiments. The
MFCs were rarely operated at full scale, implying the error was beyond 1.5 % and necessitating calibration using a soap bubble flow meter (bubbler). Dry nitrogen from a cylinder (BOC Gases, Sydney, Australia) was directed through a MFC and into the bubbler over a series of flow rates covering the full range of the MFC. The time taken for the bubble to travel a specified volume was recorded (n = 6 for each flow rate), as was room temperature and barometric pressure. The discrepancy between the use of dry air for calibration and humid air in the experiments was self-correcting if one assumes the dry gas used in calibration becomes saturated with water vapour during passage through the bubbler (Wight, 1994). This negates the discrepancy in relative humidity to a reasonable approximation. Flow rates used in all calculations were corrected to standard temperature and pressure of 100 kPa and 0 °C using Equation 2.6,
ೌೞ ்ೞ (ݓ௦ × × (2.6݈ܨ = ݓ௦௧ௗ݈ܨ ೞ ்ೌೞ where Flow is the flow rate in L min-1, P is pressure and T is temperature (K) for standardised (std) and measured (meas) values respectively. An example of the calibration of a 0 – 500 mL min-1 MFC is shown in Figure 2.12.
46 Chapter 2 Method development
0.20
0.15 ) -1 0.10 (L min (L
0.05 MFC indicated flow rate flow indicated MFC
0.00 0.00 0.05 0.10 0.15 0.20 Soap bubble meter flow rate (L min-1)
Figure 2.12. Representative calibration curve of an Aalborg mass flow controller (range 0 – 500 mL min-1 with a soap bubble flow meter at flow rates from 0.1 to c. 0.18 L min-1. Flow of dry nitrogen was directed through the MFC and into the bubbler. Error bars are %RSD of bubbler observations and incorporate both reading and timing errors. The results of regression are shown in the figure.
47 Chapter 2 Method development
2.5 Error estimation
Uncertainties in quantitative flux estimates were calculated using the propagation of errors. Error estimates for flux measurements, which comprise measurements of analyte concentration differences, flow rates and leaf areas, were applied according to
Kesselmeier et al. (1997). The relative error for the concentration calculation EC/C is determined from the precision of the calibration standard and error in the flow rates
(Equation 2.7),
ଶ ଶ ܧ ܧ௦ ܧி௪ = ඨ൬ ൰ + ൬ ൰ ݓ݈ܨ ܥ ܥ
(2.7)
where Emeas is the relative error in concentration (C) during calibration and EFlow is the relative error in flow rate (Flow) during calibration. In the laboratory experiments
(Chapters 5 and 6) hydrocarbon free, zero grade air was used. No concentration difference was found before and after the empty cuvette, though low level background levels were observed. These were extremely stable and were determined daily rather than individually for each sample. Adsorption effects for calibration gases introduced before the cuvette were negligible after 10 min. At least 20 min elapsed before instrument calibrations commenced or between changes in the concentration of calibration gases. The stability of the system meant relative calibration error terms were set conservatively 10 % for the GC.
Errors for carbonyls sampled on 2,4-dintrophenylhydrazine (DNPH) cartridges were determined from the variation of blank cartridges. For acetone, acetaldehyde and
48 Chapter 2 Method development formaldehyde these were 0.18 ± 0.03 μg cartridge-1; 0.10 ± 0.02 μg cartridge-1; and
0.09±0.02 μg cartridge-1 respectively.
In the PTR-MS the error comprises the background-corrected mass signals during calibration and measurements as well as flow rate errors. Flow rate and background signals were stable, but instrument response changes associated with detector aging
(among other factors) meant errors were determined at more frequent intervals; in this case following each series of measurements.
Errors of flux estimates (Eflux/Flux) were the combination of all sources of propagated error (Equation 2.8) and included concentration errors (EC) in addition to error estimates for flow rate (EFlow), leaf area (ELA) and leaf temperature (ET).
ଶ ଶ ଶ ଶ ܧ௨௫ ܧ ܧி௪ ܧ் ܧ = ඨ൬ ൰ + ൬ ൰ + ൬ ൰ + ൬ ൰ ܣܮ ܶ ݓ݈ܨ ܥ ݑݔ݈ܨ
(2.8)
As the sampling train was the same for all experiments, values representing the error terms for leaf area, temperature and flow rates were calculated as 0.3 %, 0.02% and 5 % respectively. Errors associated with the determination of concentration were calculated at each calibration.
2.6 Other instrumental analyses
Gas chromatography – mass spectrometry (GC-MS) was used in a number of experiments and the operating conditions for this instrument are given in later chapters.
49 Chapter 3 Characterisation of terpenoid oils in leaves
3. Characterisation of terpenoid oils in juvenile leaves of Eucalyptus globulus
3.1 Introduction
Studies of eucalyptus leaf oils have been conducted for many decades, driven in particular by their commercial value for industrial and household use. These studies have produced a voluminous body of information on variation in oil yield and composition in relation to genetic, climatic and other edaphic influences. This research has sought to answer questions relevant to the production and harvest of oil from a wide range of eucalypt species and have thus most often focussed on variation at the stand or site level (e.g. Hellyer et al., 1969; Weston, 1984; Doran et al., 1995; Dunlop et al.,
2000), or on yields of major components (Ammon et al., 1985) using commercially relevant analytical techniques (Lassak, 2002).
More recently, there has been a developing interest in ecological aspects of oil production, composition and volatilisation. This includes a better understanding of the genetic and biochemical aspects of production. For example, it is only within the last 20 years that eucalypt oil composition has been examined in plant-animal interactions (e.g.
50 Chapter 3 Characterisation of terpenoid oils in leaves
Morrow and Fox, 1980; Cork et al., 1983). So too, volatilisation of terpenoid oils from eucalypts have only recently begun to be examined in detail (Guenther et al., 1991, He et al., 2000a; Velikova et al., 2007).
Limited evidence suggests that other than isoprene, oil glands are the source of mono- and sesquiterpene emissions from eucalypts. If this is the case, greater understanding of oil gland composition, oil production and storage is required. Recently, King et al.
(2006a) examined within tree and within leaf variation of oil composition in E. polybractea, finding no evidence of variation in oil glands either within a leaf or within a tree. They found monoterpenes comprised a smaller fraction of total volatile oil composition in oil glands than in whole leaves, suggesting leaf tissue acted as a sink for volatilised oils in glands.
Numerous techniques have been applied to extract oils from plant tissue. These include
(most commonly) steam distillation (e.g. Lassak and Southwell, 1982), solvent extraction (Weston, 1984), vacuum distillation (Dunlop et al., 1997) and supercritical fluid extraction (Pourmortazavi and Hajimirsadeghi, 2007). Each is effective, but each also has inherent artefacts. The method used also contributes to conflicting reports in the literature as to the composition of volatile oils in plant tissues or even the composition of particular classes of oils. Here, analytical sensitivity as well as genetic and geographical variation play important roles.
Juvenile leaves of E. globulus were selected as the target plant material for most work in this thesis, as more developmentally advanced leaf stages occur irregularly on trees of this age and it is often difficult to distinguish between later developmental stages. I thus conducted a number of studies to characterise the terpenoid oils contained in these
51 Chapter 3 Characterisation of terpenoid oils in leaves leaves and identify those factors that might influence the conditions under which later experiments might be conducted.
The first study addressed the optimum extraction time for leaf oils from ground leaf tissue using an alkane solvent, during which the oil composition from whole leaves was also determined. The second compared the composition of leaf oil glands and whole leaves in order to identify potential sources of terpenoids in tissue beyond the oil glands. Finally, I examined whether illumination of leaves could induce changes in oil composition of the glands and whole leaves.
52 Chapter 3 Characterisation of terpenoid oils in leaves
3.2 Methods and Materials
3.2.1 Plant material
Three months old seedlings of E. globulus Labill. subsp. globulus were purchased from
New South Wales State Forests Nursery (Wagga Wagga, NSW) and grown in a glasshouse until the experiment was completed. Within two weeks of delivery, plants were transferred to 10 L pots containing sterilized potting mix consisting of vermiculite and potting mix loam in a ratio of 1:2 (v/v). Plants were fertilized with a low phosphorous, slow release fertilizer (Osmocote Plus™) and maintained under ambient light with periodic shading provided on hot days. Average diurnal temperatures ranged between 21 and 32 °C in the month before the experiment and plants were watered daily with an irrigation system. Plants were about 18 months old and 1.5 - 2 m high at the time of the experiment.
3.2.2 Determination of optimum solvent extraction time
All extractions from whole leaves used the procedures described here. Two leaves from each of 4 trees were ground together to a fine powder under liquid nitrogen using a pestle and mortar. Initially, different solvent:leaf mass ratios and injection volumes for
GC-MS analysis were tested to identify suitable protocols for analysis. Thus for the analysis of whole leaves that follows, a ratio of 100 mg of fresh leaf material in 1 mL of solvent was used. When small leaves did not yield 100 mg of material, the ratio was maintained by reducing the amount of solvent.
Pentane was chosen as the extractive solvent on the basis that it did not interfere with subsequent GC analysis, in particular with analysis of low molecular weight compounds that were quickly eluted. Owing to its high vapour pressure and thus potential to
53 Chapter 3 Characterisation of terpenoid oils in leaves confound quantification at ambient temperatures, the pentane was kept on ice. 1,2,4- trimethylbenzene was used as an internal standard and added at 50 μg mL-1. In general,
100 mg of leaf material ground under liquid nitrogen was added to a chilled 2 mL vial, to which 1 mL of chilled pentane was added and the vial sealed. Extraction times spanning 0.5, 1, 2, 4, 8, 16, 24, 48, 72 h were tested, during which vials were stored in the dark at 4 °C and periodically shaken. There were three replicates for each extraction time.
Completeness of the extraction technique was checked using a second extraction.
Following completion of the first extract, the solvent was removed and a further 1 mL of fresh pentane was added to the vial. The vial was again sealed, shaken for 30 s and stored at 4 °C for 1 h. This extract was isolated and analysed.
3.2.3 Extraction of terpenoid oils from individual oil glands
Terpenoid oil from individual glands was sampled with a glass capillary drawn to a fine point using a custom made capillary puller. A bottom-lit dissecting microscope was used to position the fine capillary tip. A leaf was placed on a Petri dish containing chilled water to prevent the lamp heat from increasing leaf temperature and volatilising oils during sampling. Tubing mounted on the wide end of the capillary was connected to a syringe that was in turn used to draw oil from a pierced gland into the tip of the capillary. Once sampling was completed, the tip was rinsed in a 2 mL vial containing a
300 μL insert filled with 40 μL of chilled pentane.
In addition, I examined spatial variation in the composition of oil glands across leaves.
Oil glands from eight regions across a leaf were sampled (Figure 3.1). Between 10 and
54 Chapter 3 Characterisation of terpenoid oils in leaves
20 glands from each region were sampled and the collected oil was placed in a single vial for subsequent analysis.
3.2.4 Effect of light on the composition of oil in glands and whole leaves
In this study, I sought to determine if the synthesis of terpenoid oils could be induced by exposure to light. In addition, and if changes in relative composition were detected, the location of these changes was examined by sampling both oil glands and whole leaves. Leaves from intact, potted trees were placed in a custom made cuvette. The walls of the cuvette remained unsealed and laboratory air was circulated through the cuvette by a fan. All leaves were placed on a heater block within the cuvette and leaf temperature was maintained at 28 ± 0.8 °C. Half of the leaves were maintained at this temperature for four hours under dark conditions while the remaining half were illuminated at 1000 μmol m-2 s-1 at the same temperature for four hours. The two opposite, distal leaves on a branch were placed in the cuvette and these results were pooled. Four branches on each of four trees were used (16 branches in total), with two branches each receiving the light and dark treatment. Each branch was considered a single sample to limit the variation inherent in inter-tree comparisons and thus facilitate the detection of a treatment effect. Following treatment application, leaves were removed and oil glands and whole leaves were sampled for analysis of oil composition.
55 Chapter 3 Characterisation of terpenoid oils in leaves
Figure 3.1. Juvenile leaf of E. globulus showing the approximate positions of the 8 regions sampled to identify differences in oil gland composition. The many small white spots observable over the entire leaf are oil glands.
3.2.5 Analysis of terpenoid oils by GC-MS
Samples of 1μL were injected via an autosampler onto a SolGel Wax fused-silica capillary column (SGE Analytical Science, Melbourne, Australia; 30m x 0.25mm id,
0.25 μm phase thickness) which was eluted with He (inlet pressure 96.5 kPa) directly into the ion source of a Thermo Polaris Q GC-ITMS (injection port 220 °C; interface
230 °C; source 200 °C). The column temperature programme began at 30 °C, held for
0.5 min; increased to 150 °C at 5 °C min-1; increased to 240 °C at 15 °C min-1 and held for 2 min. The mass spectrometer was operated in the electron impact ionisation (EI) mode with an ionisation energy of 70 eV. Mass spectra were acquired with full scans from 50 to 250 u in 0.45s. Peaks were identified by comparison of retention time and fragmentation patterns with authentic standards or, where these were not available,
56 Chapter 3 Characterisation of terpenoid oils in leaves identification was achieved by comparison with the NIST-Wiley Mass Spectra Library
(1997).
3.2.6 Statistical analyses
Differences in the spatial distribution of oils across the leaf were tested using a dissimilarity index, applied as follows. The relative oil composition for each of the 8 regions across each leaf was determined. The difference between each region on a leaf for a particular compound was then calculated in all pair-wise combinations and these values arranged in a matrix and the mean difference for each matrix determined. A one sample t-test (Minitab 12.22, Minitab Inc.,) was used to determine whether the average difference across leaves was significantly different from zero at the Į OHYHO
The induction of compositional differences in oil from glands or whole leaves following exposure to light was tested using a two-way analysis of variance (ANOVA) or, where data points were missing, a general linear model (Minitab 12.22, Minitab Inc.,). The hypothesis that the mean compositional fraction of a compound did not vary between
WLVVXHORFDWLRQVRUOLJKWOHYHOVZDVWHVWHGZLWKĮ $WZR-factor ANOVA was used in which the two factors were light treatment (light or dark), and location (gland or whole leaf). Clearly, because whole leaves included oil glands, this approach only allowed rejection of the null hypothesis on the basis of the composition of non-oil gland tissue.
57 Chapter 3 Characterisation of terpenoid oils in leaves
3.3 Results and Discussion
3.3.1 Optimum solvent extraction time
Yield is plotted against extraction time for representative mono- and sesquiterpenes in
Figure 3.2. In the first 0.5 h there was a rapid partitioning of terpenoids into the solvent phase. Concentrations of terpenoids then diminished, reaching a minimum at 4 h. After
4 h, the relative concentration of compounds in the solvent phase again increased logarithmically to a maximum between 48 h and 72 h. It took at least 48 h before the relative concentration of a given compound matched that observed in the first 0.5 h - 2 h. Hence, the selected optimum extraction time was 72 hours for all compounds.
Rapid partitioning of oil components into different phases was probably responsible for diminishing concentrations between 0.5 h and 4 h. Grinding of frozen leaves under liquid nitrogen disrupts all leaf tissues but does not necessarily break all cells. It seem likely that all, or nearly all oil glands would be disrupted, with the oil released amongst the powdered matrix. Once the powdered leaf was exposed to the pentane solvent, oil components partitioned rapidly into the solvent. The use of small vials (2 mL) with a relatively large volume (1 mL) of solvent, ensured a headspace volume of around 50%
(1 mL) and relatively fast further portioning of oil components into the headspace, thus drawing down the concentration of compounds in the liquid phase.
58 Chapter 3 Characterisation of terpenoid oils in leaves
8000000
7000000
6000000
5000000
4000000
3000000 FID response 2000000
1000000
0 0 1020304050607080 Time (h)
Figure 3.2. Effect of extraction time on yield of terpenoids from leaves of E. globulus. Leaves were ground under liquid nitrogen and extracted in cold pentane for 0.5, 1, 2, 4, 6, 8, 16, 24, 48 and 72 h. Two representative compounds are presented: Ƈ Į( -pinene; (Ŷ DOORDURPDGHQGUHQH9DOXHVDUHPHDQVRI replicate analyses ± SE.
90000000 80000000 70000000 60000000 50000000 40000000
FID response 30000000 20000000 10000000 0 0 1020304050607080 Time (h)
Figure 3.3. Comparison of extraction time and yield for the sum of limonene and eucalyptolŶ 7KH ( comparative yield following secondary extraction (Ƈ LVDOVRSUHVHQWHG
59 Chapter 3 Characterisation of terpenoid oils in leaves
Clearly, the unknown concentration of oil components in the vapour phase makes it difficult to suggest the exact reason for the observed pattern of concentrations in the liquid phase. Nevertheless, the results make clear that a period of 72 hours for extraction provided a reliable basis for further studies.
The yield of the sum of limonene and eucalyptol, that at higher concentrations partially co-HOXWH H[KLELWHG WKH VDPH EHKDYLRXU DV Į-pinene and aromadendrene (Figures 3.2,
3.3). Also presented (Figure 3.3) is the yield of a repeat extraction of 1 h duration, conducted on all tissue at the end of their appointed extraction time. Repeat extraction showed a pattern of exponential decline, unlike the pattern observed for primary extraction. The consistent reduction of recoverable terpenoids in the second extraction suggests partitioning into the solvent at a predictable rate, probably dependent solely on extant concentration in the solvent.
Steam and hydro-distillation are the most commonly applied methods for extraction of terpenoid oils from eucalypts (Lassak, 2002). This is not surprising given that these are the methods used in commercial operations. Solvent extraction is too expensive for commercial operations but ideally suited to laboratory studies, as it requires no apparatus and many extractions can be performed in parallel. Nevertheless, Weston
(1984) reported poor results using dichloromethane as solvent for extraction of oils from E. delegatensis. On the other hand, pentane proved to be an excellent solvent for this species (Winters, unpublished data). Hexane (King et al., 2004, 2006a,b) and ethanol (Brooker et al., 1988; Grayling and Brooker, 1996) have also been used with eucalypts with satisfactory results. Using ethanol, Ammon et al. (1985) found constant agitation had little effect on extraction time, but at 30 °C complete extraction required
10 h. Those authors presented data for the first 10 h at 0.5 h intervals yet found no evidence of the early ‘spikes’ observed in the present study. They used large amounts of
60 Chapter 3 Characterisation of terpenoid oils in leaves solvent and leaf material (50 mL and 4 g respectively) in vials of unknown size and it is unclear if the absence of an initially high concentration followed by rapid depletion is a consequence of choice of solvent or other conditions. While not compared with other solvents directly, the use of pentane provided very satisfactory results and its use is recommended for further studies.
3.3.2 Spatial variation in gland composition
There was no difference in the composition of oil glands across the eight sampled regions of leaves (p > 0.05). This result supports data presented by King et al. (2006a) who also found no variation in gland composition within or between leaves on the same tree. In their work, King et al. (2006a) examined the composition of individual oil glands, whereas in the present study oil from closely grouped glands was pooled for comparison. I assumed that glands within a region were formed at a developmentally similar time (Carr and Carr, 1970) and thus subject to the same suite of factors that influence loading of terpenoid oils. Studies of oil gland composition could thus reliably sample glands from any area on the leaf with the reasonable prospect that results will not be confounded by within-leaf variation. Reports of the direct sampling of oil glands from myrtaceous species are confined to the non-peer reviewed literature. Lassak
(2002) summarized that body of work and concluded this method is the least likely to produce artefacts.
61 Chapter 3 Characterisation of terpenoid oils in leaves
3.3.3 The effect of light and location on oil composition
Short-term changes in the relative composition of terpenoids were examined between oil glands and whole leaves by subjecting leaves to high light (1000 μmol m-2 s-1) for four hours at 28 °C. In chromatographic runs terminated at 25 min (Figure 3.4), up to
80 terpenoid compounds were detected. Light induced no clear difference in the relative composition of any compound, and there were no qualitative differences between leaves and oil glands. Differences were apparent between leaves, but this was invariably restricted to minor components and instrument detection limits as well as sample variation may contribute to the observed variation. Only compounds comprising more than 0.1% of the total are reported here. The large number of compounds meant positive identification of all compounds was not possible, especially given the similarity in fragmentation of terpenoids following electron ionisation (Biemann, 1962). Statistical analyses were therefore terminated at 20 min, despite the fact that C10 and C15 compounds were detected after this time.
In all samples, eucalyptol made up the greatest fraction followed by Į-pinene (Table
3.1). Only two compounds exhibited a difference in their relative composition in this study. These were the cis and trans isomers of ocimene. Position (gland versus whole leaf) produced significant differences in both compounds (p EXW OLJKW KDG QR effect and there was no significant light x position effect. Specific treatment effects were examined with post-hoc analyses. Using a Bonferroni correction for Type 1 error
(Keppel, 1991) reduced thH VLJQLILFDQFH WKUHVKROG WR Į DQG PDGH IXUWKHU comparisons non-significant.
62 Chapter 3 Characterisation of terpenoid oils in leaves
Figure 3.4. Typical chromatogram from the analysis of leaf oils of E. globulus. Numbers correspond to compounds in Table 3.1. Positive identification of many sesquiterpenes was not possible.
Nonetheless, the fraction of both isomers of ocimene was greater in whole leaf tissue relative to oil glands (see Figure 3.5 for cis-ocimene).
No significant treatment difference was observed in any other individual compound, but
Table 3.1 suggests a number of compounds are represented in greater proportion under some treatments. To examine this further, a non-parametric rank test was performed for each treatment class. Here, the treatment of a compound (light or dark, gland or whole leaf) was ranked from one to four according to its relative contribution to the
FRPSRVLWLRQXQGHUWKDWWUHDWPHQW)RUH[DPSOHĮ-pinene (row 1, Table 3.1) was highest in oil glands in the dark and assigned to rank 4, while it made the least contribution to whole leaves in the dark and assigned rank 1. These values were then used as scores for
63 Chapter 3 Characterisation of terpenoid oils in leaves
Table 3.1. The relative composition of compounds in the oil glands and whole leaf of E. globulus under light and dark conditions. Numbers in front of the compound name indicate positively identified compounds and correspond to numbers in Figure 3.2.
Oil glands Whole leaf Compound RT Light Dark Light Dark (min) Mean SE Mean SE Mean SE Mean SE 1. Į-pinene 4.16 8.43 ± 0.495 9.62 ± 0.587 9.44 ± 0.342 8.82 ± 0.355 2. ȕ-pinene 5.77 0.49 ± 0.019 0.52 ± 0.010 0.59 ± 0.020 0.52 ± 0.020 3. R-(-)-Į-phellandrene 7.17 0.06 ± 0.005 0.06 ± 0.005 0.21 ± 0.148 0.07 ± 0.010 4. myrcene 7.27 0.93 ± 0.061 0.97 ± 0.043 1.13 ± 0.081 1.01 ± 0.039 5. limonene 8.04 2.96 ± 0.091 3.09 ± 0.093 2.72 ± 0.093 3.12 ± 0.203 6. 1,8-cineole* 8.22 59.08 ± 0.895 58.55 ± 1.212 56.43 ± 0.786 58.31 ± 3.180 7. cis-ocimene 9.09 0.23 ± 0.041 0.14 ± 0.040 0.28 ± 0.054 0.32 ± 0.016 8. p-cymene 9.24 0.32 ± 0.029 0.34 ± 0.032 0.31 ± 0.023 0.35 ± 0.025 9. trans-ocimene 10.2 0.04 ± 0.005 0.04 ± 0.004 0.05 ± 0.002 0.04 ± 0.003
C10H16 10.74 0.07 ± 0.014 0.08 ± 0.015 0.07 ± 0.010 0.06 ± 0.004 10. p-Į-dimethylstyrene 14.5 0.02 ± 0.002 0.02 ± 0.001 0.03 ± 0.002 0.02 ± 0.003
C15H24 14.93 0.03 ± 0.005 0.03 ± 0.007 0.05 ± 0.006 0.04 ± 0.009
C15H24 15.13 0.60 ± 0.032 0.57 ± 0.039 0.60 ± 0.034 0.59 ± 0.055
C15H24 15.53 0.37 ± 0.055 0.35 ± 0.068 0.56 ± 0.069 0.45 ± 0.112
C15H24 15.85 0.02 ± 0.003 0.03 ± 0.003 0.03 ± 0.003 0.03 ± 0.002
C15H24 16.36 0.01 ± 0.002 0.01 ± 0.002 0.01 ± 0.003 0.01 ± 0.001
C10H16O 16.7 4.43 ± 0.246 4.19 ± 0.358 4.70 ± 0.217 4.55 ± 0.445
C15H24 16.97 0.23 ± 0.011 0.22 ± 0.020 0.26 ± 0.013 0.24 ± 0.032
C15H24 17.21 0.04 ± 0.008 0.05 ± 0.008 0.08 ± 0.008 0.05 ± 0.006
C15H24 17.37 0.05 ± 0.003 0.04 ± 0.002 0.05 ± 0.003 0.04 ± 0.011
C15H24 17.52 0.02 ± 0.002 0.02 ± 0.004 0.04 ± 0.003 0.03 ± 0.003
C15H24 17.65 0.06 ± 0.003 0.06 ± 0.006 0.07 ± 0.004 0.06 ± 0.003
C15H24 17.88 0.01 ± 0.001 0.01 ± 0.003 0.02 ± 0.002 0.02 ± 0.010 11. caryophyllene 18.22 0.65 ± 0.034 0.63 ± 0.052 0.73 ± 0.034 0.71 ± 0.003 12. trans-caryophyllene 18.35 0.03 ± 0.005 0.03 ± 0.006 0.05 ± 0.006 0.04 ± 0.084
C15H24 18.43 0.12 ± 0.008 0.12 ± 0.011 0.13 ± 0.006 0.13 ± 0.009 13. aromadendrene 18.61 5.37 ± 0.282 5.10 ± 0.276 4.90 ± 0.379 5.47 ± 0.022
C15H24 18.72 0.12 ± 0.009 0.11 ± 0.006 0.10 ± 0.005 0.12 ± 0.630
C15H24 18.83 0.24 ± 0.014 0.22 ± 0.015 0.24 ± 0.015 0.24 ± 0.019 C15H24 19.23 0.29 ± 0.012 0.30 ± 0.020 0.31 ± 0.011 0.30 ± 0.032 14. alloaromadendrene 19.53 1.61 ± 0.355 1.78 ± 0.330 1.78 ± 0.296 2.42 ± 0.035
C15H24 19.8 0.69 ± 0.362 0.72 ± 0.386 0.75 ± 0.402 0.54 ± 0.047
C15H24 19.99 0.19 ± 0.022 0.15 ± 0.022 0.17 ± 0.027 0.22 ± 0.301
C15H26O 20.11 0.06 ± 0.026 0.05 ± 0.021 0.06 ± 0.022 0.05 ± 0.013 15. a-terpineol 20.2 0.01 ± 0.002 0.02 ± 0.002 0.02 ± 0.005 0.02 ± 0.020
x Also known as eucalyptol.
64 Chapter 3 Characterisation of terpenoid oils in leaves
0.4
0.35
0.3
0.25
0.2
0.15
Percentage of oil of total Percentage 0.1
0.05
0 Gland (L) Gland (D) Whole (L) Whole (D) Treatment
Figure 3.5. Treatment effects (mean ± SE, n = 8) of light (L) or dark (D) conditions and position on the relative composition of cis-ocimene in leaves of E. globulus. Position produced a significant difference in composition for this compound, but there was no significant light effect or interaction between treatments.
4 b b
3 a a
2 Rank
1
0 Gland (L) Gland (D) Whole (L) Whole (D) Treatment
Figure 3.6. The mean relative composition of a given compound was consistently less in glands compared to whole leaves, with light (L) or dark (D) conditions having no apparent effect. In this analysis, the mean relative composition of a compound was ranked from 1 to 4, least to greatest for each treatment, and the mean rank for each treatment determined for all compounds.
65 Chapter 3 Characterisation of terpenoid oils in leaves treatment effects for each compound. Treatment effects were then summed across all compounds and subjected to ANOVA. The highly significant difference (p < 0.001) in the ranking of oil composition between oil glands and whole leaves (Figure 3.6) suggests the distribution of oils between these tissues is not random. Identifying a particular mechanism to explain these results is difficult due to the number of potential influences, many of which are not mutually exclusive.
The larger contribution of ocimene in whole leaf tissue, relative to oil gland samples and the non-random distribution of oil fractions between glands and whole leaves is consistent with the only other gland x whole leaf comparison conducted in eucalypts
(King et al., 2006). King et al. found that relative concentrations of monoterpene hydrocarbons were greater in whole leaves compared to glands. Direct comparison of the results here with those of King et al. (2006) is difficult, as the samples used in their study were harvested from the field and stored at 4 °C under unknown conditions until analysed. The treatments applied here to leaves of E. globulus were intended to induce de novo synthesis. In explaining increased concentrations of monoterpenes in whole leaf samples, King et al. (2006) argued the existence of a cuticular sink for oils mobilised from oil glands. Non-specific storage pools have been postulated and tested previously in oaks (Niinemets and Reichstein, 2002) and this may indeed be the case here but further evidence is required.
De novo synthesis may account for the differences observed in this study, but the differences between locations within leaves were only marginally statistically significant. There was no evidence for an effect of light. Little is known about short- term variation in oil composition for eucalypts. Leach and Whiffin (1989) found a single instance of an unidentified compound varying over a 24 h period, although a handful of compounds exhibited seasonal variation that was broadly correlated with
66 Chapter 3 Characterisation of terpenoid oils in leaves accompanying variation in light, temperature or humidity. Emissions of volatilised terpenoids are known to vary diurnally (Street et al., 1997) and seasonally (He et al.,
2000b) in eucalypts, but the origin of emitted compounds is yet to be identified.
It is worth noting that despite sampling individual oil glands and whole leaves for changes in composition, the analytical methods employed here are capable only of detecting changes in the oil composition of tissue outside glands. The application of light to leaves might generate four outcomes: (a) no change in composition as a result of the treatment in any region of the leaf; (b) a change in oil gland composition only; (c) a change in whole leaf composition only; or (d), a change in both oil gland and whole leaf composition. Using the analytical methods described above, the only change that could realistically be detected is one in the relative composition of oils outside the oil glands, that is, in the mesophyll tissue. Any change in oil gland composition would probably be represented in whole leaf samples, making it difficult to distinguish the source of the variation.
67 Chapter 3 Characterisation of terpenoid oils in leaves
3.4 Concluding remarks
A number of factors relevant to the sampling of terpenoid oils were examined in this study in order to provide a basis for the design and application of sampling protocols in later studies. The appropriate analytical and extraction protocols for sampling oils from leaves of E. globulus were identified, in addition to characterising compositional variation between oil glands within a leaf and the effect of short-term environmental influences.
Solvent extraction of leaf oils required at least 3 days for all compounds of interest, a period similar to previous estimates for which comparable data exists. Analytical methods using GC-MS were optimised for the analysis of a wide range of terpenoid compounds.
Oil composition did not vary between individual glands within a single leaf, consistent with previous reports for eucalypts, suggesting that the direct sampling oils from glands will probably not be confounded by within-leaf variation. Oil composition did not vary qualitatively between whole leaves or oil glands. Illumination for 4 h did not induce composition differences, but there were slight increases in the fraction of cis- and trans- ocimene in whole leaves compared to glands. Further validation of these results might identify a mechanism responsible for this response.
68 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia
4. Emissions of isoprene, monoterpene and short-chained carbonyl compounds from Eucalyptus spp. in southern Australia.
4.1 Introduction
Volatile organic compounds (VOCs) play an important role in local and regional tropospheric chemistry, affecting the concentration of hydroxyl radicals and the formation of ozone in the presence of nitrogenous oxides (Chameides et al., 1992;
Thompson, 1992). On a global scale, biogenic emissions of an estimated 1250 Tg C yr-1
(Guenther et al., 1995, 2006) constitute between 50 – 90 % of total non-methane hydrocarbon emissions (Müller, 1992; Singh and Zimmerman, 1992; Guenther et al.,
1995), emphasising the importance of accurate estimates from vegetation.
Many studies over the last four decades have demonstrated considerable variation between tree species in both the composition and strength of emissions (Guenther et al.,
1999), indicating the unreliability of taxonomic proxies for estimating emissions
(Kesselmeier and Staudt, 1999). Biogenic emissions from European and North
69 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia
American tree species, representing boreal, temperate and Mediterranean ecosystems have received most attention, but emissions from tropical forests are also of significant interest due to their potential for high rates of VOC emission (e.g. Rasmussen and
Khalil, 1988; Kuhn et al., 2004; Baker et al., 2005; Seco et al., 2008).
Eucalypts are cultivated around the world, dominate the Australian landscape and are potentially large VOC emitters, yet members of this genus have received comparatively little attention. The small number of studies has identified relatively high isoprene and monoterpene emission rates for some species (Evans et al., 1982; Benjamin et al., 1996;
Winer et al., 1989) and E. globulus specifically was used to develop models of isoprene and monoterpene emissions for tree species generally (Guenther et al., 1991, 1993).
Very few field studies have examined biogenic VOC emissions from eucalypts (e.g.
Street et al., 1997) with the majority of work conducted on relatively young, pot-grown plants. Eucalypts are well known for the storage of mono- and sesquiterpenoid oils in subdermal glands (Brophy et al., 1991) and in only one of the 15-20 species studied has isoprene not been detected (He et al., 2000a). Evidence to date suggests monoterpene emissions are primarily controlled by temperature (Guenther et al., 1991; He et al.,
2000a, b), suggesting emissions originate predominantly, if not solely, from these glands.
Carbonyl emissions from eucalypts have not yet been studied and therefore no estimates are available on their contribution to global emissions. Of the carbonyls, acetaldehyde metabolism is most well known, where under anoxic soil conditions, ethanol is produced by alcoholic fermentation in the roots, transported through the xylem to the leaves, where it is oxidised, producing acetaldehyde as an intermediate product
70 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia
(Kreuzwieser et al., 1999). By examining processes involved in carbonyl exchange, such as exchange velocities and compensation points, where net exchange of a particular compound is equal to zero as a result of ambient concentrations, the relative influence of environmental and physiological processes may be determined (Jardine et al., 2008). Global emission estimates of biogenic VOCs incorporate the scant data available from the Australian continent (Guenther et al., 2006). Clearly there is a need for more field based observations of emissions and a better understanding of the production and control of VOCs from this genus.
To this end, we examined isoprenoid and carbonyl compounds emitted by four eucalypt species: E. camaldulensis, E. globulus, E. grandis and E. viminalis in a forest plantation frequently irrigated with nutrient-rich water. These species are co-dominant in various forests of south-eastern Australia, but more significantly they are grown widely in plantations in Australia and around the world. They have also been previously studied in the few reports of biogenic emissions from eucalypts, and provide a useful comparison against glasshouse studies. Specifically, our aims were to compare isoprenoid emission rates and composition measured here with previous reports, particularly from pot-based studies, to assess the importance of field-based studies for estimating emission from these species. Secondly, we sought to quantify carbonyl emissions from eucalypts and how temperature and ambient mixing ratios influence emissions. Thirdly, we sought to identify compensation points and exchange velocities for the carbonyls emitted by these trees, particularly between sites. Finally, we tested the hypothesis that the emission of acetaldehyde, the main carbonyl emitted by numerous tree species, is positively correlated with concentrations of its metabolic precursor ethanol in the xylem sap and in turn, with rates of sap flow and transpiration.
71 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia
4.2 Material and Methods
4.2.1 Field sites and plant material
The region in which the primary study was conducted has a Mediterranean climate. The mean annual rainfall of c. 930 mm falls predominantly during the cool winter months
(data from the Australian Bureau of Meteorology). The main study site was located within the Albany Effluent Irrigation Tree Farm, 410 km south of Perth, Western
Australia (34°56’S, 117°48’E). The tree farm was established by Water Corporation
WA to evaluate the growth responses of different Eucalyptus species to secondary- treated effluent. Trees were planted at the site in 1994 and irrigated twice daily with secondary-treated effluent (Eade, 2004), increasing soil water nitrate and ammonium concentrations at 30 cm depth by c. 2-5 times relative to water irrigation alone
(Livesley et al., 2007). In the austral summer of 2004, we measured emissions from at least two individuals each of Eucalyptus camaldulensis, E. globulus, E. grandis and E. viminalis, sampling one individual per day on consecutive days. An elevated working platform was used to access the outermost, sun-exposed foliage of the tree, between 8 and 14 m above ground. Despite this, cuvettes were sometimes exposed to short periods of shading from other branches. Only adult foliage was sampled, which constituted by far the greatest proportion of leaf biomass of these trees. In a smaller study, spot measurements of carbonyls and leaf gas exchange were undertaken within the Barmah forest during the previous summer. The Barmah forest is a natural forest site in south- eastern Australia dominated by E. camaldulensis. This is a dry, open forest on inland flood plains with a mean annual rainfall of c. 430 mm yr-1 falling predominantly during the winter months. For this species carbonyl emissions were sampled under natural conditions of light and temperature using the methods described below to identify in
72 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia particular the effect of ambient carbonyl concentrations on exchange rates, as well as to compare these data with those from Albany.
4.2.2 Gas exchange systems and VOC sampling
Branch-level photosynthetic gas exchange and VOC emissions were measured with two custom-made, 0.5 L PTFE cuvettes. Air was pumped through the cuvettes at 2.5 - 3 L min-1 using PTFE-coated diaphragm pumps (KNF Neuberger, Freiburg). One cuvette contained 2 - 5 adult leaves, while the other cuvette remained empty. VOC measurements were taken from both cuvettes and the flux rate calculated as the concentration difference between cuvettes and expressed per unit leaf area and per unit leaf dry mass. To compare emission results with literature estimates on a unit mass basis, 12 leaves from three trees of each species were collected and the ratio of leaf area to dry mass was determined. An LCA-4 infra-red gas analyser (ADC Bioscientific,
Herts, UK) connected to the cuvettes was used to monitor the exchange of CO2 and
H2O. Branches were placed in the cuvette and allowed to stabilise for around 60 min before measurements commenced.
In another series of experiments, a CIRAS II (PP Systems, Hertfordshire, UK) and a Li-
6400 (LiCor Inc., Lincoln, USA), both with artificial light sources, were used to examine temperature effects on carbonyl emission rates as well as to produce standard conditions of 30 °C and 1000 ȝPROP-2 s-1 PPFD for sampling isoprenoid emissions.
Leaves were clamped in the cuvette and remained there for the entire day, and sampling commenced 1-2 h after placement in the cuvette. If initial samples showed evidence of disturbance through elevated emissions, they were excluded from analysis. Air at the
73 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia inlet of the cuvette was scrubbed using activated charcoal to avoid interference from potential sources of contamination from the gas analysers. Flow rates through these cuvettes were c. 200 - 600 mL min-1.
Isoprenoid compounds were sampled on glass cartridges packed with multi-adsorbent beds of 90 mg Carbotrap C, 60 mg Carbotrap and 60 mg Carbopack X (Supelco,
Bellafonte, PA) separated by silanised glass wool. Flow rates through these cartridges were 100 mL min-1 with sampling time between 30 and 60 min. Carbonyl compounds were sampled on 2,4-dinitrophenylhydrazone (DNPH) - coated silica gel cartridges
(Supelco, Castle Hill, Australia) at flow rates of 1 L min-1 for between 30 and 60 min.
During sampling, all cartridges were wrapped in aluminium foil to shield them from sunlight and temperature fluctuations. Immediately after sampling, cartridges were sealed and stored at 4 °C. Isoprenoid cartridges were surrounded with ice bricks, packed in insulated containers and sent to the Institute for Meteorology and Climate Research,
Garmisch-Partenkirchen, Germany. DNPH cartridges were eluted as described below before being sent for analysis to the University of Freiburg, Germany.
4.2.3 VOC analysis
Isoprenoid samples were analysed on a coupled thermal desorption gas chromatograph with flame ionisation detector (FID) and mass-selective (MS) detectors (ATD 400,
Autosystem XL gas chromatograph, Turbo-Mass, Perkin Elmer, Weiterstadt, Germany) according to Schnitzler et al. (2004). Briefly, cartridges were desorbed at 280 °C and injected onto the column using the ATD 400. The eluent was initially directed onto an aluminium oxide/potassium chloride column (10 ȝPILOPWKLFNQHVVPP ID x 30
74 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia m), capturing lighter compounds (< C6) for detection by FID. Five minutes into the run, a four-port valve was activated directing the flow onto an RTX-FROXPQ ȝPILOP thickness, 0.25 mm ID x 30 m) for analysis of all compounds > C5 by MS. The temperature programme began at 15 °C, increasing at a rate of 10 °C min-1 for 3.5 min and then slowed to 5 °C min-1 until the column reached 200 °C. Calibration was performed with gas standards either prepared by the diffusion technique after dynamic dilution or from commercially available gas standards in pressurised cylinders
LVRSUHQH ǻ2-carene, Messer, Griesheim, Germany) as described by Schnitzler et al.
(2004). ,QWKLVV\VWHPVDELQHQHLVQRWVDWLVIDFWRULO\UHVROYHGIURPȕ-pinene and any co-
HOXWHGFRPSRXQGVDUHUHSRUWHGKHUHDVȕ-pinene.
Carbonyl compounds were quantified by HPLC, following elution of the DNPH- derived hydrazones using 2 mL of acetonitrile and 1 mL H2O. Aliquots of 100 μL were injected into an HPLC (Beckman, Munich, Germany) and separation was achieved on a reverse phase C-18 Supelcosil column (5 μm pore size, 4.6 mm ID x 250 mm, Supelco,
Munich, Germany) using a water/acetonitrile gradient (Kreuzwieser et al., 1999).
Detection was via a UV/VIS detector module 166 (Beckman, Munich, Germany) at 354 nm. Calibration was achieved using authentic external DNPH - derived standards
(Supelco, Munich, Germany).
4.2.4 Sap sampling and analysis
Xylem sap was sampled from E. camaldulensis and E. globulus at Albany to relate ethanol concentration to sap flow and acetaldehyde emission. Samples were collected from branches in the vicinity of the cuvette using the protocol described by Rennenberg
75 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia et al. (1996), obtaining 2-3 mL of sap. Ethanol concentrations in sap were determined by HPLC (Dionex DX 500; Dionex, Idstein, Germany). An aliquot of 100μl of xylem sap was injected into the system for separation by reverse phase/ion exclusion column
(Hamilton PRP-X300, 7μm pore size; 4.1 mm ID x 250 mm, Hamilton, Reno, Nevada,
USA) using 75 mM perchloric acid as the eluent delivered at 1 ml min-1. Ethanol detection was by means of a pulsed amphometric detector equipped with a Pt working electrode and samples were quantified using an external ethanol standard.
Sap flow was measured using the Heat Ratio Method (HRM) on the same two species from which xylem sap was sampled. Standard measurement protocols and conversion to sap velocity are described in Burgess et al. (2001). A detailed discussion of HRM theory and instrumentation can be found in Bleby et al. (2004). Sap flow was measured half-hourly over the course of the campaign and compared with diurnal gas exchange and VOC emission measurements.
4.2.5 Error, data and statistical analyses
Error estimates incorporating calibration and sampling errors were calculated by the propagation of errors for VOC fluxes. Uncertainties for flow rate and leaf area (or dry mass) were determined as 5% and 0.3% respectively. Emission rate errors from difference measurements between cuvettes were calculated according to Doerffel
(1984) and as applied elsewhere (Kesselmeier et al., 1997; Cojocariu et al., 2004;
Rottenberger et al., 2004; Jardine et al., 2008). Relationships between controlling environmental and physiological parameters and VOC emissions were examined by regression analysis using Minitab 12 (Minitab Inc., USA) and SigmaPlot 9.0 (Systat
76 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia
Software Inc., USA). Regression analyses were used to identify compensation points and exchange velocities for the exchange of carbonyl compounds (Rottenberger et al.,
2004). Data used for these analyses were compiled from diurnal measurements, taken under ambient temperatures and carbonyl concentrations. A Mann-Whitney U test was used to test differences in unpaired transpiration rates between two sites at the 5% level.
Exchange rates (ordinate) were plotted against the ambient concentration of the same compound (abscissa), with the y-intercept of the regression indicating the compensation point and the slope of the regression showing the exchange velocity (m s-1) of the compound (Rottenberger et al., 2004; Jardine et al., 2008).
Isoprenoid fluxes sampled from leaves under standard conditions (30 &DQGȝPRO m-2 s-1) were compared with those from leaves under ambient conditions to identify how well the algorithms of Guenther et al. (1993) predicted leaf-level emissions from trees. The light- and temperature-dependent model was applied to isoprene emissions while the temperature-driven model was applied to total monoterpene emissions.
77 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia
4.3 Results and Discussion
Diurnal variations in emissions of isoprene, the sum of monoterpenes and the carbonyls formaldehyde, acetaldehyde and acetone, as well as plant physiological and meteorological parameters are shown for E. globulus (Figure 4.1; see Appendix 2 for
Supplementary Figures S4.1-S4.3 for E. viminalis, E. camaldulensis and E. grandis respectively). As measurements were made at the same site, presentation and discussion of the results will concentrate on E. globulus.
4.3.1 Isoprenoid emissions
There was considerable variation in environmental light and temperature during the campaign, producing substantial day to day differences in leaf gas exchange and VOC emission. The range of temperatures on any given day varying between 4 and 10 °C and incident radiation was also highly variable. The number of sunshine hours recorded at the nearby Bureau of Meteorology Albany Airport weather station varied between 2.8 and 10.2 h over the sampling period and cloud cover was also highly variable (data not shown). The variation in meteorological conditions produced substantial day to day differences in leaf gas exchange and VOC emission. The highest isoprene emissions were observed in E. camaldulensis, which peaked at c. 670 nmol m-2 min-1 following sustained periods of high light and temperature. Total monoterpene emission maxima ranged from 40 to c. 680 nmol m-2 min-1 in E. grandis and E. globulus respectively.
Light-dependent monoterpene emissions were not evident in the diurnal data in any of the species studied here, although the light environment was highly variable during the campaign as a result of frequent cloud cover (data not shown).
78 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia
Figure 4.1. Diurnal course of isoprene
(Ÿ DQGPRQRWHUSHQH Ɣ HPLVVLRQV $
formaldehyde (ż DFHWDOGHK\GH Ƈ DQG
acetone Ƒ ( HPLVVLRQV % QHW &2 2
assimilation (black line) and
transpiration (grey line, C) and
photosynthetic photon flux density
(PPFD, black line) and cuvette
temperature (grey line, D) from E.
globulus.
79 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia
Comparison of diurnal monoterpene emissions, particularly under low light conditions, with measurements made under standard conditions did not introduce any new compounds or appreciable changes in the relative composition of emissions, suggesting that in these species, monoterpene emissions appear to be regulated predominantly by temperature, originating in the extensive oil storage glands present in the leaves of many eucalypts, including these species.
Compared with published standard emission rates (Table 4.1), isoprene emissions from the most well studied species of the eucalypts, E. globulus, were amongst the lowest reported for this species - most similar in fact to emissions from very young leaves on pot-grown plants reported by Guenther et al. (1991). Isoprene emissions from the three remaining species were closer in range to previously published estimates, although only a single report from the literature is available in each case. In contrast to isoprene, monoterpene emissions under the same conditions were generally higher than most estimates reported in the literature (Table 4.1). Total monoterpene emissions from E. globulus for example, were only exceeded by emissions from very young leaves studied by Guenther et al. (1991) and for the remaining species, monoterpene emission rates were 2-4 times greater than those previously reported (Table 4.1). We also found marked differences between isoprene and monoterpene emissions measured under standard conditions, and standardised diurnal isoprene and monoterpene emissions calculated using the widely applied algorithms developed by Guenther et al. (1993,
Table 4.1). While these differences are important for modelling purposes, most of this variation may be accounted for by two factors in particular. The first is the environmental (light and temperature) variation encountered during 1 hour of sampling which was avoided under controlled conditions.
80 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia
Table 4.1. Comparison of Eucalyptus isoprene and monoterpene emission data from this study with that from the literature. Where available, data have been presented as emission of carbon per unit leaf area or unit leaf dry mass. Measurements have been made at a leaf temperature of 30°C and 1000 μmol m-2 s-1 PPFD unless otherwise indicated in the Notes column.
Isoprene Monoterpenes Referencea Notesb
Species (μg C m-2 h-1)(μg C g-1 h-1)(μg C m-2 h-1) (μg C g-1 h-1)
Eucalyptus spp. 38.50 6.78 1 A 561.60 1252.80 2 A, B, E 2592.00 648.00 2 A, C, E 3758.40 475.20 2 A, D, E 6436.80 2 A, B, F 2980.80 2 A, C, F 2721.60 2 A, D, F 10812.00 3 G 24.70 4 H 14.50 1.00 5 I 48.70 5.20 5 I 14.90 0.70 5 I 6091.02 56.31 729.20 4.53 6 I 4966.01 7 1306.75 5.20 2951.97 11.75 This work 443.27 1.76 3319.17 13.21 This work J E. viminalis 6.17 8 I 776.82 7.63 1274.39 12.51 This work 420.96 4.13 360.00 3.53 This work J E. grandis 4578.54 50.22 277.19 2.81 6 I 990.07 5.71 1137.60 6.56 This work 372.41 2.15 799.19 11.46 This work J E. camaldulensis 953.52 13.65 370.71 3.17 6 I 1429.67 8.68 1886.37 11.46 This work 1306.75 5.20 1180.80 11.75 This work J E. botryoides 780.90 4.36 109.42 0.99 6 I E. calophylla 4784.04 30.25 64.07 1.00 6 I E. citriodora 6773.28 45.54 129.35 1.05 6 I E. cladocalyx 608.28 5.67 94.25 0.97 6 I E. forrestiana 7102.08 33.37 6 I E. gomphocephala 1997.46 14.06 115.88 0.34 6 I E. marginata 5301.90 23.84 135.14 1.16 6 I E. robusta 4767.60 41.02 266.54 2.14 6 I E. rudis 7817.22 50.47 210.51 2.04 6 I E. sargentii 5219.70 23.43 620.50 3.91 6 I E. wandoo 805.56 4.93 517.29 1.87 6 I
81 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia
Table 4.1. continued. a References. 1: Evans et al., 1982; 2: Guenther et al., 1991; 3: Monson et al., 1991; 4: Pio et al., 1996; 5: Street et al., 1997; 6: He et al., 2000a; 7: Loreto and Delfine, 2000; 8: Winer et al., 1983. b Notes. A: measurement made with leaf temperature of 28°C ; B: leaf age < 15 days; C: leaf age 15-40 days; D: leaf age 40+ days; E: data is for 1,8-FLQHROH ) GDWDLVIRUĮ-pinene; G: measurement made with leaf temperature of 32.5°C; H: cited in Kesselmeier and Staudt, 1999; I: data standardised to 30°C; J: diurnal emissions data corrected to a leaf temperature of 30°C and 1000 μmol m-2 s-1 PPFD using the algorithms of Guenther et al. (1993).
82 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia
These effects were especially pronounced on days of intermittent cloud. The second comprises the suite of physiological and site factors that introduce variation between and within any study. Here, leaf or tree ontogeny, chemotypic variation and site nutrition are likely to account for variation in the composition of monoterpenes observed between this and other studies, including the results of Chapter 3. Genetic control of monoterpene composition in eucalypts has been known for some time (Doran and Matheson, 1994), but the extent to which environmental and nutritional factors influence gland loading and production remains less clear (e.g. Muzika, 1989). There is some evidence that stress and nutrient status can affect the size of the storage pool during the development and loading of glands in eucalypt leaves (King et al., 2004,
2006), while N availability influenced monoterpene emissions from Pseudotsuga menziesii (Lerdau et al., 1995). Nonetheless, the variation in emission rates relative to rates reported in Table 4.1, and between observed and modelled rates, emphasises the importance of field-based data from as many sites and species as possible.
Compositional differences in monoterpenes were notable between this and previous studies (Figure 4.2). Eucalyptol dominated the monoterpene composition in all species, making up between 50 - 80% of emitted monoterpenes and three compounds, Į-pinene, limonene and p-cymene were also quantitatively important in each species, collectively
83 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia
Figure 4.2. Composition of monoterpene emissions from Eucalytpus viminalis (A), E. grandis (B), E. camaldulensis (C) and E. globulus ' DW&DQGLOOXPLQDWHGDWȝPROP-2 min-1 PPFD. Letters
UHSUHVHQWFRPSRXQGVDVIROORZV $Į-SLQHQH %FDPSKHQH &ȕ-pinene; D, myrcHQH (ǻ3-carene; F, limonene; G, p-cymene; H, eucalyptol. Observations are n = 3 ± SE and total monoterpne emissions are given by the shaded bar in each panel.
84 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia making up c. 15% of emissions. Whereas Street et al. (1997) reported that ocimene comprised c. 15% of emissions and eucalyptol was nearly absent, emissions from all species in this study were dominated by eucalyptol and we did not observe ocimene.
The light dependence of ocimene emissions has been reported previously (Owen et al.,
2002; Wang et al., 2007; Loreto et al., 2000) and its absence in our study may relate to cloudy conditions during the campaign, which may also be implicated in the comparatively low rates of diurnal isoprene emission.
4.3.2 Carbonyl emissions
Formaldehyde, acetaldehyde and acetone were emitted by all eucalypt species in this study. We also observed emissions of propionaldehyde but this was detected infrequently in trace amounts and is not discussed further. Diurnal carbonyl emissions determined at the Albany site were greatest for E. globulus (Figure 4.1), peaking at 75,
34 and 12 nmol m-2 min-1 for formaldehyde, acetaldehyde and acetone respectively.
Typical diurnal carbonyl emissions for the remaining species were somewhat lower (see
Supplementary figures. S4.1-3 in Appendix 2). While there are no comparable reports of carbonyl emissions from eucalypts, diurnal emissions of formaldehyde, acetaldehyde and acetone were in the range of those reported for other species (Kesselmeier and
Staudt, 1999; Seco et al., 2007). Nevertheless, the quantitative dominance of formaldehyde in the eucalypts contrasts with observations for Norway spruce
(Cojocariu et al., 2004), where acetaldehyde, together with acetone, were the dominant short-chained carbonyls emitted over a given 24-hour period. For the same species,
Janson et al. (1999) found acetone was the dominant carbonyl. Carbonyl emissions responded inconsistently to changes in temperature in two representative species from
85 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia
Figure 4.3. Correlation of emission rate with leaf temperature for formaldehyde (A, D), acetaldehyde (B,
E) and actone (C, F) from Eucalyptus grandis (left column) and E. globulus (right column). Emission rates at five temperatures from 20° to 40°C were examined.
the Albany site, E. grandis and E. globulus (Figure 4.3). There was a significant positive correlation (p < 0.05) in formaldehyde emission rates with temperature in E. grandis (Figure 4.3a) and a near significant correlation (p = 0.055) between acetaldehyde emission and temperature in E. globulus.
86 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia
Figure 4.4. Tempearture influence on
carbonyl emission from E.
camaldulensis at the Barmah natural
forest site Ɣ ( DQG WKH ZDWHU -and
nutrient-ULFK$OEDQ\SODQWDWLRQ ǻ
87 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia
The influence of site differences on the response of carbonyls to temperature were examined between the nutrient- and water-rich Albany site, and the natural site in the
Barmah forest using E. camaldulensis (Figure 4.4). Temperature had a strong influence on emissions of all carbonyls at the Barmah forest, whereas the effect was much weaker at Albany.
While the temperature effect is well known (Kesselmeier et al., 1997; Cojocariu et al.,
2004), ambient carbonyl concentrations too have been demonstrated to influence emissions (Kesselmeier et al., 1997; Cojocariu et al., 2004; Rottenberger et al., 2004,
2005, 2008), and these were strong determinants of carbonyl flux in this study. The most interesting response was identified when comparing this effect between the two field sites, again using E. camaldulensis (Figure 4.5). The graph shows that the apparent differences in carbonyl exchange between the two sites can be explained in part by different ambient concentrations of the respective carbonyls. The low emission rates observed at the Albany site corresponded with relatively high ambient concentrations of the trace gases. Compensation points, exchange velocities, and transpiration rates are shown in Table 4.2. Compensation points and exchange velocities were similar in range to previous reports (Cojocariu et al., 2004; Rottenberger et al., 2004, 2005; Jardine et al., 2008). Despite the unusual dominance by formaldehyde of carbonyl emissions, the equivocal nature of the data here make speculation of the likely role eucalypt forests play as carbonyl sinks or sources somewhat premature.
88 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia
Table 4.2. Exchange velocities from E. camaldulensis of formaldehyde, actealdehyde and acetone at the
Barmah Forest Site and the Albany Tree Farm. Abbreviations are: EV, exchange velocity; CP, compensation point; Tr, transpiration. Transpiration rates (mean ± SD) are the average of >10 measurements on nWUHHVXQGHUVWDQGDUGFRQGLWLRQVRI 30°C and 1000 μmol m-2 s-1 PPFD and are significantly different at the 5% level.
Barmah site Albany site
EV CP R2 Tr EV CP R2 Tr
(mmol (m s-1) (ppbv) (mmol m-2 s-1) m-2 s-1) Compound (m s-1) (ppbv)
Formaldehyde -0.0078 6.84 0.79 4.70a -9.0E-5 3.87 0.30 3.51b (1.46) (0.75)
Acetaldehyde -0.0237 3.02 0.58 -0.0007 2.43 0.72
Acetone -0.0060 1.67 0.73 -0.0001 0.80 0.37
Transpiration rates were significantly lower at the Albany site. We tested whether acetaldehyde emissions might be explained by delivery of root-derived ethanol to the leaves of the trees. Acetaldehyde emission however, was not significantly related to either the mass flow of ethanol in the xylem, or with xylem ethanol concentration
(Figure 4.6a, b). In contrast, acetaldehyde exchange in E. camaldulensis and E. globulus was significantly correlated with transpiration rates (Figure 4.6c).
89 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia
Figure 4.5. The effect of ambient carbonyl concentrations on the exchange of formaldehyde (A), acetaldehyde (B) and acetone (C) from leaves of E. camaldulensis at a nutrient and water rich Albany site
ǻ DQGDQDWXUDOVLWHLQWKH%DUPDKIRUHVW Ɣ
Figure 4.6. Relationships between actealdehyde emisison and trunk xylem sap flow (A), xylem ethanol concentration (B) and leaf transpiration rates (C) in E. camaldulensis ǻ DQG E. globulus (Ɣ DW WKH
Albany plantation site.
90 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia
This strong relationship supports the hypothesis proposed by Jardine et al. (2008) that transpiration rates, as a proxy for stomatal conductance, influence the exchange rate of acetaldehyde. The finding of lower acetaldehyde emission rates (which can partially be explained by higher ambient acetaldehyde concentrations, see above) and lower transpiration rates in Albany than in Barmah supports this view. Jardine et al. (2008) suggest that physiological and environmental influences on emissions can be separated by identifying both the compensation point and exchange velocity (for acetaldehyde).
Compensation points are influenced predominantly by light and temperature, which control the processes involved in compound production and consumption. Exchange velocities however, are influenced by stomatal and mesophyll resistances, linking these values to transpiration rates. As sub-stomatal sinks exist for acetaldehyde, rapid increases in the concentration gradient between the stomatal cavity and the atmosphere as a result of stomatal closure is negated somewhat by the incorporation of acetaldehyde back into the plant tissue (Jardine et al., 2008). Thus stomata are able to exert long term control on acetaldehyde emissions. It remains unclear why transpiration rates were lower at Albany, although cloudy, cooler weather during the campaign may have influenced this. Whether formaldehyde and acetone are subject to similar controls remains to be tested, although the presence of a sink for formaldehyde has been postulated previously (MacDonald and Kimmerer, 1993; Kreuzwieser et al., 2001).
91 Chapter 4 Emissions of VOCs from Eucalyptus spp. in southern Australia
4.4 Concluding remarks
Little data is available from field studies of VOC emissions from eucalypts. We examined isoprenoid and carbonyl exchange from between the atmosphere and eucalypts at two field sites in southern Australia. The somewhat lower isoprene emission rates observed in this work compared to earlier studies can be explained by the environmental conditions (cool, cloudy) during and before the measurements (e.g.
Fuentes et al., 1999). Conversely, monoterpene emissions from these species were higher and different in composition than previously reported (Street et al., 1997; He et al., 2000a). The elevated monoterpene emissions in this study may in part reflect favourable growth conditions experienced during storage pool (oil gland) loading, while the reduced isoprene emissions may be a function of more recent environmental conditions such as climate (e.g. Fuentes et al., 1999). Given the widespread cultivation of this genus in many parts of the world, and its dominance of the Australian landscape, a more detailed understanding of the factors that introduce this variation is needed for more accurate isoprenoid estimates from these species.
Of perhaps greater need is a more detailed understanding of carbonyl fluxes from these species and the genus generally. Factors influencing emissions identified in other species have been observed here. A strong correlation between acetaldehyde and transpiration, along with examination of compensation points and exchange velocities suggested that transpiration rates may be a significant predictor of carbonyl emission rates. Together, these isoprenoid data and the novel carbonyl data allow more complete emissions inventories to be developed of VOC emissions from eucalypts.
92 Chapter 5 Light and temperature effects on terpenoid emissions
5. Temperature and lights effects on terpenoid emissions from juvenile leaves of Eucalyptus globulus
5.1 Introduction
Isoprenoids constitute the largest quantitative emissions of volatile organic compounds
(VOCs) from plants (Kesselmeier and Staudt, 1999). Very early studies identified both light and temperature as important regulators of isoprene emissions (e.g. Sanadze and
Kalandadze, 1963, cited in Tingey, 1981; Rasmussen and Jones, 1973; Tingey et al.,
1979), while temperature was thought to be the predominant driver of monoterpene emissions, particularly from stored oils in shrubs (e.g. Tyson et al., 1974; Dement et al.,
1975) and gymnosperms (e.g. Tingey et al., 1980).
Predictive models of isoprene and monoterpene emissions developed at the time used, respectively, light and temperature, and temperature alone, to derive emissions estimates (see Guenther et al., 1993 for a contemporary review). But at the same time evidence for light-dependent monoterpene emissions from some species began accumulating from labelling (Schürmann et al., 1993; Loreto et al., 1996a,b,c) and physiological (Yokouchi and Ambe, 1984; Janson, 1993; Staudt and Seufert, 1995;
93 Chapter 5 Light and temperature effects on terpenoid emissions
Staudt et al., 1997) studies, and as a consequence, the light-temperature models developed for isoprene were applied with reasonable success in the prediction of light- dependent monoterpene emissions (e.g. Schuh et al., 1997; Dindorf et al., 2005; Pio et al., 2005). Indeed light-dependent monoterpene emissions are more common than previously thought, with evidence from northern hemisphere temperate (e.g. Staudt and
Seufert, 1995; Schuh et al., 1997; Staudt et al., 1997) and Mediterranean tree species
(Owen et al., 2002) as well as some tropical species (e.g. Kuhn et al., 2002, 2004).
Moreover, a number of species that store monoterpenes in specialised structures also produce VOCs from recently assimilated carbon, particularly from the genus Pinus (e.g.
Janson, 1993; Simon et al., 1994; Staudt et al., 1997; Shao et al., 2001; Owen et al.,
2002).
Advances in the understanding of the biochemical and enzymatic controls of isoprenoid emission (Lichtenhaler et al., 1997) has provided detailed insight into the direct role light and temperature play in regulating enzymatic activity and hence the production of these compounds. In addition, concomitant progress in determining the physicochemical factors that regulate VOC emissions (Niinemets et al., 2004) has improved the power of emission models (Niinemets and Reichstein, 2002). Light and temperature remain critical drivers of biochemical processes, but the uncertainty surrounding the importance of factors, such as stomatal conductance (see review by
Kesselmeier and Staudt, 1999), seem to be adequately explained by considering the biochemical origin and physicochemical properties of these compounds and the tissues through which they pass before emission from the leaf.
Despite the increasing sophistication in our understanding of the factors that regulate
VOC emissions from plants, those models originally developed by Guenther et al.
(1991) and since refined (e.g. Guenther et al., 1995) are still among the most widely
94 Chapter 5 Light and temperature effects on terpenoid emissions applied today, mainly because the data needed to tune and run them are easily obtained and they produce acceptable predictive power.
As indicated in the previous chapter, there have been a limited number of studies on
VOC emissions from eucalypts, but only the work by Guenther et al. (1991) examined the light and temperature response of compounds from this genus in detail. This work formed the basis for the development of the widely applied isoprene and monoterpene emission algorithms, but concentrated on the two dominant monoterpenoids emitted,
HXFDO\SWRODQGĮ-pinene. The value of this work however, has been limited by the lack of fundamental data on the kinetics and sources of terpenoid VOCs emitted from eucalypts. Terpenoid emissions from eucalypts appear to originate from oil glands and thus should be regulated by temperature alone. The limited data available indicate that this is largely inferred. To redress this, this study sought to examine in more detail the effects of temperature and light, on the emission patterns of monoterpenoids from leaves of E. globulus.
95 Chapter 5 Light and temperature effects on terpenoid emissions
5.2 Methods and materials
5.2.1 Plant material
Three month old seedlings of E. globulus subsp. globulus were purchased from New
South Wales State Forests Nursery (Wagga Wagga, NSW, Australia) and transferred to
10 L pots containing sterilized potting mix. Plants were fertilized with a low phosphorous, slow release fertilizer (Osmocote Plus™) and maintained under natural light with periodic shading provided on hot days. Average diurnal temperatures ranged between 21 and 32 °C in the month before the experiment and plants were watered daily with an irrigation system. Plants were about 18 months old and 1.5 - 2 m high at the time of the experiment.
5.2.2 Gas exchange system
Gas exchange measurements were made on the distal 2-3 pairs of opposite leaves on branches of E. globulus. Healthy and apparently similar leaves were placed in a 0.75 L cuvette consisting of two chambers; a lower chamber in which the leaf was housed and an upper chamber though which water from a Julabo water bath (John Morris Scientific,
Sydney, Australia) was circulated to control leaf temperature (see Chapter 2 for a detailed cuvette description). Prior to placing the leaves in the cuvette, the stem was wrapped in polytetrafluoroethylene (PTFE) tape to limit abrasion of the stem. Leaf temperature during the experiment was manipulated using this system in 5°C steps from
20°C to 50°C. Temperatures were held constant until the monoterpene signals on the
PTR-MS had stabilised (typically equal to or greater than 1 h). Light was provided by a
250 W metal-halide lamp and incident light was monitored using a Licor Li-190SB quantum sensor (Campbell Scientific, Townsville, Australia). Light levels were held
96 Chapter 5 Light and temperature effects on terpenoid emissions constant at 500 mol m-2 s-1 for all temperature response experiments. In the light response studies, light levels were varied by adjusting the lamp height above the cuvette
WRDFKLHYHHLWKHURUȝPROP-2 s-1 PAR while leaf temperature was held at 24.9 ±
0.6 °C during stable periods. Cuvette air and leaf temperatures were monitored with
PTFE-coated Type-T thermocouples (Onetemp Pty Ltd, Paramatta, Australia) placed in the shadow of the leaf and resting either in the air (for air temperature) or against the lower surface of one leaf. Light and temperature data were collected with an NI-USB-
9211 Thermocouple Input Module and recorded at 4s intervals with NI-DAQMax software (National Instruments, Sydney, Australia). All sensors were independently calibrated.
Zero-grade air (BOC Gases, Sydney, Australia) was supplied to the cuvette at 2.68 L min-1. The air stream was humidified by directing it through a glass bottle containing distilled water held in an ice bath at 2°C + 0.1°C, achieving a stable relative humidity of
- 28 ± 2%. CO2 was added to the air stream upstream of the cuvette at around 1 mL min
1 , providing a CO2 mixing ratio of 400 ± 11 ppm. Leaf gas exchange was monitored using a CIRAS II infra red gas analyser (PP Systems, Hitchin, Hertfordshire, UK) drawing air immediately upstream and downstream of the cuvette.
5.2.3 Sampling of volatile organic compounds
VOCs were simultaneously sampled using the two complimentary systems described in
Chapter 2; the high sensitivity proton transfer reaction mass spectrometer (PTR-MS) and a gas chromatograph (GC) equipped with a flame ionisation detector (FID).
Samples for both the PTR-MS and GC were drawn at 240 mL min-1 from the exhaust side of the cuvette, from which a subsample of 75-80 mL min-1 was directed into each instrument for analysis. Calibration, background checks and operating conditions for
97 Chapter 5 Light and temperature effects on terpenoid emissions both instruments were performed as described in Chapter 2. In this experiment the PTR-
MS was used to monitor the major ions of isoprene (m/z 41 and 69) and the monoterpenes (m/z 81, 93, 95, 135, 137, 153 and 155). The ions covered the range produced by the various monoterpenes emitted by E. globulus and identified in the fragmentation studies described in Chapter 2. However, there was no difference in the pattern generated by either the parent or major fragment ions and for simplicity both isoprene and the sum of monoterpenes were calibrated against their largest ions (m/z 69 and 81 respectively) using isoprene and camphene as gas standards. Monoterpene concentrations will therefore be less accurate as the relative importance of m/z 81 varies between compounds. Obtaining accurate emission estimates when monitoring complex isobaric mixtures, such as monoterpenes emitted from plants, will remain intractably difficult without chromatographic pre-separation in the PTR-MS because of the common ions generation by many monoterpenes.
5.2.4 Verification of results and cuvette characterisation
The unusual emission patterns produced from the initial temperature response experiment described above were verified by repeating this experiment using a different cuvette. With the exception of the GC, all other aspects of the sampling and analysis system were retained. The second cuvette was constructed of glass and aluminium with c. 89 % of the internal surface area being aluminium. This contrasted with the first cuvette in which Perspex made up c. 60 % of the internal surface area and aluminium the remainder. The first cuvette had an internal volume of 0.75 L, while the second held
0.377 L. However, both shared similar internal surface areas (0.0793 and 0.0769 m2 respectively) and the flow rate was adjusted to match the exposure time of a given air volume to the surface area between both cuvettes.
98 Chapter 5 Light and temperature effects on terpenoid emissions
Adsorption and desorption processes on the internal aluminium, stainless steel, glass or
Perspex surfaces may have contributed to the observed monoterpene emission patterns.
Given the novelty of the data described below, it was important to quantify or rule out these effects. This was examined by supplying an empty cuvette with humidified zero air followed by the introduction of a monoterpenoid gas standard and manipulating cuvette wall temperature to induce desorption effects. A concentration of 47.3 ± 3.5 ppb of eucalyptol was supplied to the cuvette, which was within the range of concentrations found in the air volume of the cuvette during leaf sampling at temperatures of 20 – 30 °C. Eucalyptol was used as it is emitted in the greatest quantities by these leaves. In previous experiments, cuvette wall temperature was a few degrees below leaf temperature at low temperatures and c. 10 – 13 °C above leaf temperature at high temperatures. To exaggerate desorption effects, cuvette wall temperatures were raised by 16 and 18 °C from initial wall temperatures of 26 °C and
42 °C respectively.
5.2.5 Data analysis and error determination
Determination of experimental error was carried out as described in Chapter 2.
Statistical analyses of comparison between means used either the Wilcoxon signed rank test or Mann-Whitney U-test (according to the symmetry of the data) using Minitab version 14 (Mintab Inc., Sydney, NSW).
99 Chapter 5 Light and temperature effects on terpenoid emissions
5.3 Results
5.3.1 Temperature effects on total monoterpene emission patterns
Light and temperature effects on monoterpene emissions were monitored by both GC and PTR-MS. GC provided data on the kinetics of individual compounds at coarse time resolution, while the PTR-MS provided much higher time resolution, but was unable to resolve individual compounds.
Emission kinetics revealed by the PTR-MS showed a sharp transient monoterpene emission burst before decaying to a steady state, in response to incremental changes in temperature. A typical pattern is shown in Figure 5.1. This transient burst was repeated at each temperature increment up to 45 °C, at which point a large and sustained pulse of monoterpenes were released from the leaf. A further increase of leaf temperature up to
50 °C produced no substantial change in the emission pattern (data not shown). Thus experiments that held temperature constant until emissions had reached a steady-state were terminated at 45 °C.
The large transient bursts were both time and temperature dependent and did not display the characteristic Arrhenius-type temperature response reported for most species. Steady-state emission rates did, however, show an exponential relationship with temperature (Figure 5.2). Large variations in absolute emission rates between trees were normalised by setting the steady-state emission rate at 20 °C to a value of one and scaling emission increases to this.
100 Chapter 5 Light and temperature effects on terpenoid emissions
7000 50 45 6000 40 ) -1
5000 35 C) ° min
-2 30 4000 25 3000 20 15 2000 ( temperature Leaf Emission (nmol m 10 1000 5 0 0 08:00 09:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00 Time of day
Figure 5.1. Emission of monoterpenes (Ƈ) in response to stepwise increases in leaf temperature (solid line). The leaf was held at each temperature until the monoterpene signal reached a stable value. Leaf temperatures beyond 45 °C induced a massive burst of monoterpene emissions.
9 C
° 8 7 6 5 4 3
Proportional increase Proportional increase of 2
monoterpene emission from 20 1 0 15 20 25 30 35 40 45 50 Leaf temperature (°C)
Figure 5.2. The proportional change in steady-state emission rate resulting from an increase in leaf temperature. Emissions are proportional to those observed at 20 °C and the grey line indicates the mean of all values at that temperature. Different symbols indicate individual trees.
101 Chapter 5 Light and temperature effects on terpenoid emissions
To confirm the highly unusual monoterpene emission kinetics produced in this experiment, a second experiment was conducted, essentially replicating the first, although with some important changes. I suspected sorption effects may be responsible for these patterns and to test this, I used a different cuvette while retaining the same gas plumbing but omitting the GC and using only the PTR-MS. This cuvette was smaller
(0.377 L) than the original (c. 0.7 L) and constructed of aluminium and glass. Despite the volume differences, the second cuvette had a similar internal surface area to the first
(0.0769 m2 and 0.0793 m2 respectively). As sorptive effects were suspected, the flow rate was reduced to achieve the same ratio of residence time to surface area as in the first cuvette. In addition to these changes, the time spent at a given temperature was reduced and temperature step increases were made before emissions reached steady state. In the first experiment there was some visible evidence of leaf water stress, in particular changes in the contour of the leaf surface, at higher temperatures. Emissions therefore, may be due to both temperature and desiccation effects and to limit the latter, time spent at each temperature was shortened.
Increasing the leaf temperature before monoterpene emissions had stabilised produced the same transient emission bursts (Figure 5.3). Interestingly, the emission burst seen at
45 °C in the steady-state experiment (Figure 5.1) was reproduced by one plant in the non-steady state experiment (Figure 5.3a). The remaining four plants however, produced the pattern shown in Figure 5.3b, which essentially mimics the transient bursts seen in the steady-state study.
102 Chapter 5 Light and temperature effects on terpenoid emissions
3500 60 B 3000 50 ) -1 C)
2500 ° min 40 -2 2000 30 1500 20 1000 Leaf temperature ( temperature Leaf Emission (nmol m 500 10
0 0 10:30 11:00 11:30 12:00 12:30 13:00 13:30 14:00 14:30 15:00 15:30 16:00 Time of day
Figure 5.3. Two typical monoterpene emission patterns from leaves in which leaf temperature was increased before steady state monoterpene emissions were reached (< 1 hour). A, plant 4, showing emission exhaustion at the 45 °C temperature set point. B, plant 3 continued the pulse-decay pattern up to 50 °C, as seen in lower temperature step-changes. In each graph, monoterpene emissions are symbols and scaled against the left hand ordinate, while leaf temperature is given by the solid line and scaled against the right hand ordinate. The sharp dip in the leaf temperature traces at values above 50 °C indicates the end of the experiment and data after this do not relate to the experiment. Light levels were held at 500 ȝPROP-2 s-1 throughout.
103 Chapter 5 Light and temperature effects on terpenoid emissions
Each change in leaf temperature produced a sharply defined phase of constant acceleration in monoterpene emissions (Figure 5.3b). The acceleration in emission rate
(nmol m-2 min-1 min-1) at each temperature increment was compared to identify a temperature-dependent response. Acceleration rates increased with each step change in leaf temperature (Figure 5.4), doubling between the lowest (20 – 25 °C) and the highest
(45 – 50 °C) temperature steps in a hyperbolic trend. Despite this, there were no significant differences in rates between step changes.
3
2.5 ) -1 2 min -1 1.5 min -2 1
(nmol m 0.5
0
Rate of increasemonoterpene inRate emission 20 - 25 25 - 30 30 - 35 35 - 40 40 - 45 Leaf temperature increase step (°C)
Figure 5.4. The rate of increase (nmol m-2 min-1 min-1) in monoterpene emissions at each increment of temperature change.
104 Chapter 5 Light and temperature effects on terpenoid emissions
There was a short time lag between leaf temperature stabilisation and the peak of the emission burst and soon after the peak, emission rates decayed (Figures 5.1, 5.3a and b) in a time-dependent manner. While the emission peak coincided with stabilisation of leaf temperature, the subsequent decay was characteristically time-dependent. A single- exponential decay model explained most variance (r2 > 0.99) for most plots (Figure 5a- e). Attempts to fit a double-exponential model produced either very small or no improvement in the value of r2, suggesting the single-exponential model best describes the emission decay. The half-life time was compared between each temperature step- increase (Figure 5.6). No significant difference was found in the half-life times of emission decay between temperature steps, but a general trend was apparent with decay rates greatest at the 30 – 35 °C temperature increment, but slower rates before and after this increment (Figure 5.6).
105 Chapter 5 Light and temperature effects on terpenoid emissions
600 800 ) ) -1
A -1 B min min -2
-2 600 400
400
200 200 Emission rate (nmol m (nmol rate Emission 0 rate m (nmol Emission 0 0 500 1000 1500 2000 0 500 1000 1500 2000 Elapsed time (s) Elapsed time (s)
800 1000 ) )
-1 C -1 D
min min 800
-2 600 -2
600 400 400
200 200
Emission rate m (nmol Emission 0 rate m (nmol Emission 0 0 1000 2000 3000 0 1000 2000 3000 4000 Elapsed time (s) Elapsed time (s)
6000 )
-1 E min -2 4000
2000
Emission rate (nmol m (nmol rate Emission 0 0 1000 2000 3000 4000 Elapsed time (s)
Figure 5.5. Single-exponential models (lines) fitted to the decay phase of monoterpene emissions (symbols) at each temperature step-increase. Data are from a single plant but are typical of the response observed. Temperature step-increases are: A, 20 to 25 °C; B, 25 to 30 °C; C, 30 to 35 °C; D, 35 to 40 °C; E, 40 to 45 °C.
106 Chapter 5 Light and temperature effects on terpenoid emissions
200
150
(min) 100 1/2
50
0 20 - 25 25 - 30 30 - 35 35 - 40 40 - 45 Leaf temperature increase step (°C)
Figure 5.6. Half-life times of the time-dependent monoterpene emission decay for each temperature increase. Values are means ± SD, n = 5.
107 Chapter 5 Light and temperature effects on terpenoid emissions
5.3.2 Characterisation of cuvette sorption effects
Under isothermal conditions, sorption effects were indeed evident following introduction of the eucalyptol gas standard into the cuvette air stream (Figure 5.7). Both the introduction and removal of the gas standard from the gas stream resulted in curves that varied substantially from those predicted. This does not suggest adsorption alone explains these discrepancies, as other factors such as perfect and instantaneous mixing, and the absence of dead zones (unmixed pockets in the volume) are assumed under theoretical conditions. These assumptions would rarely be met in real reactors or cuvettes. The gas standard was introduced as a step-increase in concentration, which produced a sigmoidal response and took c. 13.1 min to reach 95 % of the final signal strength. Integration of the inverse of the area under the sorption curve indicated that
195.9 nmol of eucalyptol was adsorbed over the c. 0.0769 m2 internal surface of the cuvette. The mean residence time for a fluid element (a small quantity of gas relative to vessel size characterised by microscopic properties such as temperature, pressure, etc.) in the 0.377 L cuvette and sampling line was c. 21.5 s. A step-decrease in the presence of the eucalyptol standard produced a precipitous drop in the signal, indicating desorption effects were far less pronounced and suggesting that once saturated, desorption effects made comparatively small contribution to the signal at a constant temperature. The eucalyptol concentration took just 4.5 min to reach 5 % of the original signal.
108 Chapter 5 Light and temperature effects on terpenoid emissions
1.2 Observed 1.0 Predicted
0.8
0.6
0.4 concentration
Normalised eucalyptol Normalised 0.2
0.0 0 5 10 15 20 55 60 65 70 Elapsed time (min)
Figure 5.7. Sorption isotherm produced from the step-increase and -decrease of eucalyptol gas standard into the cuvette air stream. The solid line was that observed in the system using the gas standard and the dotted line depicts the predicted signal response given assuming no adsorption or reaction, perfect and instantaneous mixing in the cuvette and no dead zones (unmixed gas pockets). This was calculated using the turnover time of the cuvette, which was c. 21.5 s.
The effect of an increase in cuvette wall temperature on the emission signal were examined by making two large step-increases (c. 16 and 18 °C) in cuvette wall temperature from 26 °C and 42 °C respectively (Figure 5.8). Each step-increase produced a burst of eucalyptol emission, but each peaked c. 2 and 6 % respectively higher than the previous steady state concentration. The steady-state signals following the decay of each burst was between 0 – 2% higher than the previous steady state.
Given the concentration changes induced by the large temperature changes (relative to experiments on leaves) were within the error of the system and instrument, it was safe to ignore these effects.
By contrast, with leaves in the cuvette the emission dynamics at the lowest temperature step increase of 20 to 25 °C caused monoterpene concentration to double (Figure 5.9).
This is at least ten times the burst produced in the empty cuvette at a similar initial
109 Chapter 5 Light and temperature effects on terpenoid emissions
56 65 54 60 55
52 C) ° 50 50 45 48 40 46 35
44 30 Wall temperature (
Eucaltypol concentration concentration (ppb) Eucaltypol 42 25 40 20 16:45 17:00 17:15 17:30 17:45 18:00 Time
Figure 5.8. The contribution of monoterpenes desorbed from the cuvette wall to the monoterpene signal following an increase in wall temperature of the cuvette. A known concentration of eucalyptol (black line) was delivered to an empty cuvette at constant temperature (grey line). Eucalyptol concentration in the outlet airstream was monitored with PTR-MS at 0.1 Hz and the red line is a running 2 min average of this signal. Temperature step changes were 18 and 16 °C respectively, with an initial temperature of 26
°C.
140 55
120 50
45 C) 100 ° 40 80 35 60 30 40 25 Leaf temperature ( temperature Leaf 20 20 Monoteprene concentration (ppb) concentration Monoteprene 0 15 13:45 14:15 14:45 15:15 15:45 16:15 16:45 17:15 17:45 18:15 Time of day
Figure 5.9. Changes in the concentration of monoterpenes within the mixed air volume of the cuvette in response to increasing leaf temperature.
110 Chapter 5 Light and temperature effects on terpenoid emissions concentration. The same step increase at higher temperatures produced even greater increases in monoterpene concentrations. Moreover, the duration of the burst when the cuvette contains leaves was at least 6 times longer than the burst seen in the empty cuvette, yet the latter was induced by substantially larger increase in temperature.
5.3.3 Temperature effects on the emission of individual monoterpenes
Compositional changes in monoterpenes were monitored by GC during periods of steady-state emission. Ten compounds of this class were emitted by leaves of E. globulus QDPHO\ Į-SLQHQH FDPSKHQH ȕ-pinene, myrcene, p-cymene, cis-ocimene,
HXFDO\SWROOLPRQHQHȖ-terpinene and terpinolene. The two latter compounds were not detected in all plants or at all temperatures, although they did not co-elute with other compounds that may have obscured their presence. Co-elution of eucalyptol and limonene occurred depending on the concentration in the sample gas stream. At higher leaf temperatures the eucalyptol peak often obscured the latter, making the results for limonene unreliable.
Monoterpene emissions generally increased in response to increasing leaf temperature
(Figure 5.10), although absolute emission rates and the rate of increase in response to temperature were unique to each compound. Of note however, is the sigmoidal response of cis-ocimene emission to increasing temperature, peaking initially at 30-35 °C before reducing at the next temperature step-increase, and rising again at the final temperature of 50 °C.
0RQRWHUSHQRLG HPLVVLRQV ZHUH GRPLQDWHG E\ HXFDO\SWRO Į-pinene and limonene in decreasing order (Figure 5.11). All other compounds constituted a much smaller fraction of the total. The increasing dominance of eucalyptol with increasing leaf
111 Chapter 5 Light and temperature effects on terpenoid emissions
1000 80 (A) D-pinene (B) camphene ) ) -1 800 -1 60 min min -2 -2 600 40 400
20 200 Emission (nmol m Emission Emission (nmol m (nmol Emission 0 0 15 20 25 30 35 40 45 50 55 15 20 25 30 35 40 45 50 55 Leaf temperature (qC) Leaf temperature ( qC)
80 80 (C) E-pinene (D) myrcene ) ) -1 -1 60 60 min min -2 -2
40 40
20 20 Emission (nmol m (nmol Emission m (nmol Emission 0 0 15 20 25 30 35 40 45 50 55 15 20 25 30 35 40 45 50 55 Leaf temperature ( qC) Leaf temperature ( qC)
100 40 (E) p-cymene (F) ocimene ) ) -1 80 -1 30 min min -2 -2 60 20 40
10 20 Emission (nmol m (nmol Emission m (nmol Emission 0 0 15 20 25 30 35 40 45 50 55 15 20 25 30 35 40 45 50 55 Leaf temperature ( qC) Leaf temperature ( qC)
Figure 5.103DQHO$(PLVVLRQRI $ Į-SLQHQH % FDPSKHQH & ȕ-pinene; (D), myrcene; (E), p- cymene; and (F), cis-ocimene from leaves of E. globulus as a function of leaf temperature and measured by GC-FID. Symbols are the means of 5 replicate plants ± SE.
temperature obscured the proportional response of the remaining compounds.
Importantly however, there were no notable increases in the relative proportion of a given compound as temperature increased.
112 Chapter 5 Light and temperature effects on terpenoid emissions
25000 800 (G) eucalyptol (H) limonene ) ) -1 20000 -1 600 min min -2 -2 15000 400 10000
200 5000 Emission (nmol m (nmol Emission m (nmol Emission 0 0 15 20 25 30 35 40 45 50 55 15 20 25 30 35 40 45 50 55 Leaf temperature ( qC) Leaf temperature ( qC)
200 80 (I) J-terpinene (J) terpinolene ) ) -1 -1 150 60 min min -2 -2
100 40
50 20 Emission (nmol m (nmol Emission m (nmol Emission 0 0 15 20 25 30 35 40 45 50 55 15 20 25 30 35 40 45 50 55 Leaf temperature ( qC) Leaf temperature ( qC)
Figure 5.10 3DQHO % (PLVVLRQ RI * HXFDO\SWRO + OLPRQHQH , Ȗ-terpinene, and (J) terpinolene from leaves of E. globulus as a function of leaf temperature and measured by GC-FID. Symbols are the PHDQVRIUHSOLFDWHSODQWV6(,QWKHFDVHRIOLPRQHQHȖ-terpinene and terpinolene means consisted of two to five plants, although the absence of error bars indicates only one data point was available.
1 terpinolene 0.9 0.8 g-terpene 0.7 limonene 0.6 0.5 eucalyptol 0.4 ocimene 0.3 p-cymene 0.2 0.1 myrcene Proportion of total emissionProportion of 0 b-pinene 20 25 30 35 40 45 50 camphene Leaf temperature (°C) a-pinene
Figure 5.11. The relative contribution of each monoterpene to the total flux of monoterpenes from leaves of E. globulus at each temperature step measured by GC. Data are the mean of n = 5 samples; standard error omitted for clarity.
113 Chapter 5 Light and temperature effects on terpenoid emissions
5.3.4 Response of monoterpene emissions to changes in light level
Due to the nature of the cuvette and lighting system used, the light response of monoterpene emissions monitored with PTR-MS could not be separated from temperature effects. The top chamber of the cuvette was flushed with temperature- controlled water which was used to manipulate air and leaf temperature within the cuvette. The rapid removal of the light source invariably produced temperature fluctuations as a result of the thermal inertia of the cuvette system. These lasted for 10 –
15 min and caused monoterpene emissions to fluctuate. Thus it was not possible to isolate the effects of stomatal closure produced by removal of the light source from the confounding temperature changes.
However, the light response of individual monoterpenes was determined under steady- state conditions. Of the ten monoterpenes emitted, only cis-ocimene showed a significant response to changes in light levels (Figure 5.12). There were no significant differences in the emission of the remaining monoterpenes in response to changes in light levels and the data have been omitted here for brevity. Light was applied at increasing levels in a step-wise manner from 0 WRȝPROP-2 s-1 before returning to darkness. While the highest level of illumination induced a significant difference above other levels of PAR, a clear trend of increasing emission was evident. The emission of cis-ocimene remained relatively high after returning to dark conditions, despite being in darkness for 40 – 60 min before measurements were made.
114 Chapter 5 Light and temperature effects on terpenoid emissions
20 B ) -1 15 min -2 A 10 A
5 A Emission (nmol m (nmol Emission 0 0 250 500 0 PAR level (Pmol m-2 s-1)
Figure 5.12. The response of cis-ocimene to different levels of PAR (mean ± SE, n = 5). Light levels were applied in the order listed on the abscissa and emissions quantified by GC after stabilisation. Different letters above columns indicate significant differences between treatments.
5.3.5 Temperature effects on isoprene emission
The measurement of rates of isoprene emission made by GC (Chapter 2) and by the
PTR-MS proved to be in good agreement and a representative comparison is shown in
Figure 5.13. Emissions determined by GC were conducted only at the predetermined leaf temperature set point, whereas those determined by PTR-MS were continuously monitored throughout the experiment. The temperature response determined by GC
(Figure 5.14) shows emissions integrated over the 15 min sampling period and in steady state at each temperature interval. The mean isoprene emission maximum determined by GC is clearly seen at 40 °C, whereas that determined by PTR-MS shows much more variation between individual trees (Figure 5.15), with emission maxima varying over a
10° range, from 38 °C to 46 °C across five different trees. Emission rates at the temperature maxima were between 600 – 1600 nmol m-2 min-1 (Figure 5.15).
115 Chapter 5 Light and temperature effects on terpenoid emissions
.
900 800 ) -1 700 min
-2 600 500 400 300 200 Emission (nmol m 100 0 15 20 25 30 35 40 45 50 55 Leaf temperature (°C)
Figure 5.13. Representative comparison of isoprene emissions quantified using a GC (grey-filled squares) and PTR-MS (open circles). Measurements were made on an individual plant.
1800 1600 ) -1 1400 min
-2 1200 1000 800 600 400 Emission (nmol m 200 0 20 25 30 35 40 45 50 Leaf temperature (°C)
Figure 5.14. Isoprene emission rates determined by GC from leaves of E. globulus as a function of temperature (mean ± SD, n = 5).
116 Chapter 5 Light and temperature effects on terpenoid emissions
1800
1600 ) -1 1400 min -2 1200
1000
800
600
400 Isoprene emission m (nmol emission Isoprene 200
0 15 20 25 30 35 40 45 50 55 Leaf temperature (°C)
Figure 5.15. Isoprene emission in response to increasing leaf temperature measured using PTR-MS. This plot shows the compilation of the emission response from all trees in this study (n=5).
Leaf temperatures were held constant at each 5 °C interval until all masses being monitored on the PTR-MS were stable at that temperature. Below 35 °C, isoprene emissions responded directly to temperature, stabilising as temperature stabilised. At temperatures above 35 °C, emission bursts were observed. This is evident in the vertical aggregation of data points in Figures 5.13 and 5.15. The time response of the emission burst is not shown as these figures scale emission response against temperature on the abscissa. The burst was of considerably lower amplitude and shorter duration than those seen in monoterpenes, and declined to a steady state after 10 – 15 min.
Biochemical and gas exchange data that would make clearer the mechanisms driving these bursts were not collected, limiting further examination.
All trees produced similar gas exchange patterns (Figure 5.16), typical of temperature responses in eucalypts (Ngugi et al., 2003). Rates of photosynthesis and stomatal
117 Chapter 5 Light and temperature effects on terpenoid emissions conductance reached a maximum between 25 and 30 °C. Transpiration also peaked in this temperature range, declining briefly before rising again. The cuvette was flushed with air of constant absolute humidity, producing a relative humidity of 25% at the inlet. Thus increasing air (and hence leaf) temperature produced a concomitant increase the vapour pressure deficit (VPD), closing stomata and limiting photosynthesis and stomatal conductance. Transpiration also responded briefly to stomatal closing, but the increasing VPD continued to drive transpiration as temperature increased.
Carbon lost through isoprene emission, as a percentage of carbon gained through net
CO2 assimilation, (Figure 5.17) remained below five percent up to a leaf temperature of
35 °C, and remained less than one percent up to c. 45 °C. Temperatures of 45 °C and higher caused carbon assimilation to decline (Figure 5.4A); this combined with peaks in isoprene emission produced large spikes in the ratio of carbon lost through isoprene emission. Monoterpenes were excluded from the calculation of carbon loss on the assumption that emissions were derived from stored pools and thus not a product of recent carbon assimilation.
118 Chapter 5 Light and temperature effects on terpenoid emissions ) ) -1
-1 15 100 s s AB-2 -2 12 80 mol m P 9 60
6 40 assimilation(
2 3 20
0 0 Net CO 0 1 2 3 4 5 6 m (mmol conductance Stomatal 0 1 2 3 4 5 6 60 4
CD) -1 s C) -2 q 3 40
2
20 1 Leaf temperature ( temperature Leaf Transpiration (mmol m (mmol Transpiration 0 0 0 1 2 3 4 5 6 0 1 2 3 4 5 6 Elapsed time (h) 150 E
100
50
Vapour pressure deficit (mbar) deficit pressure Vapour 0 0 1 2 3 4 5 6 Elapsed time (h)
Figure 5.16. Representative patterns of gas exchange observed in leaves of E. globulus subjected to increasing temperature. Panels indicated by each letter are: A, net CO2 assimilation; B, stomatal conductance, C, leaf temperature; D, transpiration; and E, vapour pressure deficit. Measurements were PDGHXQGHUDFRQVWDQWOLJKWOHYHORIȝPROP-2 s-1.
119 Chapter 5 Light and temperature effects on terpenoid emissions
3 60 800 ) ) (×) ) ( Ƒ -1 -1 s ) __ -2 2.5 600 min m -2 2
° C) ( 40
2 mol CO 400 assimilation μ 2
20 1.5 200 Leaf temperature ( Leaf temperature Net assimilation ( Net assimilation Isoprene emission (nmol m (nmol emission Isoprene 1 0 0 012345 Elapsed time (h)
Isoprene emission / Net CO / emission Isoprene 0.5
0 20 25 30 35 40 45 50 Temperature (°C)
Figure 5.17. The ratio of carbon assimilated through photosynthesis to that lost through isoprene emission over the temperature range 20 – 50 °C. Each trace in the main figure is the ratio calculated from each tree. Inset: A representative plot comparing net CO2 assimilation, isoprene emission and leaf temperature.
120 Chapter 5 Light and temperature effects on terpenoid emissions
5.3.6 Light effects on isoprene emission
The lighting system used in this experiment limited the capacity to manipulate light
OHYHOV1RQHWKHOHVVWKUHHOLJKWOHYHOV DQGȝPROP-2 s-1 PAR) were applied to leaves at a constant leaf temperature of 28 °C (Figure 5.18). The first measurement, taken in the absence of light, was made after leaves were held overnight in darkness.
The isoprene signal in the absence of light was either absent or below the limit of detection. There was however a significant (p < 0.001) difference between emissions at the two higher light levels.
160 B ) -1 140 A min
-2 120 100 80 60 40 BDL 20 Isoprene emission m (nmol emission Isoprene 0 0 250 500 Photosynthetically active radiation (μmol m-2 s-1)
Figure 5.18. Isoprene emission from leaves of E. globulus in response to increasing levels of irradiance following overnight darkness. Data presented are means ± SD, n = 5. BDL, below detection limit. Different letters indicate a significant difference (p < 0.05) between emissions at each light level.
121 Chapter 5 Light and temperature effects on terpenoid emissions
5.4 Discussion
5.4.1 Monoterpene burst-decay emission patterns
Large bursts of monoterpene emissions were emitted by E. globulus leaves in response to increases in leaf temperature. Following stabilisation of leaf temperature, monoterpene emissions decayed in a time-dependent manner to reach a steady rate higher than the steady rate at the previous leaf temperature (5 °C lower). This pattern contrasts sharply with all previous (but limited) work on eucalypts, including that of
Guenther et al. (1991), in which the widely applied exponential temperature emission algorithm was developed (Equation 5.1, hereafter referred to as the Guenther model).
(ఉ(்ି்ೄ)) (ೄ ݁ݔ (5.1்ܯ = ܯ
The mechanism underlying this algorithm (including subsequent modifications, e.g.
Guenther et al., 1993, 1997, 2006) is a simple exponential increase in emission rate (M)
in response to temperature (T), relative to the emission rate (MTS) determined at a standard temperature (TS). The numerous studies conducted on eucalypts (Street et al.,
1997; He et al., 2000) and other species (e.g. Pio et al., 2005) have generally found a satisfactory fit between experimental and predicted data, but examples of the converse, where this response poorly fits the experimental data, also exist due, for example, to seasonal differences in production (Staudt et al., 1997; Llusiá and Peñuelas, 2000).
Nonetheless, researchers continue to search for factors that influence the temperature dependence of emission rate, ȕ (K-1) which incorporates leaf-to-leaf and seasonal variability, differences in physicochemical properties and the effect of different source- to-emission pathways (e.g. Taivarnen et al., 2005; Fillela et al., 2007). While the phenomena of transient bursts have been described recently, these typically follow the
122 Chapter 5 Light and temperature effects on terpenoid emissions removal of light, with an underlying biochemical mechanism driving the burst. For example, a pyruvate overflow mechanism may be responsible for bursts in acetaldehyde emissions following the removal of light (Holzinger et al., 2000; Karl et al., 2002). A short-OLYHG EXUVW RI Į-pinene was induced under CO2-free air and low O2 partial pressure in Q. ilex when switched from dark to light by Loreto et al. (2000).
Monoterpenes are synthesised in Q. ilex, and this burst was interpreted as a light- stimulated emission of an internal VOC pool formed during the dark. However, the temperature-induced burst-decay pattern observed in the monoterpene emissions here is unusual and raises two questions: is this response a biological phenomenon or an artefact of sample design, and if it is indeed biological, by what mechanism is it operating?
Sample design artefacts that might have influenced these results include, broadly, the improvements in instrument resolution (adsorbent cartridges versus PTR-MS) and more specifically, adsorption/desorption effects on cuvette walls or confusion between emission from recently synthesised and stored compounds.
Greater resolution in sampling frequency has been possible with sampling cartridges,
(e.g. 5 min sampling time in Loreto et al., 1996b, but at 30 min intervals), but studies on the same or similar species (e.g. Guenther et al., 1991; He et al., 2000) have sampled at longer intervals than might capture the monoterpene emission bursts. Recently, greater sampling resolution was employed by Filella et al. (2007), who used PTR-MS to determine emission temperature responses in Picea abies. Monoterpenes in this species originate from both terpene storage organs and recent biosynthesis. While their sampling methods were not explicitly stated in their study, temperature increments of 4
°C, with each step-increase held for one hour, produced emission dynamics that fit the typical exponential response to temperature and they reported no evidence of emission
123 Chapter 5 Light and temperature effects on terpenoid emissions bursts. As such, improvements in sampling resolution cannot be excluded as a factor contributing to these observations and its importance will only become apparent once more studies are repeated at finer temporal resolution.
Desorption of compounds from the cuvette walls at each temperature increment was initially suspected as contributing to the patterns observed in this study, but characterisation of these effects (section 5.3.2 above) rules this out. It is unclear if the long time taken to reach a steady signal following the introduction of gas standard into the cuvette is due to adsorption effects or poor mixing (Figure 5.7), but once steady, changes in the signal as a result of desorption are minor. A large temperature increase at the monoterpene-saturated cuvette wall produced a signal response that was at most only 5 % of the signal increase produced by leaves in response to much smaller temperature changes. While these effects are minor, it is important that sufficient time be allowed for the system to equilibrate after introducing new material into the cuvette, a process which may take many hours (Guenther et al., 1991). Nonetheless, characterising potential adsorption effects, particularly where the compounds are not well studied or known to adsorb readily on to surfaces, remains a prudent approach
(Shaub et al., 2010). It is clear in this case that desorption effects would, in the most severe case, provide a minor enhancement of the monoterpene signal.
Bursts in the biosynthesis (and emission) of some monoterpenes as a result of the step- increase in temperature may have contributed to these emission patterns. However, only one compound among ten monoterpenes, cis-ocimene, was a potential candidate. As temperatures increased, its contribution to the total monoterpene flux decreased from 6 to 1%. Thus, bursts from recently synthesised compounds are unlikely to have influenced the emission pattern.
124 Chapter 5 Light and temperature effects on terpenoid emissions
The exclusion of experimental artefacts, in addition to the repeated experiments using different cuvettes, indicates these emission phenomena are indeed authentic. Two hypotheses may explain the biological basis for the emission bursts seen in this study.
Both are based on the assumption that temperature alone is the driver of these patterns and that recently synthesised compounds play no significant part in the emission patterns. The first hypothesis proposes that stomatal regulation of VOC emissions
(Niinemets and Reichstein, 2002b, 2003) may be responsible for this pattern. In the second hypothesis, I propose that stomata may not be an important route from source to atmosphere and that instead emissions proceed directly to the atmosphere through the cuticle. Both could operate simultaneously and the case for the operation of each hypothesis in eucalypts is now addressed.
Diurnal emissions of the oxygenated monoterpenes linalool and eucalyptol closely followed diurnal variability in stomatal conductance in Pinus pinea, while limonene and trans-ȕ-ocimene were not linked to stomatal control (Niinemets et al., 2002b).
Subsequent modelling to deduce the nature of the mechanism found stomatal sensitivity of emissions was related to the Henry’s law constant of the compounds (Henry’s law describes the solubility of a gas in a liquid at a given temperature is proportional to the pressure of that gas above the liquid. The constant is the ratio of the concentration of the compound in each phase). Specifically, eucalyptol and linalool each have a lower propensity to partition from the liquid phase into the gas phase. Because of their low
Henry’s law constants, closed stomata limits emissions of these compounds until their intercellular partial pressure rises enough to penetrate the closed stomata. Conversely, those compounds with high Henry’s law constants rapidly partition from the internal liquid phase into the gas phase, maintain a high internal partial pressure and are not constrained by stomatal closure (Niinemets et al., 2002b). Stomata are demonstrably the
125 Chapter 5 Light and temperature effects on terpenoid emissions favoured route for isoprene (Fall and Monson, 1992) and monoterpene (Loreto et al.,
1996) emissions, while cuticular emissions are thought, on a theoretical basis to account for perhaps 10 – 20 % of emissions in Picea abies (Shürmann, 1993, cited in Niinemets and Reichstein, 2002b).
There is therefore, clear evidence for the stomatal control of some monoterpenes. In E. globulus, both emissions and leaf oil content is dominated by eucalyptol (60%, Figure
5.11; Chapter 3). Į-pinene also makes up a substantial fraction of the stored (Chapter 3) and emitted fraction (15 – 20 %, Figure 5.11). The latter has a Henry’s law constant
1000 times greater than that of eucalyptol (Niinemets and Reichstein, 2002a), and so should not be subject to stomatal regulation. The data here show clear effects of increasing temperature on stomatal conductance: incremental increases in cuvette temperature on inlet air of constant absolute humidity increases the vapour-pressure deficit which in turn causes rates of transpiration to increase (Figure 5.16). This increase is reflected by the increase in conductance to water vapour, which, according to the modelling of Niinemets and Reichstein (2002a), should facilitate the emission of eucalyptol. This was not discernable using the analytical techniques employed here. It may be that eucalyptol simply dominated the monoterpene signal and obscured the less-constrained HPLVVLRQV RI Į-pinene. The stomatal regulation hypothesis could be disproved by manipulating stomatal aperture through the application and removal of light under constant temperature, while simultaneously sampling individual compounds on adsorbent cartridges at short (e.g. 5 min) intervals. The experimental design used here did not allow this, but the hypothesis clearly requires testing. 126 Chapter 5 Light and temperature effects on terpenoid emissions I propose an alternative hypothesis where emissions originate directly from oil glands and traverse a relatively short pathway through a thin layer of window-like cap cells positioned above the subdermal oil gland and into the atmosphere. Under this hypothesis, the emission burst may be explained by the following process. A leaf under stable conditions emits terpenes at a constant rate. Emissions pass through cap cells, which visually, are window-like epidermal cells that sit above the oil gland, as well as a waxy cuticle, that extends over the leaf surface. When the leaf temperature increases, terpenes that saturate these cap cells (and quite probably the waxy cuticle) are rapidly driven off, producing the emission burst. These regions are depleted of terpenes faster than they can be recharged by the oil gland below, which results in the emission decay. Emissions stabilise at the new temperature once the flux of terpenes from the gland recharges the cap cells and cuticle. Evidence for this model comes from a number of sources. Firstly, evidence against a role for stomata in the regulation of monoterpene emissions from juvenile leaves of E. globulus was provided by Guenther et al. (1991), who found >75 % of monoterpenes were emitted from the adaxial leaf surface. The juvenile leaf form of E. globulus carries stomata on the abaxial surface only (Johnson, 1926), leading these authors to conclude that monoterpenes are not emitted from the stomata. The converse is true in Quercus ilex (Loreto et al., 1996b), where emissions were detected predominantly from the abaxial surface of the hypostomatous leaves, clearly implicating stomata in the emission pathway. The stomatal regulation of some VOCs is dependent on differential partitioning between liquid and gas phases within leaves, i.e. when stomata close, compounds with a low Henry’s law constant must accumulate sufficient partial pressure in order to traverse the closed stomate, while the passage of more volatile compounds remains 127 Chapter 5 Light and temperature effects on terpenoid emissions unimpeded. Double-exponential decay models were retrospectively fitted to total monoterpene fluxes from Quercus ilex to demonstrate the presence of two pools within leaves, characterised as having either fast or slow turnover rates (Niinemets and Reichstein, 2002a). These were contrasted with a poorly fitting single exponential model, which would suggest a single pool of constant turnover rate (Niinemets and Reichstein, 2002a). Exponential decay models fitted to the decay data (Figure 5.5) explained > 99% of the variation in almost all cases and the fitting of double- exponential models provided either no, or insignificant, improvement on the fit. The conclusions drawn by Guenther et al. (1991), that monoterpenes were not emitted from stomata, is supported by the fitting of the single-exponential decay model to the data described in this thesis. This implies that, at least in the case of juvenile leaves of eucalypts, emissions must travel through the cuticle. But in Quercus (Loreto et al., 1996b) and Picea (Shurmann, 1993, citied in Niinemets et al., 2002b), the cuticle was demonstrably resistant to emissions. Conversely, Schmid et al. (1992) found plant cuticles could adsorb large quantities of monoterpenes. Eucalypts are amongst the highest emitters of VOCs (Evans et al., 1982) and I argue that it is the anatomy of the eucalypt leaf – with cap cells above the oil glands – that explains these unique emission patterns and why stomata are not involved in the emission of these compounds (Guenther et al., 1991). Oil glands, the likely dominant source of monoterpene emissions, are located very close to the leaf surface. Oil gland initials are laid down while the leaf is in its embryonic state, but in its mature state, is a secretary-cell-lined cavity positioned close to the leaf surface with specialised neck and cap cells separating it from the cuticle (Carr and Carr, 1970). Cap and neck cells may be flush or protrude from the surface of the lamina and may number one, two or three layers of cells between the gland and the surface (Carr 128 Chapter 5 Light and temperature effects on terpenoid emissions and Carr, 1970). The specialised cap cells together with the cuticle form a very thin layer with a window appearance (Carr et al., 1971) and this is evident when holding leaves of many eucalypt species up to the light. Moreover, leaf surfaces of E. globulus are coated with a waxy cuticle comprised of long FKDLQ ȕ-diketones, involved in the formation and regeneration of waxy tubules (Wirthensohn and Sedgley, 1996). Wax depth is generally similar on both leaf surfaces but over the oil glands specialised concentric arrangements may develop (Louro et al., 2003). Finally, oil glands on the closely related myrtaceous species Melaleuca alternifolia were found to exude oil to the leaf surface when placed under low vacuum (<10-1 torr; List, 1995). Taken together, this evidence strongly suggests monoterpene emissions are drawn directly from the gland, through a relatively low-resistance pathway made up of specialised cap cells and waxy cuticle, into the atmosphere. But clearly more evidence is needed. A relatively simple experiment to test this hypothesis would hold leaves in the dark at constant temperature (for some hours) before repeating the step-wise increase in temperature. This experiment, together with that proposed above to further test the stomatal regulation hypothesis, would provide more compelling evidence for the operation of each mechanism. Indeed, should the latter be supported, a computationally-simple model could be applied once the character of the pathway is quantified, such as path length and permeability to different compounds, of both cap cells and the cuticle, to verify this route of emission. However, the single-exponential fit of the decay data suggests these intermediate pools – between the gland and atmosphere – act essentially as a single entity. 129 Chapter 5 Light and temperature effects on terpenoid emissions 5.4.2 Implications of monoterpene emission bursts for emission modelling The widely used model developed by Guenther et al. (see discussion above) has formed the basis for many exercises in modelling isoprenoid emissions. It applies a relatively simple temperature correction to emission rates observed under a set of standard conditions, from which it predicts isoprenoid emissions under a range of physiologically relevant temperatures. It is well recognised that factors other than light and temperature also drive of emissions and these terms are being incorporated as the evidence and the state of the art develop (Niinemets et al. 2010). The burst of monoterpenes emitted in response to changing leaf temperature have not been reported before and are obviously not represented formally in current emission models. Given that some of the emissions models, particularly that of Guenther et al. (1991, 1993), were based on measurements from eucalypts, it is possible that they include this information inherently. The question arises as to whether this behaviour produces emissions that differ significantly from those predicted by the Guenther model over longer periods, such as hours or whole days. If not, then the behaviour observed here may not need specific inclusion in later models. Initially, the Guenther model was applied to three sets of data collected during the temperature response experiments described above. These consisted of two sets in which temperature was increased before emissions had reached a steady-state (i.e. non- steady state; Figure 5.19a,b) and one where temperature was increased only after emissions had stabilised (steady state; Figure 5.19c). Total emissions at the end of each temperature step were determined for both the Guenther algorithm and observed emissions. In the case of the non-steady state emissions, the Guenther algorithm overestimated total emissions by a factor of 2 (Figure 5.19a) or underestimated total 130 Chapter 5 Light and temperature effects on terpenoid emissions emissions by a factor of 2 (Figure 5.19b). In the steady-state experiment, total emissions were underestimated by a factor of 0.3. In each case however, the Guenther algorithm underestimated emissions below 35° – 40 °C, estimated similar total emissions in this range, and beyond this either overestimated (Figure 5.19a) or underestimated (Figure 5.19b,c) emissions. Under these contrived temperature changes, it appears that the Guenther algorithm underestimates emissions below the temperature range of 35° – 40 °C, but beyond this the results are equivocal. Both the Guenther model and a rudimentary model developed from the experimental data collected in this chapter were then tested against real canopy leaf temperature data to determine putative differences between predictions. The canopy leaf temperature data were obtained from Figure 4b of Doughty and Goulden (2008; Figure 5.19d). The caveat in this test is that the model developed from these data is based on a limited range of data, constraining its validity to some extent. But it does act as a useful first step in estimating the uncertainty associated with the use of the Guenther algorithm when plants exhibit this behaviour. The model was developed by identifying a suitable function for each part of the burst- decay pattern. The curve fitting function of Graphpad Prism 5.04 (GraphPad Software, California, USA) was used and a combination of two functions was ultimately used to fit the data. The first was a Gaussian curve applied to the burst phase of emissions with the general form (௫ି)మ (ݕ = ܽ݁ (5.2 ଶమ where a is the peak height, b is the relative position of the centre of the peak and c controls peak width. A single-phase exponential decay model applied to the decay phase of emissions, with the general form 131 Chapter 5 Light and temperature effects on terpenoid emissions ିఒ௧ ܰ௧ = ܰ݁ (5.3) where Nt is the emission rate at time t, N0 LV WKH LQLWLDO HPLVVLRQ UDWH DQG Ȝ LV DQ empirically determined decay constant. Constants within each function were determined by fitting the function to each set of normalised data points and constraining it until an acceptable fit was achieved (R2 > 90%). The model was scaled against temperature changes beyond the range of those observed in the experiment by taking the mean ratio of the rate of temperature increase over time to the rate of monoterpene emission increase over time. Similarly, the height of the emission burst was constrained by the relative proportion of the temperature change and time. Unlike the Guenther model, these functions include terms representing time and temperature as proxies for underlying biological mechanisms, whose specific influence remains undetermined. In the data of Doughty and Goulden (2008), canopy leaf temperatures fluctuated between 5 °C and 10 °C rapidly (within minutes). The 10° changes were larger than those applied in the experiment. The model applied a concurrently rapid emission burst whose amplitude was three times greater than that of the Guenther model. Over the course of one hour, total emissions from my model were estimated as a factor of two to three times greater (according to the integration method) than those predicted by the Guenther algorithm. This is a large difference between models and certainly warrants the further development of a model to capture this behaviour. 132 Chapter 5 Light and temperature effects on terpenoid emissions Figure 5.19. Comparison of the Guenther monoterpene emission model with burst-decay emission patterns as leaf temperature was increased from 20° to 50 °C. Panel A and B, temperature increases before emissions had stabilised. Panel C, leaf temperature increased after emissions had stabilised. Panels A-C share the same legend: leaf temperature (broken line), observed emissions (thick solid line), fitted Guenther model (thin solid line). Panel D, fitting of both the Guenther model and a model developed from the empirical data represented in Panels A-C, to canopy leaf temperature data from Doughty and Gouldan, 2008 (see text for details). Curves represent leaf temperature (broken line), Guenther model fit (thin solid line) and model derived from this experiment (thick broken line). 133 Chapter 5 Light and temperature effects on terpenoid emissions 5.4.3 Evidence for light- and temperature-dependent emission of cis-ocimene Of the ten monoterpenes emitted from leaves in this study, only one, cis-ocimene, suggested evidence of an enzymatic origin within the leaf. Although high background levels were evident in emissions during darkness, illumination stimulated an increase in emissions beyond the background level. In addition, the temperature response is distinctly different from other monoterpenes for which observations across all plants were available. The temperature response showed a peak at 30 – 35 °C before declining between 40 – 45 °C. Emissions again rose at 50 °C. This pattern appears to be generated by a combination of recent biosynthesis and volatilisation from storage pools. However, the contribution of this compound to total monoterpenes declined with temperature from 6 % at the lowest temperatures to 1 % at the highest temperatures (Figure 5.11). I did not observe ocimene emissions from E. globulus in the field study described in Chapter 4, but they were reported by Street et al. (1997). Owen et al. (2002) noted the light and temperature dependence of ocimene emissions from a number of Mediterranean species, although trans-ȕ-ocimene emissions from Hevea brasiliensis were more tightly linked to circadian rhythms rather than light and temperature per se (Wang et al., 2007). 5.4.4 Isoprene emissions from eucalypts The temperature optimum for isoprene emissions at around 40 °C accords with optima observed in other species (Monson et al., 1992; Lerdau and Keller, 1997). At temperatures < 35 °C, isoprene emissions did not produce bursts in the manner observed in monoterpene emissions, instead remaining closely tied to temperature. At temperatures > 35 °C, small bursts were evident, but the steady-state rate following decay from the peak was less than 5% of the peak emission rate, and the effect was not 134 Chapter 5 Light and temperature effects on terpenoid emissions consistent between trees. However, at temperatures of 40 – 50 °C the effect was pronounced and probably indicates significant stress on the isoprene biosynthetic apparatus. Isoprene bursts at similar temperatures have been observed in Quercus rubra Singsaas and Sharkey (2000), who argued the decay in emission at high temperatures represented regulation of synthesis rather than denaturation of the enzyme. Conversely, the decrease in emissions at very high temperatures probably does represent irreversible denaturation, as leaf surfaces typically looked flaccid after 10 – 20 min at these temperatures. The light response was normal, but the limited intervals in light level yielded no new information about emission rates at saturating levels. The loss of carbon through isoprene emission was notably low compared to previous reports in the literature. At 45 °C, the ratio of carbon lost through isoprene emission did not exceed 1 % of carbon gain through photosynthesis. This ratio tripled at 50 °C, but this temperature was highly stressful for the leaf and the results are less robust than at lower temperatures. Leaves of Populus tremuloides subjected to temperatures over the range 20 – 40 °C lost between c. 0.5 – 8 % of recently assimilated carbon as isoprene. Whether the lower carbon loss observed in E. globulus relates to inadvertent adaptation of these specific plants, or is representative of the species more generally is unknown. Peak isoprene emission rates were slightly higher than those observed in the field study described in Chapter 4, but still at the lower end of the range of emissions observed in other studies (See Table 3.1 in Chapter 3). There was considerable variation in the temperature at which emissions peaks and as with other emission behaviours observed between plants in this study, the reasons underlying these differences are unclear. As leaf age was not strictly accounted for, this cannot be excluded. Indeed leaf age and leaf form clearly require further study. 135 Chapter 5 Light and temperature effects on terpenoid emissions 5.5 Concluding remarks Contrary to previous reports of temperature-dependent monoterpene emissions from plants, and eucalypts in particular, these plants produced large bursts of monoterpenes in response to 5 °C increases in leaf temperature. Once leaf temperature stabilised, these bursts decayed in a time-dependent manner. The temperature increase clearly stimulated the released of compounds from storage pools, to which de novo synthesis appears not to have contributed to any significant degree. The mechanism(s) responsible for regulation of the burst is unclear, but stomatal regulation or direct emission from oil glands to the atmosphere are probable explanations. I argue that emission from oil glands is the most likely explanation. Understanding the mechanism responsible will be critical for accurately estimating emissions from these trees. A rudimentary model fitted to the emission bursts suggests that the phenomenon may be captured by models already in use under limited temperature ranges. Where high temperatures or large temperature fluctuations are experienced, current models may not capture these effects on emission rates. Further studies will certainly reduce the uncertainty of the algorithm applied here and help to identify whether the uncertainty associated with this phenomenon should be considered more rigorously when scaling emissions from these trees. 136 Chapter 6 Origin of terpenoids from leaves of E. globulus 6. The origin of terpenoid emissions from juvenile leaves of 13 Eucalyptus globulus revealed by CO2 fumigation 6.1 Introduction Terpenoid emissions from the leaves of plants are widely known to originate from either specific storage organs, or to be the product of recent biosynthesis, and either one or both sources may exist within a single leaf. For example, Quercus ilex synthesises but does not store monoterpenes (Loreto et al., 1996c) while emissions from Picea abies (Shürmann et al., 1993) originate from both stored and recently synthesised sources. Additionally, Niinements et al. (2002b) have shown that all plants store terpenoids to some extent before emission to the atmosphere. That more than one origin of terpenoids may exist in a single leaf has a potentially important bearing on the prediction of emissions from a given species, particularly when formulating emissions inventories for various forest types. Eucalypts, along with other members of the Myrtaceae, are characterised by the presence of (often large numbers of) oil glands within their leaves. The handful of studies conducted previously on this genus suggests oil glands are the sole source of 137 Chapter 6 Origin of terpenoids from leaves of E. globulus terpenoid emissions. The exception of course is isoprene, known universally to be the product of recent biosynthesis. In plants that emit terpenes from specific storage organs, gas exchange studies in which light and temperature are manipulated generally provide the first indication that de novo synthesis may take place (e.g. Owen et al., 2002). However gas exchange studies alone are an equivocal indicator of de novo synthesis, as factors related to physicochemical properties, such as solubility or vapour pressure, may instead produce a response that could inadvertently be interpreted as one dependent on recent biosynthesis. 13 Fumigation of leaves with C-labelled CO2 provides unequivocal evidence for the incorporation of recently assimilated carbon into emitted VOCs. This method has been applied widely over the last 20 years to a number of plant species (e.g. Schürmann et al., 1993) and is a definitive demonstration of de novo synthesis. Labelled fumigation experiments on plants that do not possess specific storage organs have shown that 100% incorporation of the labelled carbon is never achieved (Delwhiche and Sharkey, 1993; Loreto et al., 1996c, 2000), and that therefore other sources of non-labelled carbon are being drawn upon during synthesis (Kreuzwieser et al., 2002; Schnitlzler et al., 2004). This presents problems when trying to separately estimate the contribution to VOC emissions of stored versus newly synthesised VOCs (Shao et al., 2001; Dindorf et al., 2005). Data from previous chapters suggests that cis-ocimene, in particular, may be synthesised from recently assimilated carbon. To test this leaves of E. globulus were 13 fumigated with CO2 to: (1) identify if terpenoid compounds emitted from the leaves of this species are produced de novo; (2) identify the origin of de novo monoterpenes through the incorporation of 13C into terpenoids in individual oil glands, gas phase 138 Chapter 6 Origin of terpenoids from leaves of E. globulus emissions and whole leaves; and (3) determine how the activity of monoterpene synthases relates to monoterpene emissions. 139 Chapter 6 Origin of terpenoids from leaves of E. globulus 6.2 Methods and materials 6.2.1 Plant material The plants were from the same cohort described in chapter 5 and were grown under the same conditions. 6.2.2 Gas exchange system Gas exchange measurements were performed as described in chapter 5 and used the 0.7 L cuvette described previously. Light was provided by the same 250 W metal-halide lamp and held constant at 500 ȝmol m-2 s-1 for the duration of the experiment. Leaf temperature was held at 27.9 ± 1.3 °C for all experiments. Humidified zero air (c. 28 % relative humidity) mixed with CO2 of natural isotopic abundance to produce a CO2 concentration of 400 ppm was supplied to the cuvette. Exchange of H2O and CO2 were monitored with a CIRAS II infra red gas analyser (PP Systems, Hitchin, Hertfordshire, UK) sampling air before and after the cuvette. While infra red gas analysers are less 13 sensitive to CO2, flow rates were identical for each CO2 source. 6.2.3 Sampling of volatile organic compounds VOCs were simultaneously sampled using two methods. The high- sensitivity proton transfer reaction mass spectrometer (PTR-MS) was used to monitor, in real time, changes in the isotopic signal of terpenoid compounds. In addition, charcoal-filled adsorbent cartridges were used to trap the VOCs for later identification of the individual monoterpenes via GC-MS and by gas chromatography (GC equipped with a flame ionisation detector; FID). Samples for both the PTR-MS and the traps were drawn at 240 mL min-1 from the exhaust side of the cuvette, from which a sub-sample of 75-80 140 Chapter 6 Origin of terpenoids from leaves of E. globulus mL min-1 was directed into each instrument for analysis. Calibration, background checks and operating conditions for both instruments were performed as described in Chapter 2. The PTR-MS was used to monitor the molecular and fragment ions of isoprene (m/z 69 - 74) and the monoterpenes (m/z 81 - 95; m/z 135 - 145, m/z 155 - 165), but these ranges were modified during the experiment to monitor ions that showed a response to labelling (identified in the fragmentation studies described in Chapter 2). 6.2.4 Charcoal adsorption cartridges Adsorbent cartridges were custom-made for collecting gas-phase monoterpenes for subsequent analysis of isotopic distribution by GC-MS. Borosilicate glass tubes 7 cm long with an internal diameter of 3.2 mm were loaded with 5 mg of 20-60 mesh activated charcoal (Sigma Aldrich, Sydney) and plugged at either end with silanised glass wool. Charcoal was pre-treated by heating to 450 °C while passing helium through the bed at 80 mL min-1 for 4 hours. Tests were conducted to determine the quantitative recovery of four representative terpenoids spiked onto the charcoal at concentrations similar to those expected. Compound recovery varied between 50 – 160 % which was deemed unacceptable for quantitative purposes and results using these data are qualitative only. Sample air was drawn through the cartridge at 0.5 L min-1 for 60 min and after sampling, a glass wool plug was removed from one end and the charcoal transferred to the ȝ/insert of a 2 mL glass vial. Carbon disulphide (50 ȝ/ was immediately added and tapped briefly to remove air bubbles before standing for 30 min at room temperature. Tests indicated extraction times greater than 30 min did not improve analyte recovery. After 30 min the extract was transferred into a second vial and stored at 4 °C until analyses. Once extracted, compounds were very 141 Chapter 6 Origin of terpenoids from leaves of E. globulus stable, with no difference between air tight vials tested eight weeks apart. Charcoal beds were used only once. 6.2.5 Identification of 13C-labelled terpenoids within leaf tissue Potential sites of terpenoid biosynthesis within leaves were assessed by sampling 13 individual glands and whole leaf tissue immediately after fumigating leaves with CO2 for 6 hours. The methods described in Chapter 3 (section 3.2.2-5) were used in this study to sample terpenoid oils from individual oil glands and whole leaf tissue. Following fumigation, leaves were removed from the cuvette and immediately cooled on ice. Of the four leaves from the cuvette, two were selected for whole leaf extraction and transferred to liquid nitrogen. The remaining two leaves were selected for oil gland sampling using the methods described in Chapter 3 (section 3.2.3) followed by whole leaf oil extraction. 6.2.6 GC-MS analysis of individual terpenes Samples from individual oil glands, extracts of whole leaf tissue and extracts from adsorbent cartridges were all analysed using the GC-MS method described in Chapter 3 (3.2.5). 6.2.7 Extraction and temperature assay of monoterpene synthase Leaves from the trees used in the gas exchange study, in addition to similarly aged leaves from specimens of E. viminalis grown under identical conditions, were assayed for terpenoid synthase activity at the Institute for Meteorology and Climate Research, Garmisch-Partenkirchen, Germany. Healthy juvenile leaves were instantly frozen in liquid N2 and packed in solid CO2 for transport. On arrival they were stored at -80 °C 142 Chapter 6 Origin of terpenoids from leaves of E. globulus until assayed, approximately 3 – 4 weeks later. Proteins were isolated using modified protocols previously developed for Quercus ilex (Fischbach et al. 2000). Initial testing suggested standard protocols developed for Q. ilex would also work for eucalypts. Subsequent assays indicated terpenoid oils were carried into the protein extract, and various attempts to separate the oil from the extract proved unsuccessful. Synthase activity was therefore assayed by temperature response to identify synthase activity that might be identifiable above the high background level. Leaves were ground under liquid N2 and 100 mg (fresh weight) of the powder was suspended in 5 mL of chilled extraction buffer (0.7 M MOPS, pH 7.3, 1.5 % PEG, 20 mM MgCl2, 1 % PVP-30 (w/v), 8.3 % (w/v) Dowex 1 x 2, 0.5 M Na-Ascorbate, 0.5 M Na2S2O5, 0.5 M DTT). Remaining steps in the protocol were conducted at 4 °C or less. Following stirring for 15 min, the homogenate was centrifuged at 18 000 g for 20 min and 2.5 mL of the supernatant was desalted on PD-10 columns (Amersham-Pharmacia, Freiburg, Germany) with elution buffer (50 mM KPi, pH 7.3, 10 % glycerol (v/v) and antioxidants (200 mM Na-DVFRUEDWH P0 ȕ–mercaptoethanol). From the resulting P/ RI SURWHLQ H[WUDFW ȝ/ ZDV WUDQVIHUUHG LQWR P/ JDV-tight crimp vials, IROORZHGE\ȝ/RI00J&O27KHHQ]\PHUHDFWLRQZDVVWDUWHGE\DGGLQJȝ/RI P0JHUDQ\OGLSKRVSKDWH *'3 ILQDOFRQFHQWUDWLRQ ȝ0 DQGYLDOVZHUHLQFXEDWHG for 60 min at one of eight 5 °C temperature increments between 25 °C and 60 °C. The reaction was stopped by removing the reaction mixture with a syringe and rinsing the YLDO ZLWK ȝ/ RI GLVWLOOHG ZDWHU 0RQRWHUSHQHV JHQHUDWHG E\ WKH UHDFWLRQ RI monoterpene synthases with the GDP substrate were sampled from the headspace analysed by gas chromatography (GC). A Chrompack CP gas chromatograph (GC) (Frankfurt/M., Germany) equipped with a headspace-volume-autosampler (Gerstel, Mülheim, Germany) and temperature- 143 Chapter 6 Origin of terpenoids from leaves of E. globulus programmed injection system (KAS 3, Gerstel, Mülheim, Germany) was used for analysis. Headspace injections of 1 mL were concentrated on a precolumn trap containing Tenax TA (60/80 mesh) at 27 °C. Following desorption at 240 °C, compounds were carried onto a capillary column (DB-P[PP,'ȝP film thickness; J&W Scientific, Folsom, CA, USA) with the temperature program beginning at 35 °C, increasing after 0.5 min to 78 °C at 30 °C min-1, held for 4 min then increased at 9 °C min-1 to 160 °C, then increased again at 35 °C min-1 to 250 °C. Products were detected with a FID operating at 270 °C. Compounds were identified by retention time comparison with authentic standards, but were not quantified given the high background levels. However, protein extracts incubated without the GDP substrate were used to correct for degradation of the enzyme. 6.2.8 Data analysis and error determination Determination of experimental error was carried out as described in Chapter 2. Half-life time of the decay of the unlabelled signal of compounds detected by PTR-MS were = (determined from the time evolution of this signal according to the equations ܰ(ݐ ିఒఛ ܰ݁ DQGIJ(1/2) =ln(2)/ߣ where N0 is the calibrated instrument signal at t = 0. Half- life time calculations were made using GraphPad Prism 5 (GraphPad Software, California, USA). 144 Chapter 6 Origin of terpenoids from leaves of E. globulus 6.3 Results 6.3.1 Origin of 13C-labelled terpenoid compounds 13 -2 -1 Following six hours of CO2 IXPLJDWLRQXQGHU FRQVWDQWOLJKW ȝPROP s ) and temperature (28 °C), only four of the more than 40 compounds detected had 13 incorporated C. These were isoprene (C5H8), isovaleraldehyde (C5H10O, tentatively identified), cis-ocimene (C10H16) and trans-caryophyllene (C15H24). All were collected from the gas phase and there was no evidence of 13C uptake in any terpenoid compounds within the oil glands or whole leaves. 6.3.2 Identification and kinetics of 13C-labelled compounds Isoprene and isovaleraldehyde were only detected by PTR-MS while cis-ocimene and trans-caryophyllene were detected only by GC-MS, following desorption from the charcoal cartridges. Monitoring of the molecular and fragment ions of mono- or sesquiterpenes with PTR-MS could not be used to follow the incorporation of 13C, because these are low abundance ions which need to be monitored against a high background signal from the stored terpenoids. Isoprene and iso-valeraldehyde were not detected by GC-MS as these are very low boiling point compounds and the option of cryogenic injections onto the column was not available to us. PTR-MS data showed 13C being incorporated into isoprene from approximately 10 min 13 after commencement of fumigation with CO2 (Figure 6.1) and the level of incorporation reached a maximum of 82 % by mass after 2.5 h. After reverting to CO2 at natural isotopic abundance, the observed 13C incorporation declined at a rate similar to its incorporation. A second compound, later tentatively identified as iso- valeraldehyde, demonstrated similar 13C incorporation kinetics, with 13C incorporation 145 Chapter 6 Origin of terpenoids from leaves of E. globulus 200 12CO 13CO 12CO 2 2 2 m/z 69 ) -1 m/z 70 150 min m/z 71 -2 m/z 72 100 m/z 73 m/z 74 50 Sum Emission (nmol m (nmol Emission 0 0 50 100 150 200 250 300 350 400 450 Elapsed time (min) Figure 6.1 Time course of 13C incorporation and wash-out into isoprene emitted from leaves of E. globulus. Isotopes are m/z 69, [12C]isoprene; m/z 70, isoprene with one 13C; m/z 71, isoprene with two 13C and so on up to m/z 74 with all five carbons labelled with 13C. The vertical dotted lines indicate switching 13 between 1.1 % and 99.5 % CO2. The small peaks at 235 min were produced by an interruption to the JDVIORZ/HDYHVZHUHKHOGDW&XQGHUȝPROP-2 s-1 PAR for the duration of the experiment. The data represent a typical isotope profile of 4 replicate plants. 100 12CO 13CO 2 2 m/z 85 80 m/z 86 m/z 87 60 m/z 88 m/z 89 40 m/z 90 Arbitrary units Arbitrary m/z 91 20 m/z 92 0 0 50 100 150 200 250 300 350 400 450 Elapsed time (min) Figure 6.2. Time course of 13C labelling for the m/z range 85 – 92. Ions at m/z 87 and m/z 85 declined in 13 response to fumigation with CO2. Transient increases and declines were observed in the abundances of m/z 86, 88 and 89 and sustained increases in m/z 91and 92, corresponding with the fumigation with 13 13 CO2. This indicated C incorporation into a five carbon VOC, later tentatively identified as iso- 13 valeraldehyde (C5H10O). The vertical dotted lines indicate switching between 1.1 % and 99.5 % CO2. Experimental conditions were identical to those in Figure 6.1. 146 Chapter 6 Origin of terpenoids from leaves of E. globulus observed about 15 min after fumigation started (Figure 6.2) and reaching a maximum of 82 % by mass after 5 h. The rate of 13C incorporation into the parent ion occurred slightly faster in isoprene (m/z IJ1/2 = 4.47 min) than in isovaleraldehyde (m/z IJ1/2 = 5.11 min; m/z IJ1/2 = 5.94 min). While scanning for 13C labelling into the monoterpenes over the range m/z 81 to m/z 95, a shift of 5 mass units corresponding to the incorporation of five 13C atoms was noted for ions occurring at m/z 85 and m/z 87 (Figure 6.2), which simultaneously declined in response to the introduction of 13C. In addition, incrementally higher masses showed transient peaks following the sequential replacement of 12C with 13C atoms in the molecule, akin to the labelling pattern of isoprene (Figure 6.1). This indicated the molecule which was a C5 compound of molecular mass 86, with ionisation in the PTR- MS inducing single-proton addition (m/z 87) and removal (m/z 85). A molecular mass of 86 corresponds to the plant volatiles iso-valeraldehyde and methyl butenol, both with the structural formula C5H10O, but the signal may also have resulted from adduct formation of another labelled compound during ionisation. Mass scans in the range m/z 81 – 95 ruled out monoterpenes as potential contributors to this pattern. Isoprene shows no ion fragments at these masses and was also excluded. p-Cymene produces ion fragments at m/z 93 (Tani et al., 2004; Chapter 2 in this thesis), but also showed no 13 response to CO2 fumigation (data not shown). Iso-valeraldehyde, detected previously in the analysis of leaf oils from a number of eucalypt species (Brophy et al., 1991) and two alcohols: 3-methyl-3-buten-1-ol, emitted by Quercus ilex (Loreto et al., 1996c) and 2-methyl-3-buten-2-ol, emitted by a number of Pinus species (Harley et al., 1998; Baker et al., 2001; Gray et al., 2005) were considered candidates as all share a common formula and molecular mass. Qualitative gas standards, produced by dilution of neat liquid standards into N2, were used to 147 Chapter 6 Origin of terpenoids from leaves of E. globulus examine the fragmentation spectra of each compound generated by the PTR-MS. Iso- valeraldehyde produced fragments at m/z 85 and m/z 87 in similar proportions to those seen in the labelling study (Figure 6.3A), while the methyl butenol (MBO) compounds did not (Figure 6.3B). Further evidence was sought by running these compounds on the chromatographic columns used in this experiment (Solgel WAX) and in the temperature – light response experiment of Chapter 5 (BP-1 100 % polydimethylsiloxane). Neither matched the retention time of unknown peaks in the Solgel WAX column, while iso- valeraldehyde coincided with unknown peaks from the GC runs in Chapter 5 (data not shown). However, iso-valeraldehyde was characterised by two distinct but poorly shaped peaks because the GC system lacked cryogenic focussing for very volatile compounds. A further attempt was made to confirm the identification of this compound by sampling cuvette air onto a DNPH cartridge as described in Chapter 4, for subsequent analysis by LC-MS. A DNPH derivative of iso-valeraldehyde could not be obtained, and analysis by LC-MS proved inconclusive. 148 Chapter 6 Origin of terpenoids from leaves of E. globulus Figure 6.3. Fragmentation spectra generated by PTR-06$ȝ/RIHDFKZDVGLOXWHGLQWRLQGLYLGXDO/ Tedlar bags and filled with ultrapure N2. Spectra are the background-subtracted average of 10 – 15 cycles. A, iso-valeraldehyde. B, 2-methyl-3-buten-2-ol. Prominent ions are indicated next to the bars. 149 Chapter 6 Origin of terpenoids from leaves of E. globulus The monoterpene (C10) cis-ocimene, and the sesquiterpene (C15) trans-caryophyllene were the largest terpenoids to incorporate 13C as identified by mass spectrometry (Figure 6.4). An isomeric form of ocimene, tentatively identified as trans-ocimene from an authentic standard, also incorporated 13C, but the compound was not consistently detected and was excluded from further analysis. The degree of incorporation of labelled carbon into these C10 and C15 compounds was determined by comparison of the mass spectrum of the respective compound over the course of the labelling experiment, with ions selected according to their abundance in the spectrum or their relationship to the molecular ion. Although the cis-ocimene molecular ion (M+., m/z 136) was < 2% relative abundance, the corresponding ion at m/z 146, representing the incorporation of ten 13C atoms was clearly discernable (Figure 6.4A). This level of incorporation was confirmed by analysis of the [M-15]+. ion (m/z 121, 18%) where the incorporation of 9 13C atoms could be clearly observed in the corresponding labelled ion at m/z 130. The incorporation of 13C into the [M-15]+. ion reached a maximum of approximately 77 % after 2.5 h (Figure 6.5A), similar values to those reported above for isoprene. The of trans-caryophyllene molecular ion (M+., m/z 204) had a 6% abundance and incorporation of fifteen 13C atoms could be clearly observed in the corresponding ion at m/z 219 (Figure 6.5B). Incorporation of 13C into trans-caryophyllene appeared to stabilise at 15 – 20 % after 5 – 6 h. 150 Chapter 6 Origin of terpenoids from leaves of E. globulus Figure 6.4. Mass spectra of A, cis-ocimene and B, trans-caryophyllene, emitted from leaves of E. globulus showing the shift in fragmentation resulting from incorporation 13C into the molecule. The 13C labelled spectra (red) were captured 6 h after labelling began and are compared with the unlabelled spectra (black) showing natural isotopic abundance. 151 Chapter 6 Origin of terpenoids from leaves of E. globulus A 13 CO2 m/z 98:91 100 m/z 113:105 m/z 130:121 ratio (%) ratio 50 m/z 0 0 1 2 3 4 5 6 7 8 Elapsed time (h) 40 B 13 CO2 m/z 98:91 m/z 143:133 30 m/z 203:189 m/z 219:204 20 ratio (%) ratio m/z 10 0 0 1 2 3 4 5 6 7 8 Elapsed time (h) Figure 6.5. Time course of 13C incorporation into A, cis-ocimene and B, trans-caryophyllene, emitted from leaves of E. globulus for the ion fragments listed in the legend. Relative incorporation of 13C was calculated as [m/z (labelled)/(m/z unlabelled + m/z labelled)] x 100 using ions from electron ionised mass spectra averaged across the total ion chromatogram for a given compound at each time point. Duration of 13 CO2 fumigation is indicated on each graph. Data are from a single tree but are typical of the pattern observed in four trees. 152 Chapter 6 Origin of terpenoids from leaves of E. globulus 6.3.3 Monoterpene synthase activity Monoterpene synthase activity was determined by headspace analysis following incubation of proteins extracted from eucalyptus leaves with geranyl diphosphate (GDP). The assay proved problematic because stored oils were carried through the protein extraction process. This was demonstrated when headspace analysis of denatured (boiled) extracts, produced essentially similar results. Variations in the protocol, including salting out extracts and radioactive phosphatase assays using [1 – 3H]-GDP (Fischbach et al., 2000) failed to resolve this problem. However, incremental increases in the incubation temperature resulted in the increased production of cis- ocimene. The normalised response was detectable above the background monoterpene levels. This phenomenon was produced in both E. globulus and E. viminalis (Figure 6.6) and in both species maximum enzymatic production of cis-ocimene occurred between 50 °C and 55 °C, while denaturation appears to occur above this temperature range. 2WKHUPRQRWHUSHQHVVXFKDVĮ-pinene, showed no response to temperature (data not shown). 153 Chapter 6 Origin of terpenoids from leaves of E. globulus 3 2 1 Normalised activity Normalised 0 20 25 30 35 40 45 50 55 60 65 Temperature (qC) Figure 6.6. Production of cis-ocimene in response to incrementally higher incubation temperatures during the monoterpene synthase assay for leaves of E. globulus (squares) and E. viminalis (circles). Each symbol is the mean of two assays at that temperature. 154 Chapter 6 Origin of terpenoids from leaves of E. globulus 6.4 Discussion 13 Exposure of E. globulus leaves to CO2 revealed that both the monoterpene, cis- ocimene, and the sesquiterpene, trans-caryophyllene, incorporate recently synthesised carbon and are thus subject to regulation by light as well as temperature. In addition, isoprene and the tentatively identified iso-valeraldehyde, also incorporated recently fixed carbon. 6.4.1 The origin of labelled terpenes Terpenes emitted by E. globulus appear to originate from two sources within the leaf: the oil glands and the mesophyll tissue. Moreover, emissions of stored and de novo synthesised compounds appear to be compartmentalised respectively into these two sites. The absence of labelled carbon in compounds stored in the oil glands seems to exclude the possibility that synthesis took place in the gland epithelial cells and the products deposited into the gland. It is unlikely that labelled compounds were too dilute to detect in the oil glands given the number of glands sampled and the sensitivity of the instruments. Labelled compounds were also absent in the whole leaf tissue, indicating their absence from tissue outside the oil glands. However, clearly compounds were labelled, but only seen in emissions, and given the similarity in labelling patterns between the three terpenoids and iso-valeraldehyde, it seems likely that the chloroplasts of the mesophyll cells are the route through which carbon was fixed and made available for terpenoid synthesis. Detailed biochemical analysis is needed to verify this circumstantial evidence. Nonetheless, an identical pattern to that 13 reported here in was found in Norway spruce following CO2 fumigation (Schürmann et al., 1993). However, 14C labelling in Maritime pine detected monoterpene synthesis in the epithelial cells of the growing part of the needle 155 Chapter 6 Origin of terpenoids from leaves of E. globulus (Bernard-Dagan et al., 1979), while older parts of the needle were significantly diminished in labelled carbon. In addition,labelled sesquiterpenes were detected only in the whole leaf tissue surround the resin ducts. 13 C appeared rapidly in isoprene following exposure to labelled CO2, but the temporal resolution afforded by the use of charcoal adsorbent meant the appearance of 13C in cis-ocimene and trans-caryophyllene could not be determined as accurately. Despite this, the time taken for 13C enrichment to saturate and the final fraction of the molecule enriched with 13C was remarkably similar between isoprene and cis- 13 ocimene, with both reaching around 80 % enrichment after 2.5 h exposure to CO2. Labelling of trans-caryophyllene occurred at a slower rate and was less complete than in isoprene and cis-ocimene. This pattern appears to be the result of the rapid incorporation of labelled carbon into the C5 and C10 terpene substrates dimethylallyl diphosphate (DMADP) and geranyl diphosphate (GDP) respectively. The rate of incorporation observed in emissions is much slower in E. globulus than monoterpene labelling in Q. ilex (Loreto et al., 1996c), which saturated at around 80 % after 20 – 30 min. Differences in leaf architecture, such as bulk leaf density, may explain this effect. The slower and more limited labelling of trans-caryophyllene relative to the C5 and C10 terpenes also fits with the export of isopentenyl diphosphate (IPP) from the plastid into the cytosol, where carbon from the mevalonic pathway also contributes to the synthesis of sesquiterpenes (Laule et al., 2003). The link between the two compartments has been demonstrated previously in E. globulus, where deuterated deoxyxylulose was incorporated into both ocimene and caryophyllene at 83 % (Piel et al., 1998), although it is not clear over what time frame this occurred. 156 Chapter 6 Origin of terpenoids from leaves of E. globulus 6.4.2 Identification of 13C labelling The labelling experiments confirm unequivocally the incorporation of recently fixed carbon into cis-ocimene and trans-caryophyllene, but the extent of this incorporation can be obscured by emissions from stored sources. This is particularly so, when analysing samples by GC-MS, and where the molecular ion is of low abundance or is non-existent. To determine whether smaller ion fragments could be reliably used to examine labelling kinetics, Loreto et al. (1996c) tested a statistical null model that assumed the random distribution of C atoms in the terpene molecule against the labelling they observed. Their conclusion that all carbons were probably randomly labelled is now known not be true, with carbon atoms drawn from specific substrates (Kreuzwieser et al., 2002). However, in the absence of a suitably abundant molecular + ion, the [M – CH3] · ion could be used to determine incorporation kinetics, providing the number of carbons in the molecule is sufficiently high. Terpene emissions from stored sources could pose a greater problem for interpretation if they are large relative to the de novo fraction as the incorporation of the 13C label would need to be determined against a large endogenous background. The light response experiments in Chapter 5 found cis-ocimene emissions were six WLPHVJUHDWHUZKHQH[SRVHGWRȝPROP-2 s-1 PAR (15 nmol m-2 min-1) compared to darkness (2.5 nmol m-2 min-1), where dark emissions represent those from the stored pool. This light:dark emission ratio is therefore around 6:1, but as temperature was 3 °C warmer in this study (with the same light level), this ratio might be slightly smaller and a more conservative ratio of 5:1 is assumed. Therefore, 20% of the 12C signal (at saturated enrichment of the de novo fraction) is due to emission from stored oils. Correcting for this produced a slight increase from 77 % to 79 % in the ratio of 13C incorporation, which is within the error range of these estimates. Using the same 157 Chapter 6 Origin of terpenoids from leaves of E. globulus spectral data, but changing the emissions from stored pools to 70 % of de novo emissions, saturated enrichment would be corrected to 93 %, representing a large change. As an alternative, Ghirardo et al. (2010) dealt with this using PTR-MS by assuming the labelling seen in the monoterpenes must reflect that seen in isoprene, given their proximity in the biosynthetic pathway. The identification of iso-valeraldehyde requires confirmation. However, on the basis of the data described in this thesis it is a reasonable candidate for the ions observed at m/z 85 and 87, particularly given previous reports of iso-valeraldehyde in the oil extracts of a number of eucalypt species, including E. globulus (Brophy et al., 1991). However, caution is warranted given the rate of labelling seems fast relative to its putative source within the leaf. The few reports of iso-valeraldehyde in the plant literature (Dalal et al., 1968; Nimitkeatkai et al., 2005) suggest its emissions results from the action of pyruvate decarboxylase on pyruvate during the catabolism of L- leucine (Yoshimoto et al., 2001; Nimitkeatkai et al., 2005). If this is indeed the source of iso-valeraldehyde from eucalypt leaves then it seems at odds with the rapid incorporation of 13C into the molecule. Conversely, 2-methyl-3-buten-2-ol and various structural isomers have been reported in the literature, mostly in pines (Harley et al., 1998; Baker et al., 1999; Gray et al., 2003, 2005) but also in Quercus ilex (Loreto et al., 1996c). While retention time matching by gas chromatography proved inconclusive, analysis of the iso-valeraldehyde fragmentation by PTR-MS generated a 13 pattern (Figure 6.3) that matched the ions seen during fumigation with CO2 (Figure 6.2). Although more ion fragments were seen in the standard, it provided a better fit than methyl butenol, which has not been reported from eucalypts. Regardless, this compound warrants positive identification to determine its importance in emission from this species. 158 Chapter 6 Origin of terpenoids from leaves of E. globulus 6.4.3 The putative importance of de novo synthesis While the monoterpene synthase assays were limited by the passage of terpene oils from the ground leaves into the protein extract, the incubation assay using an incremental rise in temperature was able to discriminate contaminant oils from those synthesised during the incubation period, revealing only cis-ocimene was produced enzymatically. The temperature optimum at 50 °C was around 10 °C higher than found in Q. ilex (Fischbach et al., 2000) but similar in range to isoprene synthase temperature optima from leaves of E. globulus (Winters, Schnitzler and Zimmer, unpublished data). These data confirm that a fraction of cis-ocimene emitted from these leaves is indeed from a de novo source. Importantly, no other monoterpenes were synthesised during the assay, however the presence of low levels of de novo monoterpenes would have been very difficult to detect against the background of the oils carried-over from the protein extraction. The absence of 13C labelling in any other monoterpene, either emitted or stored, suggests monoterpene synthesis may be subject to temporal regulation within these leaves. Seasonal regulation of monoterpenoid synthesis has been demonstrated previously in Quercus ilex (Fischbach et al., 2002) which does not store terpenes, and in Pinus sylvestris (Hakola et al., 2005) which both stores and syntheses terpenes. In peppermint (Mentha x piperita L.) monoterpene synthases are active during leaf development but inactive in the mature leaf (Turner et al., 1999; McConkey et al., 2000). These data suggest that once oil glands have been filled during leaf expansion, the epithelial cells become either dormant or degenerate and the oil produced at that point will be the only oil for the remainder of the leaf’s 2-4 year life span. Yet biosynthesis of non-stored terpenes persists within the mesophyll for the life of the plant. A similar pattern of development is observed in Maritime pine (Bernard-Dagan et al., 159 Chapter 6 Origin of terpenoids from leaves of E. globulus 1979). Preliminary evidence for this comes from an unpublished comparison of emissions between young and old juvenile leaves of E. globulus, where young leaves emit eucalyptol two to three orders of magnitude greater than cis-ocimene, while in the aged leaf this had dropped to 1:1. 160 Chapter 6 Origin of terpenoids from leaves of E. globulus 6.5 Concluding remarks With the exception of isoprene, the few studies of terpenoid emission from eucalypts suggested oil glands were the source of these emissions and thus regulated by temperature alone. Given the predominance of oil glands in the leaves of eucalypts, these are a logical source of emissions. This study has shown that while most terpenoids emitted from the juvenile leaf form of E. globulus are emitted from stored pools, the monoterpene cis-ocimene and the sesquiterpene trans-caryophyllene originate from de novo synthesis. In addition to isoprene, an oxygenated C5 compound tentatively identified as iso-valeraldehyde was also emitted directly from de novo synthesis. Future work needs to identify how these emissions respond to higher levels of light and temperature, particularly given the high levels of each encountered in the habitat of many eucalypts. Similarly, the positive identification, quantification and response to environmental variables of iso-valeraldeyhde require attention. This is further warranted given iso-valeraldehyde produces a large fragment within the PTR-MS at m/z 69, causing a potentially serious error in isoprene flux estimates. 161 Chapter 7 Conclusions 7. Conclusions This thesis examined some of the factors that regulate volatile organic compound emissions from Eucalyptus globulus subsp. globulus. This widely cultivated species has been the most well studied of the eucalypts, yet fundamental questions concerning emission regulation, such as the source and route of emissions from within the leaf, to date remain unanswered. Three primary questions were examined as a contribution to redressing this. 1. What range of compounds and at what rates are terpenoids and carbonyls emitted from field-grown eucalypts cultivated under conditions of non-limiting nutrient and soil water availability, and how do environmental factors influence these emissions? 2. How do temperature and light influence terpenoid emissions from E. globulus, given there has been no overlap between detailed laboratory-based studies of a few compounds with field-based studies covering a greater spectrum of compounds? 162 Chapter 7 Conclusions 3. Do de novo synthesised terpenes make any contribution to emissions from E. globulus? The few field-based observations of VOC emissions from eucalypts prompted a study to characterise the emission of isoprene, monoterpenes and the carbonyl compounds formaldehyde, acetaldehyde and acetone from a mixed eucalypt plantation in south western Australia (Chapter 4). Carbonyl emissions were also sampled from a second site in eastern Australia. Isoprene and total monoterpene emissions were of a similar absolute range between the four species of eucalypt studied. However, despite the non- limiting nutrient and water status at the site, isoprene emissions from adult leaves were at the low end of the range of emissions reported previously. Conversely, total monoterpene emissions were at the high end of the range of previously reported emission rates. The underlying reasons for these results could not be explicitly identified, but is it hypothesised that cool temperatures and low light levels at the time of sampling may have resulted in lowered levels of isoprene emissions. Monoterpene emissions on the other hand, presumed to originate predominantly or solely from oil glands within the leaf, may have developed large pool sizes due to favourable growing conditions at the site, and thus exhibited higher rates than previously observed. Carbonyl emissions, believed to be reported for the first time from eucalypts here, were similar in magnitude to reports from European species. Low transpiration rates in south-western Australia, relative to the eastern Australian site, were believed responsible for the large differences in acetaldehyde emission between the two sites. The influence of temperature and light on isoprenoid emissions were examined under controlled laboratory conditions on juvenile leaves of E. globulus (Chapter 5). By far the most important finding in this study was the large transient burst of total monoterpene emission that followed incremental increases in leaf temperature. Bursts at 163 Chapter 7 Conclusions all temperatures exceeded the steady-state emission rate for that temperature, which typically took more than one hour to achieve. The burst phase was linear and related to temperature while the decay of emissions following the burst was time dependent, to which a single exponential model provided the best fit. This suggested a single pool was the source of emissions and differed from two-pool models which have been observed in the non-terpene storing species such as Quercus ilex. Adsorption effects in the cuvette were ruled out as a potential source of these bursts. The precise mechanism regulating the large transient bursts is unknown, but I argue that various lines of evidence exclude stomata as a major control and instead hypothesise that emissions originate directly from subdermal oil cavities. In contrast, isoprene emissions closely followed temperature at all but the highest (> 40 °C) leaf temperatures, where small bursts were related to temperature effects on biosynthesis. At steady states, the emission of individual monoterpenes generated an exponential response to increasing leaf temperature. Fluctuating leaf temperatures on warm sunny days are likely to produce emissions that are not captured by any of the current models. Further elucidation of the regulating mechanism is needed before these bursts can be satisfactorily modelled. The light dependence of cis-ocimene emissions were evident, but its small contribution to total emissions in these leaves at least, means it is unlikely to be responsible for the transient burst of monoterpene emission. 13 The origin of monoterpene emissions was examined by fumigating leaves with CO2 and monitoring the isotopic composition of terpenoid compounds evolved during this process (Chapter 6). Over the time course of the experiment no labelled compounds were detected only in emitted compounds. Oil glands and leaf tissue were sampled but labelled terpenes were detected. Four compounds incorporated 13C: isoprene (C5), iso- valeraldehyde (C5), cis-ocimene (C10) and trans-caryophyllene (C15). Iso- 164 Chapter 7 Conclusions valeraldehyde was only tentatively identified. Of the remaining compounds, the final level of incorporation of 13C and the time taken to reach this level, were similar between isoprene and cis-ocimene (c. 80% and 2.5 h respectively), while the final fraction of 13C in trans-caryophyllene (and the time taken to reach these values) were considerably greater in both respects). This reflects the transport of the terpenoid precursor isopentenyl pyrophosphate into the cytosol where it is used for sesquiterpene synthesis. The ratio of de novo synthesised mono- and sesquiterpenes was small relative to the flux from stored pools, but this may change dramatically as leaves age, or indeed with leaf form. Arneth et al. (2008) recently asked why, in contrast to isoprene, were global estimates of monoterpene emissions so dissimilar. While their paper was addressing the discrepancies between modelled emission estimates at landscape and global scales, the question has immediate relevance to the work presented in this thesis. The preceding chapters present new evidence that demonstrates terpenoid emissions from E. globulus, and perhaps the genus more generally, are more complex than previously thought. For example, monoterpene emissions are far more susceptible to temperature fluctuation than observations have previously suggested. Similarly, while the emission of de novo mono- and sesquiterpenes has been positively identified, their significance over the typical two- to four-year life of a leaf remains entirely unknown. Also unclear is how transferrable these results are to other species of eucalypts, or indeed other oil gland - bearing species. Foliar oil glands are present in many eucalypt species, but considerable variation in oil content and composition can exist between individuals of a species (Brooker et al., 2002; King et al., 2006). The environmental and genetic effects that influence oil content and composition will also influence the formation, composition and degradation of the cuticular waxes of these leaves. Again, the nature of these 165 Chapter 7 Conclusions processes and their influence on terpenoid emissions from these taxa is unknown. While many plant species possess some form of oil storage structure or organ, it seems likely that only those that are separated from the atmosphere by a relatively thick membrane will exhibit this burst-decay behaviour. Taxa that share these features include members of the Myrtaceae, such the widely distributed genera Melaleuca and Leptospermum. Other families in the Australian flora that may be candidates for this behaviour include the Rutaceae, (e.g. Zieria ingramii) and Sapindaceae (e.g. Dodonea viscosa). Therefore, development of the work presented in this thesis in the immediate future should focus on the points below to address some of the questions generated by this this work. Expanding the range of species and locations from which emissions are sampled would continue to improve emission estimates and their environmental controls. Clearly more data on emissions of non-terpenoid VOCs are also required. Emission mechanisms are at the heart of emission estimates. Further development of work in this thesis would most profitably identify the precise mechanism regulating the transient emissions from E. globulus through gas exchange studies manipulating light with temperature precision not possible in this experiment. This would be complimented by sampling monoterpenes during the emission burst with a finer time resolution. The stomatal regulation and direct gland emission hypotheses may explain these transient dynamics. With the evidence presented in Chapter 5 I argued that direct emission was more likely, but both may be operating. Determining compositional differences at finer time scales, during changes in light and temperature will help to resolve this. 166 Chapter 7 Conclusions The significance of de novo synthesised mono- and sesquiterpenes over both the life of an individual leaf and different leaf forms needs to be resolved. Both gas exchange and terpene synthase studies will provide these answers. The fate of oil once in the glands remains very unclear. Clearly it is emitted, but other processes, such as catabolism have been proposed yet the evidence is patchy. Resolving these questions may go some way to providing better estimates of VOCs from the Australian landscape. 167 References Alessio, G.A., Peñuelas, J., de Lillis, M., Llusia, J., 2008a. Implications of foliar terpene content and hydration on leaf flammability of Quercus ilex and Pinus halepensis. Plant Biology 10: 123-128. Alessio, G.A., Peñuelas, J., Llusia, J., Ogaya, R., Estiarte, M., de Lillis, M., 2008b. Influence of water and terpenes on flammability in some dominant Mediterranean species. International Journal of Wildland Fire 17: 274-286. Altincicek, B., Duin, E.C., Reichenberg, A., Hedderich, R., Kollas, A.-K., Hintz, M., Wagner, S., Wiesener, J., Beck, E., Jomaa, H., 2002. LytB protein catalyzes the terminal step of the 2-Cmethyl-D-erythritol-4-phosphate pathway of isoprenoid biosynthesis. FEBS Letters 532: 437-440. Ammann, C., Spirig, C., Neftel, A., Steinbacher, M., Komend, M., Schaub, A., 2004. Application of PTR-MS for measurements of biogenic VOC in a deciduous forest. International Journal of Mass Spectrometry 239: 87-101. Ammon, D.G., Barton, A.F.M., Clarke, D.A., Tjandra, J., 1985. Rapid and accurate determination of terpenes in the leaves of Eucalyptus species. Analyst 110: 921-924. Apel, E.C., Calvert, J.G., Gilpin, T.M., Fehsenfeld, F., Lonneman, W.A., 2003. Nonmethane Hydrocarbon Intercomparison Experiment (NOMHICE): Task 4, ambient air. Journal of Geophysical Research 108 (D9): DOI:10.1029/2002JD002936. Arneth A., Monson R.K., Schurgers G., Niinemets Ü., Palmer P.I., 2008. Why are estimates of global terrestrial isoprene emissions so similar (and why is this not so for monoterpenes)? Atmospheric Chemistry and Physics (8): 4605–4620. Ashton, D.H., Martin, D.G., 1996. Regeneration in a pole-stage forest of Eucalyptus regnans subjected to different fire intensities in 1982. Australian Journal of Botany 44: 393-410. Atkinson, R., 2000. Atmospheric chemistry of VOCs and NOx. Atmospheric Environment 34: 20632101. 168 Baker, B., Bai, J.-H., Johnson, C., Cai, Z.-T., Li, Q.-J., Wang, Y.-F., Guenther, A., Greenberg, J., Klinger, L., Geron, C., Rasmussen, R., 2005. Wet and dry season ecosystem level fluxes of isoprene and monoterpenes from a sout-heast Asian secondary forest and rubber tree plantation. Atmospheric Environment 39: 381-390. Baker, B., Guenther, A., Greenberg, J., Goldstein, A., Fall, R., 1999. Canopy fluxes of 2-methyl-3buten-2-ol over a ponderosa pine forest by relaxed eddy accumulation: field data and model comparison. Journal of Geophysical Research 104 (D21): 26107-26114. Baker B., Guenther A., Greenberg J., Fall R., 2001. Canopy level fluxes of 2-methyl-3-buten-2-ol, acetone, and methanol by a portable relaxed eddy accumulation system. Environmental Science and Technology 35 (9): 1701-1708. Ballesta, P., Field, R., De Saeger, E., 2001. Interlaboratory exercises for volatile organic compounds determination. Atmospheric Environment 35: 5729–5740. Beerling, D.J., Hewitt, C.N., Pyle, J.A., Raven, J.A., 2007. Critical issues in trace gas biogeochemistry and global change. Philosophical Transactions of the Royal Society of London A 365: 1629-1642. Benjamin, M.T., Sudol, M., Bloch, L., Winer, A.M., 1996. Low-emitting urban forests: a taxonomic methodology for assigning isoprene and monoterpene emission rates. Atmospheric Environment 30: 1437-1452. Bernard-Dagan C., Carde J.P., Gleizes M., 1979. Etude des composés terpéniques au cours de la croissance des aiguilles du Pin maritime: comparaison de données biochimiques et ultrastructurales. Canadian Journal of Botany 57: 255-263. Bertin, N., Staudt, M., 1996. Effect of water stress on monoterpene emissions from young potted Holm oak (Quercus ilex L) trees, Oecologia, 107(4), 456–462. Biemann, K., 1962. Mass spectrometry: organic chemical applications. McGraw Hill Book Company, Inc, New York. 169 Blanch, J., Penuelas, J., Llusia, J., 2007. Sensitivity of terpene emissions to drought and fertilization in terpene-storing Pinus halepensis and non-storing Quercus ilex. Physiologica Plantarum 131: 211–225. Bleby, T.M., Burgess, S.S.O., Adams, M.A., 2004. A validation, comparison and error analysis of two heat-pulse methods for measuring sap flow in Eucalyptus marginata saplings. Functional Plant Biology 31: 645-658. Boland, D.J., Brophy, J.J., House, A.P.N., 1991. Eucalyptus leaf oils: use, chemistry, distillation and marketing. Inkata Press, Melbourne. Bouvier, F., Rahier, A., Camara, B., 2005. Biogenesis, molecular regulation and function of plant isoprenoids. Progress in Lipid Research 44: 357-429. Boyd, R.K., Basic, C., Bethem, R.A., 2008. Trace quantitative analysis by mass spectrometry. John Wiley and Sons, West Sussex, England. Brooker, M.I.H., 2002. Botany of the eucalypts, in: Coppin, J.J.W., (Ed.), Eucalyptus: the genus Eucalyptus. Taylor and Francis, London, pp 3-35. Brooker, M.I.H., Barton, A.F.M., Rockel, B.A., Tjandra, J., 1988. The cineole content and taxonomy of Eucalyptus kochii Maiden and Blakely and E. plenissima (Gardner) Brooker, with an appendix establishing these two taxa as subspecies. Australian Journal of Botany 36: 119-129. Brooker, M.I.H, Kleinig, D.A., 1999. Field guide to Eucalypts. Volume 1, South-eastern Australia. Bloomings Books, Victoria, Australia. Brophy, J.J., House, A.P.N., Boland, D.J., Lassak, E.V., 1991. Digests of the essential oils of 111 species from northern and eastern Australia, in: Boland, D.J. et al. (Eds.), Eucalyptus leaf oils: use, chemistry, distillation and marketing. Inkarta Press, Melbourne, pp 29-156. 170 Burgess, S.S.O., Adams, M.A., Turner, N.C., Beverly, C.R., Ong, C.K., Khan, A.A.H., Bleby, T.M., 2001. An improved heat pulse method to measure low and reverse rates of sap flow in woody plants. Tree Physiology 21: 589-598. Carr, S.G.M., Carr, D.J., 1970. Oil glands and ducts in Eucalyptus L’Hérit. II. Development and structure of oil glands in the embryo. Australian Journal of Botany 18, 191-212. Carr S.G.M., Milkovits L., Carr D.J., 1971. Eucalypt phytoglyphs: the microanatomical features of the epidermis in relation to taxonomy. Australian Journal of Botany 19: 173-190. Chameides, W.L., Fehsenfeld, F., Rodger, M.O., Cardelino, C., Martinex, J., Parish, D., Lonneman, W., Lawson, D.R., Rasmussen, R.A., Zimmerman, P., Greenberg, J., Middleton, P., Wang, T., 1992. Ozone precursor relationships in the ambient atmosphere. Journal of Geophysical Research 97: 6037-6055. Cojocariu, C., Kreuzwieser, J., Rennenberg, H., 2004. Correlation of short-chained carbonyls emitted from Picea abies with physiological and environmental parameters. New Phytologist, 162, 717-727. Cork, S.J., Hume, I.D., Dawson, T.J., 1983. Digestion and metabolism of a natural foliar diet (Eucalyptus punctata) by an arboreal marsupial, the koala (Phascolarctos cinereus). Journal of Comparative Physiology 153: 181-190. Cruz, C.N., Pandis, S.N., 1997. A study of the ability of pure secondary organic aerosol to act as cloud condensation nuclei. Atmospheric Environment 31: 2205-2214. Dalal, K.B., Salunkhe, D.K., Olson, L.E., Do, J.Y., Yu, M.H., 1968. Volatile components of developing tomato fruit grown under field and greenhouse conditions. Plant and Cell Physiology 9: 389-400. 13 13 Delwiche C.F., Sharkey T.D., 1993. Rapid appearance of C in biogenic isoprene when CO2 is fed to intact leaves. Plant, Cell and Environment 16: 587-591. Dement, W.A., Tyson, B.J., Mooney, H.A., 1975. Mechanism of monoterpene volatilization in Salvia mellifera. Phytochemistry 1975: 2555-2557. 171 Dindorf, T., Kuhn, U., Ganzeveld, L., Schebeske, G., Ciccioli, P., Holzke, C., Köble, R., Seufert, G., Kesselmeier, J., 2005. Emission of monoterpenes from European beech (Fagus sylvatica L.) as a function of light and temperature. Biogeosciences Discussions 2: 137-182. Doran, J.C., Matheson, A.C., 1994. Genetic parameters and expected gains from selection for monoterpene yields in Petford Eucalyptus camaldulensis. New Forests 8: 155-167. Doran, J.C., Williams, E.R., Brophy, J.J., 1995. Patterns of variation in the seedling leaf oils of Eucalyptus urophylla, E. pellita and E. scias. Australian Journal of Botany 43: 327-336. Doughty, C.E., Goulden, M.L., 2008. Are tropical forests near a high temperature threshold? Journal of Geophysical Research 113, G00B07, doi:10.1029/2007JG000632. Dudai, N., Larkov, O., Ravid, U., Putievsky, E., Lewinsohn, E., 2001. Developmental control of monoterpene content and composition in Micromeria fruticosa (L.) Druce. Annals of Botany 88: 349-354. Dudareva, N., Andersson, S., Orlova, I., Gatto, N., Reichelt, M., Rhodes, D., Boland, W., Gerschenzon, J., 2005. The non-mevalonate pathway supports both monoterpene and sesquiterpene formation in snapdragon flowers. Proceedings of the National Academy of Science of the USA 102: 933-938. Dunlop, P.J., Bignell, C.M., Hibbert, D.B., 1997. Use of gas chromatograms of the essential leaf oils of the genus Eucalyptus for taxonomic purposes. Australian Journal of Botany 45: 1-13. Dunlop, P.J., Bignell, C.M., Hibbert, D.B., 2000. Use of gas chromatograms of essential leaf oils to compare clones of Eucalyptus camaldulensis. Biochemical Systematics and Ecology 28: 383-391. Eade, K., 2004. Albany Effluent Irrigation Treefarm Triennial Report 2004. Water Corporation, Western Australia. 172 Evans, R. C., Tingey, D.T., Gumpertz, M.L., Burns, W.F., 1982. Estimates of isoprene and monoterpene emission rates in plants. Botanical Gazette 143: 304-310. Eyles, A., Davies, N.W., Mohammed, C. M., 2004. Traumatic oil glands induced by pruning in the wound-associated phloem of Eucalyptus globulus: chemistry and histology. Trees 18: 204-210. Fall, R., 2003. Abundant oxygenates in the atmosphere: a biochemical perspective. Chemical Reviews 103: 4941-4951. Fall, R., Monson, R.K., 1992. Isoprene emission rate in relation to stomatal distribution and stomatal conductance. Plant Physiology 100: 987-992. Fang, C.W., Monson, R. K., Cowling, E.B., 1996. Isoprene emission, photosynthesis, and growth in sweetgum (Liquidambar styraciflua) seedlings exposed to short- and long-term drying cycles. Tree Physiology 16: 441-446. Fehsenfeld, F., Calvert, J., Fall, R., Goldan, R., Guenther, A.B., Nicholas Hewitt, C., Lamb, B., Liu, S., Trainer, M., Westberg, H., Zimmerman, P., 1992. Emissions of volatile organic compounds from vegetation and the implications for atmospheric chemistry. Global Biogeochemical Cycles 6: 389-430. Filella, I., Wilkinson, M.J., Llusià, J., Hewitt, N.C., Peñuelas, J., 2007. Volatile organic compounds emissions in Norway spruce (Picea abies) in response to temperature changes. Physiologia Plantarum 130, 58-66. Finlayson-Pitts, B.J., Pitts, J.N., 2000. Chemistry of the upper and lower atmosphere. Academic Press, San Diego. Fischbach, R.J., Staudt, M., Zimmer, I., Rambal, S., Schnitzler, J.-P., 2002. Seasonal pattern of monoterpene synthase activities in leaves of the evergreen tree Quercus ilex L. Physiologia Plantarum 114: 354-360. 173 Fischbach, R. J., Zimmer, I., Steinbrecher, R., Pfichner, A., Schnitzler, J.-P., 2000. Monoterpene synthase activities in leaves of Picea abies (L.) and Quercus ilex L. Phytochemistry 54: 257-265. Fuentes, J.D., Wang, D., Gu, L., 1999. Seasonal variations of isoprene emissions from a boreal forest. Journal of Applied Meteorology 38: 855-869. Funk J.L., Giardina, C.P., Knohl, A., Lerdau, M.T., 2006. Influence of nutrient availability, stand age, and canopy structure on isoprene flux in a Eucalyptus saligna experimental forest. Journal of Geophysical Research 111: G02012.1-G02012.10. Ghirardo, A., Koch, K., Taipale, R., Zimmer, I., Schnitzler, J.-P., Rinne, J., 2010. Determination of de novo and pool emissions of terpenes from four common boreal/alpine trees by 13 CO2 labelling and PTR-MS analysis. Plant, Cell and Environment, DOI: 10.1111/j.13643040.2009.02104.x. Giese M., Bauer-Doranth U., Langebartels C., Sandermann H., 1994. Detoxification of formaldehyde by the spider plant (Chlorophytum comosum L.) and by soybean (Glycine max L.) cell-suspension cultures. Plant Physiology 104: 1301 -1309. Goodwin, T.W. and Mercer, E.I., 1972. Introduction to Plant Biochemistry. Pergamon Press Ltd, Oxford. de Gouw, J., Warneke, C., Karl, T., Eerdekens, G., van der Veen, C., Fall R., 2003. Sensitivity and specificity of atmospheric trace gas detection by proton-transfer-reaction mass spectrometry. International Journal of Mass Spectrometry 223-224: 365-382. Gray, D. W., Goldstein, A. H., Lerdau, M.T., 2005. The influence of light environment on photosynthesis and basal methylbutenol emission from Pinus ponderosa. Plant, Cell and Environment 28: 1463-1474. Gray, D.W., Lerdau, M.T., Goldstein, A.H., 2003. Influences of temperature history, water stress and needle age on methyl butenol emissions. Ecology 84: 765-776. 174 Grayling, P.M., Brooker, M.I.H., 1996. Evidence for the identity of the hybrid Eucalyptus ‘brachyphylla’ (Myrtaceae) from morphology and essential oil composition. Australian Journal of Botany 44: 1-13. Guenther, A., Baugh, B., Brasseur, G., Greenberg, J., Harley, P., Klinger, L., Serça, D., Vierling, L., 1999. Isoprene emission estimates and uncertainties for the Central African EXPRESSO study domain. Journal of Geophysical Research 104: 30625-30639. Guenther, A., Greenberg, J., Harley, P., Helmig, D., Klinger, L., Vierling, L., Zimmerman, P., Geron, C., 1996. Leaf, branch, stand and landscape scale measurements of volatile organic compound fluxes from U.S. woodlands. Tree Physiology 16: 17-24. Guenther, A.B., Hewitt, C.N., Erickson, D., Fall, R., Geron, C., Graedel, T., Harley, P.C., Klinger, L., Lerdau, M., McKay, W.A., Pierce, T., Scholes, B., Steinbrecher, R., Tallamraju, R., Taylor, J., Zimmerman, P., 1995. A global model of natural volatile organic compound emissions. Journal of Geophysical Research 100: 8873-8892. Guenther, A., Karl, T., Harley, P., Wiedinmyer, C., Palmer, P.I., Geron, C., 2006. Estimates of global terrestrial isoprene emissions using MEGAN (Model of Emissions of Gases and Aerosols from Nature). Atmospheric Chemistry and Physics 6: 3181-3210. Guenther, A., Monson, R.K., Fall, R., 1991. Isoprene and monoterpene emission rate variability: observations with Eucalyptus and emission rate algorithm development. Journal of Geophysical Research 96: 10799-10808. Guenther, A., Zimmerman, P., Harley, P., Monson, R.K., Fall, R., 1993. Isoprene and monoterpene emission rate variability: model evaluations and sensitivity analyses. Journal of Geophysical Research 98: 12609-12617. Hakola, H., Tarvainen, V., Bäck, J., Ranta, H., Bonn, B., Rinne, J., Kulmala, M., 2005. Seasonal variation of mono-and sesquiterpene emission rates of Scots pine. Biogeosciences Discussions 2: 1697-1717. 175 Hansel, A., Jordan, A., Holzinger, R., Prazeller, P., Vogel, W., Lindinger, W., 1995. Proton transfer reaction mass spectrometry: on-line trace gas analysis at the ppb level. International Journal of Mass Spectrometry and Ion Processes 149/150: 609-619. Harley, P., Fridd-Stroud, V., Greenberg, J., Guenther, A., Vasconcellos, P., 1998. Emission of 2methyl-3-buten-2-ol by pines: a potentially large natural source of reactive carbon to the atmosphere. Journal of Geophysical Research 103 (D19): 25479-25486. Harrison, D., Hunter, M.C., Lewis, A.C., Seakins, P.W., Nunes, T., Pio, C., 2001a. Isoprene and monoterpene emission from the coniferous species Abies Borisii-regis: implications for regional air chemistry in Greece. Atmospheric Environment 35: 4687-4698. Harrison, D., Hunter, M.C., Lewis, A.C., Seakins, P.W., Bonsang, B., Gros, V., Kanakidou, M., Touaty, M., Kavouras, I., Mihalopoulos, N., Stephanou, E., Alves, C., Nunes, T., Pio, C., 2001b. Ambient isoprene and monoterpene concentrations in a Greek fir (Abies Borisii-regis) forest. Reconciliation with emissions measurements and effects on measured OH concentrations. Atmospheric Environment 35: 4699-4711. Harrison, D., Seakins, P.W., Lewis, A.C., 2000. Simultaneous monitoring of atmospheric methane and speciated nonmethane hydrocarbon concentrations using Peltier effect sub-ambient pre-concentration and gas chromatography. Journal of Environmental Monitoring 2: 59-63. Hatakeyama, S., Izumi, K., Fukuyama, T., Akimoto, H., 1989. Reactions of ozone with Į-pinene in air: yields of gaseous and particulate products. Journal of Geophysical Research 94: 1301313024. He, C., Murray, F., Lyons, T., 2000a. Monoterpene and isoprene emissions from 15 Eucalyptus species in Australia. Atmospheric Environment 34: 645-655. He, C., Murray, F., Lyons, T., 2000b. Seasonal variations in monoterpene emissions from Eucalyptus species. Chemosphere -Global Change Science 2: 65-76. Heldt, H.-W., 2005. Plant Biochemistry. Elsevier Academic Press, London, p. 403. 176 Hellyer, R.O., Lassak, E.V., McKern, H.H.G., Willis, J.L., 1969. Chemical variation within Eucalyptus dives. Phytochemistry 8: 1513-1514. Helmig, D., 1999. Air analysis by gas chromatography. Journal of Chromatography A 843: 129-146. Henery, M.L., Wallis, I.R., Stone, C., Foley, W.J., 2008. Methyl jasmonate does not induce changes in Eucalyptus grandis leaves that alter the effect of constitutive defences on larvae of a specialist herbivore. Oecologia DOI: 10.1007/s00442-008-1042-x. Henrich, L.H., 1995. Detectors and data handling, in Grob, R.L. (Ed.), Modern practice of gas chromatography. John Wiley and Sons, Inc., New York. Hewitt, C.N., Haywood, S., Tani, A., 2003. The application of proton transfer reaction-mass spectrometry (PTR-MS) to the monitoring and analysis of volatile organic compounds in the atmosphere. Journal of Environmental Monitoring 5: 1-7. Hoffmann, T., Odum, J.R., Bowman, F., Collins, D., Klockow, D., Flagan, R.C., Seinfeld, J. H., 1997. Formation of organic aerosols from the oxidation of biogenic hydrocarbons, Journal of Atmospheric Chemistry 26: 189–222. Holzinger, R., Sandoval-Soto, L., Rottenberger, S., Crutzen, P.J., Kesselmeier, J., 2000. Emissions of volatile organic compounds from Quercus ilex L. measured by Proton Transfer Reaction Mass Spectrometry under different environmental conditions. Journal of Geophysical Research 105(D16): 20573–20579. Janson, R.W., 1993. Monoterpene emission from Scots pine and Norwegian spruce. Journal of Geophysical Research 98: 2839-2850. Janson, R., De Serves, C., Romero R., 1999. Emission of isoprene and carbonyl compounds from a boreal forest and wetland in Sweden. Agricultural and Forest Meteorology 98, 671-681. 177 Jaoui, M., Leungsakul, S., Kamens, R.M., 2003. Gas and particle products distribution from the UHDFWLRQ RI ȕ-caryophyllene with ozone. Journal of Atmospheric Chemistry 45: 261–287. Jardine, K., Harley, P., Karl, T., Guenther, A., Lerdau, M., Mak, J.E., 2008. Plant physiological and environmental controls over the exchange of acetaldehyde between forest canopies and the atmosphere. Biogeosciences 5: 1559-1572. Johnson, E.D., 1926. A Comparison of the Juvenile and Adult Leaves of Eucalyptus globulus. New Phytologist 25 (3): 202-212. Karl, T., Curtis, A.J., Rosenstiel, T.N., Monson, R.K., Fall, R., 2002. Transient releases of acetaldehyde from tree leaves – products of a pyruvate overflow mechanism? Plant, Cell and Environment 25: 1121-1131. Kavouras, I.G., Mihalopoulos, N., Stephanou, E.G., 1998. Formation of atmospheric particles from organic acids produced by forests. Nature 395: 683-686. Kesselmeier, J., 2001. Exchange of short-chain oxygenated volatile organic compounds (VOCs) between plants and the atmosphere: a compilation of field and laboratory studies. Journal of Atmospheric Chemistry 39: 219–233. Kesselmeier, J., Bode, K., Hofmann, U., Müller, H., Schäfer, L., Wolf, A., Ciccioli, P., Brancaleoni, E., Cecinato, A., Frattoni, M., Foster, P., Ferrari, C., Jacob, V., Fugit, J.L., Dutaur, L., Simon, V., Torres, L., 1997. Emission of short chained organic acids, aldehydes and monoterpenes from Quercus ilex L. and Pinus pinea L. in relation to physiological activities, carbon budget and emission algorithms. Atmospheric Environment 31: 119-133. Kesselmeier, J., Staudt, M., 1999. Biogenic volatile organic compounds (VOC): an overview on emission, physiology and ecology. Journal of Atmospheric Chemistry 33: 23-88. Keppel, G., 1991. Design and Analysis: A Researcher’s Handbook. Prentice Hall, Inc., Englewood Cliffs, New Jersey, pp. 167-169. 178 King, D.J., Gleadow, R.M., Woodrow, I.E., 2004. Terpene deployment in Eucalyptus polybractea; relationships with leaf structure, environmental stresses and growth. Functional Plant Biology 31: 451-460. King, D.J., Gleadow, R.M., Woodrow, I.E., 2006a. Regulation of oil accumulation in single glands of Eucalyptus polybractea. New Phytologist 172: 440-451. King, D.J., Gleadow, R.M., Woodrow, I.E., 2006b. The accumulation of terpenoid oils does not incur a growth cost in Eucalyptus polybractea seedlings. Functional Plant Biology 33: 497-505. Kreuzwieser, J., Scheerer, U., Rennenberg, H., 1999. Metabolic origin of acetaldehyde emitted by poplar (Populus tremula x P. alba) trees. Journal of Experimental Botany 50: 757-765. Kreuzwieser, J., Harren, F.J.M., Laarhoven, L.J.J, Boamfa, I., te Lintel-Hekkert, S., Scheerera, U., Hüglin, C., Rennenberg, H., 2001. Acetaldehyde emission by the leaves of trees – correlation with physiological and environmental parameters. Physiologia Plantarum 113: 41-49. Kreuzwieser, J., Graus, M., Wisthaler, A., Hansel, A., Rennenberg, H., Schnitzler J.P., 2002. Xylem-transported glucose as an additional carbon source for leaf isoprene formation in Quercus robur. New Phytologist 156: 171–178. Kuhn, U., Rottenberger, S., Biesenthal, T., Wolf, A., Schebeske, G., Ciccioli, P., Brancaleoni, E., Frattoni, M., Tavares, T.M., Kesselmeier, J. 2002. Isoprene and monoterpene emissions of Amazonian tree species during the wet season: direct and indirect investigations on controlling environmental functions. Journal of Geophysical Research 107, doi: 10.1029/2001JD000978. 179 Kuhn, U., Rottenberger, S., Biesenthal, T., Wolf, A., Schebeske, G., Ciccioli, P., Brancaleoni, E., Frattoni, M., Tavares, T.M., Kesselmeier, J. 2004. Seasonal differences in isoprene and light-dependent monoterpene emission by Amazon tree species. Global Change Biology 10: 663-682. Lassak, E.V., 2002. Research trends and future prospects. In: Eucalyptus: the genus Eucalyptus. Coppen, J.J.W. (ed). Taylor and Francis Group, London, pp. 384-404. Lassak, E.V., Southwell, I.A., 1982. The steam volatile leaf oils of some species of Eucalyptus subseries STRICTINAE. Phytochemistry 21: 2257-2261. Laule, O., Führholz, A., Chang, H.S., Zhu, T., Wang, X., Heifetz, P.B., Gruissem, W., Lange B.M., 2003. Crosstalk between cytosolic and plastidial pathways of isoprenoid biosynthesis in Arabidopsis thaliana. Proceedings of the National Academy of Science of the USA 100 (11): 6866-6871. Lavoir, A.-V., Staudt, M., Schnitzler, J.-P., Landais, D., Massol, F., Rocheteau, A., Rodriguez, R., Zimmer, I., Rambal, S., 2009. Drought reduced monoterpene emissions from the evergreen Mediterranean oak Quercus ilex: results from a throughfall displacement experiment. Biogeosciences 6, 1167-1180. Leach, G.J., Whiffin, T., 1989. Ontogenetic, seasonal and diurnal variation in leaf volatile oils and leaf phenolics of Angophora costata. Australian Systematic Botany 2: 99-111. Lerdau, M., Keller, M., 1997. Controls on isoprene emission from trees in a subtropical dry forest. Plant Cell and Environment 20: 569-578. Lerdau, M., Matson, P., Fall, R., Monson, R., 1995. Ecological controls over monoterpene emissions from Douglas Fir (Pseudotsuga menziesii). Ecology 76: 2640-2647. Li, H., Madden, J.L., Davies, N.W., 1994. Variation in Leaf Oils of Eucalyptus nitens and E. denticulata. Biochemical Systematics and Ecology 22 (6): 631-640. 180 Li, H., Madden, J.L., Potts, B.M., 1995. Variation in Volatile Leaf Oils of the Tasmanian Eucalyptus Species – I. Subgenus Monocalyptus. Biochemical Systematics and Ecology 23 (3): 299-318. Li, H., Madden, J.L., Potts, B.M., 1996. Variation in Volatile Leaf Oils of the Tasmanian Eucalyptus Species II. Subgenus Symphyomyrtus. Biochemical Systematics and Ecology 24 (6): 547-569. Lichtenthaler, H.K., Schwender, J., Disch, A., Rohmer, M., 1997. Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via a mevalonate-independent pathway. FEBS Letters 400: 271-274. de Lillis, M., Bianco, P.M., Loreto, F., 2009. The influence of leaf water content and isoprenoids on flammability of some Mediterranean woody species. International Journal of Wildland Fire 18: 203-212. Lindinger, W., Hansel, A., Jordan, A., 1998. On-line monitoring of volatile organic compounds at pptv levels by means of Proton-Transfer-Reaction Mass Spectrometry (PTR-MS): Medical applications, food control and environmental research. International Journal of Mass Spectrometry and Ion Processes 173: 191-241. List, S., Brown, P.H., Walsh, K.B., 1995. Functional anatomy of the oil glands of Melaleuca alternifolia (Myrtaceae). Australian Journal of Botany 43: 629-641. Livesley, S.J., Adams, M.A., Grierson, P.F., 2007. Soil water nitrate and ammonium dynamics under a sewage effluent-irrigated eucalypt plantation. Journal of Environmental Quality 36: 18831894. Llusiá, J., Peñuelas, J., 2000. Seasonal patterns of terpene concentration and emission from seven Mediterranean woody species in field conditions. American Journal of Botany 87: 133-140. 181 Logan, B.A., Monson, R.K., 1999. Thermotolerance of leaf discs from four isoprene-emitting species is not enhanced by exposure to exogenous isoprene. Plant Physiology 120: 821-825. Loivamaki, M., Loius, S., Cinege, G., Zimmer, I., Fischbach, R.J., Schnitzler, J.-P., 2007. Circadian rhythms of isoprene biosynthesis in Grey poplar leaves. Plant Physiology 143: 540-551. Loreto, F., Ciccioli, P., Cecinato, A., Brancaleoni, E., Frattoni, M., Fabozzi, C, Tricoli, D., 1996a. Evidence of the photosynthetic origin of monoterpenes emitted by Quercus ilex L. leaves by 13C labelling. Plant Physiology 110: 1317-1322. Loreto, F., Ciccioli, P., Cecinato, A., Brancaleoni, E., Frattoni, M., Tricoli, D., 1996b. Influence RIHQYLURQPHQWDOIDFWRUVDQGDLUFRPSRVLWLRQRQWKHHPLVVLRQRIĮ-pinene from Quercus ilex leaves. Plant Physiology 110: 267-275. Loreto, F., Ciccioli, P., Brancaleoni, E., Cecinato, A., Frattoni, M., Sharkey, T.D., 1996c. Different sources of reduced carbon contribute to form three classes of terpenoid emitted by Quercus ilex L. Leaves. Proceedings of the National Academy of Science of the USA 93: 9966-9969. Loreto, F., Ciccioli, P., Brancaleoni, E., Frattoni, M., Delfine, S., 2000. Incomplete 13C labelling RIĮ-pinene content of Quercus ilex leaves and appearance of unlabelled C in Į-pinene emission in the dark. Plant, Cell and Environment 23: 229-234. Loreto, F., Delfine, S., 2000. Emission of isoprene from salt-stressed Eucalyptus globulus leaves. Plant Physiology 123: 1605-1610. Loreto, F., Mannozzi M., Maris C., Nascetti P., Ferranti F., Pasqualini S., 2001. Ozone quenching properties of isoprene and its antioxidant role in leaves. Plant Physiology 126: 993–1000. 182 Loreto F., Velikova V., 2001. Isoprene produced by leaves protects the photosynthetic apparatus against ozone damage, quenches ozone products, and reduces lipid peroxidation of cellular membranes. Plant Physiology 127: 1781–1787. Louro, R.P., Dos Santos, A.V., Machado, R.D., 2003. Ultrastructure of Eucalyptus grandis and Eucalyptus urophylla. I. Shoots cultivated in vitro in multiplication and elongation-rooting media. International Journal of Plant Sciences 160 (2): 217–227. MacDonald, R.C., Fall, R., 1993. Detection of substantial emissions of methanol from plants to the atmosphere. Atmospheric Environment 27A: 1709-1713. MacDonald, R.C., Kimmerer, T.W., 1993. Metabolism of transpired ethanol by eastern cottonwood (Populus deltoides Bartr.). Plant Physiology 102: 173-179. Maleknia, S., Bell, T.L., Adams, M.A., 2007. PTR-MS analysis of reference and plant-emitted volatile organic compounds. International Journal of Mass Spectrometry 262: 203–210. Martin, D., Tholl, D., Gershenzon, J., Bohlmann, J., 2002. Methyl jasmonate induces traumatic resin ducts, terpenoid resin biosynthesis, and terpenoid accumulation in developing xylem of Norway spruce stems. Plant Physiology 129: 1003-1018. May, F.E., Ash, J.E., 1990. An assessment of the allelopathic potential of Eucalyptus. Australian Journal of Botany 38: 245-254. McCaskill, D., Croteau, R., 1995. Monoterpene and sesquiterpene biosynthesis in glandular trichomes of peppermint (Mentha x piperita) rely exclusively on plastid-derived isopentenyl diphosphate. Planta 197: 49-56. McConkey M.W., Gerschenzon, J., Croteau, R.B., 2000. Developmental regulation of monoterpene biosynthesis in the glandular trichomes of peppermint. Plant Physiology 122: 215-224. Monson, R.K., Harley, P.C., Litvak, M.E., Wildermuth, M., Guenther, A.B., Zimmerman, P.R., Fall, R., 1994. Environmental and developmental controls over the seasonal pattern of isoprene emission from aspen leaves. Oecologia 99: 260-270. 183 Monson, R.K., Hill, A.J., Zimmerman, P.R., Fall, R.R., 1991. Studies of the relationship between isoprene emission rate and CO2 or photon-flux density using a real time isoprene analyser. Plant, Cell and Environment 14: 517-523. Monson, R. K., Jaeger, C. H., Adams, W. W., Driggers, E. M., Silver, G. M., Fall, R. (1992). Relationships between isoprene emission rate, photosynthesis, and isoprene synthase activity as influenced by temperature. Plant Physiology 98: 1175-1180. del Moral, R., Muller, C.H., 1970. The allelopathic effects of Eucalyptus camaldulensis. American Midland Naturalist 83: 254-82. del Moral, R., Willis, R.J., Ashton, D.H., 1978. Suppression of coastal heath vegetation by Eucalyptus baxteri. Australian Journal of Botany 26: 203-19. Morrow, P.A., Fox, L.R., 1980. Effects of variation in Eucalyptus essential oil yield on insect growth and grazing damage. Oecologia 45, 209-219. Müller, J.-F., 1992. Geographical distribution and seasonal variation of surface emissions and deposition velocities of atmospheric trace gases. Journal of Geophysical Research 97: 37873804. Muzika, R.-M., 1989. Terpenes and phenolics in response to nitrogen fertilization: a test of the carbon/nutrient balance hypothesis. Chemoecology 4, 3-7. Ngugi, M.R., Hunt, M.A., Doley, D., Ryan, P., Dart, P.J., 2003. Photosynthetic light and temperature responses of Eucalyptus cloeziana and Eucalyptus argophloia. Australian Journal of Botany 51: 573-583. Niinemets, Ü., Monson, R.K., Arneth, A., Ciccioli, P., Kesselmeier, J., Kuhn, U., Noe, S.M., Peñuelas, J., Staudt, M., 2010. The leaf-level emission factor of volatile isoprenoids: caveats, model algorithms, response shaping and scaling. Biogeosciences 7: 1809-1832. Niinemets, Ü., Loreto, F., Reichstein, M., 2004. Physiological and physicochemical controls on foliar volatile organic compound emissions. Trends in Plant Sciences 9: 180-186. Niinemets, Ü., Hauff, K., Bertin, N., Tenhunen, J.D., Steinbrecher R., Seufert, G., 2002a. 184 Monoterpene emissions in relation to foliar photosynthetic and structural variables in Mediterranean evergreen Quercus species. New Phytologist 153, 243-256. Niinemets, Ü., Reichstein, M., 2002. A model analysis of the effects of nonspecific monoterpenoid storage in leaf tissues on emission kinetics and composition in Mediterranean sclerophyllous Quercus species. Global Biogeochemical Cycles 16, 4, 1110, doi: 10.1029/2002GB001927. Niinemets, Ü., Reichstein, M., Staudt, M., Seufert, G., Tenhunen, J.D., 2002b. Stomatal constraints may affect emission of oxygenated monoterpenoids from the foliage of Pinus pinea. Plant Physiology 130: 1371-1385. Niinemets, Ü., Reichstein, M., 2003. Controls on the emission of plant volatiles through stomata: Differential sensitivity of emission rates to stomatal closure explained. Journal of Geophysical Research 108 (D7), 4208, doi:10.1029/2002JD002620. Niinemets Ü., Seufert, G., Steinbrecher, R., Tenhunen, J.D., 2002c. A model coupling foliar monoterpene emissions to leaf photosynthetic characteristics in Mediterranean evergreen Quercus species. New Phytologist 153: 257-275. Nimitkeatkai, H., Ueda, Y., Furukawa, H., Inamoto, K., Doi, M., 2005. Emission of methylbutyric acid from Gypsophila paniculata L. during bud opening: changes in amino acid catabolism. Scientia Horticulturae 106: 370-380. Owen, S.M., Harley, P., Guenther, A., Hewitt, C.N., 2002. Light dependency of VOC emissions from selected Mediterranean plant species. Atmospheric Environment 36: 3147-3159. Peñuelas, J., Llusia, J., Asensio, D., Munne-Bosch, S., 2005. Linking isoprene with plant thermotolerance, antioxidants and monoterpene emissions. Plant, Cell and Environment 28: 278-286. Pichersky, E., 2009. Plant Specialised (Secondary) Metabolism: Genetics, Biochemistry and Evolution. School of Biology Workshop, Australian National University, Canberra. 15-17 April 2009. 185 Piel, J., Donath, J., Bandemer, K., Boland, W., 1998. Mevalonate-independent biosynthesis of terpenoid volatiles in plants: induced and constitutive emission of volatiles. Angewandte Chemie International Edition 37 (18): 2478-2481. Pier, P.A., McDuffie, C., 1997. Seasonal isoprene emission rates and model comparisons using whole-tree emissions from white oak. Journal of Geophysical Research 102 (D20): 2396323971. Pio, C.A., Nunes, T.V., Valente, A. R., 1996. Biogenic hydrocarbon emissions from vegetation in a southern European environment, in Borrell, P.M. et al. (Eds.), The Proceedings of EUROTRAC Symposium 1996, Computational Mechanics Publications, Southhampton, pp. 35–43. Pio, C.A., Silva, P.A., Cerqueira, M.A., Nunes, T.V., 2005. Diurnal and seasonal emissions of volatile organic compounds from cork oak (Quercus suber) trees. Atmospheric Environment 39: 1817-1827. Pourmortazavi, S.M., Hajimirsadeghi, S.S., 2007. Supercritical fluid extraction in plant essential and volatile oil analysis. Journal of Chromatography A 1163: 2-24. Rapley, L.R., Allen, G.R., Potts, B.M., Davies, N.W., 2008. Constitutive or induced defences – how does Eucalyptus globulus defend itself from larval feeding? Chemoecology 17: 235-243. Rasmussen, R., Khalil, M., 1988. Isoprene over the Amazon basin. Journal of Atmospheric Chemistry 93: 1417-1421. Rasmussen, R.A., Jones, C.A., 1973. Emission of isoprene from leaf discs of Hamamelis. Phytochemistry 12: 15-19. Rennenberg, H., Schneider, S., Weber, P., 1996. Analysis of uptake and allocation of nitrogen and sulphur by trees in the field. Journal of Experimental Botany 47: 1491-1498. 186 Röckmann, T., Brenninkmeijer, C.A.M., Neeb, P., Crutzen, P.J., 1998. Ozonolysis of nonmethane hydrocarbons as a source of the observed mass independent oxygen isotope enrichment in tropospheric CO. Journal of Geophysical Research 103: 1463-1470. Roderick, M.L., Farquhar, G.D., Berry, S.L., Noble, I.R., 2001. On the direct effect of clouds and atmospheric particles on the productivity and structure of vegetation. Oecologia 129: 21-30. Rottenberger, S., Kleiss, B., Kuhn, U., Wolf, A., Piedade, M.T.F., Junk, W., Kesselmeier, J., 2008. The effect of flooding on the exchange of the volatile C2-componds ethanol, acetaldehyde and acetic acid between leaves of Amazonian floodplain tree species and the atmosphere. Biogeosciences 5: 1085-1100. Rottenberger, S., Kuhn, U., Wolf, A., Schebeske, G., Olivia, S. T., Tavares, T.M., Kesselmeier, J., 2004. Exchange of short-chain aldehydes between Amazonian vegetation and the atmosphere. Ecological Applications 14: S247-S262. Rottenberger, S., Kuhn, U., Wolf, A., Schebeske, G., Olivia, S. T., Tavares, T.M., Kesselmeier, J., 2005. Formaldehyde and acetaldehyde exchange during leaf development of the Amazonian deciduous tree species Hymenaea courbaril. Atmospheric Environment 39: 2275-2279. Rosenstiel, T.N., Potosnak, M.J., Griffen, K.L., Fall, R., Monson, R.K., 2003. Increased CO2 uncouples growth from isoprene emission in an agriforest ecosystem. Nature 421: 256-259. Ruuskanen, T.M., Kolari, P., Bäck, P., Kulmala, M., Rinne, J., Hakola, H., Taipale, R., Raivonen, M., Altimir, N., Hari, P., 2005. On-line field measurements of monoterpene emissions from Scots pine by proton-transfer-reaction mass spectrometry. Boreal Environment Research 10: 553-567. 187 Sapir-Mir, M., Mett, A., Belausov, E., Tal-Mshulam, S., Frydman, A., Gidoni, D., Eyal, Y., 2008. Peroxisomal localization of Arabidopsis isopentenyl diphosphate isomerases suggests that part of the plant isoprenoid mevalonic acid pathway is compartmentalized to peroxisomes. Plant Physiology 148: 1219-1228. Schaub, A., Blande, J.D., Graus, M., Oksanend, E., Holopainen, J.K., Hansel, A., 2010. Real-time monitoring of herbivore induced volatile emissions in the field. Physiologia Plantarum 138: 123-133. Schmid, C., Steinbrecher, R., Ziegler, H., 1992. Partition coefficients of plant cuticles for monoterpenes. Trees 6: 32-36. Schnitzler, J.-P., Steinbrecher, R., Zimmer, I., Steigner, D., Fladung, M., 2004. Hybridization of European oaks (Quercus ilex x Q. robur) results in a mixed isoprenoid emitter type. Plant, Cell and Environment 27: 585-593. Schürmann, E., Ziegler, H., Kotzias, D., Schönwitz, R, Steinbrecher, R., (1993). Emission of biosynthesised monoterpenes from needles of Norway spruce. Naturwissenschaften 80: 276-278. Schuh, G., Heiden, A.C., Hoffman, T., Karl, J., Rockel, P., Rudolph, J., Wildt, J., 1997. Emission of volatile organic compounds from sunflower and beech: dependence on temperature and light intensity. Journal of Atmospheric Chemistry 27: 291-318. Seco, R., Peñuelas, Filella, I., 2007. Short-chained oxygenated VOCs: Emission and uptake by plants and atmospheric sources, sinks and concentrations. Atmospheric Environment 41: 2477-2499. Seigler, D.S., 1998. Plant Secondary Metabolism. Kluwer Academic Publishers, Norwell, Massachusetts. Shao, M., Czapiewski, K.V., Heiden, A.C., Kobel, K., Komenda, M., Koppmann, R., Wildt, J., 2001. Volatile organic compound emissions from Scots Pine: mechanisms and description by algorithms. Journal of Geophysical Research 106 (D17), 20483-20491. 188 Sharkey, T.D., 1996. Emission of low molecular mass hydrocarbons from plants. Trends in Plant Science 1: 78-82. Sharkey, T.D., Chen, X., Yeh, S., 2001. Isoprene increases thermotolerance of fosmidomycin-fed leaves. Plant Physiology 125: 2001-2006. Shaw, D.J., 1992. Introduction to colloid and surface chemistry. Butterworth-Heinemann, Oxford. Shelton, D, Aitken, KS, Doimo, L, Leach, DN, Baverstock, PR & Henry, RJ, 2002. Genetic control of monoterpene composition in the essential oil of Melaleuca alternifolia (Cheel). Theoretical and Applied Genetics 105: 377-383. Simon, V., Clement, B., Riba, M.L., Torres, L., 1994. The Landes Experiment – monoterpenes emitted from the maritime pine. Journal of Geophysical Research 99 (D8): 16501-16510. Simmons, D., Parsons, R.F., 1987. Seasonal Variation in the Volatile Leaf Oils of Two Eucalyptus Species. Biochemical Systematics and Ecology 15 (2): 204-215. Singh, H.B., Zimmerman, P.R., 1992. Atmospheric distribution and sources of nonmethane hydrocarbons, in: Nriagu, J.O. et al. (Eds.), Gaseous pollutants: characterization and cycling. John Wiley and Sons, New York. Singsaas, E.L., Sharkey, T.D., 2000. The effects of high temperature on isoprene synthesis in oak leaves. Plant, Cell and Environment 23: 751–757. Singsaas, E.L., Lerdau, M., Winter, K., Sharkey, T.D., 1997. Isoprene increases thermotolerance of isoprene emitting species. Plant Physiology 115: 1413-1420. Siwko, M.E., Marrink, S.J., de Vries, A.H., Kozubek, A., Uiterkamp, A.J.M.S., Mark, A.E., 2007. Does isoprene protect plant membranes against thermal shock? A molecular dynamics study. Biochimica et Biophysica Acta 1768: 198-206. 189 Staudt, M., Bertin, N., Hansen, U., Seufert, G., Ciccioli, P., Foster, P., Frenzel, B., Fugit, J.L., 1997. Seasonal and diurnal patterns of monoterpene emissions from Pinus pinea (L.) under field conditions. Atmospheric Environment 31 (S1): 145-156. Staudt, M., Seufert, G., 1995. Light-dependent emission of monoterpenes by holm oak (Quercus ilex L.) leaves. Plant, Cell and Environment 21: 385-395. Staudt, M., Ennajah, A., Mouillot, F., and Joffre, R., 2008. Do volatile organic compound emissions of Tunisian cork oak populations originating from contrasting climatic conditions differ in their responses to summer drought? Canadian Journal of Forest Research 38: 2965–2975. Steeghs, M.M.L., Crespo, E., Harren, F.J.M., 2007. Collision induced dissociation study of 10 monoterpenes for identification in trace gas measurements using the newly developed proton-transfer reaction ion trap mass spectrometer. International Journal of Mass Spectrometry 263: 204–212. Steinbacher, M., Dommena, J., Ammann, C., Spirig, C., Neftel, A., Prevota, A.S.H., 2004. Performance characteristics of a proton-transfer-reaction mass spectrometer (PTR-MS) derived from laboratory. International Journal of Mass Spectrometry 239: 117–128. Steinbrecher, R., Eichstädter, G., Schürmann, W., Torres, L., Clement, B., Simon, V., Kotzias, D., Daiber, R., van Eijk, J., 1994. Monoterpenes in air samples: European intercomparison experiments. International Journal of Environmental and Analytical Chemistry 54: 283-297. Street, R.A., Hewitt, C.N., Mennicken, S., 1997. Isoprene and monoterpene emissions from a Eucalyptus plantation in Portugal. Journal of Geophysical Research 102: 15875-15888. Taipale, R., Ruuskanen, T.M., Rinne, J., Kajos, M.K., Hakola, H., Pohja, T., Kulmala, M., 2008. Quantitative long-term measurements of VOC concentrations by PTR-MS – measurement, calibration, and volume mixing ratio calculation methods. Atmospheric Chemistry and Physics 8: 6681–6698. 190 Taivarnen, V., Hakola, H., Hellén, H., Bäck, J., Hari, P., Kulmala, M., (2005). Temperature and light dependence of the VOC emissions of Scots pine. Atmospheric Chemistry and Physics (5): 989–998. Tani, A., Hayward S., Hansel A., Hewitta C.N., 2004. Effect of water vapour pressure on monoterpene measurements using proton transfer reaction-mass spectrometry (PTR-MS). International Journal of Mass Spectrometry 239: 161–169. Tani, A., Hayward, S., Hewitt, C.N. (2003). Measurement of monoterpenes and related compounds by proton transfer reaction-mass spectrometry (PTR-MS). International Journal of Mass Spectrometry 223–224: 561–578. Tanner, D., Helmig, D., Hueber, J., Goldan, P., 2006. Gas chromatography system for the automated, unattended, and cryogen-free monitoring of C2 to C6 non-methane hydrocarbons in the remote troposphere. Journal of Chromatography A 1111: 76-88. Tholl, D., Boland, W., Hansel, A., Loreto, F., Röse, U.S.R., Schnitzler, J.-P., 2006. Practical approaches to plant volatile analysis. The Plant Journal 45: 540–560. Thompson, A.M., 1992. The oxidising capacity of the Earth’s atmosphere: probable past and future changes. Science 256: 1157-1165. Tingey, D. T., 1981. The effect of environmental factors on the emission of biogenic hydrocarbons from live oak and slash pine, in Bufalini, J.L. and Arnts, R.R. (Eds.), Atmospheric Biogenic Hydrocarbons, Volume 1, Emissions, Ann Arbor Science Publishers, Inc., Michigan, pp. 53-80. Tingey, D., Manning, M., Grothaus, L., Burns, W. 1979. Influence of light and temperature on isoprene emission rates from live oak. Physiologia Plantarum 47: 112-118. Tingey, D.T., Manning, M., Grothaus, L.C., Burns, W.F., 1980. The influence of light and temperature on monoterpene emission rates from slash pine. Plant Physiology 65: 797-801. 191 Tingey, D.T., Turner, D.P., Weber, J.A., 1991. Factors controlling the emissions of monoterpenes and other volatile organic compounds, in: Sharkey, T.D., et al., (Eds), Trace gas emissions by plants. Academic Press, San Diego, pp. 93-119. Turnbull, J.W. and Booth, T.H., 2002. Eucalypts in cultivation: an overview, in: Coppin, J.J.W., (Ed.), Eucalyptus: the genus Eucalyptus. Taylor and Francis, London, pp 52-74. Turner, G., Gerschenzon, J., Nielson, E.E., Froehlich, J.E., Croteau, R., 1999. Limonene synthase, the enzyme responsible for monoterpene biosynthesis in peppermint, is localised to leucoplasts of oil gland secretory cells. Plant Physiology 120: 879-886. Turner, J.G., Ellis, C., Devoto, A., 2002. The Jasmonate Signal Pathway. The Plant Cell 14: 153-164. Tyson, B.J., Dement, W.A., Mooney, H.A., 1974. Volatilisation of terpenes from Salvia mellifera. Nature 252: 119-120. Valentini, R., Greco, S., Seufert, G., Bertin, N., Ciccioloi, P., Cecinato, A., Brancaleoni, E., Frattoni, M., 1997. Fluxes of biogenic VOC from Mediterranean vegetation by trap enrichment relaxed eddy accumulation. Atmospheric Environment 31: 229-238. Velikova, V., Loreto, F., Stefanov, D., Yordanov, I., 2007. Characterization of juvenile and adult leaves of Eucalyptus globulus showing distinct heteroblastic development: photosynthesis and volatile isoprenoids. Plant Biology DOI 10.1055/s-2007-964964. Vernet, P., Gouyon, P.H., Valdeyron, G., 1986. Genetic control of the oil content in Thymus vulgaris L.: a case of polymorphism in a biosynthetic chain. Genetica 69: 227 – 231. Vickers, C.E., Possell, M., Cojocariu, C.I., Velikova, V.B., Loathawornkitkul, J., Ryan, A., Mullineaux, P.M., Hewitt, C. N., 2009. Isoprene synthesis protects transgenic tobacco plants from oxidative stress. Plant, Cell and Environment 32: 520-531. Wang, Y.-F., Owen, S.M., Li, Q.-J., Peñuelas, J., 2007. Monoterpene emissions from rubber trees (Hevea brasiliensis) in a changing landscape and climate: chemical speciation and environmental control. Global Change Biology 13: 2270-2282. 192 Weston, R.J., 1984. Composition of essential oil from leaves of Eucalyptus delegatensis. Phytochemistry 23: 1943-1945. Wight G.D. 1994. Fundamentals of air sampling. Lewis Publishers, New York. Wilkinson, M.J., Owen, S.M., Possell, M., Hartwell, J., Gould, P., Hall, A., Vickers, C., Hewitt, C.N., 2006. Circadian control of isoprene emissions from oil palm (Elaeis guineensis). Plant Journal 47: 960-968. Winer, A.M., Fitz, D.R., Miller, P.R., Atkinson, R., Brown, D.E., Carter, W.P.L., Dodd, M. C., Johnson, C. W., Myers, M. A., Neisess, K. R., Poe, M. P., Stephens, E.R., 1983. Investigation of the role of natural hydrocarbons in photochemical smog formation in California. Final Report to California Air Resources Board. Contract No. A0-056-32. Wirthensohn, M.G., Sedgley, M., 1996. Epicuticular wax structure and regeneration on developing juvenile Eucalyptus leaves. Australian Journal of Botany 44: 691-704. Yokouchi, Y., Ambe, Y., 1984. Factors affecting the emission of monoterpenes from Red Pine (Pinus densiflora). Plant Physiology 75: 1009-1012. Yoshimoto, H., Fukushige, T., Yonezawa, T., Sakai, Y., Okawa, K., Iwamatsu, A., Sone, H., Tamai, Y., 2001. Pyruvate decarboxylase encoded by the PDC1 gene contributes, at OHDVW SDUWLDOO\ WR WKH GHFDUER[\ODWLRQ RI Į-ketoisocaproate for isoamyl alcohol formation in Saccharomyces cerevisiae. Journal of Bioscience and Bioengineering 92: 83-85. Zeidler, J., Schwender, J., Müller, C., Wiesner, J., Weidenmeyer, C., Beck, E., Jomaa, H., Lichtenthaler, H.K., 1998. Inhibition of the non-mevalonate 1-deoxy -D-xylulose-5-phosphate pathway of plant isoprenoid biosynthesis by fosmidomycin. Zeitschrift fuer Naturforschung 53c: 980-986. Zimmer, W., Brüggemann, N., Emeis, S., Giersch, C., Lehning, A., Steinbrecher, R., Schnitzler, J.-P., 2000. Process-based modelling of isoprene emission by oak leaves. Plant, Cell and Environment 23: 585-595. 193 Appendix 1 Fragmentation spectra generated in the PTR-MS under the conditions described in Chapter 2. 100 69 3-methyl-2-buten-1-ol 100 93 MW 86 119 80 80 p-cymene MW 134 60 60 40 40 134 41 20 Relative intensity (%) 20 Relative intensity (%) 0 0 0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 120 140 160 180 200 m/z m/z 100 100 81 137 camphor 153 MW 152 80 80 eucalyptol MW 154 60 60 40 40 20 20 Relative intensity (%) Relative intensity (%) 0 0 0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 120 140 160 180 200 m/z m/z 100 103 iso-valeric acid 100 102 101 80 57 80 60 pinacolone (3,3-dimethyl-2-butanone) 60 57 MW 100 40 40 20 Relative intensity (%) 20 Relative intensity (%) 0 0 20 40 60 80 100 120 140 160 180 200 0 m/z 0 20 40 60 80 100 120 140 160 180 200 m/z 194 Appendix 1 Fragmentation spectra generated in the PTR-MS under the conditions described in Chapter 2. 100 100 42 acetonitrile 93 toluene MW 42 MW 92 80 80 60 60 40 40 20 20 Relative intensity (%) Relative intensity (%) 0 0 0 20 40 60 80 100 120 140 160 180 200 0 20 40 60 80 100 120 140 160 180 200 m/z m/z 100 1,2,4-trimethylbenzene 120 MW120 80 60 40 20 Relative intensity (%) 0 0 20 40 60 80 100 120 140 160 180 200 m/z 195 Appendix 2 Supplementary Figure S4.1 Diurnal course of isoprene (Ÿ DQGPRQRWHUSHQH Ɣ HPLVVLRQV $ IRUPDOGHK\GH ż DFHWDOGHK\GH Ƈ DQG acetone (Ƒ HPLVVLRQV % QHW&22 assimilation (black line) and transpiration (grey line, C) and photosynthetic photon flux density (PPFD, black line) and cuvette temperature (grey line, D) from E. viminalis. 196 Supplementary Figure S4.2 Diurnal course of isoprene (Ÿ DQGPRQRWHUSHQH Ɣ HPLVVLRQV $ IRUPDOGHK\GH ż DFHWDOGHK\GH Ƈ DQG acetone (Ƒ HPLVVLRQV % QHW&22 assimilation (black line) and transpiration (grey line, C) and photosynthetic photon flux density (PPFD, black line) and cuvette temperature (grey line, D) from E. camaldulensis. 197 Supplementary Figure S4.3 Diurnal course of isoprene (Ÿ DQGPRQRWHUSHQH Ɣ HPLVVLRQV $ IRUPDOGHK\GH ż DFHWDOGHK\GH Ƈ DQG acetone (Ƒ HPLVVLRQV % QHW&22 assimilation (black line) and transpiration (grey line, C) and photosynthetic photon flux density (PPFD, black line) and cuvette temperature (grey line, D) from E. grandis. 198