Role of Plastid Terminal Oxidase (PTOX) As a Safety Valve for Electrons

Role of Plastid Terminal Oxidase (PTOX) as a safety valve for electrons

in Hordeum vulgare (Barley) plants.

A thesis submitted to The University of Manchester for the degree of

Doctor of Philosophy in Plant Sciences in the Faculty of Science and

Engineering and

2018

Mariela P. Aguilera Miranda

School of Earth and Environmental Sciences

Table of Contents

List of figure ...... 7

List of tables ...... 10

Abbreviations ...... 11

Abstract ...... 14

Declaration ...... 15

Acknowledgements ...... 18

Chapter 1 ...... 19

General Introduction ...... 19

Introduction ...... 20

1. Photosynthesis ...... 21

1.1 Light reactions ...... 21

1.2 The Calvin Benson Bassham cycle ...... 23

2. Reactive Oxygen Species production ...... 26

3. Mitigation of ROS through the antioxidant system ...... 27

3.1 ROS scavenging enzymatic systems ...... 28

3.2 ROS scavenging non enzymatic system ...... 32

4. Prevention of ROS production ...... 33

4.1 Non-photochemical quenching (NPQ) ...... 33

4.2 Cyclic electron transport (CET) ...... 34

4.3 Mehler reaction ...... 35

5. The Plastid Terminal Oxidase (PTOX) ...... 35

5.1 Discovery of PTOX ...... 36

5.2 PTOX role in the carotenoid biosynthesis ...... 38

5.3 Chloroplast biogenesis ...... 41

5.4 Chlororespiration ...... 41

5.5 PTOX as a safety valve for electron transport ...... 42

5.6 PTOX localization and characterization ...... 43

5.7 PTOX activity regulation ...... 45

6. Aims and Objectives ...... 46

Chapter 2 ...... 48

Role of Plastid Terminal Oxidase (PTOX) as alternative electron sink in

Hordeum vulgare ...... 48

Introduction ...... 49

Materials and Methods...... 53

Plant growth ...... 53

Measuring gas exchange ...... 53

Measuring chlorophyll fluorescence and electron transport to oxygen...... 53

Chlorophyll content ...... 56

Immunoblot analysis...... 56

Reverse transcription polymerase chain reaction (rt-PCR) ...... 58

Specific leaf area (SLA) ...... 59

Statistical analysis ...... 59

Results ...... 60

Temperature and oxygen effects on P700 and chlorophyll fluorescence

parameters ...... 60

Effect of light intensities on chlorophyll fluorescence and P700 oxidation

parameters across different temperatures and oxygen concentrations...... 62

PSII electron transport is sensitive to Plastid Terminal Oxidase inhibitor n-

Propyl Gallate...... 66

Characterization of developmental acclimation of H. vulgare at two different

grown temperature, 10°C and 20°C...... 70

Temperature and oxygen effects on chlorophyll fluorescence and P700

parameters of plants grown at 10 ºC ...... 71

Effect of light intensity on chlorophyll fluorescence and P700 parameters

across different temperatures and two O2 concentrations in plants grown at

10°C ...... 75

Barley developmental acclimation to cold does not induce Plastid Terminal

Oxidase electron sink activity ...... 77

Discussion ...... 79

Conclusion ...... 85

Chapter 3 ...... 86

Role of Plastid Terminal Oxidase (PTOX) as alternative electron sink in water restricted and salt-treated Hordeum vulgare plants ...... 86

Introduction ...... 87

Materials and Methods...... 91

Plant growth ...... 91

Measuring gas exchange ...... 91

Measuring chlorophyll fluorescence and electron transport to oxygen...... 91

Chlorophyll content ...... 94

Immunoblot analysis...... 94

Quantitative polymerase chain reaction (Q-PCR) ...... 96

Specific leaf area (SLA) ...... 97

Relative Water Content (RCW) ...... 97

Statistical analysis ...... 98

Results ...... 99

Characterization of acclimation of H. vulgare to water limitation or salinity 99

Effect of light intensity on chlorophyll fluorescence and P700 oxidation

parameters at two O2 concentration in water restricted plants...... 103

PSII electron transport is sensitive to n-propyl gallate (n-PG), a Plastid

Terminal Oxidase inhibitor, in water restricted plants ...... 105

Effect of light intensities on chlorophyll fluorescence and P700 oxidation

parameters at two O2 concentration in salt treated plants...... 108

PSII electron transport is sensitive to n-propyl gallate (n-PG), a Plastid

Terminal Oxidase inhibitor, in plants watered with 150 mM NaCl ...... 111

Discussion ...... 114

Conclusion ...... 119

Chapter 4 ...... 120

Dynamic responses of Plastid Terminal Oxidase (PTOX) to changing environmental conditions in Hordeum vulgare plants ...... 120

Introduction ...... 121

Materials and Methods...... 124

Plant growth ...... 124

Measuring chlorophyll fluorescence and electron transport to oxygen. .... 124

Immunoblot analysis...... 127

Trypsin treatment of thylakoid isolation extract ...... 127

Isolation of thylakoid at different pH ...... 127

Thylakoid extraction method ...... 128

Statistical analysis ...... 130

Results ...... 131

Dynamic responses of chlorophyll fluorescence and P700 oxidation

parameters to two sequences with a different alternation of O2

concentration...... 131

Localization of Plastid terminal oxidase (PTOX) in barley plants after 7 days

of watering with 150mM NaCl and control plants...... 134

Variation of PTOX binding to the thylakoid membrane in barley plants

exposed to high light or pH during the extraction...... 136

Discussion ...... 141

Conclusion ...... 146

Chapter 5 ...... 147

Discussion ...... 147

References ...... 156

Final Word count: 37,716

List of figure

Figure 1.1. Chloroplast structure…………………………………………………….22

Figure 1.2 .Schematic diagram of electron and proton flow through thylakoids...... 24

Figure 1.3. Schematic diagram of the three phases of the Calvin Benson

Bassham, which are carboxylation, reduction and regeneration…………….….25

Figure 1.4. ROS production pathways in chloroplasts………………..…………..28

Figure 1.5. ROS and antioxidants defence mechanism………………………….31

Figure 1.6. Electron flow in electron chain transport…………………………...…37

Figure 1.7. immutans plant with a variegated phenotype………………………...38

Figure 1.8. Carotenoid biosynthesis and related pathways…………………….. 39

Figure 1.9. Structural model of PTOX………………………………………………45

Figure 2.1. P700 and fluorescence signals……………………………………….55

Figure 2.2. The O2 sensitivity of PSI and PSII parameters across a range of temperatures…………………………………………………………………………..61

Figure 2.3. Irradiance response of PSII parameters……………………….……..63

Figure 2.4. Irradiance response of P700 parameters…………………………..…65

Figure 2.5. The effect of 1mM n-PG on chlorophyll fluorescence and PSI parameters…………………………………………………………………………….68

Figure 2.6. The effect of 1mM n-PG on chlorophyll fluorescence and PSI parameters…………………………………………………………………………….69

Figure 2.7. Developmental acclimation of barley to cold…………………………72

Figure 2.8. The O2 sensitivity of chlorophyll fluorescence parameters across a range of temperatures………………………………………………………………..74

Figure 2.9. Irradiance response of PSII and P700 parameters in barley plants grown at 10 ºC…………………………………………………………………….…..76

Figure 2.10. The effect of 1mM n-PG on chlorophyll fluorescence and PSI parameters…………………………………………………………………………….78

Figure 3.1. P700 and fluorescence signals………………………………………..93

Figure 3.2. The effect of water limitation and salinity on the relative water content

(RWC, a and b) and Specific Leaf area (SLA, c and d) in Barley leaves…….100

Figure 3.3. The effect of water limitation and salinity on gas exchange parameters…………………………………………………………………………...101

Figure 3.4. The effect of water limitation and salinity on the ratio of chlorophyll a to b (a and b) and the total chlorophyll content (a and b)…………………….…102

Figure 3.5. The effect of water limitation and salinity on PTOX expression and

PTOX abundance……………………………………………………………………102

Figure 3.6. Irradiance response of PSII electron transport rate (a) and NPQ

(b)…………………………………………………………………………………..…104

Figure 3.7. Irradiance response of P700 electron transport rate (a), k (b) and the proportion of P700 oxidised (c)…………………………………………………….105

Figure 3.8.The effect of 5mM n-PG on PSII ETR (a) and NPQ (b)……………106

Figure 3.9. The effect of 5mM n-PG on PSI ETR (a), k (b) and proportional oxidised (c)…………………………………………………………………………...107

Figure 3.10. Irradiance response of PSII electron transport rate (a) and NPQ

(b)……………………………………………………………………………………..109

Figure 3.11. Irradiance response of P700 electron transport rate (a), k (b) and the proportion of P700 oxidised (c)………………………………………………..110

Figure 3.12. The effect of 5mM n-PG on PSII ETR (a) and NPQ (b)………….112

Figure 3.13. The effect of 5mM n-PG on PSI ETR (a), k (b) and proportional oxidase (c)……………………………………………………………………………113

Figure 4.1. P700 and fluorescence signals………………………………………126

Figure 4.2. The O2 sensitivity of PSII parameters across a sequence with a different order in the application O2 concentration……………………………….132

Figure 4.3. The O2 sensitivity of P700 parameters across a sequence with a different order in the application O2 concentration…………………………..…..133

Figure 4.4. Effect of salinity in the localisation of PTOX protein in barley plants…………………………………………………………………………………135

Figure 4.5. Effect of salinity in the localisation of PTOX protein in barley plants………………………………………………………………………………….136

Figure 4.6. Effect of salinity and water restriction in the binding of PTOX protein to thylakoid membranes in barley plants………………………………………....137

Figure 4.7. Effect of salinity in the localisation of PTOX protein in barley plants………………………………………………………………………………….139

Figure 4.8. Effect of salinity in the binding of PTOX protein to thylakoid membranes in barley plants………………………………………………………..140

List of tables

Table 2.1 PTOX primer sequence used in rt-PCR……………….……………….58

Table 3.1 PTOX primer sequence used in rt-PCR………………………………...97

Abbreviations

ATP Adenosine triphosphate ATP  subunit of ATP synthase APX Ascorbate peroxidase AsA Ascorbic acid Asp Aspartic acid BT-T broken thylakoid treated with water BT+T broken thylakoid treated with trypsin Car Carotenoids CET cyclic electron transport CBB Calvin Benson Bassham cycle Chl Chlorophyll CN Cyanide 3Chl triplet-state chlorophyll Cyt b6f cyctochrome b6f complex CuZn-SOD CuZn superoxide dismutase DHAR Dehydroascorbate reductase DMAPP, dimethylallyl diphosphate DXP, 1-deoxy-d-xylulose 5-phosphate ETR electron transport rate ETC electron transport chain Fd ferredoxin FNR ferredoxin NADP+ oxidoreductase gh ghost GAP glyceraldehyde 3-phosphate GGPP geranylgeranyl pyrophosphate Glu Glutamate GSH Glutathione GR Glutathione reductase His Histidine

H2O2 hydrogen peroxide IPP isopentenyl diphosphate IT-T Intact thylakoid treated with water IT+T Intact thylakoid treated with trypsin

11 im immutans HL high Light LED light emitter diode Leu Leucine LHCI light harvesting complex I LHCII light harvesting complex II LL low Light MDHAR Monodehydroascorbatereductase NADP oxidized nicotinamide adenine dinucleotide phosphate NADPH reduced nicotinamide adenine dinucleotide phosphate NDH NAD(P)H dehydrogenase nPG n-propyl gallate NPQ non-photochemical quenching MEP 2-C-methyl-d-erythritol 4-phosphate

O2 molecular oxygen OH. hydroxyl radicals

3 O2 oxygen triplet-state

1 O2 singlet oxygen

-. O2 superoxide anion radicals PDS Phytoene desaturase PFD photon flux density PsaC PSI-C core subunit of photosystem protein PC plastocyanin PGR5 proton gradient regulation 5 proteins PGRL1 PGR5-like 1 proteins PSI photosystem I PSII photosystem II PsbQ oxygen-evolving complex PSII protein PTOX plastid terminal oxidase PQ plastoquinone PQ.- plastosemiquinone PQH2 plastoquinol 3-PGA 3-phosphoglycerate qI Photoinhibitory quenching qT State-transition quenching qE Energy-dependent quenching RC reaction centre

ROS reactive oxygen species RuBisCo ribulose-bisphosphate carboxylase/oxygenase RuBP Ribulose 1,5 bisphosphate SOD superoxide dismutase UQH2 ubiquinol Tyr Tyrosine ΦPSII quantum yield of photosystem II ΔpH gradient of pH µg microgram

Abstract

Photosynthesis is a primary target of different abiotic stresses. Under sub- optimal conditions, the imbalance between a low-efficiency carbon fixation and excessive light absorbed can generate an overburdening of the photosynthetic apparatus. This can lead to the production of reactive oxygen species (ROS), which can damage cellular components. Plastid Terminal Oxidase (PTOX) has been proposed to play a role in some higher plants, acting as a safety valve for electrons, resulting in protection of the plastoquinone pool from over-reduction under environmental stress conditions. Due to PTOX ability to transport electrons from plastoquinol to molecular oxygen, generating water. The regulation of the enzyme is still unclear, however, it has been hypothesised to be sensitive to stromal pH variation and recent findings suggest that a translocation of the protein to the grana membranes could be involved. The protective role of PTOX as alternative electron sink in Hordeum vulgare plants was explored, in particular during cold acclimation and against water limitation and high salinity conditions. Additionally, the regulation of the activity of this enzyme was studied. Plastid terminal oxidase (PTOX) seems to account for the diversion of up to 25% of electrons to oxygen in control plants, acting as an important safety valve for electron transport potentially protecting barley leaves from an overreduction of the plastoquinol pool. In cold acclimated plants, PTOX does not appear to be important, however PTOX protein and transcript were still detected. In water restricted and salt treated plants, evidence of PTOX acting as a safety valve for electrons was seen. This plastoquinone oxidase seem to be diverting up to 31% of the total amount of electron transported by PSII at 21% O2 in water restricted plants and up to 45% in salt-treated leaves. This was supported by the presence of increased PTOX protein in both cases and also an increase in PTOX transcript either transiently, in water restricted plants, or in a sustained way in salt treated plants. Additionally, a dynamic and reversible response to O2 concentration was seen in the photosystem (PSII) electroon transport chain (ETR) and non-photochemical quenching (NPQ). Therefore it seems to be unlikely that PTOX is inhibited or in some way regulated by a previous oxygen concentration. It was not possible to confirm the translocation of PTOX to the grana in barley plants, however, direct or indirect variation in the pH has an effect on PTOX protein recovery in the salt-treated plants, with increasing detection of PTOX at more alkaline pH.

Declaration

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Acknowledgements

I would like to express my gratitude to my supervisor, Dr Giles Johnson and all members of my laboratory due to the enormous help that they have been giving me during all the process of my PhD. Additionally, I am very grateful to the human team in plants science for the experimental techniques and personal support provided. Also, I would also like to acknowledge CONICYT for funding this PhD.

On the other hand, at the personal level, I just need to express my infinite love and gratitude for my family, which always have been pushing me to improve.

Especially, to the women in my family, which always have been inspiring me to embrace living my life and encouraging me to go for the challenges, which will allow me to pursue my dreams.

During this journey, I have been experienced the most extraordinary learning process academically and even more transcendentally as a human being. My apologies for not naming all of you one by one, but the life has been so generous with me, that I hope to thank you in person.

Anyway, thank you for teaching me to build up a family and a home from scratch, being an immigrant definitely has been reframing my identity in the most overwhelming way due to all of you surrounding me.

Chapter 1

General Introduction

Introduction

In climate change scenarios, the temperature is expected to increase, and this is expected to have a negative impact on crop yields. For example, a study using

Kansas wheat variety field trial outcomes, predicts a net wheat yield reduction

[1]. A similar tendency has been predicted for the US in crops like soybean and maize, in response to the potential increase in the temperature as a part of global warming [2]. In addition, Lesk and collaborators in 2016 [3] showed that a reduction of around 10% of cereal production in 177 countries resulted from drought and extreme heat due to climate change. Another study has estimated that per each Celsius degree increase, the global yield of soybean, rice, maize, and wheat are reduced in 3.1%, 3.2%, 7.4% and 6.0% respectively [4].

The uncertainties about the extent of climate change in the near future and its detrimental effect on crop production and food security, highlight the necessity to understand better how plants overcome stress and generate the opportunity to utilize this knowledge to improve crop production [5], [6]. At the centre of plant responses to environmental stress is the process of photosynthesis, which is the ultimate factor determining plant growth [7], [8].

In this chapter, an overview of photosynthesis is presented, as a principal process on which crop productivity depends and how stress can negatively affect its efficiency is discussed [9]. Mechanisms for the alleviation of and protection against stress will be covered. I will then explore in more detail the role of a protein called the Plastid Terminal Oxidase (PTOX) as a protective mechanism against stress. This protein has been proposed to act as a safety valve for photosynthetic electron transport, potentially reducing the risks of overburden of the photosynthetic apparatus when harvested energy exceeds the energy utilized in photochemistry [6], [11]. Understanding the role of the PTOX

20 protein in the important crop Hordeum vulgare (barley) will be the main focus of this thesis.

1. Photosynthesis

Photosynthesis is the process by which plants capture light energy required to drive growth. It is often described as having two stages; the light and dark reactions and, in eukaryotic photosynthetic organisms, photosynthesis takes place in the chloroplast [16], [17]. These organelles contain a system of internal membranes, which are known as thylakoids, where photosynthetic pigments are located: chlorophylls and carotenoids [18]. Thylakoids form stacked membranes which associate closely with each other, forming structures known as grana.

Where the membrane system is not stacked, this is called the stroma lamellae.

The non-membranous inside space of the chloroplast is known as the stroma

(Figure 1.1, [19]).

1.1 Light reactions

The term “Light reactions” refers to the reactions occurring in the thylakoid membranes, while the “dark reactions” take place in the stroma [16], [17]. During the light reactions, photons are absorbed by antenna complexes, which are proteins binding chlorophylls and carotenoids [20]. These absorbed photons generate an excited state in pigment molecules. The energy can then be transferred between pigments and the electronic excited states migrate from one molecule to another. This mechanism is a consequence of the spatial arrangement of antenna systems such that the peripheral pigments absorb light with shorter wavelengths and higher excitation energy, in comparison to central

21 pigments. This results in the movement of excitation energy towards the core area of the antenna system and later its delivery to the reaction centres [21].

The reaction centres are pigment-protein complexes, which are composed of proteins binding chlorophylls, carotenoids and other cofactors, including quinones and iron sulphur centres. In the reaction centres, dimers of pigments, called special pairs, act as primary electron donors which, when they receive energy from the antenna system, are able to donate an electron to another molecule. The photosynthetic apparatus in higher plants has two reaction centres, known as photosystem II (PSII) and photosystem I (PSI), containing the special pairs P680 and P700, respectively. When the special pair of chlorophylls are excited by a photon to form an excited electronic state, this is transformed into a strongly reducing species, transferring an electron rapidly to a primary electron acceptor molecule [22]. This has the effect of converting excitation energy into chemical energy.

Figure 1.1. Chloroplast structure. Electron micrograph of a chloroplast from tobacco (A). Schematic diagram of a chloroplast, where the envelope membranes, thylakoids (grana and stroma lamellae) and stroma are shown (B).

Figure from Blankenshp, 2014 [23].

Subsequent to the first photochemical reaction, a charges separation, the primary donor pigment becomes a highly oxidizing species. In the case of PSII, this is able to oxidize water, releasing O2 and protons into the thylakoid lumen.

For each bimolecular oxygen molecule that is expelled, 4 electrons are extracted from 2 water molecules, which then flow through the electron transport chain.

These electrons are used by the PSII reaction centres to reduce a molecule of plastoquinone (PQ), which takes up two protons from stroma, forming PQH2 [22],

[24]. Subsequently, the latter can be reoxidised by a complex called Cytochrome b6f, which, in turn, reduces a small soluble protein called plastocyanin (PC) [25].

The reduced PC donates electrons to oxidized P700 in PSI. When P700 absorbs a photon, electrons are transferred to ferredoxin (Fd) and then forwarded to ferredoxin NADP+ oxidoreductase (FNR), which is able to carry out the electron reduction of NADP+, resulting in NADPH (Figure 1.2, [26]).

1.2 The Calvin Benson Bassham cycle

The Calvin Benson Bassham cycle fixes CO2 from the atmosphere and produces sugars within the chloroplast stroma. The carboxylation reaction is carried out by the enzyme ribulose-bisphosphate carboxylase/oxygenase

(RuBisCo), which catalyses the reaction of CO2 with Ribulose 1,5 bisphosphate

(RuBP). The mechanism of this reaction results in formation of two molecules of

3-phosphoglycerate (3-PGA). Subsequently, the reduction phase is carried out, where PGA undergoes a phosphorylation and its product is reduced, to form a triose phosphate.

Figure 1.2. Schematic diagram of electron and proton flow through thylakoids.

Water oxidation produces protons which are released into the lumen and simultaneously Photosystem II (PSII) reduces plastoquinone (PQ). The latter reduces the cytochrome b6f and additionally transfers protons from stroma to lumen. Subsequently, plastocyanin (PC) oxidises the cytochrome b6f and in a second stage reduces PSI. Electrons flow from PSI to ferredoxin (Fd) and then to ferredoxin NADP+ oxidoreductase (FNR), which mediates NADPH formation.

Electron transport results in the formation of a proton gradient across the thylakoid membrane, with protons being released into the lumen by the water splitting complex of PSII and the cytochrome b6f complex. This proton gradient is used by ATP synthase in order to produce ATP, which is released in stroma.

Both, ATP and NADPH are used in the Calvin Benson Bassham cycle (CBB).

Finally, a regeneration phase consists of a series of reactions, which start from the triose phosphates and regenerate RuBP through a rearrangement of the carbon skeleton and its phosphorylation state [27], [28]. For every six molecules of PGA formed by RuBisCo, five will be used to regenerate RuBP, while one will leave the cycle and be used to synthesise sugars (Figure 1.3).

Figure 1.3. Schematic diagram of the three phases of the Calvin Benson

Bassham, which are carboxylation, reduction and regeneration. In the carboxylation phase, ribulose-bisphosphate carboxylase/oxygenase (RuBisCo) fixes a CO2 to Ribulose 1,5 bisphosphate (RuBP) resulting in the formation of two molecules of 3-phosphoglycerate (3-PGA). Through simultaneously reduction and phosphorylation 3-PGA will result in a triose phosphate in a phase called Reduction. The regeneration phase involves through a rearrangement of the carbon skeleton and the phosphorylation of triose phosphates to regenerate

RuBP.

2. Reactive Oxygen Species production

Light tends to be captured by photosynthetic apparatus in an optimal way, except in situations where the capacity for its assimilation is exceeded and harvested energy is greater than that which can be used in photochemistry. The consequences of overburdening the photosynthetic apparatus include photoinhibition, which can be caused by a blockage of electron transfer and decreases in assimilation. Chlorophyll molecules and thylakoid structure can be damaged in extreme cases, however, PSII seems to be the most affected by photoinhibition, while the activity of PSI is inhibited to a lesser extent [29], [30].

This phenomenon can result from oxidative lesions, due to the production of reactive oxygen species (ROS) coupled to the transfer of electrons in the electron transport chain [31], [32]. Additionally, significant amounts of ROS are released from both PSII and PSI, although predominantly in the latter [33].

1 -. In PSII, singlet oxygen ( O2), superoxide anion radicals (O2 ), hydrogen peroxide

. 1 (H2O2) and hydroxyl radicals (OH ) are formed (Figure1.4). O2 generation requires an interaction between triplet-state chlorophyll (3Chl) and oxygen triplet-

3 state ( O2; Eqn 1). This process is more important in PSII, where charge recombinations from the first ion radical pair in PSII (P680+.Phe-.) or from

+ - P680 QA . occur. The generation of triplet chlorophyll can be increased due to double reduction and protonation of QA [34]–[41].

3 3 1 * Chl* + O2→Chl + O2 (Eqn 1)

-. -. -. - O2 is produced due to the reduction of O2 by Phe , QA and QB ; while H2O2 is

-. formed due to either spontaneous or SOD-catalysed dismutation of O2 [36],

[39], [42].

ROS is also generated in PSI, through a process called the Mehler reaction,

-. which consists in a photoreduction of molecular oxygen to O2 . Subsequently, the latter is disproportionated by CuZn superoxide dismutase (CuZn-SOD) to

H2O2 and O2, after which H2O2 can be converted to water by means of enzymes including ascorbate and glutathione peroxidases. In other words, in this cycle the electrons flow from water in PSII to water in PSI, so this pathway is often referred to as the Water-Water cycle [35], [36], [40], [43]–[45].

.- In addition, O2 can also be produced spontaneously by the reduction of molecular oxygen (O2) by plastosemiquinone (Figure 1.4). This reaction is promoted by illumination, due to the potential alkalinisation of stroma and an

.- increase in the plastosemiquinone (PQ ) concentration, facilitating O2 reduction.

Subsequently, plastohydroquinone (PQH2) reduces the superoxide, generating hydrogen peroxide (H2O2) [46]. The estimation of this alternative electron transport at high light is that it could be up to 60% of total electron transport and it has been suggested to be important at both moderate and high temperatures

[47].

Another spontaneous pathway of superoxide production has been proposed to occur in the Cyt b6f complex (Figure 1.4, [48], [49]).

3. Mitigation of ROS through the antioxidant system

Overloading of the photosynthetic apparatus can result in ROS production. The negative effects of this are however attenuated in plants by protective systems of antioxidants, which scavenge ROS. These can be enzymatic or non- enzymatic. Examples of constituents of the enzymatic antioxidant system are: superoxide dismutases (SOD), ascorbate peroxidase (APX), monodehydroascorbatereductase (MDHAR), dehydroascorbate reductase

(DHAR) and glutathione reductase (GR). The non-enzymatic antioxidant system

27 includes glutathione) (GSH), ascorbic acid (AsA), carotenoids and tocopherols

[36], [40], [50], [51].

Figure 1.4. ROS production pathways in chloroplasts. In PSI molecular oxygen

-. (O2) is reduced to O2 after which CuZn-SOD generates H2O2. In PSII, the

3 3 1 P680* interacts with an oxygen triplet-state ( O2) forming singlet oxygen ( O2).

-. Spontaneous reduction of molecular oxygen (O2) to O2 may also occur in the

Cyt b6f complex or plastoquinone.

3.1 ROS scavenging enzymatic systems

One of the best-studied examples of an antioxidant enzyme is superoxide dismutase (SOD). This is a metalloenzyme component of the ROS scavenging enzymatic system, and forms of this are located in most subcellular compartments that produce activated oxygen. This metalloenzyme is able to

-. -. catalyse the dismutation of two molecules of O2 , one O2 being reduced to

H2O2 and another oxidized to O2 (Eqn 2, [36]).

-. -. + O2 + O2 + 2H H2O2 + O2 (Eqn 2) SOD

-. SOD catalysis can result in a reduction in O2 concentration, which can lead to a decreased in the probability of OH. formation by Haber-Weiss reaction. (Figure

1.5, [45], [52]–[55]). However, through the Fenton reaction, H2O2 can generate

ROS as OH. [56]. The overproduction of this SOD enzyme has been suggested to result in a higher tolerance of oxidative stress [57]. SOD has 3 isoenzymes in plants, of which 2 are detected in the chloroplast, being the Fe-SOD isozymes commonly found in chloroplast fractions [54] and the Cu/Zn-SOD isozymes present in chloroplast and also cytosolic fractions [58].

The Ascorbate peroxidase (APX), is a peroxidase taking part in the water-water and Ascorbate-Glutathione (AsA-GSH) cycles, scavenging H2O2. This enzyme makes use of ascorbate (AsA) to reduce H2O2 with concomitant release of two molecules of water and monodehydroascorbate radical (MDHA, Eqn 3).

Furthermore, the overproduction of APX seems to raise the activity of other antioxidants enzymes, resulting in a strengthening of ROS scavenging and oxidative stress tolerance [51].

H2O2 + 2AsA 2H2O + 2MDHA (Eqn 3) APX

Another peroxidase, glutathione peroxidase (GPX) also catalyzes the reduction of H2O2 to H2O in the chloroplast and also of lipid peroxides to their corresponding alcohols (Eqn 4). This enzyme has been suggested to increase its concentration, when plants experience a fungal infection, water deficit, and metal stress, indicating that GPX could be induced in response to stress [59].

H2O2 + 2GSH H2O + GSSG (Eqn 4) GPX

Ascorbic acid (AsA) is important in maintaining the redox homeostasis, scavenging ROS.

The enzyme monodehydroascorbate reductase (MDHAR) regenerates AsA, by the reduction of monodehydroascorbate (MDHA, Eq 5) and utilizes as an electron donor either NADH or NADPH, however, this enzyme has a higher affinity for NADH than NADPH [60]. The activity of MDHA seems to rise in plants that experience environmental stress [61].

MDHAR MDHA + NAD(P)H AsA + NAD(P)+ (Eqn 5)

In addition, dehydroascorbate (DHA) is produced by the disproportionation of

MDHA carried out by ferredoxin (Fd) [60]. Dehydroascorbate reductase (DHAR) plays a crucial role in the conservation of the level of AsA in its reduced state, because of its capacity to reduce DHA to Asa, through the utilization of glutathione (GSH) as reducing substrate (Eqn 6, [62] )

DHA + 2GSH AsA + GSSG (Eqn 6) DHAR

The redox state of AsA is key in order to tolerate different abiotic stresses, which produce ROS. It has been suggested that DHAR overexpression can increase stress tolerance in plants subjected to diverse types of stresses [40].

Glutathione reductase (GR) is a flavoprotein oxidoreductase, located mainly in chloroplasts which catalyses the reduction of the disulphide bond in GSSG, to form two molecules of GSH (Eqn 7). Thus this enzyme participates in the maintenance of the GSH pool, as the latter molecule is involved in the

30 antioxidant process and metabolic regulation. It has been suggested that GR and GSH can participate in the determination of the tolerance level of plants under various stresses. Additionally, GR is a involved in the ascorbate- glutathione (AsA-GSH) cycle and can have in a crucial role in the defence system against ROS [63]–[65].

GR GSSG + NAD(P)H 2GSH + NAD(P)+ (Eqn 7)

Figure 1.5. ROS and antioxidants defence mechanism. Figure from Gill and

Tuteja, 2010 [36].

3.2 ROS scavenging non enzymatic system

In addition to antioxidant co-factors of enzymes, several species can act as antioxidants in an enzyme independent manner. Glutathione (GSH) is considered to be a metabolite with a relevant role in the defence of the oxidative damage generated by ROS. It is located in the majority of subcellular compartments in the reduced state and it acts as an antioxidant in diverse ways.

Due to this, the molecule can act as a free radical scavenger, reacting directly

-. . with ROS such as O2 , OH and H2O2 [66]–[68] .

Ascorbic acid (AsA) has the capacity to donate electrons in both enzymatic and non-enzymatic reactions. Therefore, this is suggested to be a powerful ROS

-. scavenger, which offers membrane protection as a result of scavenging O2 ,

. H2O2, OH and being involved in the regeneration of α-tocopherol from tocopheroxyl radicals [50], [68]. In the chloroplast, AsA plays a role as a cofactor for violaxanthin de-epoxidase thus sustaining dissipation of excess excitation energy [69].

Carotenoids (Car) can prevent damage to the photosynthetic apparatus through dissipating excess excitation energy, in other words, these are able to quench a

1 chlorophyll triplet and thus to prevent the formation of O2 (Eqn 8 [70], [71]).

Car + 3Chl* 3Car* +Chl (Eqn 8)

Tocopherols are lipid-soluble antioxidants, which protect biomembranes and

1 lipids due to quenching O2 in the chloroplast, resulting in a prevention of damage in structure a function of PSII [67], [68].

4. Prevention of ROS production

Antioxidant systems can alleviate the negative effect of ROS by reacting with them and converting them to less reactive molecules. This tends however to be energetically demanding, due to the necessity of having high levels of antioxidants and the associated cost of synthesis and regeneration [44].

Therefore, plants display simultaneously other strategies to prevent ROS production through the regulation of light harvesting and electron transport processes [7], [39], [72].

4.1 Non-photochemical quenching (NPQ)

An important example of modulation of photosynthetic electron transport is Non-

Photochemical Quenching (NPQ), one of the faster mechanisms offering photoprotection.

NPQ relieves the surplus of excitation from the light-harvesting complex (LHC II) antenna as heat (Figure 1.6), through various component: qI, qT and qE, the latter being the major dissipator of the energy [73], [74]. The generation of a pH gradient across the thylakoid membrane triggers NPQ, due to the acidification of the lumen mediating the protonation of PsbS protein [75]. It is suggested that

PsbS could be necessary to make the LHCII antenna more sensitive to the gradient of pH (ΔpH) and also have a role as a switch, triggered by ΔpH, involved with conformational changes associated with lumenal intermolecular interconnection [76], increasing the mobility PSII complex and the speed in the generation of NPQ and its relaxation [77].

At the same time, the lumenal acidification activates the xanthophyll cycle, by activating the enzyme violaxanthin de-epoxidase. This enzyme catalyzes two successive de-epoxidations, firstly on the violaxanthin converting it to

33 antheraxanthin and later producing zeaxanthin, allowing the relaxation of the energy as heat [78]. Zeaxanthin seems to be able to modify the sensitivity of

LHCII to luminal pH changes as well as delaying the relaxation of NPQ.

Therefore, zeaxanthin has been proposed not just to trigger the photoprotective state in LHCII, but also offer protection under fluctuating light [79].

4.2 Cyclic electron transport (CET)

While Linear electron transport (LET; Section 1.1) produces ATP and NADPH for utilization in the CCB cycle, PSI cyclic electron transport (CET) just generates ATP [80]. Two pathways for PSI CET have been described, the most important of which involves the proton gradient regulation 5 (PGR5) and PGR5- like 1 (PGRL1) proteins, transferring electrons to plastoquinone [81]. The secondary pathway utilizes a protein complex called the NADPH dehydrogenase

(NDH) complex (Figure 1.6) to transfer electrons from PSI back to PQ. In both pathways, the electrons are transported from ferredoxin (Fd) to plastoquinone

(PQ), generating pH gradient across of thylakoid (ΔpH) without generation of

NADPH [82], [83]. The CET has been suggested to play a crucial role in the balancing of ATP/NADPH production and this pathway seems to protect both photosystems by preventing the stromal overreduction [84], [85].

In addition to a possible role in controlling ATP:NADPH, CET has been recognised to have a key role in photosynthesis of higher plants, due to its participation in controlling light harvesting, particularly when plants are exposed to strong illumination. When CET occurs, an increase in the proton gradient across the thylakoid membrane is generated, which triggers NPQ protecting PSII against light excess [86]–[88].

4.3 Mehler reaction

The Mehler reaction or water–water cycle is another alternative electron transport pathway, which has been suggested to play a role in photoprotection

(Figure 1.6, [89]). In this cycle, the electrons flow from water at PSII to oxygen at

PSI, diverting excess electrons from the photosynthetic electron transport chain.

The water-water cycle has been described as an important alternative electron sink, diverting the excessive electrons under stress conditions [90]–[92].

However, the Mehler reaction also generates ROS, so its protective function remains contraverisal. It has been estimated to account for up to 10% of the electrons transported through the photosynthetic electron transport chain [93].

5. The Plastid Terminal Oxidase (PTOX)

The pathways and protein complexes discussed above have been studied for many years. A much more recently discovered protein involved in chloroplast electron transport is the Plastid terminal oxidase (PTOX). This protein is thought to take part in an alternative electron transport pathway and has been proposed to have an important role as an alternative electron sink in some higher plants, when they experience environmental stress, protecting the photosynthetic apparatus from an overburn, through the prevention of plastoquinol pool overreduction, due to its ability to divert electron from plastoquinol pool (PQH2) to oxygen with concomitant production of water (Figure 1.5, Eqn 9, [10], [11],

[14], [94], [95]).

PTOX PQH2 + O2 PQ+ H2O (Eqn 9)

5.1 Discovery of PTOX

PTOX was first identified in mutant plants showing a variegated phenotype, immutants (im) in Arabidopsis thaliana (Figue 1.7 a, [96], [97]) and ghost (gh) in tomato mutants plants (Figure 1.6 b, [98]). These variegation mutants, which show white patches in their vegetative and reproductive tissues, have been shown to contain green areas where cells contain normal functional chloroplasts but also to have yellow or white sectors having abnormal looking chloroplasts.

Also, some white heteroplastic areas are observed with a mixture of normal and abnormal chloroplasts in the same cells, with impaired chloroplasts being predominant [96], [99]. Similarities between the IM protein sequence and

Mitochondrial Alternative Oxidase (AOX) protein were found through databases.

In the plant mitochondrial electron transport chain, AOX plays a role as an alternative electron transport carrier, bypassing electron flow from ubiquinol

(UQH2) to O2. In vitro enzyme assays showed the plastoquinol oxidase activity of

PTOX and, due to its homology and location in the chloroplast, PTOX was named Plastid Terminal Oxidase. Also, both proteins share the cyanide (CN) insensitivity and inhibition in their activities mediated n-propyl gallate (n-PG) and n-octyl gallate [100].

The development of the patchy phenotype in PTOX mutants is not developmentally pre-determined. For example, high temperature and high light seem to have an effect over the im Arabidopsis mutant, inducing an increase in the extent of white sectors, whilst a low light intensity allows the development of green sectors, such that a completely green plant can develop when the irradiance is low enough [101]. Additionally, the palisade layer appears to modify its thickness in im plants, with the green areas being thicker and the white sectors thinner than in wild-type plants, possibly resulting from the variation in the elongation of the palisade layer cells [99].

The anomalous chloroplasts in white areas show an absence of the organization in the lammela structures and in these sectors the accumulation of phytoene, a carotenoid intermediate was reported, suggesting a defective activity of phytoene desaturase [102].

Figure 1.6 .Electron flow in electron chain transport, highlighting the water-water cycle carried out by plastid terminal oxidase (PTOX, green), as a result of the transfer of electrons from the reduced PQ to molecular oxygen. Additionally, chlororespiration is a process, where firstly the PQ is reduced in a non- photochemical way through NAD(P)H-dehydrogenase (NDH) and subsequently, this is oxidised by PTOX action, associated to water formation (pink). Moreover,

Mehler reaction is shown, being a photoreduction of oxygen to O2- in PSI.

Afterwards, this is disproportionated by CuZn superoxide dismutase (CuZn-

SOD) to H2O2, which is converted in water, by means of ascorbate peroxidase

(APX, lilac). Show an arrow form FNR to cyt b6f to indicate cyclic (orange). In blue the Non-photochemical quenching, a dissipation of excessive energy as heat.

Figure 1.7. immutans plant with a variegated phenotype, from Aluru and

Rodermel, 2004 (a, [103]). Leaves, flowers and fruit from ghost mutant tomato, from Carol and Kuntz, 2001 (b, [102]) Organs from variegated plants show green and white sectors in tissue that is normally green, PTOX gene is impaired in these organisms.

5.2 PTOX role in the carotenoid biosynthesis

Carotenoids are ubiquitously present in photosynthetic organisms and have an essential role in the photosynthetic process, being part of reaction centres and accessory pigments, stabilizing the lipid phase of thylakoid and also playing a role in photoprotection. Plants lacking carotenoids experience photooxidative damage and show an albino phenotype [104].

Carotenoids are terpenoids, the biosynthesis of which involves isoprenoid biosynthesis as a first step, which later generates phytoene (Figure 1.8), a colourless C40 carotenoid precursor. After two consecutive desaturations, the phytoene turns in to lycopene, a red compound common to both α and β carotenoid synthesis [105].

Figure 1.8. Carotenoid biosynthesis and related pathways. Carotenoids are written in the colour they appear. GAP, glyceraldehyde 3-phosphate; DXS, 1- deoxy-d-xylulose 5-phosphate synthase; DXP, 1-deoxy-d-xylulose 5-phosphate;

MEP, 2-C-methyl-d-erythritol 4-phosphate; IPP, isopentenyl diphosphate;

DMAPP, dimethylallyl diphosphate; GGPP: geranylgeranyl pyrophosphate.

Figure modified from Wei et al., 2014 [105].

Phytoene desaturase (PDS) is a key step in carotenoid biosynthesis and its catalytic activity seems to be a rate-limiting step. In order for this desaturation to occur, the plastoquinone pool acts as a crucial electron acceptor in higher plants

[102]. In immutans plants, within the white sectors, it has been observed that an accumulation of colourless C40 phytoene occurs, while, in the green areas, normal chloroplasts and concentrations of carotenoid were observed,

39 accompanied by a well functioning PDS [96]. It seems to be likely that PTOX plays a role in oxidizing the plastoquinol pool, supporting PDS activity and carotenoid biosynthesis, by preventing an overreduction of plastoquinone pool, under conditions where photosynthetic electron transport flow does not provide enough available electron acceptors for the normal functioning of PDS [102],

[106].

PTOX activity is probably most relevant at the beginning of chloroplast biogenesis, when the components of the electron transport chain are just starting to be synthesized and linear electron flow is not yet fully active. At this stage, in an im plant, it is likely that carotenoid biosynthesis gets blocked by the absence of electron acceptors to oxidase PDS, leading to photodamage and white sectors. However, once the chloroplasts mature, PTOX is dispensable, due to a fully working linear electron transport chain, which at moderate light can manage to keep the plastoquinol at least partially oxidised. Additionally, it seems to be important to consider that there appears to be a threshold of the minimal electron transport needed to avoid the failure of carotenoid production and therefore the white areas [107].

In the case of gh, the fruit also keep the colour of the branch which originates them. While young fruit attached to green branches are green, white branches generate white fruit. These can turn yellowish, containing much lower levels of lycopene than the red fruit developed in green areas or wild-type plants. This suggests that, at some point, probably through a different pathway than PTOX, the availability of electron acceptors was solved, however, this mechanism is still unclear [98].

5.3 Chloroplast biogenesis

As was already discussed, im and gh plants exhibit a variegated phenotype, with white areas showing abnormal chloroplasts, implying differences in morphology and number of this type of organelle with respect to wild-type. It seems to be that im cotyledons possess fewer chloroplasts, and that the variegated sectors appear early in the leaf development [96], [107]. Additionally, it had been suggested that, in plants experiencing high light during their early development,

PTOX activity could be important for the normal development and organisation of thylakoid structure, due to its activity modulating the ROS produced by an immature photosynthetic apparatus. Thus, the absence of PTOX could remove an alternative pathway for dissipation of light, helping to prevent the overreduction of the plastoquinone pool, reducing the risk of photooxidative stress in an immature organelle. This could be potentially protective of the negative effect due to the excess of energy, over the maturation process of the nascent chloroplas [99], [107], [108].

5.4 Chlororespiration

Chlororespiration has been suggested to be a respiratory electron transport pathway occurring in the dark in chloroplasts. This pathway might involve a light- independent reduction of the PQ pool through the activity of NADPH dehydrogenase (NDH), followed by an oxidation of plastoquinol (PQH2) mediated by PTOX and simultaneous generation of water (Figure 1.5). It has been reported that reduced ferredoxin could be the initial electron donor for the

NDH complex [109]–[111]. The absence of Photosystem (PS) II in tobacco mutant lines, provides evidence supporting a role for PTOX in chlororespiration, with a substantial increase in abundance of PTOX protein and NDH complexes being seen in such mutants [112]. An experiment performed with n-propyl

41 gallate (n-PG, a PTOX activity inhibitor) in PTOX overexpressor tobacco line, also provides evidence of PTOX playing a role in chlororespiration by oxidizing the plastoquinol in absence of light [113].

Under abiotic stress, chlororespiration seems to be triggered in some photosynthetic organisms, acting as an alternative electron transport pathway.

For instance, there is evidence of NDH and PTOX increasing their activity and/or abundance when plants experience heat, high light and water stress [13], [15].

5.5 PTOX as a safety valve for electron transport

In addition to its possible roles in carotenoid biosynthesis and chlororespiration, it has been suggested that PTOX can act as an alternative electron sink from

PSII in some higher plants, due to its ability to divert electrons from plastoquinol pool to oxygen, generating water (Figure 1.5), so protecting the plastoquinol pool from overreduction when plants experience abiotic stress [11], [12], [113], [114].

PTOX has been reported to have a protective role against a variety of stresses.

A clear example is seen when Eutrema salsugineum is exposed to high salinity.

In this condition this salt tolerant plant increased its abundance of PTOX protein and diverted up to 30% of electrons from PSII [10]. Another example is Avena sativa, where PTOX protein increased its abundance under heat and high light conditions [94]. Additionally, this protein seems to have a role when plants experience cold temperatures, as seen in lodgepole pine [14] and in Ranunculus glacialis, where application of high light increased protein and possible activity

[11], [12]. This induction in the amount of this quinol oxidase protein and an increase in its catalytic activity was also observed in Rosa meillinae when on the top of high light and heat, the water restriction is applied [15]. Also, the same

42 conditions triggered a rise in the abundance of the enzyme in Crysanthemun morifolium and Spathiphyllum wallisi [13].

Research performed by Clarke and Johnson (2001, [115]) in Hordeum vulgare

(barley) leaves reported an O2 sensitivity in PSII across a range of temperatures, which was intensified at high temperature and was accompanied by a lack of O2 effect on PSI [115]. This oxygen sensitivity was explained by the authors as a result from oxygen acting as an electron acceptor from PSII via an unknown mechanism. This work was carried out before the discovery of PTOX, and a role for PTOX in this process has not been shown in this species.

5.6 PTOX localization and characterization

PTOX is a nuclear-encoded protein. It is also present in photosynthetic prokaryotic cyanobacteria and in the majority of eukaryotic algae, which typically possess two copies in the genome, as seen for instance in Chlamydomonas reinhardtii, with the proteins performing different functions [111]. PTOX 1 is thought to be involved in carotenoid biosynthesis whilst PTOX 2 is important in chlororespiration [116]. Higher plants so far described have just one copy in their genome [100].

Depending on the species, PTOX has a molecular weight between 40 and 50 kDa. In eukaryotes it also possesses an N-terminal transit sequence, which targets the protein to the chloroplast and which is cleaved after the enzyme is imported into this organelle [117], [118].

The initial PTOX localization experiments, were carried out in isolated and purified chloroplasts from spinach. PTOX was detected through western blot analysis and was absent in samples treated with protease indicating the protein binding to the stromal phase of the thylakoid membrane [119]. The utilisation of

43 different fractions of the thylakoid membrane allowed the identification of the location of this protein as being the stroma lamellae [106], [111], [119]. In contrast, new evidence describes that PTOX can also be localized in grana in

Eutrema salsugineum, potentially as a part of the activation of this enzyme, which involves its translocation from the stroma thylakoids [120].

PTOX has been characterized as a member of the diiron carboxylate quinol oxidase (DOX) class of proteins, catalyzing the oxidation of the plastoquinol pool (PQH2) and reducing molecular oxygen (O2), with concomitant generation of water [121]. The prediction for this quinol oxidase describes it as an interfacial membrane protein, having an active site with four-helix bundle and the enzyme is attached to the thylakoid membrane by a fifth α- helix. Also, its active centre encapsulates a di-iron centre, where two iron atoms are bound through two glutamate and four histidines residue [114], [122]. Through a site- directed mutagenesis of PTOX in planta and in vitro, the six Fe-binding sites were demonstrated to be essential for this quinol oxidase being active (Figure.

1.9, [123]) In addition, the stability and the activity of this protein were shown to be dependent on a specific sequence of amino acids belonging to Exon 8 and 5 additional sites (Leu135, His 151, Tyr212, Tyr234, and Asp295) were described to be indispensable for PTOX activity in vitro and in planta (Figure 1.9, [122]).

The activity of PTOX recombinant protein expressed in E. coli has also been studied using in vitro assays, which showed that while n-propyl gallate and n- octyl gallate seem to have an inhibitory effect over PTOX activity, its catalysis is insensitive to CN [124]. Additionally, this quinol oxidase has been reported to have a catalytic activity substrate specific, showing a high affinity for plastoquinol, oxidizing it but not UQH2, duroquinol or benzoquinone [111], [124].

Figure 1.9. Structural model of PTOX. PTOX is proposed to have four α-helices and a diiron centre. Exon 8, which is present only in PTOX, is indicated in bold.

Amino acid residues important for PTOX function in vitro (Leu-135, His-151, and

Tyr-212) are shown in grey and those essential for in vitro and in vivo function

(Tyr-234, Asp-295, and the six Glu and His residues in the catalytic site) are shown in black [122].

5.7 PTOX activity regulation

Most studies on higher plants have suggested that PTOX is only a minor component of the stroma thylakoid, however, in several species of plants this quinone oxidase increases its abundance following exposure to stressful conditions such as drought, cold or salinity [10], [11], [15].

The possibility of PTOX competing for the electrons from PSII with the electron transport chain has been questioned however, and in many cases, the transport of electrons mediated by this enzyme seems to be marginal with respect to the total amount of electrons transported. As a result, some authors have

45 questioned the likelihood that PTOX can act as a safety valve, offering protection from an overreduction of the plastoquinol pool [111], [125].

In tobacco, overexpression of PTOX resulted in plants which were more susceptible to photoinhibition than wild-type lines. It seems to be that PTOX was generating superoxide as a side effect of its catalytic activity, acting as a prooxidant and failing in increase the protection against photoinhibition in the overexpressor lines [126].

The ability of PTOX to act as an antioxidant or a prooxidant has been suggested to depend on the abundance of the quinol substrate. Superoxide radicals are produced even in high substrate concentrations at pH 8, whilst at pH 6-6.5 this is only seen at limited quinol concentrations [127], [128].

Recently, in a study on the species Eutrema salsugineum, which was reported to show high PTOX activity, it was seen that PTOX overexpression does not result in catalytic activity by itself, suggesting that other factors or proteins are necessary to give rise to activity. When PTOX overexpressing plants were exposed to salt, these exhibited a faster induction and higher final level of PTOX catalytic activity. The authors proposed that the activity of this quinol oxidase requires the translocation of PTOX from stroma thylakoid to grana thylakoid

[120].

6. Aims and Objectives

The questions still open in the literature and the recent evidence of PTOX induction and its regulation show that there is a need to further understand this protein. This thesis focuses on the possible role and functioning of PTOX in barley plants.

The first chapter explores whether the temperature dependent O2 sensitivity of

PSII electron transport seen in Hordeum vulgare (barley) plants [115] could result from PTOX activity. Additionally, the effect of cold acclimation on PTOX induction is investigated, as a possible protective mechanism against low temperatures in this plant. This research found that PTOX activity is significant in barley leaves but that it is not an important defence against low-temperature in cold acclimated plants. In order to expand our knowledge about the protecting effect of PTOX against stress in barley plants, the second chapter addresses whether PTOX could be playing a protective role against water restriction and salinity in barley plants. We show that PTOX activity is significant in barley leaves exposed to water limitation, however, it is more important in the defence against salt stress.

Finally, in the third chapter, the dynamic responses of PTOX protein and its activity are explored, in order to understand better the regulation of this enzyme in H. vulgare. Through testing the effect of alternating the O2 concentrations over

PTOX activity, and other stimuli like high light or different pH conditions, possible variations in abundance or localisation of PTOX in different fractions of the thylakoid membrane are examined. PTOX activity seems to respond dynamically and reversibly to variation in oxygen concentration. At the same time, an increase in the recovery of PTOX protein observed is observed in water restricted and salt-treated plants, which is consistent with this quinol oxidase possibly being increasingly associated to the thylakoid depending on variation in stromal pH induced by saturating light.

Chapter 2

Role of Plastid Terminal

Oxidase (PTOX) as alternative

electron sink in Hordeum

vulgare

Introduction

Climate change is a concern across the world, due to its potential negative effects on crop production and therefore food security [5], [129], [130]. A lot of effort is being invested to understand how extreme environmental conditions impact the metabolism of plants and the protective mechanisms used by them to overcome this stress [131]–[134].

Photosynthesis is one of the primary process affected by many types of abiotic stress [135], [136]. Environmental conditions, such as drought, high irradiance, extreme temperatures and high salinity, can reduce the efficiency of carbon fixation [137]–[142]. Under some conditions, the light absorbed by light- harvesting complexes exceeds the energy processed by photochemistry through the electron transport chain and later used in CO2 fixation by the Calvin-Benson cycle [73]. This surplus of energy can result in an overburden of the photosynthetic apparatus having as a consequence reactive oxygen species

(ROS) production, which can lead to oxidative lesions in cellular components and photoinhibition [7], [136], [143].

Plants avoid or attenuate the negative effects of ROS using antioxidant systems, which scavenge them; nonetheless, these systems are metabolically demanding

[36], [40], [144]. Simultaneously, alternative strategies prevent ROS production through the modulation of photosynthetic electron transport, which at the same time is energetically less demanding [145].

Modulation of photosynthetic electron transport includes processes like non- photochemical quenching (NPQ), which involves the dissipation of energy as heat [146]–[148], and alternative electron transport pathways, for instance, cyclic electron transport (CET) [80], [86], Mehler reaction [36], [40], [149],

49 chlororespiration [13], [15], [88], [150] and, it has been proposed, activity of the plastid terminal oxidase (PTOX) activity [10]–[12], [100], [118].

PTOX, or plastoquinone oxygen oxidoreductase, is an enzyme which has been suggested by various authors [10], [11], [13], [15], [95], [118] to have a relevant role in some higher plants, acting as an electron safety valve, protecting the photosynthetic apparatus from an overburden, due to its ability to divert electron from the plastoquinol pool to O2, generating water [113], [114], [151]. This mechanism has been suggested to be potentially photoprotective, because it contributes to avoiding or at least reducing the blockage of the acceptor side of

PSI, decreasing the probability of ROS formation, whilst at the same time reducing excitation pressure on PSII, minimising photoinhibition.

Due to its ability to oxidase the plastoquinol pool, PTOX has been reported to act as a cofactor in carotenoid biosynthesis, playing a role in chloroplast biogenesis [102], [106], [110], [111]. In some species, however, an increase in its abundance and activity has been seen when plants experience environmental stress. Investigations performed in Eutrema salsugineum (previously Thellungilla halophila) [10], Ranunculus glacialis [11], [12] and Pinus contorta [14] provide evidence of PTOX being induced by environmental stress conditions, increasing its abundance and activity, diverting electron from linear electron transport (LET) to O2.

Induction of PTOX activity in plants exposed to stress can be seen as part of a plant acclimation process [12]. Acclimation involves changes in gene expression, resulting in a modification of the organism’s phenotype as a response to variation in environmental conditions [80]. Acclimation can result from the influence of the environmental conditions experienced either during the development of leaf, developmental acclimation, or after the leaf is fully

50 developed, dynamic acclimation [152]. Acclimation contrasts to fast regulatory responses such as NPQ, however, modifications in regulatory processes can be the result of the acclimation process.

In many species a decrease in the sensitivity to photoinhibition is seen to be a consequence of cold acclimation. Some cold acclimated cereals increase

Ribulose 1,5-bisphosphate (RuBP)-regeneration in Calvin-Benson cycle, increasing the electron flow being used to mediate CO2 fixation and resulting in an increment in photosynthetic capacity [153]. Leaf growth in spinach at colder temperatures is reported to be slower, and leaf area of some plants reduced, when they experience a decrease in temperature.

Some authors claim that cold acclimation involves adjustment at low temperatures and higher light at the same time [154]. Under high irradiance, plants often show a reduction in the relative concentration of light-harvesting complex (LHC) and in grana stacks in thylakoid membranes, resulting in photosynthesis saturating at higher light intensities [155]. Additionally, the adjustment tends to involve a rise in electron transport complexes, photosystems, ATP synthase and Rubisco, resulting in a rise in the maximum photosynthetic rate [156], [157].

PTOX activity and abundance have been reported to increase in Ranunculus glacialis when this species experience high light and low temperatures [11], [12], in Lodgepole pine after a cold acclimation [14] and in Avena sativa, PTOX protein increases under heat and highlight conditions [94].

Research performed by Clarke and Johnson (2001) in Hordeum vulgare (Barley) leaves reported an O2 sensitivity in PSII across a range of temperatures, which was intensifiedat high temperature and was accompanied by a lack of O2 effect

51 on PSI [115]. This O2 sensitivity is consistent with the activity of an alternative electron transport pathway such as PTOX.

This chapter investigates whether PTOX is responsible for the O2 sensitivity shown by PSII in barley plants dependent on temperature. Also, the effect of cold developmental acclimation on PTOX induction is explored, as a possible protective mechanism against low temperatures in barley. In this chapter, I show that PTOX activity is significant in barley leaves but that it is not an important defence against low temperature stress in this plant.

Materials and Methods

Plant growth

Seeds of Hordeum vulgare were germinated in a cabinet with controlled conditions of 100 µmolm-2 s-1 of light (warm white LEDs, colour temperature

3000-3200 K) 12 hours of photoperiod and either 20°C day/16ºC night temperature or 10°C day/6ºC night temperature. After two weeks of development, measurements were taken in the first leaf.

Measuring gas exchange

Gas exchange measurements were carried out using an infrared gas analyser

(Licor, LI-6400XT, LI-COR Bioscience) at 25°C. Leaves were clamped side by side in the cuvette to fill the chamber. Leaves were left for 5 minutes in darkness to acclimitise to the chamber and the rate of gas exchange was measured. After,

-2 -1 leaves were illuminated (800 µmolm s white light) for 25 minutes, to measure

-1 the capacity for photosynthesis in plants exposed to 2000 µLL of CO2.

Measuring chlorophyll fluorescence and electron transport to oxygen.

Chlorophyll fluorescence and P700 oxidation were simultaneously measured using PAM-101 chlorophyll fluorimeters (Heinz Walz, Effeltrich, Germany).

First, the maximum variation in the absorbance at 830 nm was measured to estimate the redox state of P700. Dark-adapted leave were exposed to continuous far-red light for 20-30 s (FR), with a saturating flash of white light

(200 ms, 4500 µmolm-2s-1) to fully oxidise of P700 (P700 total, Figure 2.1a).

Fo (initial fluorescence level) was then measured. The maximum fluorescence level, Fm (maximum fluorescence) was measured by applying a saturating flash of light (4500 µmolm-2s-1) for 1 second. The actinic light was switched on for 25 minutes and Fm’ in the light measured by giving flashes at regular intervals

(Figura 2.1e).

Fluorescence parameters were calculated using the following equations

(Maxwell and Johnson, 2000):

Efficiency of Photosystem II (PSII) = (Fm'-Ft)/ Fm'

ETR = ΦPSII × PFD (photon flux density)

Non-photochemical quenching (NPQ) = (Fm- Fm')/ Fm'

Where Ft is the fluorescence level measured immediately before application of a saturating flash, Fm the maximal fluorescence measured in the dark-adapted leaf and Fm’ the value measured in the light.

After 25 minutes, a series of light-dark transitions were applied, with an accumulation of 60 measurements being made using 100 ms dark periods, performed at 10s intervals (Figure 2.1c). in order to measure the kinetics of

P700 turn over and the proportion of P700 oxidised. A single exponential curve was fitted (Figure 2.1b ) to the resulting decay to determine the rate constant for the re-reduction of P700 (k) and the proportion of P700 oxidase (P700+[prop]).

The residuals of the exponential fitting were ramdomly distributed arround zero showing that the model describes the data well, representing a pseudo-first order reaction (Figure 2.1d).

The P700 electron transport rate was calculated as:

P700 ETR = (P700+ [prop] x k ) s-1

Figure 2.1. P700 and fluorescence signals. P700 fully oxidised signal, induced by Far Red (FR, a), Dark period inducing the reduction of P700 (c), fitting of a monoexponential function to the decay of P700 oxidase (b), the residuals of the monoexponential fitting to P700 oxidase decay (d).

The sequence of fluorescence signal (e), the zero level of fluorescence (F0) is measured switching on the measuring light (ML). Later, the maximal fluorescence (Fm) level is measured by applying a saturating pulse (SP). Then, the actinic light (AT), a light able to trigger photosynthesis, is switched on and after some time the maximal fluorescence (Fm') in the light is measured after the application of a saturating flash light (SP). Additionally, Ft corresponds to the level of fluorescence before the application of the second SP.

Samples were supplied with saturating CO2 and either 21% or 2% O2 during mesurments. This gas was provided by bubbling either 2% compressed oxygen from a cylinder (BOC Gases) or laboratory air through a solution of 1M

Na2CO3/NaHCO3 (pH 9; ~5% CO2).

A water bath (Bath 2219 multitemp II, LKB Bromna) was used to control the temperature in the leaf-chamber and a thermocouple located under the leaf allowed monitoring of the leaf-chamber temperature during the measurements.

Chlorophyll content

A leaf of known area was ground with 2 ml of 80% v/v acetone in a mortar. This extract was made up to a final volume of 10 ml. The solution was centrifuged, at

3000g for 5 minutes and subsequently, the supernatant was placed in a glass cuvette and the absorbance measured, using an Ocean Optic USB4000 spectrophotometer. The chlorophyll content was calculated as:

Chlorophyll a = [13.71 x (A663)] – [2.85 x (A646)]

Chlorophyll b = [22.39 x (A646)] – [5.42 x (A663)]

[158]

Immunoblot analysis

Protein extraction was carried out following the method of Cuello and Quiles

[159].

Protein content of extracts was estimated by performing a Bradford assay [160] and a standard curve was made with known concentrations of bovine serum albumin. One volume of loading buffer (LDSN sample buffer 4x nupage,

Invitrogen) plus 1/10 volume of DTT was added to 3 volumes of protein sample,

20l of this solution containing 20 g of protein was loaded per well of a pre-cast

Nupage SDS gels (Nupage 4-12% Precise Bis-Tris gel, Invitrogen) and at least 1 well per gel was loaded with 20ul of 50% molecular weight marker (Precision

56 plus protein dual Xtra standards. A running buffer was made from MES (2.5mM),

SDS (0.005%), Tris base (2.5mM) and EDTA (0.05mM), plus antioxidant (1%,

Nupage antioxidant, Invitrogen). Gels were run at 80V in a cold room for 90 minutes. One of these Nupage SDS gels was stained with 20 ml of Coomassie brilliant blue R250 (0.1%), glacial acetic acid (10%) and methanol (45%). Gels were then destained using methanol (50%) and acetic acid (10%), for 2 hours on a rocking table, this being repeated several times.

A second gel was used for western blot analysis. This was put into a tray containing transfer buffer made from Bicine (2.5mM), Bis-Tris (2.5 mM) and

EDTA (0.05mM), Methanol (10%) plus antioxidant (1%,Nupage antioxidant,

Invitrogen) with 2 sheets Western blotting filter paper (0.83 mm, Thermo

Scientific) and nitrocellulose transfer membrane (Whatman, PROTRAN) which were wetted with sufficient buffer. A sandwich was assembled on the bottom of a cassette (XCell II Blot module), in the anode side were located 2 blotting pads, 1 sheet of filter paper, followed by the membrane, gel, 1 sheet of filter paper and 2 blotting pads. A blot roller was to use to remove air bubbles and lock the top of the cassette into place and slide the cassette into the control unit. Proteins were transferred to nitrocellulose membrane at 30V for 60 minutes. Blots were checked by Ponceau S staining to ensure even transfer.

Membranes were blocked with 3% BSA in TBS made from Tris-HCl (20 mM, pH

7.6), NaCl (125 mM) for 90 minutes at room temperature, subsequently, incubated overnight at 4C with primary antibody against PTOX (kindly provided by Dr. M. Kuntz, Universite´ Joseph Fourier, Grenoble, France). This was detected using the anti-rabbit HRP (Agrisera) in TBS-Tween (0.1%) through incubation for 2 hours. A mix of 2 volumes western blot chemiluminescent substrate 3 volumes of ECL (Pierce ECL Substrate, Thermo Scientific) and 1 volume of Femto (SuperSignal West Femto Maximum Sensitivity Substrate,

Thermo Scientific), was used to cover the membrane and incubated it for 1 min.

Later the membrane was developed using a CL-Xposure film (CL-Xposure film,

Thermo Scientific) with 10 seconds of exposure time.

Reverse transcription polymerase chain reaction (rt-PCR)

Barely leaves were flash frozen in growth conditions using liquid nitrogen. mRNA was extracted using RNAeasy plant mini kit (Qiagen N.V). Genomic DNA was removed with DNase I (DNase I amplification grade, Thermo Scientific) and reverse transcription was carried out with a cDNA synthesis kit (Tetro cDNA synthesis kit, Bioline). The sequence of the H. vulgare Plastid Terminal Oxidase

(PTOX, transcript name: HORVU2Hr1G122660.10) transcript was obtained from

Phytozome v12.1 (University of California) and the same database was used to

BLAST the primer sequences to check specificity. The loading control was Actin transcript (Accession number: AY145451.1) obtained from a publication of

Ferdous and collaborators [161]. The rt-PCR was performed using My Taq DNA polymerase (Bioline) according to the recommendation of the supplier using an annealing temperature of 60°C and a thermal cycler (T1000, BIO-RAD).

Table 2.1 PTOX primer sequence used in rt-PCR.

Primers (5’-3’) Tm (°C)

PTOX Forward GTT-CTC-CTC-ACT-CCG-TGC-AGA-GC 55.5

Reverse GCA-CGG-AGG-TAC-ACA-ACT-GGT-C 53.5

ACTIN Forward CCA-CGA-GAC-GAC-CTA-CAAC 60.0

Reverse CAC-TGA-GCA-CGA-TGT-TTC-C 58.0

Specific leaf area (SLA)

Leaf segments were scanned using a flat bed scanner at 300 dpi (Lide110,

Canon) and images analysed using Image J program to estimate the leaf area.

Leaf pieces were dried for 3 days at 60°C to constant weight to obtain the dry mass.

The SLA was calculated as:

SLA = leaf area / leaf dry mass

Statistical analysis

Two-way ANOVAs and Tukey's post hoc test were performed, using Graph Pad

Prism 7.

Results

Temperature and oxygen effects on P700 and chlorophyll fluorescence parameters

To gain a better understanding of the temperature response of oxygen sensitivity, P700 oxidation and chlorophyll fluorescence parameters were measured in 14 day-old barley plants grown at 20°C, across a range of temperatures from 10°C to 40° C at an actinic light intensity of 800 µmol m-2s-1.

The measurements were carried out either at 21% or 2% O2 concentration at saturating CO2 (Figure 2.2a-f).

With increasing temperature, PSII ETR and ФPSII, increased, to a maximum at

35 ºC (Figure 2.2 a and c). Between 15°C and 35°C, low O2 resulted in a decrease in PSII ETR (dotted line). Oxygen sensitivity, becames larger with increasing temperature up to 35°C, consistent with the activity of an alternative electron flow from PSII to O2. In contrast, the level of NPQ was steady through all range of temperatures tested and independent of the O2 concentration.

P700 ETR was estimated as the product of the amount of oxidised P700

(P700+[prop]) and the rate constant for the re-reduction of P700 (k). The electron transport of P700 (Figure 2.2b) increased slightly going from 10°C and 20°C and fell again at the highest temperatures, but was generally insensitive to the O2 level across the range of temperatures.

Although the rate of PSI turnover was insensitive to O2, effects could be seen in the parameters used to calculate this. While k exhibited a positive relationship with the temperature below 45°C, the proportion of P700 oxidised did not show a clear tendency. Both parameters showed some O2 sensitivity.

Figure 2.2. The O2 sensitivity of PSI and PSII parameters across a range of temperatures. PSII electron transport rate (a), PSII quantum yield (c), NPQ (e),

P700 ETR (b), pseudo-rate constant for the re-reduction of P700 (d) and proportion of P700 oxidised (f) measured either at 2% O2 (red dotted line) or

2 1 21% O2 (black solid line) at 25°C and 800 µmol m- s- of actinic light. The measurements were performed in the first leaf of 14 days old barley plants, grown at 20°C and 100 µmol m-2 s-1 of light The error bars represent the standard error of at least 4 replicates.

Values of k were lower at low O2 than at ambient, in a manner that correlated with the response of PSII. Changes in k were however compensated for by a change in P700 oxidation, resulting in no evidence supporting an effect of either low O2 concentration on P700 ETR.

Effect of light intensities on chlorophyll fluorescence and P700 oxidation parameters across different temperatures and oxygen concentrations.

Light intensity curves were measured in barley plants grown at 20°C and at 100

2 -1 µmol m- s of light. The measurements were conducted either at 21% or 2% O2 concentration, at saturating CO2 and at three different temperatures: 15°C, 25°C and 35°C.

At the three temperatures, PSII ETR (Figure 2.3a-c) rose with increasing light intensity up to saturation in both plants exposed to 21% O2 (black solid line) and

2% O2 (red dotted line), except at 2% O2 at the highest temperatures, where saturation was not reached. PSII ETR saturated more rapidly at the lowest temperatures. Nonetheless, the PSII ETR saturation was achieved at the highest light intensity when the parameters were measured at 35°C and 21% O2.

At 10 ºC, PSII ETR and Ф PSII were insensitive to O2 across the range of PFD investigated (Figure 2.3 a and b). However, with increasing temperature, O2 sensitivity in PSII ETR and ФPSII was seen (Figure 2.3b-f), with this effect being largest at the highest temperature tested (Figure 2.3c and 2.3f). This O2 effect suggests electron transport to an alternative pathway dependent on O2, which was intensified at higher temperatures.

Measurements of NPQ exhibited an O2 sensitivity at 10°C (Figure 2.3g), being lower at 2% O2. At moderate and higher temperatures, NPQ was largely

62 independent of the O2 level in most of the range of light intensities (Figure 2.3h and i).

Figure 2.3. Irradiance response of PSII parameters. PSII electron transport rate

(a-c), ФPSII (d-f) and NPQ (g-i) were measured either at 2%O2 (red dotted line) or 21% O2 (black solid line), at 15°C, 25°C and 35°C. The measurements were performed in the first leaf of 14 days old barley plants, grown at 20°C and 100

µmol m-2 s-1 of light. The error bars represent the standard error of at least 4 replicates.

P700 electron transport showed no O2 sensitivity at 15°C and 25°C (Figure 2.4a- b). At 35°C, 2% O2 triggered a slight decline in P700 ETR (Figure 2.4c). As the

ETR is exclusively dependent on O2 in PSII (Figure 2.4b) when the measurements were performed at 25°C, the data suggest an alternative electron pathway diverting electron only from PSII to O2. At higher temperature, 35°C, the

ETR in both photosystems shown some level of sensitivity to O2 concentration

(Figures 2.3c and 2.4c).

P700 ETR is estimated as the product of the amount of oxidised P700 and the rate constant for the re-reduction of P700 (k). Values of k were clearly affected by the oxygen level, when the temperatures were in the extreme of the range tested, 15°C and 35°C, by this experiment (Figures 2.4 e-f). At 25 C, k was less sensitive to O2 concentration.

Regardless of temperature, the proportion of P700 oxidised showed only marginal O2 sensitivity.

In summary, there is evidence of an alternative electron pathway diverting electrons from PSII to O2 at 25°C, compatible with PTOX activity. At 35°C, the flow of electrons to oxygen seems to involve both PSI and PSII.

Figure 2.4.Irradiance response of P700 parameters. P 700 electron transport rate (a-c), k (d-f) and the proportion of P700 oxidised (g-i) were measured either at 2%O2 (red dotted line) or 21% O2 (black solid line), at 15°C, 25°C and 35°C.

The measurements were performed in the first leaf of 14 days old barley plants, grown at 20°C and 100 µmol m-2 s-1 of light. The error bars represent the standard error of at least 4 replicates.

PSII electron transport is sensitive to Plastid Terminal Oxidase inhibitor n-

Propyl Gallate

In order to explore in more detail if PTOX was responsible for or a partial contributor to the alternative electron transport to O2 shown exclusively by PSII at 25°C or both photosystems at 35°C, experiments using n-Propyl Gallate (n-

PG), a PTOX inhibitor, were performed. Measurements were conducted in 14 day old barley leaves, which were vacuum infiltrated either with water or with a

1mM n-PG solution.

When measurements were performed at 25°C, PSII ETR (Figure 2.5a) and

ФPSII (Figure 2.5c) were sensitive to n-PG, suggesting that PTOX is acting as an electron sink for PSII. In the absence of inhibitor, in leaves vacuum infiltrated just with water, the additional electron transport through PSII was 25% higher compared to PSII ETR in leaves where PTOX was inhibited using 1mM n-PG.

Additionally, NPQ (Figure 2.5e) and P700 parameters (Figure 2.5b, d and f) were independent of the presence of the inhibitor.

On the other hand, vacuum infiltration of barley leaves induced a significant oxygen sensitivity of both PSII and PSI electron transport, which was independent of n-PG. This implies damage is being caused by the infiltration, which induces a Mehler-type reaction.

At 35°C in the light response curve, the O2 sensitivity was larger than at lower temperature, however, the additional turnover resulting from PTOX activity achieved lower values that at 25°C. In this case, the supplementary flow of electron due to PTOX diverting electrons was up 22% of the total amount of electrons transported by PSII (Figure 2.6a and c). NPQ and the parameters of

P700 were independent of O2, making unlikely the possibility of Mehler reaction diverting electron from PSI.

In summary, there is evidence of PTOX acting as an electron transport sink at moderate and high temperature in H. vulgare but not at low temperature.

Figure 2.5. The effect of 1mM n-PG on chlorophyll fluorescence and PSI parameters. PSII electron transport rate (a), PSII quantum yield (c), NPQ (e),

P700 ETR (b), k (d) and proportion of P700oxidised (f) were measured either at

2 1 2% O2 or 21% O2 at 25°C and 800 µmol m- s- of actinic light. The measurements were performed in the first leaf of 14 days old barley plants, grown at 20°C and 100 µmol m-2 s-1of light. The error bars represent the standard error of at least 4 replicates. Two-way ANOVA (p≤0.05) and Tukey's post hoc tests were performed, these results are presented with letters over columns in the graph where the same letters represent no significant differences between mean.

Figure 2.6. The effect of 1mM n-PG on chlorophyll fluorescence and PSI parameters. PSII electron transport rate (a), PSII quantum yield (c), NPQ (e),

P700 ETR (b), rate constant for the re-reduction of P700 (k, d) and proportion of

P700 oxidised (f) measured either at 2%O2 or 21%O2 at 35°C and 800 µmol m-

2s-1 of actinic light. The measurements were performed in the first leaf of 14 days old barley plants, grown at 20°C and 100 µmol m-2 s-1of light. The error bars represent the standard error of at least 7 replicates. Two-way ANOVA

(p≤0.05) and Tukey's post hoc tests were performed, these results are presented with lowercase letters over columns in the graph where the same letter represents no significant differences between mean.

Characterization of developmental acclimation of H. vulgare at two different grown temperature, 10°C and 20°C.

When plants of H. vulgare grown at 20 ºC were exposed for short periods to

10ºC, no evidence of electron transport to oxygen was observed. Other studies have shown induction of PTOX activity at low temperature [11], [12]. It is possible therefore that an increase in the period of time that plants are exposed to cold temperature could result in a developmental acclimation, with induction of

PTOX activity as part of the process.

Barley plants were grown at 20 or 10 ºC and 100 µmol m-2 s-1 for 14 days. The total chlorophyll content (Figure 2.7a) was higher in plants grown at 10°C, as was the ratio of chlorophyll a:b (Figure 2.7a), suggesting that acclimation does indeed occur.

Gas exchange parameters were measured in order to have information about how the CO2 fixation process was affected in plants subjected to lower growth temperature conditions. Measurements of gas exchange were performed at saturating conditions of light and CO2 thus avoiding photorespiration. A higher stomatal conductance (Figure 2.6d) was observed in plants grown at 10°C, which was accompanied by a higher level of maximum photosynthesis (Pmax,

Figure 2.7b).

The specific leaf area (SLA; Figure 2.6c) corresponds to the ratio of leaf area to dry mass, [162], [163]. SLA was significantly lower for leaves growing at 10°C, which is consistent with leaves being denser or thicker.

Finally, the presence of the PTOX protein (Figure 2.7f) was confirmed using western blots and by measurement of the PTOX transcript expression using reverse transcription PCR (Figure 2.7e). Both measurements indicate an expression of PTOX that was similar at low and moderate temperatures.

Therefore, if there is any difference in electron transport at these temperatures, likely it would result from the modulation in the activity of the protein rather than overexpression of PTOX transcript and higher protein concentration.

The colder temperature had an effect on the growth of barley plants, slowing down their leaf growth (not shown). However, these plants were able to acclimate to this lower temperature, In the adjustment shown by plants grown at a colder temperature, the level of PTOX transcript expression and PTOX protein presence do not seem to change. Therefore if PTOX is playing a role in helping to overcome the thermal stress in barley, it is more likely to result from the differential activity of the protein that a clear increase in the protein.

Temperature and oxygen effects on chlorophyll fluorescence and P700 parameters of plants grown at 10 ºC

Chlorophyll fluorescence and P700 parameters were tested in barley plants grown at 10°C, across a range of temperatures from 10°C to 40° C, at saturating actinic light (800 µmol m-2 s-1). The measurements were carried out either at

21% or 2% O2 concentration at saturating CO2.

PSII ETR (Figure 2.8a) and ФPSII (Figure 2.8b) were dependent on the O2 concentration in plants grown at 10°C. Dependence, which was seen between

15°C and 35°C, but shifted from a warmer thermal range by ~5°C to a colder range, when compared to the O2 dependence shown by plants grown at 20°C.

There is evidence therefore of an electron diversion from PSII to O2 playing a role in H. vulgare plants developed at 10°C.

Figure 2.7. Developmental acclimation of barley to cold. The ratio of chlorophyll a to b and total chlorophyll content (a), capacity for photosynthesis (Pmax, b), specific leaf area (SLA, c), stomatal conductance (d), the expression of PTC transcript (e) and the presence of PTOX protein (f) were tested in the first leaf of barley plants grown either at 10°C or 20°C and 100 µmol m-2 s-1of light. All measurements were carried out at 25°C and in the case of gas exchange quantifications, after 25 minutes of 800 µmol m-2 s-1 of actinic light. The error bars represent the standard error of at least 8 replicates.Two-way ANOVA

(p≤0.05) and Tukey's post hoc tests were performed in a-d, and different numbers of stars represent significant differences between mean ( * p≤0.05,** p≤0,01,*** p≤0,001,**** p≤0,0001). PCR gel (e) represents three independent replications and actin was used as the loading control, the protein blot (f) is representative of two technical replicates.

With respect to NPQ (Figure 2.8e), this parameter was insensitive to O2 concentration except between 25°C and 30°C, when the PSII ETR was the largest in the thermal range tested. Thus, there is evidence of the higher level of saturation and ΔpH achieved at the low O2 concentration in this temperature range, contrasting with the complete O2 independence is played by plants developed at 20°C.

To explore the contribution of alternative electron transport to O2 from PSI, P700

ETR (Figure 2.8b), k (Figure 2.8d) and proportion of P700 oxidised (Figure 2.8f) were determined. P700 ETR did not have a simple tendency, with no consistent evidence of an effect of the temperature on the ETR from PSI.

P700 ETR depends on the integrated effect of k (rate constant for the re- reduction of P700) and proportion of P700 oxidised (P700+). Figure 2.8d shows a positive relationship between the rate constant for the re-reduction of P700 and temperature between 10°C and 30°C, which reversed above 30°C, correlating with PSII ETR. In contrast, the proportion of P700 oxidised decreased up to 35°C and later increased. Changes in these parameters counteract each other, resulting in little overall change in P700 ETR.

Figure 2.8. The O2 sensitivity of chlorophyll fluorescence parameters across a range of temperatures. PSII electron transport rate (a), PSII quantum yield (c),

NPQ (e), P700 ETR (b), k (d) and proportion of P700 oxidised (f) measured either at 2%O2 (red dotted line) or 21% O2 (black solid line) at 25°C and 800

µmol m-2s-1 of actinic light. The measurements were performed in the first leaf of

14 days old barley plants, grown at 10°C and 100 µmol m-2 s-1 of light. The error bars represent the standard error of at least 4 replicates and GT the grown temperatures.

Effect of light intensity on chlorophyll fluorescence and P700 parameters across different temperatures and two O2 concentrations in plants grown at 10°C

Light intensity curves were measured on barley plants grown at 10°C and at 100

-2 -1 µmol m s of light. The measurements were conducted either at 21% or 2% O2 concentration, at saturating CO2 and at 25°C.

PSII ETR had a positive relationship with the PFD until a saturation level around

-2 -1 1000 µmol m s of light, for leaves exposed to both atmospheric and low O2 concentration. This parameter also showed an O2 dependence between 700 and

-2 -1 1500 µmol m s of light. Consistently, ФPSII seems to be dependent on O2 concentration.

Increasing PFD resulted in an increase of NPQ. Low O2 resulted in higher NPQ at the two initial points of the light intensity curve but NPQ saturated at the same level.

P700 ETR was insensitive to the oxygen concentration used in the experiments.

While, P700 ETR, k and proportion oxidase all showed a positive relationship with PFD, k and oxidised proportion were both oxygen sensitive.

In summary, there is evidence of an alternative electron pathway in PSII but not acting in PSI.

Figure 2.9. Irradiance response of PSII and P700 parameters in barley plants grown at 10 ºC. PSII electron transport rate (a), PSII quantum yield (c), NPQ

(e), P 700 electron transport rate (b), k (d) and the proportion of P700 oxidised

(f) were measured either at 2%O2 (red dotted line) or 21% O2 (black solid line), at 25°C. The measurements were performed in the first leaf of 14 days old barley plants, grown at 20°C and 100 µmol m2 s-1 of light. The error bars represent the standard error of at least 5 replicates.

Barley developmental acclimation to cold does not induce Plastid Terminal

Oxidase electron sink activity

Vacuum infiltrations of 14 day old barley plants, grown at 10°C and 100 µmol m-2 s-1 of light, were performed either with water or 1mM n-PG, a PTOX inhibitor, at

25°C, to elucidate, whether the electron transport dependence on O2 shown by

PSII in previous experiments could be attributable either in full or some extent to

PTOX acting as a safety valve for electrons. In the case of PTOX acting as electron sink diverting electrons from PSII, this additional turnover would be expected to be suppressed after the application of n-PG and therefore, to decrease PSII ETR.

Vacuum infiltration with 1mM n-PG did not have any effect on the chlorophyll fluorescence parameters PSII ETR (Figure 2.10a), Ф PSII (Figure 2.10c) and

NPQ (Figure 2.10e). In other words, at least with measurements being conducted with 1mM n-PG, there is no evidence that the additional turnover of electrons in PSII.results from PTOX activity diverting electrons to O2.

Regarding parameters describing PSI, ETR P700 (Figure 2.10b), k (Figure

2.10d) and Proportion of P700 oxidised (Figure 2.10f), the tendency was similar to PSII. These parameters were insensitive to 1mM n-PG.

In summary, even though PSII shows an oxygen dependence, PTOX seems not to be responsible for it and therefore, there is no evidence of PTOX being induced in barley, during developmental acclimation to cold.

Figure 2.10. The effect of 1mM n-PG on chlorophyll fluorescence and PSI parameters. PSII electron transport rate (a), PSII quantum yield (c), NPQ (e),

P700 ETR (b), k (d) and proportion of P700 oxidised (f) measured either at 2%

-2 -1 O2 or 21% O2 at 25°C and 1000 µmol m s of actinic light. The measurements were performed in the first leaf of 14 days old barley plants, grown at 10°C and

100 µmol m-2 s-1of light. The error bars represent the standard error of at least

4replicates. Two-way ANOVA (p≤0.05) and Tukey's post hoc tests were performed, these results are presented with upper case letters over columns in the graph where different letters represent significant differences between mean.

Discussion

The effects of temperature and oxygen were explored in terms of P700 and chlorophyll fluorescence parameters in barley plants. Oxygen sensitivity in PSII

ETR was observed between 20C and 35C (Figure 2.2a), and increased with rising temperature. In contrast, there was no oxygen effect on P700 ETR (Figure

2.2b) and NPQ (Figure 2.2c). This additional electron transport in PSII at moderate and high temperatures in barley leaves suggests the activity of an alternative electron transport pathway, dependent on oxygen. There are various pathways utilising oxygen as an electron sink, examples of these including photorespiration [92], [164], [165], Mehler reaction [166]–[168], direct reduction of O2 by plastoquinol pool (PQH2, [46], [47]) and Plastid terminal oxidase [10],

[12], [15].

Photorespiration is a side reaction of carbon fixation, where RuBP carboxylase oxygenase (rubisco) rather than fixing CO2, instead uses O2 as a substrate, expending energy. The relative oxygen affinity of the enzyme increases with the temperature and the rate of this reaction increases at a higher O2 or low internal

CO2 [169], [170]. However, it seems unlikely that photorespiration could be responsible for oxygen acting as an electron sink here, since all the measurements were performed at saturating CO2, suppressing the risk of competition between both substrates.

Mehler reaction, or the water-water cycle, takes electrons arriving at PSI from

PSII and diverts them to oxygen [44], [45] and therefore when this electron pathway is active, it is expected to observe an oxygen effect in the ETR of both photosystems. The oxygen effect on PSII ETR in the light intensity curve is enhanced by rising temperature rise and, at 25°C, the magnitude of the oxygen sensitivity of PSII ETR was stable across a wide range of irradiances, which

79 resulted in a saturation of PSII ETR (Fig 2.3 b). At 35°C PSII ETR showed saturation in the presence of 21% O2 but did not saturate at low O2 (Figure 2.3 c). Nonetheless, PSI ETR showed no sensitivity to O2 at moderated temperatures and only a slight sensitivity at 35°C (Figure 2.4 b and c). This tends to speak against the Mehler reaction acting as a significant electron sink at a moderated temperature, although, at 35°C Mehler reaction potentially could explain part of the oxygen dependence.

Since PSI ETR is largely insensitive to O2 concentration, it seems most likely that this is acting as an electron acceptor prior to PSI. This might occur as a spontaneous reaction of dioxygen (O2) being reduced by plastosemiquinone or through direct O2 reduction for example at the PSII acceptor side.

The spontaneous reduction of molecular oxygen (O2) by plastosemiquinone to

.- superoxide (O2 ) is promoted by illumination, which can result in an increase in

.- stromal pH and plastosemiquinone (PQ ) concentration, facilitating the O2 reduction. Later, the plastohydroquinone (PQH2) reduces the superoxide, generating hydrogen peroxide (H2O2) [46]. The estimation of this alternative electron transport could achieve 60% of the total amount of electron transported by electron transport rate under high light [47]. The alternative electron transport through PSII dependent on oxygen in moderate to high temperature could be potentially being explained by this pathway.

Another possibility, which could result in the oxygen sensitivity in PSII, is the

.- reduction of molecular oxygen (O2) to superoxide (O ) in the acceptor side of

PSII. This O2 reduction tends to occur when there is no plastoquinone pool available or it is fully reduced. The generation of superoxide is maintained either by pheophytin, tightly bound to QA, loosely bound QB or cyt b559 [33], [171].

In addition, the superoxide (O.-) also has been proposing to be produced in Cyt b6f complex, which also could be another pathway contributing in the generation of oxygen sensitivity in PSII ETR [48], [49].

Although we cannot exclude the possibility of direct reduction of O2 from PSII or the electron transport chain, we also need to consider the possibility that PTOX is acting as an alternative electron acceptor in barley at higher temperatures. We were able to detect a significant amount of PTOX protein, using Western Blot analysis, and expression, based on PCR product, suggesting that it is expressed in mature leaves. To test for activity of PTOX, leaves were vacuum infiltrated with n-PG (PTOX inhibitor, [124]). Our findings show a clear effect of n-PG, suggesting that PTOX could be diverting up to 25 % of the total amount of electron transport at 25°C and 22% at 35°C at high light. PTOX has been claimed previously to act as a protective mechanism against stress in species like Ranunculus glacialis, Pinus contorta and Eutrema salsugineum [10]–[12],

[14]. Our data are consistent with a significant activity of PTOX in barley. At temperatures higher than 35°C the PSII ETR drops down drastically and the oxygen sensitivity reduces (Figure 2.2). It seems to be that PTOX is not able to keep its activity at that temperature, not being able to contribute to the electron transport from a probably highly inactivated PSII [172]. On the other hand, Feilke and collaborators [173] observed that to raise the level of CO2, at 2000 PPM, in tobacco lines overexpressing PTOX from Chlamydomonas (Cr-PTOX1) resulted in an increase of the CO2 assimilation to a similar level than the wildtype. This was interpreted by the author as a result of an increase in electron transport with respect to measurements performed at the normal condition of CO2 (400 PPM).

Because of the abolishment of PTOX activity due to the unbind of PTOX to thylakoid membrane, resulting from the acidification of the stroma by the high level of CO2. However, in a previous study published by Stepien and Johnson

-1 [10], measurements were performed at saturating CO2 (2000 mL L ) in Eutrema salsugineum plants shown an bigger oxygen sensitivity in salt-treated plants.

This oxygn effect was considered by the researcher as an indirect indication of

PTOX activity, which was confirmed later using n-PG. Therefore, it seem to be that PTOX protein could be activly diverting electron even at saturating level of

CO2.

On the other hand, at 15°C the electron transfer through both PSI and II are notably smaller than at higher temperatures indicating that electron transport becomes limited at low temperature, potentially increasing the risk of photoinhibition. However, the oxygen effect in PSII ETR does not seem to be important at 15°C or lower temperatures in either photosystem, except possibly in PSII when the light intensity is extremely high, around 2000 µmol m-2s-1

(Figure 2.3a). This absence of evidence for diversion of electrons to oxygen makes unlikely the possibility of PTOX acting as an electron sink.

Therefore, PTOX appears to act as a significant electron sink at higher temperatures, but not in the cold. This is perhaps surprising, as electron transport is more readily saturated at low temperature. Studies on R. glacialis and P. contorta suggest an important role for PTOX at low temperature.

Previous work has shown that many plant species are capable of acclimation to growth at different temperatures, altering the relative abundance of electron transport complexes and enzymes to optimise their growth in different conditions. This led us to consider whether the role of PTOX would still be important in low temperature-grown plants.

When barley was grown at low temperature (10°C), there was a significant increase in photosynthetic capacity (Figure 2.7 b) and stomatal conductance

(Figure 2.7 d). Barley leaves also have a higher total amount of chlorophyll per

82 unit area and a higher ratio of chlorophyll a/b than those grown in warmer conditions. This indicates a reduction in the abundance of light-harvesting complexes (LHC) relative to reaction centres in thylakoid membranes, and possibly a change in the PSI: PSII ratio. Such changes have been reported to be part of the modifications seen during the cold acclimation, increasing photosystem capacity to overcome high irradiance [154], [155].

Cold acclimated leaves of barley show oxygen sensitivity at lower temperatures than in warm developed plants, so that the PSII ETR v/s temperature response was shifted (Figure 2.8). The increase in the oxygen dependence at lower temperatures could reflect a more relevant role of an alternative electron sink as a protective mechanism against the excessive energy harvested at colder conditions. In species like Ranunculus glacialis, PTOX has been observed to act as an alternative electron sink at cold and high light conditions. The light intensity curve performed at 25°C in cold developed plants shows oxygen sensitivity just in PSII ETR (Figure 2.9a), while PSI ETR is fully insensitive

(Figure 2.9b). The absence of oxygen sensitivity in PSI ETR makes unlikely

Mehler reaction being responsible by the transport of electron to oxygen.

Perhaps, three pathways which can lead to reactive oxygen species production as superoxide can not be excluded. The spontaneous reduction of oxygen by plastoquinone or the acceptor side of PSII or Cyt b6f reducing oxygen can potentially result in the oxygen dependence observed in PSII ETR, while there is not evidence of PTOX activity to vacuum infiltrate barley leaves with PTOX inhibitor (Figure 2.10). This potential disappearance of PTOX activity when barley plants are developed at cold temperatures could be related to the modifications experienced by plants during the acclimation process. However also the absence of evidence of PTOX activity when the measurement is performed at cold temperatures could indicate that the effect of temperature can

83 interfere with the needed conditions for PTOX being active. Especially considering that western blots against PTOX protein show its presence in the samples, nonetheless, it is apparently not diverting electrons. A possibility could be that low temperature increases the viscosity of the thylakoid membrane [131],

[174], which can have a negative effect over diffusion of the even smallest electron transport component as plastoquinol. Potencially, limiting its availability to bind to PTOX in a high protein density grana and being the plastoquinone diffusion suggested being highly restricted to microdomains [175]. Another possibility could be that the n-PG penetrates less well in leaf tissue of barley plants grown at low temperatures, preventing its action over the PTOX activity or a potential disturbance of the samples resulting from the vaccum infiltration.

Conclusion

Barley leaves grown at 20oC show an oxygen dependence in PSII ETR, which increases with increasing temperature and at high temperatures, it is intensified by the irradiance. Our finding showed that plastid terminal oxidase (PTOX) seems to contribute with this diversion of electrons to oxygen at moderated and high temperature, acting as an important safety valve of electrons potentially protecting barley leaves from an overreduction of the plastoquinol pool. At the same time, it is supported by the presence of transcript and protein of PTOX in this plants. However, at lower temperatures, PTOX does not appear to be relevant.

As PTOX was not triggered at cold temperature, barley plants were developmentally acclimated to cold to explore whether PTOX could be part of cold-acclimated mechanisms. The effect of PTOX inhibitor in vacuum infiltrated leaves was not significant, making unlikely that PTOX playing a role as a mechanism of developmental cold acclimation in barley leaves.

Chapter 3

Role of Plastid Terminal

Oxidase (PTOX) as alternative electron sink in water restricted

and salt-treated Hordeum

vulgare plants

Introduction

Understand better the mechanisms involved in stress tolerance in plants seems to be a suitable complementary strategy to mitigate the potentially detrimental consequences of climate change [5], [6] by decreasing the vulnerability of crop production and food security.

In general, light is captured by the photosynthetic apparatus of plants in an optimal way, except in situations where the capacity for assimilation of fixed carbon is exceeded and harvested energy is, therefore, greater than can be used in photochemistry [45], [176]. This state can be triggered when plants experience high light intensities, drought, extreme temperatures or high salinity, due to a reduction in the efficiency of carbon fixation [138]–[140], [177]. The consequences of overburdening the photosynthetic apparatus include the production of reactive oxygen species (ROS), which can result in oxidative lesions including photoinhibition the specific damage of the photosystem II (PSII) reaction centre [31], [32], [178].

ROS are thought to cause damage in a wide variety of cellular components, including membranes, proteins and DNA [36], [56], [143], [179]. For instance, chlorophyll molecules and thylakoid structure can be damaged in extreme cases, however, PSII is the most affected by photoinhibition, while the activity of

Photosystem I (PSI) is inhibited to a lesser extent [29], [30]. It has been suggested that ROS is able to cleave the D1 protein (PsbA) in the PSII reaction centre [31], [32].

The negative effects of ROS can be prevented or reduced in plants by a protective system of antioxidants, which scavenge ROS. These can be enzymatic or non-enzymatic, however, the necessity of having high

87 concentrations of antioxidants means this system is energetically demanding

[44].

At the same time, plants display alternative strategies to prevent ROS production, modulating photosynthetic electron transport [145], which can be metabolically more economical. Non-Photochemical Quenching (NPQ; energy dissipation as heat, [146], [147]) and alternative electron transport pathways have been proposed as possible strategies. These mechanisms of photoprotection include PSI cyclic electron transport (CET, [86], [180]), Mehler reaction [36], [40], [149], chlororespiration [13], [88], [95], [181], and Plastid terminal oxidase (PTOX, [10]–[12], [100], [118]).

PTOX is an enzyme, which has been proposed to play an important role in higher plants acting as an alternative sink of electrons, due to its ability to transport electrons from PQ to molecular oxygen, with concomitant generation of water [10], [11], [13], [15], [95], [118]. Its mechanism seems to be carried out when the acceptor side of the PSI is blocked, therefore it can be potentially photoprotective, due to a reduction in the production of ROS. For instance,

PTOX has been proposed to have a protective role against stress in Eutrema salsugineum (previously Thellungiella halophile, [10]), Ranunculus glacialis [11],

[12] and Pinus contorta [14]. In these three species, there is evidence for an electron diversion from the linear electron transport, dependent on molecular oxygen and associated with an increase in PTOX concentration, during stress situations. Information collected from many studies has also led to the proposal that PTOX could have other important roles in chloroplast biogenesis, as a cofactor of carotenoid biosynthesis [106], [110], [111], [182], [183].

Salinity stress can lead to nutrient imbalance and ion toxicity [184]–[186].

Moreover, plants exposed to water or soil high in salt could experience a

88 reduction of their ability to take up water from the soil, developing water stress.

Thus, salt and water stress have common consequences and therefore responses in plants. Both types of stresses can lead to a decrease in relative water content (RWC), dehydration, potentially resulting in osmotic stress due to the mobilisation of water from the cytoplasm and vacuole to the extracellular space [187]–[191]. Cellular dehydration triggered by these two stresses has been reported that can potentially lead to a reduction in cellular volume and leaf expansion, resulting in a decrease of the growth rate [186], [190], [192], [193].

As water limitation is a phenomenon observed in these two stresses, the early responses trigger by both stresses can be equivalent. To avoid losing water the stomata are closed, generating a concomitant limitation on evapotranspiration and the CO2 uptake, resulting in a decreasing of photosynthesis [177], [187],

[194], [195]. The reduction in carbon fixation has the potential capacity to develop an imbalance with the yield of the electron transport chain (ETC), making these plants more liable to generate ROS. In fact, ROS production is another shared symptom of both kinds of stresses, which could result in a detrimental effect on the metabolism and cellular components [187], [196], [197].

In addition to acting as an alternative electron sink, PTOX has been suggested to play an important role in acclimation processes [12]. Acclimation is an adjustment in the composition of tissues in response to environmental changes.

These modifications in plants correspond to phenotypic adjustment resulting from variation in gene expression. Fast regulatory responses (e.g. NPQ) are not part of acclimation, as they do not involve alterations of gene expression, although the capacity for such regulation can change as a part of acclimation

[80].

PTOX seems to have a protective role against salt stress, in Eutrema salsugineum, diverting up to 30% of the total amount of electron transported by

PSII[10]. In the case of Rosa meillandina, whether plants experience high light and heat PTOX was equivalent to control level, however, when water restriction was applied simultaneously, PTOX activity and abundance increase, offering protection from an overeducation of plastoquinol pool [15]. Applying the same three conditions to Chrysanthemum morifolium and Spathiphyllum wallisii a

PTOX protein increment was induced, especially, in the shade tolerant

Spathiphyllum wallisii [13]. Also, PTOX transcripts were reported to be up- regulated in soybean seedlings after 6 hours of water stress [109].

An O2 sensitivity in PSII ETR was observed in Hordeum vulgare (Barley) leaves in a range of temperatures [115]. In Chapter II, evidence was found that PTOX activity was induced at moderate and higher temperatures, potentially acting as a protective mechanism.

Here, I investigate whether PTOX could be playing a protective role against stress due to water restriction and salinity in barley plants. In this chapter, I show that PTOX activity is significant in barley leaves exposed to water limitation and salinity, being more important in the defence against salt stress.

Materials and Methods

Plant growth

Seeds of Hordeum vulgare were germinated in a cabinet with controlled conditions of 100 µmol m-2 s-1 of light (warm white LEDs, colour temperature

3000-3200 K) 12 hour photoperiod and 20°C day/16ºC night temperature. After

7 days of development, plants were exposed to differential watering regimes for

7 days, which could be either an absence of watering or a usual amount of watering but with a 150 mM NaCl solution. Measurements were conducted on the first leaves of two-week old barley plants.

Measuring gas exchange

Gas exchange measurements were carried out using an infrared gas analyser

(Licor, LI-6400XT, LI-COR Bioscience) at 25°C. Leaves were clamped side by side in the cuvette to fill the chamber. Leaves were left for 5 minutes in darkness to acclimatise to the chamber and the rate of gas exchange was measured.

-2 -1 After, leaves were illuminated (800 µmol m s white light) for 25 minutes, to measure the capacity for photosynthesis, stomatal conductance and

-1 evapotranspiration in plants exposed to 2000 µL L of CO2.

Measuring chlorophyll fluorescence and electron transport to oxygen.

Chlorophyll fluorescence and P700 oxidation were simultaneously measured using PAM-101 chlorophyll fluorimeters (Heinz Walz, Effeltrich, Germany).

(200 ms, 4500 µmolm-2s-1) to fully oxidise of P700 (P700 total, Figure 3.1a).

Fo (initial fluorescence level) was then measured. The maximum fluorescence level, Fm (maximum fluorescence) was measured by applying a saturating flash

91 of light (4500 µmolm-2s-1) for 1 second. The actinic light was switched on for 25 minutes and Fm’ in the light measured by giving flashes at regular intervals

(Figura 3.1e).

Fluorescence parameters were calculated using the following equations

(Maxwell and Johnson, 2000):

Efficiency of Photosystem II (PSII) = (Fm'-Ft)/ Fm'

ETR = ΦPSII × PFD (photon flux density)

Non-photochemical quenching (NPQ) = (Fm- Fm')/ Fm'

P700 turn over and the proportion of P700 oxidised. A single exponential curve was fitted (Figure 3.1b ) to the resulting decay to determine the rate constant for the re-reduction of P700 (k) and the proportion of P700 oxidase (P700+[prop]).

The residuals of the exponential fitting were ramdomly distributed arround zero showing that the model describes the data well, representing a pseudo-first order reaction (Figure 3.1d).

The P700 electron transport rate was calculated as:

P700 ETR = (P700+ [prop] x k ) s-1

Figure 3.1. P700 and fluorescence signals. P700 fully oxidised signal, induced by Far Red (FR, a), Dark period inducing the reduction of P700 (c), fitting of a monoexponential function to the decay of P700 oxidase (b), the residuals of the monoexponential fitting to P700 oxidase decay (d).

Samples were supplied during the process with saturating CO2 and either 21% or 2% O2. This gas was provided by bubbling either 2% compressed oxygen from a cylinder (BOC Gases) or laboratory air through a solution of 1M

Na2CO3/NaHCO3 (pH 9; ~5% CO2).

Chlorophyll content

A leaf of known area was ground with 2 ml of 80% v/v acetone in a mortar. This extract was made up to a final volume of 10 ml. The solution was centrifuged, at

Chlorophyll a = [13.71 x (A663)] – [2.85 x (A646)]

Chlorophyll b = [22.39 x (A646)] – [5.42 x (A663)]

[158]

Immunoblot analysis

Protein extraction was carried out following the method of Cuello and Quiles

[159].

Invitrogen) plus 1/10 volume of DTT was added to 3 volumes of protein sample,

20l of this solution containing 20 g of protein was loaded per well of a pre-cast

Nupage SDS gels (Nupage 4-12% Precise Bis-Tris gel, Invitrogen) and at least 1 well per gel was loaded with 20ul of 50% molecular weight marker (Precision

94 plus protein dual Xtra standards. A running buffer was made from MES (2.5mM),

SDS (0.005%), Tris base (2.5mM) and EDTA (0.05mM), plus antioxidant (1%,

A second gel was used for western blot analysis. This was put into a tray containing transfer buffer made from Bicine (2.5mM), Bis-Tris (2.5 mM) and

EDTA (0.05mM), Methanol (10%) plus antioxidant (1%,Nupage antioxidant,

Invitrogen) with 2 sheets Western blotting filter paper (0.83 mm, Thermo

Scientific) and nitrocellulose transfer membrane (Whatman, PROTRAN) which were wetted with buffer mentioned before. A sandwich was assembled on the bottom of a cassette (XCell II Blot module), in the anode side were located 2 blotting pads, 1 sheet of filter paper, followed by the membrane, gel, 1 sheet of filter paper and 2 blotting pads. A blot roller was to use to remove air bubbles and lock the top of the cassette into place and slide the cassette into the control unit. Proteins were transferred to nitrocellulose membrane at 30V for 60 minutes. Blots were checked by Ponceau S staining to ensure even transfer.

Membranes were blocked with 3% BSA in TBS made from Tris-HCl (20 mM, pH

Thermo Scientific), was used to cover the membrane and incubated it for 1 min.

Later the membrane was developed using a CL-Xposure film (CL-Xposure film,

Thermo Scientific) with 10 seconds of exposure time.

Quantitative polymerase chain reaction (Q-PCR)

Leaves after 1, 3, 5, 7, 8 days started the treatment were flash frozen in growth conditions using liquid nitrogen. mRNA was extracted using Rneasy plant mini kit (Quiagen N.V). Genomic DNA was removed with DNase I (DNase I amplification grade, Thermo Scientific) and reverse transcription was carried out with a cDNA synthesis kit (Tetro cDNA synthesis kit, Bioline). The sequence of the H. vulgare Plastid Terminal Oxidase (PTOX, transcript name:

HORVU2Hr1G122660.10) transcript was obtained from Phytozome v12.1

(University of California) and the same database was used to blast the primer sequences to check specificity. The primers for the reference gene, ADP- ribosylation factor 1-like protein (Accession number: AJ508228,2), was obtained from work reported by Ferdous and collaborators [161] Primer sequences used are given in Table 2.1.

A relative standard curve quantification of PTOX was performed by Real-Time

Quantitative PCR, using Fast Start Universal SYBR Green Master (Rox) according to the recommendation of the supplier using an annealing temperature of 60°C and a Real-Time PCR System (Step One, Applied

Biosystems).

Table 3.1 PTOX primer sequence used in rt-PCR.

Gen Primers 5’ – 3’ Tm (°C)

Forward GTT-CTC-CTC-ACT-CCG-TGC-AGA-GC 55.5 PTOX Reverse GCA-CGG-AGG-TAC-ACA-ACT-GGT-C 53.5

Forward GCT-CTC-CAA-CAA-CAT-TGC-CAA-C 49.7 ADP Reverse GAG-ACA-TCC-AGC-ATC-ATT-CAT-TCC 50.6

Specific leaf area (SLA)

Leaf segments were scanned using a flat bed scanner at 300 dpi (Lide110,

Canon) and images analysed using Image J program to estimate the leaf area.

Leaf pieces were dried for 3 days at 60°C to constant weight to obtain the dry mass.

The SLA was calculated as:

SLA = leaf area / leaf dry mass

Relative Water Content (RCW)

Both extremes of barley leaves were cut, leaving 6 cm length central section. the leaf weights (fresh weight, FW) were recorded and the leaves were located inside of a plastic bag keeping a vertical position and being cover completely by water, exceeding for 1cm of water the highest point of the leaves. The plastic bags containing the leaves were placed at 4°C in darkness for 48 hours. After this period leaves were dried with a paper towel and weighed (turgid weight,

TW). Later leaves were placed inside a labelled envelope and dried at 60°C in an oven for 48 hours until the mass was constant. Finally, the samples were reweighted (dry weight, DW).

The RWC was calculated as:

RWC (%) = [(FW-DW) / (TW-DW)] x 100

Statistical analysis

Two-way ANOVAs, Tukey's post hoc test and T-test were performed, using

Graph Pad Prism 7.

Results

Characterization of acclimation of H. vulgare to water limitation or salinity

The findings in Chapter II showed that PTOX seems to have a significant activity at moderate and high temperature, nonetheless, this protein appears not to have an important role protecting barley leaves against low temperatures. In order to extend the knowledge of PTOX functioning, barley plants were acclimated for one week either to water limitation or salinity (watered with 150 mM NaCl).

The relative water content (RWC) was calculated for all the treatments. This parameter seems not to be affected by the water restriction experienced by the plants (Figure 3.2a), however, barley exposure to a saline solution induced a significant reduction the relative water content (Figure 3.2b). In addition, the specific leaf area (SLA), which corresponds to the ratio of leaf area to dry mass, did not show variation due to the treatments (Figure 3.2 c and d). Therefore, the treatments do not appear to affect the density or thicknesses of leaves.

Measurements of gas exchange were performed at saturating levels of light and

CO2, in order to prevent photorespiration interfering with the measurements and thus to explore whether the carbon fixation was altered by the acclimation process to watering treatments. Higher photosynthesis maxima were observed in salt-treated (Figure 3.3a ) and water limited plants (Figure 3.3b). Treatments did not affect the evapotranspiration (Figure 3.3 c and d) and stomatal conductance (Figure 3.3 e and f). The adjustment shown in Pmax suggests that plants acclimated, but the absence of a reduction in the other parameters implies that the treated plants experienced only mild stress.

Additionally, the ratio of chlorophyll a to b and the total chlorophyll content did not show differences between the control plants and two treated groups (Figure

3.4). This supports that treated plants experienced a mild stress.

Besides, both treatments seemed to induce the expression of PTOX, however, it was transient in water restricted plants, while in salt-treated plants the transcript of PTOX was kept higher than the control group later on (Figure 3.5 a). PTOX protein was confirmed using a western blot in control, water restricted and salt treated barley plants, being 1.2 fold higher in plants exposed to water limitation and 1.6 fold higher in plants watered with 150 mM NaCl compared to the control subjects.

Figure 3.2. The effect of water limitation and salinity on the relative water content

(RWC, a and b) and Specific Leaf area (SLA, c and d) in Barley leaves. All the measurements were carried out on two-week old barley leaves, which were exposed for one week to their respective treatment. The error bars represent the standard error of at least 6 replicates for RWC and 8 replicates for SLA. Data were analysed using T-Tests (p≤0.05), different numbers of stars represent significant differences between mean (ns – not significant, * p≤0.05,** p≤0,01,*** p≤0,001,**** p≤0,0001).

100

Figure 3.3. The effect of water limitation and salinity on gas exchange parameters. Capacity for photosynthesis (Pmax, a and b), evapotranspiration (E, c and e) and stomatal conductance (G, e and f). All the measurements were carried out at two weeks old barley leaves, which were exposed for one week to their respective treatment. The error bars represent the standard error of at least

4 replicates. Test-t (p≤0.05), different numbers of stars represent significant differences between mean (ns- not significant, * p≤0.05,** p≤0,01,*** p≤0,001,**** p≤0,0001).

101

Figure 3.4. The effect of water limitation and salinity on the ratio of chlorophyll a to b (a and b) and the total chlorophyll content (a and b). All the measurements were carried out at two weeks old barley leaves, which were exposed for one week to their respective treatment. The error bars represent the standard error of at least 4 replicates. Test-t (p≤0.05), different numbers of stars represent significant differences between mean (ns-not significant, * p≤0.05,** p≤0,01,*** p≤0,001,**** p≤0,0001).

Figure 3.5 The effect of water limitation and salinity on PTOX expression and

PTOX protein abundance. Relative standard curve quantification of PTOX transcript relative to ADP-ribosylation factor 1-like protein, error bars represent the standard error of at least 3 replicates (a) and western blot of PTOX protein

(b), representative of two technical replicates. All the measurements were carried out on two-week old barley leaves, which were exposed for one week to their respective treatment.

102

Effect of light intensity on chlorophyll fluorescence and P700 oxidation parameters at two O2 concentration in water restricted plants.

Light intensity curves were conducted in barley leaves either at 2% or 21% O2 concentration and saturating CO2. Measurements were carried out in barley leaves of plants grown at 20°C and 100 µmol m-2 s-1 of light and exposed during the last developmental week either as before for control or to the absence of watering for the treated plants.

The photosystem (PS) II electron transport rate (ETR, Figure 3.6 a) increased to saturation with the increase in irradiance in control and water limited plants at both O2 concentrations. While PSII ETR was almost the same in both groups of plants at 2% O2, at atmospheric concentration, plants restricted in the watering show that PSII ETR achieved a higher level of saturation compared to the control group. This increase in the level of saturation in treated plants results in a clearly larger oxygen sensitivity in leaves of plants experienced water restriction, suggesting this treatment induces a bigger transport of electrons mediated by one o several alternative electron pathways to O2 than controls.

Regarding NPQ (Figure 3.6b), this parameter exhibited an increment with the increasing light intensity up to the steady state. At lower O2 concentration, the two less intense irradiances induced a higher NPQ, while, the opposite situation occurred at higher light intensities.

No O2 sensitivity was observed in the P700 ETR at the lower and middle irradiances used. An O2 effect appeared in the highest two irradiances applied.

Additionally, k and proportion oxidised were O2 sensitive and all three parameters showed a positive relationship with the light intensity.

103

Figure 3.6. Irradiance response of PSII electron transport rate (a) and NPQ (b).

Measurements were performed either at 2%O2 (red dotted and solid line) or 21%

O2 (black dotted and solid line), in the first leaf of 14 days old barley plants, at

25°C. Plants were grown at 20°C and 100 µmol m-2s-1 of light and exposed during the last developmental week either to before mentioned condition for control or to the absence of watering for the treated plants. The error bars represent the standard error of at least 4 replicates.

104

Figure 3.7. Irradiance response of P700 electron transport rate (a), k (b) and the proportion of P700 oxidised (c). Measured were performed either at 2%O2 (red dotted and solid line) or 21% O2 (black dotted and solid line), in the first leaf of 14 days old barley plants, at 25°C. Plants were grown at 20°C and 100 µmol m-2s-1 of light and exposed either to before mentioned or for the last developmental week to the absence of watering. The error bars represent the standard error of at least 4 replicates.

PSII electron transport is sensitive to n-propyl gallate (n-PG), a Plastid

Terminal Oxidase inhibitor, in water restricted plants

As an O2 dependence was observed in PSII ETR, which could result from the activity of an alternative electron transport to O2, vacuum infiltration of barley leaves was conducted either with 5 mM n-PG or water for control, to test the possibility of PTOX being at least partially responsible for the O2 effect in this group of plants. These measurements were performed in 14 days old of barley

105 leaves from water restricted plants, at 25°C and at two O2 concentrations (Figure

3.8).

When the measurements are performed at 21% O2 in presence of n-PG, there is a 31% reduction in the PSII ETR, measuring at 2% O2 there is no evidence of such n-PG effect. This n-PG effect over PSII ETR suggests PTOX diversion up the 31% of the total amount of electrons transport by PSII. Similarly, NPQ seems to be insensitive to n-PG at 2% of O2, contrary to the reduction observed in this parameter at 21% O2.

Figure 3.8. The effect of 5mM n-PG on PSII ETR (a) and NPQ (b).

Measurements were performed either at 2%O2 or 21% O2, in the first leaf of 14 days old barley plants, at 25°C. Plants were grown at 20°C and 100 µmol m-2 s-1 of light and exposed either to before mentioned or for the last developmental week to the absence of watering. The error bars represent the standard error of at least 4 replicates. Two-way ANOVA (p≤0.05) and Tukey's post hoc tests were performed, these results are presented with letters over columns in the graph where the same letters represent no significant differences between mean.

106

In the case of P700, there is no evidence of this parameter being sensitive to n-

PG, contrary to what was seen for k and proportion P700 oxidised. These sensitivities cancelled out each other being imperceptible in P700 (Figure 3.9).

Figure 3.9. The effect of 5mM n-PG on PSI ETR (a), k (b) and proportional oxidised (c). Measurements were performed either at 2%O2 or 21% O2, in the first leaf of 14 days old barley plants, at 25°C. Plants were grown at 20°C and

100 µmol m-2s-1 of light and exposed either to before mentioned or for the last developmental week to the absence of watering. The error bars represent the standard error of at least 4 replicates. Two-way ANOVA (p≤0.05) and Tukey's post hoc tests were performed, these results are presented with letters over columns in the graph where the same letters represent no significant differences between mean.

107

Effect of light intensities on chlorophyll fluorescence and P700 oxidation parameters at two O2 concentration in salt treated plants.

Measurements were performed in leaves of barley plants exposed to 7 days of watering with a solution of 150 mM NaCl and control conditions. Both groups of plants were grown at 20°C and 100 µmol m-2 s-1 of light for two weeks and light intensity curves were conducted at 25°C, saturating levels of CO2 and either at

2% or 21% O2 concentration (Figure 3.10).

The PSII ETR increased with PFD until reaching a plateau at both O2 concentrations (Figure 3.10a). Besides, this parameter showed a rise in salt- treated plants with respect to the controls, being minor at 2% O2 but clearly noticeable at atmospheric O2 concentrations. The reduction in the electron transport achieved by PSII at a lower O2 concentration could be interpreted as a signal of one or several alternative electron pathways, diverting electron from

PSII to O2. This O2 sensitivity shown by PSII ETR appears to be larger in salt- treated plants than controls, suggesting that this additional flow of electrons was more highly induced by the salt treatment. In NPQ also had a positive relationship with PFD up to saturation, being higher at the three lower irradiances at 2% O2, an effect which was inverted at the highest light intensity

(Figure 3.10b).

While up to moderate irradiances P700 did not show O2 dependence, at the two highest light intensities an O2 effect was observed. This was accompanied by an

O2 sensitivity presented by k and proportion oxidised.

108

Figure 3.10. Irradiance response of PSII electron transport rate (a) and NPQ (b).

Measured were performed either at 2%O2 (red dotted and solid line) or 21% O2

(black dotted and solid line), in the first leaf of 14 days old barley plants, at 25°C.

Plants were grown at 20°C and 100 µmol m-2s-1of light and exposed the last developmental week either to before mentioned condition for control or to be watered with 150 mM NaCl solution for the treated plants. The error bars represent the standard error of at least 6 replicates.

109

Figure 3.11. Irradiance response of P700 electron transport rate (a), k (b) and the proportion of P700 oxidised (c). Measured were performed either at 2%O2

(red dotted and solid line) or 21% O2 (black dotted and solid line), in the first leaf of 14 days old barley plants, at 25°C. Plants were grown at 20°C and 100 µmol m-2 s-1of light and exposed the last developmental week either to before mentioned condition for control or to be watered with 150 mM NaCl solution for the treated plants. The error bars represent the standard error of at least 6 replicates.

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PSII electron transport is sensitive to n-propyl gallate (n-PG), a Plastid

Terminal Oxidase inhibitor, in plants watered with 150 mM NaCl

In the previous section, an O2 sensitivity was observed in PSII ETR, which could be an indication of the activity of an alternative electron transport from PSII to O2 in plants watered with a salt solution. With the objective to explore if PTOX could be diverting this additional electron or a portion of them from PSII, barley leaves were vacuum infiltrated either with 5 mM n-PG (PTOX inhibitor) or water for control. Besides, plants which did not experience the vacuum infiltration were added to the measurements, as a vacuum infiltration control, performed at 25°C and at 2% or 21% O2.

After a vacuum infiltration with 5 mM n-PG, at 21% O2 the PSII ETR was 45% lower than the same parameter measured in water infiltrated leaves. However, the same measurement carried out at 2% O2 did not show any significant difference in this parameter between leaves infiltrated with water or with n-PG. the reduction of the electron transport experienced by PSII in presence of n-PG suggests that diverting up 45% of the total amount of electrons transported by

PS. On the other hand, the NPQ was slightly higher in presence of n-PG at 21%

O2.

Additionally, n-PG did not seem to have any effect over the P700 ETR, k and proportional oxidase at either O2 concentrations.

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Figure 3.12. The effect of 5mM n-PG on PSII ETR (a) and NPQ (b). Measured were performed either at 2% O2 or 21% O2, in the first leaf of 14 days old barley plants, at 25°C. Plants were grown at 20°C and 100 µmol m-2 s-1of light and exposed either to before mentioned or to be watered with 150 mM NaCl solution for the treated plants. The error bars represent the standard error of at least 6 replicates. Two-way ANOVA (p≤0.05) and Tukey's post hoc tests were performed, these results are presented with letters over columns in the graph where the same letters represent no significant differences between mean.

112

Figure 3.13. The effect of 5mM n-PG on PSI ETR (a), k (b) and proportional oxidase (c). Measured were performed either at 2%O2 or 21% O2, in the first leaf of 14 days old barley plants, at 25°C. Plants were grown at 20°C and 100 µmol m-2s-1of light and exposed either to before mentioned or to be watered with 150 mM NaCl solution for the treated plants. The error bars represent the standard error of at least 6 replicates. Two-way ANOVA (p≤0.05) and Tukey's post hoc tests were performed, these results are presented with letters over columns in the graph where the same letters represent no significant differences between mean.

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Discussion

The responses of barley plants to 7 days of exposure to an absence of watering or to salinity were explored. While the relative water content (RWC) of water- limited plants did not show variation, the salt treated plants reduced their RWC, indicating a slight dehydration of their leaf tissues (Figure 3.2 a and b) probably due to the osmotic stress that salt induced in these plants [188], [190], [192].

However, the stress induced by both treatments was mild because it did not trigger a significant alteration in SLA, a parameter which also tends to respond to the leaf dehydration [198]–[200].

In addition, a small but significant increase in the capacity for photosynthesis in salt-treated and water restricted plants was observed (Figure 3.3 a and b). This suggests, that both treatments lead to acclimation of photosynthesis, resulting in a higher level of carbon assimilation. It seems to be unlikely that the larger level of Pmax could be caused by closing the stomata because stomata conductance

(G) and evapotranspiration (E) were not significantly different to control plants.

Neither the chlorophyll content nor chlorophyll a:b ratio were modified by the treatments, making it unlikely that the components of the electron transport chain have altered stoichiometry which explains the rise in Pmax.

Water deficit results in the irradiance response of PSII ETR showing an O2 sensitivity, which is higher than that seen in control plants, in most of the range of light above saturation (Figure 3.6a). A similar tendency was observed in salt- treated plants, however, in this case, the O2 sensitivity was even larger than in water-restricted plants. This sensitivity suggesting the activity of an alternative electron pathway dependent on O2, diverting electrons from PSII. The electron could be transported to O2 mediated by several alternative pathways like photorespiration [92], [164], [165], Mehler reaction [36], [40], [166], [168], Plastid

114

Terminal oxidase (PTOX, [11], [12], [15]), or by spontaneous reduction of plastoquinol pool [46], [47], Cyt b [48], [49] or aceptor side PSII [33], [171].

An alternative electron transport to O2 mediated by photorespiration seems to be unlikely in subjects exposed to both treatments, due to the measurements being performed at the saturated levels of O2.

As photorespiration does not seem to be an explanation for the additional electron transport experienced by water limited and salt-treated plants, the

Mehler reaction or water-water cycle potentially could be an alternative explanation. The latter electron pathway transports the electrons from PSI to O2, and therefore, when this pathway is active, we expect to observe an O2 sensitivity in ETR of both photosystems simultaneously. Plants exposed to water deficit and salt treatment exhibit a simultaneous O2 sensitivity in ETR in both PS only at the highest light intensities, between 1500 and 2000 µmol m-2 s-1. This provides evidence of a potential contribution of electron transport to O2 being mediated by the Mehler reaction. It seems to be that the Mehler reaction could be triggered at extremely high irradiance, when the PSII ETR in both treatments shown a hardly noticeable drop which could be result of a mild photoinhibition, especially considering the apparent saturation of NPQ from moderated to the highest irradiance, probably being unable to provide additional protection to photosynthetic apparatus by a safe dissipation of the excessive energy at extremely high irradiance in both groups of plants [146]–[148].

While the Mehler reaction could be a plausible explanation for the O2 dependence at extremely high light intensities, at moderate and low intensities this seems to be unlikely. In this range of irradiances, it appears to be more likely to involve the action of one or more pathways diverting the electrons in a

115 previous step of the electron transport chain, likely the direct reduction of O2 at the acceptor side of PSII or in Cyt b6f complex from the PQ pool or via PTOX.

Although we should consider the possibility of these three direct O2 reductions could be collaborating with the alternative electron transport measure in PSII to

O2 , Plastid Terminal Oxidase also could be involved. In fact, there is evidence of the PTOX transcript being express in a higher level in salt-treated plants and water-limited plants than controls. However, the expression of PTOX transcript was transient in water restricted plants, while in salt-treated plants, the transcript level was kept higher even after the decline in water restricted plants (Figure

3.5). PTOX protein also was detected in samples of barley plants being 1.2 folds higher in subjects exposed to water limitation and 1.6 folds higher in plants watered with a salt solution than the control group. In order to explore whether

PTOX was a contributor to the diversion of electrons to O2, vacuum infiltrations were performed in barley leaves with n-PG (PTOX inhibitor, [124].

Measurements carried out in the presence of n-PG show, a significant reduction up to 31% in water restricted subjects and up to 45% in salt-treated leaves with respect to the total amount of electron transport by PSII at 21% O2, providing evidence of PTOX acting as a safety valve of electrons when plants experience a water deficit or salinity. All the vacuum infiltration were performed at moderate illumination, a condition where there is no evidence for Mehler reaction only for

PTOX activity. Despite the clear evidence of PTOX activity, it is necessary to consider that the vacuum infiltration process affected leaves in other ways.

Also the effect of saturating CO2 (2000 PPM) on PTOX activity needs to be considered. In a transgenic line of tobacco overexpressing PTOX from

Chlamydomonas (Cr-PTOX1), increasing the CO2 resulted in higher carbon assimilation [173]. This was explained as a result of an abolition of PTOX activity in the Tobacco overexpressor, due to the dissociation of PTOX from the

116 thylakoid membrane, mediated by the decrease of pH in the stroma due to CO2 acidification. However, this hypothesis was not experimentaly tested and in contrast, in salt treated Eutrema plants at saturating CO2 conditions, a clear oxygen sensitivity was reported [10]. This indirect evidence of PTOX activity and also the utilization of n-PG confirmed PTOX activity [10], providing strong evidence of PTOX being responsible for the increase in electron transport at

21% of O2 even at saturating CO2 conditions.

Also, our findings are supported by the literature about PTOX, which has provided evidence of this protein acting as a protective mechanism against stress, in particular, involving drought, for example, in Rosa meillandina increasing PTOX activity and abundance [15], in Chrysanthemum morifolium and Spathiphyllum wallisii where PTOX protein was [13]. Besides, there is evidence of salinity inducing PTOX activity and an increment in the amount of protein in Eutrema salsugineum.

There are some responses and signalling in plants common to several types of stresses [201], [202], however, it seems to be unlikely that this applies to PTOX, as it was not induced by cold in barley plants (Chapter 2). Nonetheless, plants exposed to water or soil higher in salt could result in a reduction of their ability to take up water from the soil, developing water stress. Thus, salt and water stress have common consequences and therefore responses in plants [177], [187],

[191], [192] and maybe PTOX could be part of the protective mechanism against to osmotic stress generated as a consequence of the dehydration that plants exposed to these two type of stresses. Especially considering that salt-treated plants presented the higher level of reduction in the relative water content

(RWC) and the higher level of PTOX activity in comparison with plants experience water deficiency. Although, salt stress also has consequences not shared with water stress as nutrient unbalance and ion toxicity [184]–[186], [189]

117 and which could be having an additional negative impact in the subjects and inducing PTOX activity more highly as a compensation.

118

Conclusion

Barley leaves exposed to salinity show a significant reduction in the relative water content (RWC) and besides water-limited and salt-treated plants experience a higher level of photosynthetic capacity (Pmax) than the control.

This suggests that both groups of plants have experienced a mild stress and they are able to acclimate to that stress.

The water deficit results in an irradiance response of PSII ETR showing an O2 sensitivity, which seems to be higher than in control plants and a similar tendency was observed in salt-treated plants, however, in this case, the O2 sensitivity was even larger than in water-restricted plants. At the highest irradiance, there is evidence of Melher reaction being at least partially responsible for the alternative electron to O2, while at moderated light intensities,

Plastid Terminal Oxidase seem to be involved diverting up to 31% in water restricted subjects and up to 45% in salt-treated leaves with respect to the total amount of electron transported by PSII at 21% O2. Providing evidence of PTOX acting as a safety valve of electrons when plants are experienced water deficit and salinity.

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Chapter 4

Dynamic responses of Plastid

Terminal Oxidase (PTOX) to

changing environmental conditions in Hordeum vulgare

plants

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Introduction

Photosynthesis is a primary target of different abiotic stresses [203] . Under sub- optimal conditions, an imbalance can be generated between a low efficiency carbon fixation and excessive light harvested, overburdening the photosynthetic apparatus and potentially leading to production of reactive oxygen species

(ROS, [143]). ROS can damage cellular components [31], [32]; however, to avoid their detrimental effects, they are scavenged through an energetically demanding antioxidant system [36], [40], [44], [45]. An alternative strategy implemented by plants to prevent ROS generation is a modulation of the electron transport [145] and one example of this could be an alternative electron pathway like Plastid Terminal Oxidase (PTOX) activity [10]–[12], [120].

PTOX was the first identified in variegated plants, immutans (im) in A. thaliana

[96], [97] and ghost (gh) in tomato [98]. In the variegated phenotype, which presents green and white sectors in tissues that are normally green, the PTOX gene is impaired. It has been documented that cells of white spots possess abnormal plastids, chloroplast biogenesis is blocked, and there is an absence of pigment and a plastid accumulation of phytoene [99], [102].

PTOX is a nuclear-encoded protein, coded for by two genes in the case of some eukaryotic algae or by one gene in higher plants [117]. Depending on the species, PTOX has a molecular weight between 40 and 50 kDa and is targeted to the chloroplast by a transit peptide [100], [117]. Once in the chloroplast, it binds to the stromal side of the thylakoid membranes in the stromal thylakoid or lamellae (non-appressed) region [119] and in Eutrema salsugineum the enzyme is translocated to the lumen as part of its activation process [120].

It has been described that PTOX is a member of diiron carboxylate quinol oxidase (DOX) class of proteins, being able to oxidize the plastoquinol

121 pool (PQH2) and reducing molecular oxygen, resulting in water production [106],

[121].This quinol oxidase has been predicted as an interfacial membrane protein with a four-helix bundle located from thylakoid to the stroma side in its active site and encapsulating a di-iron centre. The two iron atoms are ligated by four histidines and two glutamate residues [114], [122]. Site-directed mutagenesis of

PTOX in vitro and in planta, has shown that the six Fe-binding sites are indispensable for the activity of the protein [114]. Also, an amino acid sequence located in exon 8, is essential for the activity and stability of the enzyme.

Additionally, 5 sites (Leu135, His 151, Tyr212, Tyr234, and Asp295) more were identified to be indispensable for PTOX activity in vitro and in planta [122]. PTOX recombinant protein expressed in E. coli and utilised in an in vitro assay of

PTOX activity found that while the enzyme is active in the presence on CN, it is sensitive to n-propyl gallate and n-octyl gallate [124]. At the same time, PTOX was described to be substrate specific in its catalytic activity, almost exclusively oxidizing Plastoquinol and not UQH2, duroquinol or benzoquinone [111], [124].

PTOX has been reported to act as an antioxidant or a prooxidant depending on the abundance of the quinol. Thus, while superoxide radicals are produced even in high substrate concentration at pH=8, at pH 6-6.5 this is just at limited quinol concentrations [127], [128].

The potential ROS production by PTOX and the possibility of this protein competing with the linear electron transport [204], led to Krieger-Lizskay and

Feilke (2016, [204]) proposing an hypothesis for PTOX activity regulation. They suggested that under high light conditions, as the pH in the stroma turns alkaline, PTOX binds to the membrane giving it access to plastoquinol pool and making it active. When the light conditions are not saturating, the pH will be lower, keeping PTOX in a soluble and inactive condition [204]. This pH dependent model could allow us to understand the discrepancies in the literature

122 about PTOX activity and all the evidence about this enzyme acting as a safety valve of electrons in some higher plants. Which is due to its ability to divert electrons from plastoquinol pool to oxygen, generating water, having as a result, the protection of the plastoquinol pool from an overreduction when plants experience abiotic stress [11], [12], [120].

In fact, PTOX has been reported to have a protective role against a variety of stresses [110], for instance salt stress, increasing its abundance and diverting up to 30% of electrons from PSII in Eutrema salsugineum [10], [120] and 45% in

Hordeum vulgare (Chapter 3). The same induction in the amount of protein and an increase in the activity was observed in Rosa meillinae on the top of high light and heat, the water restriction is applied [15]. Also, the same conditions triggered a rise in the abundance of the enzyme in Crysanthemun morifolium and Spathiphyllum wallisi. Another example is Avena sativa, where PTOX protein increase its abundance in heat and high light conditions [94].

Additionally, This protein also seems to have a role when plants experience cold temperature in lodgepole, inducing the accumulation of the enzyme [14] and in

Ranunculus glacialis where application of high light increased in activity [11],

[12].

This chapter addresses the dynamic responses of the PTOX protein and its activity, in order to understand better the regulation of this enzyme in Hordeum vulgare. Through of testing the effect of alternating the O2 concentrations over

PTOX activity, and other stimuli like high light or different pH’s possible variation in abundance or localisation of PTOX in different fractions of the thylakoid membrane are examined.

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Materials and Methods

Plant growth

Seeds of Hordeum vulgare were germinated in a cabinet with controlled conditions of 100 µmolm-2 s-1 of light (warm white LEDs, colour temperature

3000-3200 K) 12-hour photoperiod and 20°C day/16ºC night temperature. After

7 days of development, plants were exposed to differential watering regimes for

7 days, which could be either an absence of watering or a usual amount of watering but with a 150 mM NaCl solution. Measurements were conducted on the first leaf of two-week-old barley plants.

Measuring chlorophyll fluorescence and electron transport to oxygen.

Chlorophyll fluorescence and P700 oxidation were simultaneously measured using PAM-101 chlorophyll fluorimeters (Heinz Walz, Effeltrich, Germany).

(200 ms, 4500 µmolm-2s-1) to fully oxidise of P700 (P700 total, Figure 4.1a).

(Figura 4.1e).

Fluorescence parameters were calculated using the following equations

(Maxwell and Johnson, 2000):

Efficiency of Photosystem II (PSII) = (Fm'-Ft)/ Fm'

ETR = ΦPSII × PFD (photon flux density)

Non-photochemical quenching (NPQ) = (Fm- Fm')/ Fm'

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P700 turn over and the proportion of P700 oxidised. A single exponential curve was fitted (Figure 4.1b ) to the resulting decay to determine the rate constant for the re-reduction of P700 (k) and the proportion of P700 oxidase (P700+[prop]).

The residuals of the exponential fitting were ramdomly distributed arround zero showing that the model describes the data well, representing a pseudo-first order reaction (Figure 4.1d).

The P700 electron transport rate was calculated as:

P700 ETR = (P700+ [prop] x k ) s-1

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Figure 4.1. P700 and fluorescence signals. P700 fully oxidised signal, induced by Far Red (FR, a), Dark period inducing the reduction of P700 (c), fitting of a monoexponential function to the decay of P700 oxidase (b), the residuals of the monoexponential fitting to P700 oxidase decay (d).

126

Samples were supplied during the process with saturating CO2 and either 21% or 2% O2. This gas was provided by bubbling either 98% N2/2% O2 from a cylinder (BOC Gases) or laboratory air through a solution of 1M

Na2CO3/NaHCO3 (pH 9; ~5% CO2).

Immunoblot analysis

Protein extraction was carried out following the method of Cuello and Quiles

[159].

Trypsin treatment of thylakoid isolation extract

The half of extract (1ml) was exposed to three cycles of freeze and thaw using liquid nitrogen and both, 500 µl broken and 500 µl unbroken thylakoid extracts were treated with 250 µl of trypsin (2mg/ml, trypsin from porcine pancreas tablet,

Sigma), vortex and later incubated at 4ºC for 30 minutes. After this time, the reaction was stopped with 7,6 µl of phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 1M. The controls, samples without trypsin, keeping the same volumes received 250 µl of water and later were exposed to the same treatment.

Isolation of thylakoid at different pH

Again the protein extraction was carried out following the method of Cuello and

Quiles [159], however, before the chloroplasts isolation step (See in Cuello and

Quiles method, step number 5) the extract was divided into equal volumes, which where processed in the same form just with buffers adjusted to either pH=8 or pH=6.

Also, a second method of isolating thylakoid was performed.

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Thylakoid extraction method

Barley leaves were washed and then wiped with paper, and then placed in a blender with a slurry grinding buffer containing sorbitol (330 mM), MgCl2 (5mM),

NaCl (1M) and HEPES pH=8 (20mM) or MES pH=6 (20mM). The solution was filtered through two layers of muslin and then through 1 layer of absorbent cotton surrounded by 2 layers of muslin. The filtrate was centrifuged at 3500g for 5 minutes and the supernatant was decanted. Using a brush the pellet was resuspended in shocking buffer, MgCl2 (5mM) and either HEPES pH=8 (20mM)

MES pH=6 (20mM), and subsequently, it was centrifuged at 3500g for 5 minutes at 4oC. The supernatant was decanted again and the pellet was resuspended in grinding buffer using a brush, then the suspension was centrifuged at 3500g for

10 minutes. Finally, the supernatant is discarded and the pellet is resuspended in 1.5 ml of grinding buffer.

Invitrogen) plus 1/10 volume of DTT was added to 3 volumes of protein sample,

20l of this solution containing 20 g of protein was loaded per well of a pre-cast

Nupage SDS gel (Nupage 4-12% Precise Bis-Tris gel, Invitrogen) and at least 1 well per gel was loaded with 20ul of 50% molecular weight marker (Precision plus protein dual Xtra standards. A running buffer was made from MES (2.5mM),

SDS (0.005%), Tris base (2.5mM) and EDTA (0.05mM), plus antioxidant (1%,

128

A second gel was used for western blot analysis. This was put into a tray containing transfer buffer made from Bicine (2.5mM), Bis-Tris (2.5 mM) and

EDTA (0.05mM), Methanol (10%) plus antioxidant (1%,Nupage antioxidant,

Invitrogen) with 2 sheets Western blotting filter paper (0.83 mm, Thermo

Membranes were blocked with 3% BSA in TBS made from Tris-HCl (20 mM, pH

7.6), NaCl (125 mM) for 90 minutes at room temperature. Subsequently, the membrane was incubated either overnight at 4C with primary antibody against

PTOX (kindly provided by Dr. M. Kuntz, Universite´ Joseph Fourier, Grenoble,

France) or 60 minutes at room temperature with primary antibody against a protein of the oxygen-evolving complex of PSII (PsbQ, 1:10000, Agrisera) or 60 minutes at room temperature with primary antibody against PSI-C core subunit of photosystem (PsaC, 3:10000, Agrisera) or 60 minutes at room temperature with primary antibody against  subunit of ATP synthase (ATP, 1:5000,

Agrisera). This was detected using the anti-rabbit HRP (3.3:10000, Agrisera) in

TBS-Tween (0.1%) through incubation for 2 hours. A mix of 2 volumes western blot chemiluminescent substrate 3 volumes of ECL (Pierce ECL Substrate,

Thermo Scientific) and 1 volume of Femto (SuperSignal West Femto Maximum

Sensitivity Substrate, Thermo Scientific) for PTOX detection, 6 volumes of ECL and 1 volume of Femto for PsbQ, 6 volumes of ECL and 1 volume of Femto for

129

PsaC, 1 volume of ECL and 1 volume of Pico (SuperSignal West Pico PLUS

Chemiluminescent Substrate) for ATP. These solutions were used to cover the membrane and incubated it for 1 min. Later the membrane was developed using a CL-Xposure film (CL-Xposure film, Thermo Scientific) with 10 seconds of exposure time for each antibody.

Statistical analysis

Graphs and images were elaborated using Graph Pad Prism 7.

130

Results

Dynamic responses of chlorophyll fluorescence and P700 oxidation parameters to two sequences with a different alternation of O2 concentration.

The findings of Chapters 2 and 3 show that PTOX activity was significant at moderate and high temperatures in control plants, and was induced by a water deficit or salinity in barley leaves. In order to broaden the understanding of how

PTOX dynamically responds to variation in O2 concentration, PSII parameters were measured in leaves exposed to two different sequences of 3 intervals of 30 minutes either starting with 2%, followed by 21% to finalize with 2% of O2 or the opposite. The measurements were conducted in plants exposed to the last week of their development being watered with a 150 mM of NaCl solution.

The electron transport rate (ETR) of photosystem (PS) II (Figure 4.2a) seems to exhibit a similar oxygen effect at 2% and 21% of O2, in barley leaves experiencing either sequence of O2 concentration. This indicates that the activity of PTOX, is reversibleand not being affected by the previous O2 conditions. In the case of NPQ (Figure 4.2b), this parameter appears to slightly increase with the time that the leaf is being exposed to the measurement but also to increase slightly at low O2 concentrations, suggesting that the presence of atmospheric

O2 inhibits pH.

In contrast to PSII, measurements of P700 ETR (Figure 4.3a) indicate that starting the sequence with 2% O2 seems to result in higher values throughout the sequence than seen in leaves measured using the sequence initiated with

21% O2. In both cases, the level of P700 ETR was slightly lower at the end of the third interval in comparison to that observed in the first interval for both sequences. The O2 sensitivity shown by k (Figure 4.3b) and proportion oxidase

131

(4.3c) appears to be reversible, however, the behaviour shown by these two parameters is opposite and offset each other, to result in an O2 independence in

P700 ETR.

Figure 4.2. The O2 sensitivity of PSII parameters across a sequence with a different order in the application O2 concentration. PSII electron transport rate

(a), PSII quantum yield (c), NPQ measured in a sequence of either at 2%, 21% and 2% O2 (red line) or 21, 2% and 21% O2 (black line) at 25°C and 1000 µmol m-2s-1 of actinic light. The measurements were performed in the first leaf of 14 days old barley plants, at 25°C. Plants were grown at 20°C and 100 µmol m-2s-1 of light and watered during the last developmental week with 150 mM NaCl. The error bars represent the standard error of at least 4 replicates and dotted blue lines show when the O2 concentration was switched.

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Figure 4.3. The O2 sensitivity of P700 parameters across a sequence with a different order in the application O2 concentration. P700 electron transport rate

(a), k (b) and the proportion of P700 oxidised (c) were measured in a sequence of either at 2%, 21% and 2% O2 (red line) or 21%, 2% and 21% O2 (black line) at

25°C and 1000 µmol m-2s-1 of actinic light. The measurements were performed in the first leaf of 14 days old barley plants, at 25°C. Plants were grown at 20°C and 100 µmol m-2s-1 of light and watered during the last developmental week with 150 mM NaCl. The error bars represent the standard error of at least 4 replicates.

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Localization of Plastid terminal oxidase (PTOX) in barley plants after 7 days of watering with 150mM NaCl and control plants.

Western blots were performed on samples of isolated thylakoid from barley leaves salt treated for 7 days or on control plants. In order to explore the possibility of PTOX protein location being modified not only in the stromal side but also to the lumenal side of the thylakoid, by the induction of the higher activity of the protein due to the salt treatment. Samples of intact isolated thylakoid membranes (IT) and thylakoid broken by freeze-thaw treatment (BT) were treated either with trypsin (+T) or with water (-T).

Figure 4.4 shows an example blot incubated with a specific antibody against

PTOX protein. PTOX shows a similar behaviour to that of PsbQ, a protein located on the lumenal side of thylakoid. Both proteins were detected through bands just in the lane loaded with samples of broken and intact isolated thylakoid not treated with trypsin. Regarding ATPβ, a protein located on the stromal side of thylakoid, the blot exhibited protein bands in all the lanes, except in broken thylakoid treated with trypsin in control plants. This suggests a protection of the protein binding to the stromal side of thylakoid.

The possible modification of PTOX location between stroma thylakoid and grana due to salt treatment were explored (Figure 4.5). Samples of different fractions of thylakoid of salt treated barley plants were blotted using a specific antibody against PTOX protein. This protein was detected in the stromal thylakoid fraction

(lane 1 and 3), however, the band of this specific protein was absent in grana fraction when the loading was 10µg of the total amount of protein or less.

Nonetheless, if the loading is increased the protein band for PTOX was detected, indicating that trace amounts of PTOX were present in the grana fraction.

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Figure 4.4 Effect of salinity in the localisation of PTOX protein in barley plants.

Western blot of PTOX, ATP β (stroma side of thylakoid) and PsbQ (lumen side of thylakoid) protein of thylakoid isolation of salt treated and control barley plants. The wells from left to right were loaded with 20µg of the total amount of the protein of samples of Intact thylakoid treated with water (IT-T), intact thylakoid treated with trypsin (IT+T), broken thylakoid treated with water (BT-T) and broken thylakoid treated with trypsin (BT+T). Samples were extracted from the first leaf of 14 days old barley plants, grown at 20°C and 100 µmol m-2s-1 of light and exposed during the last developmental week to be watered with 150 mM NaCl solution or water (control). The blot is representative of two technical replicates.

135

Figure 4.5 Effect of salinity in the localisation of PTOX protein in barley plants.

Western blot of PTOX protein in samples of isolated stroma thylakoid and isolated grana of salt treated barley plants. The wells from left to right were loaded with 5µg (lane 1 and 2) and 10 µg (lane 3 and 4) of the total amount of the protein of stroma thylakoid and grana samples respectively and with 173µg of the total amount of protein of grana sample. Samples were extracted from the first leaf of 14 days old barley plants, grown at 20°C and 100 µmol m-2s-1 of light and exposed during the last developmental week to be watered with 150 mM

NaCl solution. The blot is representative of one technical replicate.

Variation of PTOX binding to the thylakoid membrane in barley plants exposed to high light or pH during the extraction.

Western blots were performed in samples of isolated thylakoid of barley leaves of 7 days salt treated or control plants. In order to explore the potential effect of light in PTOX protein binding to the thylakoid membrane in salt treated, water restricted and control plants. Samples exposed to 4 hours of 1000 µmol m-2s-1 of light or dark were used to perform a western blot (Figure 4.6). While salt-treated and water restricted plant extracts seem to have a higher abundance of PTOX when these subjects have been exposed to high light in comparison to dark exposed plants, the control group appear to shown the opposite effect.

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Figure 4.6 Effect of salinity and water restriction in the binding of PTOX protein to thylakoid membranes in barley plants. The wells from left to right were loaded with 20µg of the total amount of the protein of control plants exposed to dark

(lane 1), control plants exposed to 4 hours of high light (Control HL, lane 2), water restricted plants expose to dark (H2O Rest. Dark, lane 3), water restricted plants expose to 4 hours of high light (H2O Rest. HL, lane 4), salt-treated plants exposed to dark (Salt Dark, lane 5) and salt-treated plants exposed to 4 hours of high light (Salt HL, lane 6). Samples were extracted from the first leaf of 14 days old barley plants, grown at 20°C and 100 µmol m-2 s-1 of light and exposed during the last developmental week to being watered with 150 mM NaCl solution or water (control). The densitrometry was performed using the ATP β protein as loading control and the blot is representative of two replicates.

To broaden the understanding of the light effect apparently increasing PTOX protein binding to the thylakoid membrane in salt-treated plants, these extracts were treated either with trypsin or water to investigate whether PTOX protein distribution was changed by the irradiance. Again, PTOX protein exhibited a similar behaviour than the protein located in the lumenal side of thylakoid, PsbQ

137

(Figure 4.7). Being bands for both proteins detected just in the lane loaded with samples of broken and intact isolated thylakoid not treated with trypsin. This time a different protein located in the stromal side of thylakoid was used, PsaC, to discard that the protection observed of the protein located in stromal was a particularity of ATP β (Figure 4.4). In spite of the change in the protein tested, a similar result to the blot previously presented was observed, being PsaC protein protected by the trypsin action in all the extracts no matter if the were exposed to high light or not.

Krieger-Liszkay and Feilke [204] postulated that PTOX becomes associated with thylakoid membrane when the stromal pH is alkaline under high light and potentially being soluble under not saturated light condition. In order to try to have more evidence whether PTOX activity could be being regulated in barley plants in this way, thylakoid isolation was performed at different pH’s using two different methodologies. The extract of salt-treated plants resulted from more alkaline extraction, using both methodologies, seem to have more abundant

PTOX protein that the extract performed at pH=6 (Figure 4.8). While in control plants the higher detection of PTOX appears to be at less alkaline pH extraction.

138

Figure 4.7 Effect of salinity in the localisation of PTOX protein in barley plants.

Western blot of PTOX, PsaC (stroma side of thylakoid) and PsbQ (lumen side of thylakoid) protein of thylakoid isolation of salt treated. The wells from left to right were loaded with samples of Intact thylakoid treated with water (IT-T), intact thylakoid treated with trypsin (IT+T), broken thylakoid treated with water (BT-T) and broken thylakoid treated with trypsin (BT+T). The wells were loaded with

20µg of the total amount of the protein of control plants exposed to dark salinity or 4 hours of high light. Samples were extracted from the first leaf of 14 days old barley plants, grown at 20°C and 100 µmol m-2s-1 of light and exposed during the last developmental week to be watered with 150 mM NaCl solution or water

(control). The blot is representative of two replicates.

139

Figure 4.8 Effect of salinity in the binding of PTOX protein to thylakoid membranes in barley plants. Samples extracted by the method described by

Cuello and Quiles in 2014 (a, [159]) and samples extracted by spinach extraction method (b). The wells were loaded with a volume of extract with corresponding to 10µg of chlorophyll in control salt-treated plants. Samples were extracted from the first leaf of 14 days old barley plants, grown at 20°C and 100

µmol m-2s-1 of light and exposed during the last developmental week to be watered with 150 mM NaCl solution or water (control). The densitrometry was performed using the ATP β protein as loading control and the blot is representative of two replicates.

140

Discussion

The effect of applying two different sequences, alternating two O2 concentrations during three intervals on chlorophyll fluorescence and P700 parameters in barley plants were explored. PSII ETR, as an indirect evidence of PTOX activity, and

NPQ does not seem to show any effect when plants were exposed to a different sequence of O2 concentrations. Thereby, there is evidence that PTOX activity responds dynamically to O2 % modification in a fully reversible way, suggesting an absence of inhibition of the enzyme due to a previous exposure to a specific

O2 concentration. In order to analyze this measurement, it should be considered that there maybe an effect of saturating concentration of CO2, as it has been seen that increasing the level of CO2 increases assimilation of Tobacco transgenic lines overexpressing PTOX from Chlamydomonas (Cr-PTOX1) compared to wildtype level. These findings were explained as an indirect indication of the abolition of PTOX activity in the tobacco overexpressor, due to the dissociation of PTOX from the thylakoid membrane resulting from the decrease of pH in the stroma due to saturating CO2 [204]. Nonetheless, this explanation to the adjustment in CO2 assimilation was not experimentally tested by the authors and at the same time, Stepien and Johnson reported a significant

O2 sensitivity in salt-treated Eutrema salsugineum plants under saturating conditions of CO2. This O2 sensitivity was considered indirect evidence of PTOX electron diversion from the linear electron transport. PTOX activity was later confirmed using a PTOX inhibitor, therefore, it seems to be unlikely that there is a complete inactivation of PTOX at 2000 PPM [205].

In the case of NPQ (Figure 4.1b), also seem to be responding slightly to the O2 concentration, which could indicate that the variation in O2 is slightly affecting the gradient of pH across the thylakoid [75]–[77]. It is relevant to considerer that the

141 gradient of pH could be partially generated by the cyclic electron flow [80], [86], even though experiments to test this were not performed.

Regarding P700 ETR, this parameter appears to be affected by the application of different concentrations of O2 during the initial interval of the sequence. The measurements performed applying the sequence of O2 concentration starting at

21%, seem to show lower values of PSI ETR during the three intervals. This effect of the previous O2 concentration is not consistent with the activity of

Mehler reaction due to that this alternative electron pathway, it is more likely to increase the electron transport in PSI at 21% of O2. [44], [45].

In previous work, PTOX has been reported to be located on the stromal side of the thylakoid in the stroma lamellae (non-appressed area, [119]. However, this

PTOX protein detection was performed in Arabidopsis thaliana, a species that has been reported not to have an important PTOX activity [10]. Recent work in the salt tolerant species Eutrema salsuginuem a species in which PTOX is thought to be an important PSII electron sink, has shown that the location of this may change in response to salt, moving the the grana stacks [120]. Therefore, we tested whether PTOX protein could modify its location in barley plants watered with 150mM with respect to control plants. Regardless of the treatment,

PsbQ, a protein located on the lumenal side of thylakoid [206] was found to be sensitive to trypsin (Figure 4.4). This suggests that the samples that we expected to be intact thylakoid were broken during the extraction, meaning we were unable to determine the presence of PTOX in the lumen. At the same time, two separate stromal side marker proteins, ATP [207] and PsbC [208] were clearly present in all of the samples of both treatments. including the supposedly broken thylakoids, however, the protein detected in broken thylakoid treated with trypsin (BT+T) seem to be somewhat less abundant than in the rest of the this way, the stromal side proteins, which in theory should be exposed to

142 the action of trypsin even in the intact thylakoid will get protected. However, if that is the explanation for the pattern of the presence of both proteins, the doubt about why PTOX is behaving as a lumenal side protein and not as its counterpart located in the stromal side is not clear. Potentially the small abundance of PTOX could allow a faster digestion, so that the majority of PTOX is digested before of exchange the orientation of the thylakoid.

The possible presence of PTOX protein in the grana thylakoid in salt-treated barley plants was tested (Figure 4.5), to see if relocation of the protein inside of grana was induced by stress [120]. In this condition, a relocation of the enzyme could be advantageous, making it easier for the plastoquinol to ineract with

PTOX and potentially increasing its activity and reducing the probability of ROS production. Only trace amounts of PTOX could be detected in grana fractions.

Only if large amounts of protein are loaded could PTOX be detected in the fraction of grana. It is likley that this represents contamination from stroma proteins. It is important to examine the distribution of marker proteins in different fractions to confirm this, especially given the unexpected results from trypsin experiments.

The regulation of PTOX has been hypothesized to be regulated by the pH in the stromal side of the thylakoid [204], with the saturating light being an inducer turning the pH more alkaline, allowing the PTOX association with the thylakoid membrane or keeping it soluble in the opposite situation. The abundance of

PTOX isolated with the thylakoid fraction was tested, in salt-treated, water restricted and control barley plants after a 4 hours extreme high light application or just dark (Figure 4.6). The blot shows an increase in the abundance of the protein in salt-treated and water restricted plants isolated following exposure of plants to high light, which could be the result of the potential modification in the

143 pH in the stromal side of the thylakoid. However, control plants showed a contrasted response, which could not be explained by this hypothesis.

The effect of light on PTOX recovery in the thylakoid may be driven by pH, however it is a sustained effect, which survives the isolation of membranes from leaves. It has recenlty been suggested that PTOX may under some circumstances relocate to the thylakoid lumen [120], a process which could potentally be modulated by pH. Isolation of thylakoid membranes from salt- treated plants exposed to a 4 hours extreme light previous to the extraction or just a dark period was performed, and samples exposed to trypsin treatment

(Figure 4.7). The pattern of bands exhibited by PsaC (stromal side protein),

PsbQ (lumenal side protein) and PTOX was the same than in Figure 4.4.

Suggesting that again the thylakoid were probably broken during the extraction process and potentially the membrane immediately turn inside out. It is important to highlight that in this occasion, the western blotting was performed with a different protein located in the stromal side than in the experiment shown Figure

4.4. With the objective, to discard that the behaviour observed in the protein located outside the grana, was a specific issue for that protein and not representative of the localisation of the rest of proteins from the stromal side of thylakoid. Therefore, from this blot it is not possible to extract reliable information about if PTOX could be relocalized in some extent to the lumen side of thylakoid due to the period of high light.

Finally, the effect in PTOX abundance of performing the isolation of the thylakoid at two different pH’s (Figure 4.8 a and b) was tested, using two different methods of protein extraction. Both methods showed a similar result, having higher PTOX detection at more alkaline pH in salt-treated plants, while the control group exhibited a lower PTOX abundance at the same pH. It is interesting that if we take into account that high light could result in a more

144 alkaline stroma, the results present in Figure 4.8 and 4.6 suggest a similar effect of the alkaline pH or high light, inducing a more alkaline pH, in salt-treated plants and control group. However, only in salt-treated plants did the PTOX abundance develop in an expected more alkaline stroma, in agreement with the hypothesis that PTOX binds more to the membrane under these conditions [204]. In the control plants, the result is contrary to the hypothesis, but consistent between different the Figure 4.6 and 4.8.

145

Conclusion

The response of O2 sensitivity in PSII ETR, an indirect evidence of PTOX activity, and of P700 parameters to a different order in the alternation of O2 contraction in salt-treated plants, was tested. A dynamic and reversible response was seen in the PSII ETR O2 sensitivity and NPQ. Therefore it seems to be unlikely that PTOX is inhibited or in some way regulated by a previous oxygen concentration.

On the other hand, high light induced a higher detection of the PTOX enzyme in salt-treated and water restricted plants, which could be the result of more alkaline conditions in the stroma, developed by the saturating light. Consistent with this, when the protein extraction of isolated thylakoid was performed at more alkaline pH, the result seems to be the same as in the salt-treated plant, recovering the highest amount of PTOX protein at this more alkaline pH.

Additionally, the control groups showed an opposite tendency to salt-treated plants, with a higher detection of the enzyme at less alkaline stromal pH and in dark isolated thylakoids.

146

Chapter 5

Discussion

147

Discussion

The uncertainties about the negative impacts of the climate change on crop production and food security are a constant concern [5], [129], [130].

Understanding better the mechanisms that plants display to protect themselves from abiotic stress [132]–[134], could open new strategies to face the vulnerability of food security, using our knowledge to improve crop tolerance stress stress and increase productivity [8]. Plastid terminal oxidase (PTOX) has been proposed to be a strong candidate for manipulation, to improve the stress tolerance of plants, especially, if we are able to clarify our knowledge as to its regulation [118]. PTOX has been suggested to act as a safety valve for electrons from the electron transport chain in some plants, potentially protecting them against abiotic stresses such as drought, high salinity or extreme temperatures

[10], [12]–[15], [94], [120]. With its ability to divert electrons from plastoquinol pool (PQH2) to oxygen (O2), generating water (H2O) [106], [121], PTOX can help prevent plastoquinone pool overreduction and therefore minimise the negative effect of reactive oxygen species (ROS) production [100].

In the particular case of Hordeum vulgare (barley), it was previously reported that PS (photosystem) II electron transport shows an O2 sensitivity, which increases with the temperature, while PSI was independent of O2 concentration

[115]. As these findings are consistent with PTOX activity and with barley being an important crop, we decided to investigate whether PTOX is playing a role as an alternative electron sink in this species.

Data presented in this thesis points to PTOX playing an important role in barley.

Activity is seen to be associated with high temperatures (Chapter 2) and to be increased as a response to water and salt stress (Chapter 3). Sensitivity of PSII to O2 suggests the activity of an alternative electron transport such as

148 photorespiration [92], [164], [165], Mehler reaction [36], [40], [166], [168], spontaneous reduction of molecular oxygen or PTOX activity [11], [12], [15].

Saturation by CO2 allowed us to exclude photorespiration and the insensitivity of

PSI electron transpor to O2 in most conditions suggested that Mehler reaction does not explain all effects seen, though it probably contributes under some conditions.

Although spontaneous O2 reduction cannot be discarded, PTOX activity was further tested, utilizing n-Propyl Gallate (n-PG) a PTOX inhibitor [124]. These experiments provide evidence of n-PG decreasing the total amount of electrons transported by PSII, consistent with PTOX activity. PTOX activity does not however appear to be important at low temperature, in contrast to conclusions in some other species [11], [12], [14].

When barley plants were grown at low temperature, they showed significant differences in physiological and morphological parameters compared to plants grown at 20C, indicating that they undergo developmental acclimate to cold.

Although there was no evidence of PTOX activity in plants measured at low temperature, these cold acclimated leaves of barley showed oxygen sensitivity at lower temperatures than warm developed plants, with the PSII ETR v/s temperature response curve being shifted to lower temperature (Figure 2.8). The increase in oxygen dependence at lower temperatures could reflect a more relevant role of alternative electron sinks as a protective mechanism against the excess energy in naturally varying conditions. In species like Ranunculus glacialis, PTOX has been observed to act as an alternative electron sink under cold and high light conditions [11], [12]. In plants grown at both 20°C and 10°C, both PTOX transcript and protein were detected, making it likely that the absence of PTOX activity at lower temperatures is related to the inactivity of the

149 enzyme, rather than its absence. This suggests that the protein activity is regulated in a way that is not simply related to its substrate concentration.

The regulation of this plastoquinol oxidase is still unclear, but it has been hypothesized to be pH dependent [204] and new evidence has been shown that, in Eutrema salsugineum, PTOX activity involves the translocation of the protein to grana lamellae [120]. Maybe the absence of these activation steps could explain the absence of PTOX activity when the protein is detected in cold- acclimated subjects. However, it is difficult to speculate in the case of barley, due to the incomplete evidence about PTOX protein localisation, though no evidence was found for a relocalisation in this species (Chapter 4).

The lack of activity of the protein in cold could be related to the effect of the low temperature on the membrane, increasing the viscosity of the thylakoid [131],

[174], reducing the mobility of and therefore availability of small electron transfer molecules like plastoquinone [175].

Even though cold temperature seems not to induce PTOX activity, another type of stress, for instance, due to water restriction could trigger that. Barley plants were exposed to drought, resulting in a small but significant increase in the photosynthetic capacity (Chapter 3), suggesting that the subjects were exposed to a mild stress, which allows them to acclimate to water-restriction increasing the carbon assimilation. The O2 sensitivity observed in the irradiance response of PSII ETR seems to be larger in water restricted plants than in control plants, in most of the range of light above saturation. This O2 effect suggests an alternative electron transport dependent on O2. Mehler reaction could be a possible collaborator in the diversion of electrons [36], [40], [149], but just at

-2 -1 highest light intensities, above 1500 µmol m s , where an O2 effect was detected in P700 ETR.

150

Even though spontaneous O2 reduction could not be discarded, PTOX activity was further examined, through vacuum infiltrations with either n-PG [124] or water. These experiments seem to provide evidence about the effect of n-PG reducing up to 21% of the total amount of electrons transported by PSII (Figure

3.8 a). This finding makes likely the possibility of PTOX diverting these electrons and acting as a safety valve of electrons under the conditions of water restriction as seen in other species like Rosa meillandina increasing PTOX activity and abundance [15] and in Chrysanthemum morifolium and

Spathiphyllum wallisii where PTOX protein was incremented [13]. This interpretation is also supported by the detection of PTOX protein in the sample

(Figure 3.5b) and a transient increase in the expression of the transcript during the period of watering restriction (Figure 3.5a).

In order to expand more the knowledge about PTOX acting as an alternative electron sink in barley, experiments were performed in plants watered with NaCl.

The subjects showed an adjustment probably just to a mild salt stress because measurements of relative water content indicating only a slight dehydration of their leaf tissues, probably due to the osmotic stress that salt induced in these plants [188], [190], [192]. These subjects also increased significantly the photosynthetic capacity (Figure 3.3 b), indicating that the salt treatment induced a rise in the carbon assimilation capacity and therefore acclimation.

O2 dependence of PSII ETR was found to depend on irradiance , suggesting the activity of an alternative electron transport at high light only. The O2 effect was observed in P700 ETR only at highest light intensities, above 1500 µmol m-2 s-1.

It is possible that Mehler reaction could be diverting electron probably in this range of irradiance [36], [40], [149]. The spontaneous O2 reduction also could be potentially contributing with the diversion of electrons from the linear electron transport [33], [46], [48], [49], [171]. However, as with drought, specific

151 measurements were performed to examine PTOX activity using n-PG [124].

These revealed an effect of n-PG over PSII ETR, reducing the transport of electron by 45%. This evidence suggests that PTOX plays an even greater role than in water restricted plants. Consistent with this, plastoquinol oxidase has been suggested to have a role as a safety valve of electrons in Eutrema salsugineum, diverting up 30% of PSII electron transport [205].

To summarise PTOX seem to have a protective role in barley at moderate and high temperature and also it seems to be induced by the absence of watering or salinity. However, cold developmental acclimation fails to induce the activity of the protein even though, when the protein was present in the tissue and the

PTOX transcript being express. The regulation of the protein needs to be understood in order for PTOX to be a viable candidate to manipulate in crops to improve their stress tolerance. It has been reported that higher expression of

PTOX transcript does not result in higher activity of the quinol oxidase [118].

Therefore, the dynamic responses of PTOX protein and its activity were explored, in order to understand better the regulation of this enzyme in H. vulgare.

The reversibility of PTOX activity through the alternation of O2 concentration was evaluated. The response of PSII ETR to variation in the O2 was dynamic (Figure

4.2), adjusting to the variation in the O2 concentrations, and at the same time reversible, not being altered by the O2 concentration experienced by the subjects previously. So, this could be an indirect indication of PTOX protein not being irreversibly affected by oxygen variation. NPQ also seems to be respond slightly to the O2 concentration, which could indicate that the variation in O2 is slightly affecting the gradient of pH across the thylakoid [75]–[77]. It is relevant to considerer that the pH gradient could be generated by cyclic electron flow [80],

[86], even though experiments to test that were not performed.

152

For a long time, PTOX was reported to be located exclusively in the stromal side of the thylakoid in the stroma lamellae (non appressed thylakoids, [119]) new findings have also shown that the protein is relocated to the grana and may be translocated to the lumen after a salt treatment in Eutrema salsugineum, and this process seems to be involved in the activation of the plastoquinone oxidase

[120].

The effect of salt treatment by itself (Figure 4.4) plus a short period of high light previous to the extraction (Figure 4.7), on the orientation of PTOX in the thylakoid was tested. Regardless of salinity or light treatment, the lumenal protein, PsbQ [206], was digested in the presence of trypsin in the broken and intact thylakoid samples. This finding indicates that thylakoid membrane was broken previous to be treated with trypsin. If this assumption was true, it was logical to expect the protein located in the stromal side of the thylakoid, ATP

[207] and PsbC [208], to show the same pattern of trypsin sensitivity as the lumenal protein. On the contrary, both proteins were found in all the samples, showing protection from trypsinwhich could potentially result from a simultaneous breaking of the thylakoid with a turning inside out of the membrane. However, this does not explain PTOX protein having the same pattern of presence that the lumenal side protein and not as its counterpart located in the stromal side. It could be that the reduced level of PTOX protein allowed an extremely fast trypsin digestion, being able to be mostly complete before exchange the orientation of the thylakoid.

The antioxidant or prooxidant activity shown by PTOX depending on pH [127],

[182], led to the suggestion that the regulation of PTOX is by the pH gradient.

Saturating light could trigger an alkalinisation of the stromal side of the thylakoid, allowing the PTOX binding to the thylakoid membrane or keeping it soluble

[204]. The effect of a short period of high light or darkness before the extraction

153 on the abundance of PTOX protein was investigated in control, water-restricted and salt-treated plants (Figure 4.6). High light seems to increase the recovery of

PTOX protein in water- restricted and salt-treated plants. It is possible that the short period of high light raises the saturation level in the photosynthetic apparatus in the treated plants. Inducing an alkalinisation of stromal space, increasing the association of the PTOX protein to the thylakoid. However, in control plants, the effect of high light on PTOX recovery was the opposite. In addition, the effect of isolating thylakoids at different pH values was explored in salt-treated plants. In these subjests, the recovery of PTOX protein was higher at more alkaline pH, consistent with the hypothesis of PTOX binding being regulated by pH. However, control plants again showed the opposite result. It is interesting that salt-treated plants exposed to high light, which we expect to develop a more alkaline stromal space, exhibit the same tendency as plants extracted directly in more alkaline pH. Besides the same tendency is observed between control plants, which at the same time is the opposite to salt treated plants.

In spite of this there is not enough evidence to discard or confirm the translocation of PTOX in barley plants presenting an active quinol oxidase, it seems to be that direct or indirect variation in the pH has an effect on PTOX protein recovery in the salt-treated plants, which has been shown to develop activity of the enzyme.

Much is still unknown about PTOX regulation which should be clarified, before

PTOX protein goes from being a potential to a real candidate to manipulate crop stress tolerance and productivity. In this field a possible good approach could be to develop a GFP-tagged PTOX protein in a plant species that naturally develops PTOX activity under conditions, allowing the tracking of the protein to determining its flow through sub-cellular fractions. Also, these plants could be

154 useful to examine the different abundance of PTOX in different developmental stages and plants tissue. Complementary information could extracted from experiments using proteomics, to explore the possibility of any post-translational modifications which the enzyme could experience during its activation.

In particular, in the case of barley, proteomics would be useful to reduce the uncertainties concerning the presence of PTOX in the grana in treated plants which are able to develop PTOX activity. Barley being an important crop, it would be relevant to understand better PTOX regulation in this species, for later generation of transgenic lines to explore whether the increase in PTOX protection really affects the productivity of the crop. If this is the situation, investigating PTOX activity in wheat could be relevant, because of the two species being closely related, with wheat being generally more stress sensitive

[209], [210].

155

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