Effects of ultraviolet radiation on Hibiscus rosa-sinensis, Beta vulgaris and -t. Helianthus annuus

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loannls Panagopoulos

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Dapartmtnt of Plant Physiology University of Lund, Sweden Lund 1992 LUND UNIVERSITY DOCTORAL DISSERTATION Date of MM Department of Plant Physiology October 1992 P.O. Box 7007 S-220 07 LUND ffifUSs / NBFB-1025 / 1-39 /1992 Arffcorf.) T"-"""'••••"•""• Ioannis Panagopoulos TtthwrtrtiMi Effaces of ultraviolet radiation on Hibiscus rosa-sinensis, Beta vulparis and Helianthus annuus

' It is believed that increased levels of ultraviolet-B radiation (UV-B; 280-320 nm) will result in serious , threat to plants. In the present study the effects of UV (particularly UV-B) were studied on chlorophyll fluorescence, ultraweak luminescence (UL) and plant growth. Parameters related to light emission were ' determined, and the effects of UV-B on hypocotyl elongation and levels of free IAA were examined. Hibiscus rosa-sinensis L, Beta vulgaris L. and Helianthus annuus L. were used as material. They were - grown in greenhouse or in grc *th chambers and exposed to short or long term UV-B simulating different levels of ozone depletion, abort exposure of Hibiscus leaves to UV resulted in a gradual increase in both : UL and peroxidase activity followed by a decline after 72 h and a decrease in variable chlorophyll fluorescence. The action of UV-B on sugar beet plants depended on light quality and irradiance and : infection by Cercospora beticola Sacc. The interaction between UV-B and the disease resulted in a large ; reduction of dry weight and enhanced UL The lowest Chi a and growth was found in plants grown under 1 low irradiance and exposed to UV-B supplemented with UV-A (320-400 nm). UVB also inhibited photosystem II, increased UL and peroxidase activity. Under relatively high PAR, UV-B increased dry . weight of laminae and UL but had no effect on Chi content. Sugar beet plants grown with light depleted in ' the 320-400 nm region of the spectrum and exposed to UV-B died. Low levels of UV-B did neither affected [ hypocotyl elongation nor amounts of free IAA in sunflower plants grown under low (LL; 143 umol m"2 s"1) or high PAR (HL; 800 nmol m*2 s*1). Three times more daily U V-B increased the amount of free IAA, but inhibited hypocotyl elongation. Sunflower plants exposed to HL with low UV-B had more leaves and larger leaf area than those grown under HL only, although these differences lessened by the end of the > experiment. Higher Fy/F,,^ and F690/F735, Chi a and carotenoids were found in plants exposed to low UV-B. Indeed, UV-B can be harmful but may also have enhancing effects on plants. The action of UV-B is : highly dependent upon the growing conditions, such as the daily fluences of UV-B, the background irradiance and its quality, the plant species and whether the plants are vigorous or stressed.

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Ort. Effects of ultraviolet radiation on Hibiscus rosa-sinensis, Beta vulgaris and Helianthus annuus

Ioannis Panagopoulos

Department of Plant Physiology University of Lund, Sweden

Academic Dissertation 5 1 t

permission of the Faculty of Science, University of Lund to be defended at the Department of Plant Physiology, Sölvegatan 35, Lund, 24 October at 10:00 AM for the degree of Doctor of Philosophy

Faculty Opponent: Prof Alan H. Teramura, Department of Botany, 1210 H. J. Patterson Hall, University of Maryland, College Park, Maryland, USA

Lund 1992

CODEN: LUNBDS/NBFB-1025/1-39/1992 ISBN 91-971353-4-8 APPENDAGE Papers I and III are reprinted with the kind permission of the Physiologia Plantarum. Paper II is reprinted with the kind permission of Elsevier Sequoia, Lausanne, Switzerland, publishers of the Journal of Photochemistry and Photobiology B: Biology.

Errata Paper II: 3. Results, 3.1, Plant growth (page 77): "Plants grown under YL+UV-B died after 3 weeks" should be "Plants grown under YL+UV-B died after 10 days." i Table 5 (page 81). The F^ for WL+UVA+UVB is 3.4± 0.2 instead of 3 ± 0. Effects of ultraviolet radiation on Hibiscus rosa-si ne nsis, Beta vulgaris and Helianthus annuus

f i loannis Panagopoulos

Department of Plant Physiology University of Lund, Sweden

Lund 1992 I

|> "ΥΠΕΡ ΕΚΑΣΤΟΥ ΔΕ ΕΓΠΝ Ο ΛΟΓΟΣ Ο ΤΗΝ '"* ΑΙΤΙΑΝ ΕΧΩΝ, ΗΝ ΔΕΙ ΜΗ ΛΑΝΘΑΝΕΙΝ Ο ; ΓΑΡ ΑΝΕΥ ΤΑΥΤΗΣ ΠΟΙΩΝ ΚΑΙ ΤΟ ΕΘΕΙ ΚΑΙ ' ΤΟΙΣ ΣΥΜΒΑΙΝΟΥΣΙΝ ΚΑΤΑΚΟΛΟΥΘΩΝ *· ΚΑΤΟΡΘΟΙ ΜΕΝ ΙΣΩΣ, ΟΥΚ ΟΙΔΕΝ ΔΕ... ΤΟ ΔΕ ΤΕΛΕΙΟΝ ΕΞ ΑΜΦΟΙΝ. ΟΣΟΙ ΔΕ ΚΑΙ ΤΟ ΘΕΩΡΕΙΝ ΜΑΛΛΟΝ ΑΓΑΠΩΣΙΝ, ΑΥΤΟ ΤΟΥΤΟ Λ- ΙΔΙΟΝ ΤΟΥ ΛΟΓΟΥ ΚΑΙ ΤΗΣ ΑΙΤΙΑΣ."

"About each procedure there is the account that gives its reason, and the reason must not escape us. For the man who carries out the procedure in ignorance of the reason, guided by habit and by event may perhaps succeed, but he does not know... and complete possession of the v comes •**•• • from both. As for those who have a greater love for understanding comes only when we have the account and the reason." I

THEOPHRASTUS, DE CAUSIS PLANTARUM, BOOK in, 2.3 Table of contents List of separate publications 1 Abbreviations 2 A. Introduction 3 A.I Solar radiation 3 A.2 Factors affecting solar UV-B radiation reaching Earth 5 A.2.1 The hole in the sky 5 A.3 The biological importance of solar UV-B radiation 6 A.4 Action of UV-B radiation on some important plant molecules 8 A.5 General plant action spectrum of UV-B radiation 8 A.6 Examples of plant response to UV-B radiation 10 B. The present study 14 B.I Objectives 14 B.2 Experimental material and conditions 15 t B.3 Parameters measured 15 B.3.1 Light emission 15 B.3.1.1 Chlorophyll fluorescence 15 B.3.1.2 Ultraweak luminescence 17 i B.3.2 Peroxidase activity and lipid peroxidation 19 B.3.3 Photosynthetic pigments 20 B.3.4 Stomatal resistance 20 B.3.5 Plant growth 20 B.4 Results and discussion 21 B .4.1 Chlorophyll fluorescence, photosynthetic pigments and stomatal resistance 21 B.4.2 Ultraweak luminescence, peroxidase activity and lipid peroxidation 22 B.4.3 Plant growth 24 C. Concluding remarks 26 Acknowledgements 28 References 30 Paper I-IV 41 «*••

List of separate publications

This thesis is based on the following publications, which will be referred to by their Roman numerals.

I. Panagopoulos, I., Bornman, J. F. and Björn, L. O. 1989. The effects of UV-B and UV-C radiation on Hibiscus leaves determined by ultraweak luminescence and fluorescence induction.- Physiol Plant. 76: 461-465.

II: Panagopoulos, I., Bornman, J. F. and Björn, L. 0.1990. Effects of ultraviolet radiation and visible light on growth, fluorescence induction, ultraweak luminescence and peroxidase activity in sugar beet plants.- J. Photochem. Photobiol. B: Biol. 8: 73-87.

III. Panagopoulos, I., Bornman, J. F. and Björn, L. O. 1992. Response of sugar beet plants to ultraviolet-B (280-320 nm) radiation and Cercospora leaf spot disease.- Physiol. Plant. 84: 140-145.

IV. Panagopoulos, I., Sundberg, B., Bornman, J. F. and Björn, L. 0. 1992. Effects of ultraviolet-B (280-320 nm) radiation on growth, auxin level and chlorophyll fluorescence in Helianthus annuus. Manuscript. Abbreviations:

ABA, abscisic acid Chi, chlorophyll IAA, indoleacetic acid

Fo, initial fluorescence

Fmax, maximum fluorescence Frv = rFma x - rFo F690, fluorescence at 690 nm F735, fluorescence at 735 nm MDA, malondialdehyde PAR, photosynthetic active radiation PS, photosystem TBA, thiobarbituric acid UL, ultraweak luminescence UV, ultraviolet

UV-BBE, biologically effective ultraviolet-B radiation weighted with Cald well's spectrum (1971) and nonnalized to 300 nm WL, white light A. Introduction

A.I Solar radiation

The solar radiation, based on wavelength, is divided into: - infrared radiation (>700 nm) - visible radiation (400-700 nm) - ultraviolet radiation (<400 nm). The ultraviolet radiation is further subdivided into: - UV-A or near-UV radiation (400-320 nm) - UV-B or mid-UV radiation (320-280 nm) - UV-C or far-UV radiation (< 280 nm) In the stratosphere, molecular oxygen (O2) absorbs solar radiation in the Schumann-Runge bands (175-205 nm) and the Herzberg continuum (190-242 nm). The radiation energy causes the 0-0 bond to break and once released, atomic oxygen (O) rapidly combines with O2 to form ozone (O3) by the reaction la, O+O2 + M—>O3 + M (la) where M denotes any third body molecule such as N2 and O2. The O3 formed in reaction la further reacts with oxygen atoms (reaction lb) or absorbs incoming solar radiation in the region of 215-295 nm (reaction lc).

O3 + O —> 2 O2 (lb)

O3 + hv —> O2 + O

Thus, ozone is constantly produced and broken down in the stratosphere, and if it could be compressed at standard pressure and temperature at sea level, the total amount of ozone in the stratosphere would occupy a layer only 2-5 mm in thickness. Although ozone is a minor component of the atmosphere, its presence is essential to life on earth. Ozone absorbs so strongly in the UV-C region of the spectrum (Fig. 1), that even if stratospheric ozone were reduced by as much as 90 %, wavelengths below 280 nm would contain less energy than 10"3 W nr2 nm'1 (Caldwell 1977). Consequently, life is protected from the lethal action of UV-C radiation, the range where the key molecules of life, DNA and RNA, absorb strongly. In contrast, ozone absorbs weakly in the region of UV-A radiation (Fig. 1). This region is less harmful than UV-C, since nucleic acids and proteins do not absorb significantly (Jagger 1985). Furthermore, living organisms use UV-A radiation in photoreactivation and photoprotection. Plants and fungi also many responses in this region of the electromagnetic spectrum, which are regulated by an unknown photoreceptor called cryptochrome or the blue/UV-A receptor (Senger and Schmidt 1986). In the UV-B region of the spectrum, the absorption cross section for ozone drops steeply. Although atmospheric ozone prevents most of the UV-B radiation from reaching Earth, a small but biologically significant portion of this radiation reaches the biosphere. A decrease of the ozone concentration in the stratosphere will increase the amount of UV-B radiation reaching our planet. Such a perspective has caused many scientists in the world to stress the importance of maintaining the stratospheric ozone layer, and to investigate possible biological effects of increased UV-B radiation.

1200 tio n

se c t 1000 v

ecu l 800 cro s

uoi : o 600 a s i 400 o •ii C o 200 O N O 150 200 250 300 Wavelength, nm Fig. 1. Absorption cross section of ozone at 226 K (solid line) and relative DNA absorption (dotted line) in the region 185-320 nm. The values of absorption cross section of ozone were extracted from Molina and Molina (1986). The absorption values of DNA were extracted from Rubert (1964) and normalized to 100 at 260 nm. A.2 Factors affecting solar UV-B radiation reaching the Earth

The sun angle and the amount of ozone in the atmosphere are the two primary factors that determine the solar UV-B radiation reaching the Earth's surface. The solar radiation has the shortest path through the atmosphere when the sun is directly overhead and for this reason most UV-B radiation is received during the four hours centred around solar noon. The bulk of the ozone is produced above the equator where the solar radiation has the maximum intensity and stratospheric currents flowing towards the poles carry the ozone to higher latitudes. As a result, the highest global ozone concentration appears near the North pole and at about 60 degrees south latitude (Stolarski, 1988). Near the equator, the seasonal variation of UV-B radiation reaching the Earth's surface is small because of the small seasonal ozone fluctuation and changes in sun angle. In temperate regions, the ozone concentration is higher in late winter and in the beginning of spring and lowest in late summer and early autumn. Thus UV-B radiation shows strong seasonal dependence. Other processes in the atmosphere, such as scattering and absorption by molecules and aerosols cause attenuation of solar UV radiation (Caldwell 1981). Additionally, local conditions such as cloud cover, haze and dust, altitude and air pollution, including sulphur dioxide and tropospheric ozone, influence the amount of UV-B radiation reaching the biosphere.

A.2.1 The hole in the sky In 1974, Molina and Rowland suggested in an article published in Nature that chlorofluorocarbons destroy the stratospheric ozone: "Chlorofluoromethanes are being added to the environment in steadily increasing amounts. These compounds are chemically inert and may remain in the atmosphere for 40-150 years, and concentrations can be expected to reach 10 to 30 times present levels. Photodissociation of the chlorofluoromethanes in the stratosphere produces significant amounts of chlorine atoms, and leads to 1 the destruction of atmospheric ozone". Eleven years later, atmospheric I < scientists from the British Antarctic Survey reported that the springtime amount of ozone in the atmosphere over Halley Bay, Antarctica, had decreased by more than 40 % between 1977 and 1984 (Farman et al. 1985). Since then, the Antarctic ozone hole has been recorded for the years 1987, 1989, 1990 and 1991 (Stolarski et al. 1992), and many articles have confirmed the role of the chlorofluorocarbons in the destruction of stratospheric ozone. Chlorofluorocarbons are man-made substances widely used as aerosol propellants, refrigerants, cleaning solvents for electronic components and foaming agents for plastics. Once these molecules have been transported to the stratosphere, photodissociation produces catalytic chlorine atoms (reaction 2.1a).

CC12F2 + hv —-> -CC1F + O (2.1 a) The chlorine atoms produced can react with ozone molecules (reaction 2.1b). 5 Cl» + 03 —-> CIO- + O2 (2.1b)

The CIO» radicals often react with atomic oxygen (reaction 2.1c), especially at altitudes above 30 km, releasing Cl* again and completing one cycle of the ClOx catalytic chain.

CIO-+ O —-> Cl« + O2 (2.1c)

On average, each Cl» released into the stratosphere at an altitude of 30 km icmoves several thousands of ozone molecules before arriving back in the troposphere as HCl many months later (Rowland 1982). Further destruction of ozone may be caused by oxides of nitrogen which are produced in combustion processes (reactions 2.Id and 2.1e).

NO + O3 —> NO2 + O2 (2.1 d)

NO2 + O—>NO + O2 (

Apart from the hole in the sky above Antarctica, there is now evidence which suggests that ozone destruction also takes place above the Northern hemisphere. A gradual long-term downward trend has been reported at north mid-latitudes (National Aeronautics and Space Administration 1988). For Sweden, Hofmann et al. (1989) concluded that a small ozone hole might have been present over Kiruna (68 °N, 21 °E). In the morning of January 29 of 1992 the ozone value observed above Norrköping was 200-210 Dobson units. This was the lowest amount of total ozone ever recorded in Sweden.

A.3 The biological importance of solar UV-B radiation

The effects of UV-B radiation on living organisms occur because of photochemical absorption by molecules such as nucleic acids, proteins, porphyrins, carotenoids, steroids and quinones. According to Jagger (1985), there is a transition in the mechanism of UV-B action around 305 nm. For wavelengths below 305 nm the mechanism is almost the same as that for UV- , C. The DNA absorbance in the UV-B region increases exponentially with ^ decreasing wavelengths. Table 1 shows the relative effectiveness of photons in the region 290-320 nm for altering DNA. UV-B absorption leads to DNA • damage and the formation of cyclobutane pyrimidine dimers (Ellison and \ Childs 1981, Harm 1969). Recently, it was found that UV-B induces J photoreactivation in Arabidopsis thaliana (Pang and Hays 1991), a finding - which suggests that pyrimidine dimers are formed in plants exposed to UV-B radiation. In addition, Quaite et al. (1992) quantified the production of pyrimidine dimers in Medicago sativa exposed to UV of different 6 wavelengths. The resulting action spectrum had a maximum value at 280 nm and extended to 365 nm.

Table 1. Relative effectiveness of photons in the UV-B region for altering DNA, extracted from Harm (1980).

Wavelength, nm Relative effectiveness 290 160.00 295 60.00 300 15.00 305 2.60 310 0.60 315 0.10 320 0.03

At wavelengths above 305 nm, there is a contribution by another mechanism similar to that of UV-A radiation: photodynamic action and the production of i peroxides. A photodynamic action requires molecular oxygen, a sensitising agent and excitation light. The sensitizer absorbs light and is converted from V the ground state (°S) to the first singlet state (*S). Intersystem crossing and 3 f spin inversion will result in the less energetic but longer lived triplet ( S) i excited state. The 3S may react with molecular oxygen in several ways (Foote 1976). Consequently, oxidation of fatty acids, sugars, cellulose, guanine, amino acids such as cysteine, histidine, methionine, tryptophane, tyrosine, and damage to DNA can occur, leading to inactivation of enzymes and destruction of cell membranes (Elstner 1987, Foote 1976, Halliwell and Gutteridge 1989, Spikes 1989). Miguel and Tyrrell (1983) provided evidence that lethality in Escherichia coli strains was oxygen-dependent at 313 nm. UV-B-induced photoperoxidation of lipids has been documented (Sakanashi et al. 1986, Salmon et al. 1990). Photosensitization of NADH and NADPH by monochromatic radiation in the range of 290 to 405 nm resulting in the formation of superoxide anions was also observed (Cunningham et al. 1985). Kramer et al. (1991) found that UV-B exposure resulted in significant increase of lipid peroxidation (measured with the thiobarbituric test and expressed as malondialdehyde) in cotyledons and leaves of two cucumber cultivars. A.4 Action of UV-B radiation on some important plant molecules

In plants, a polypeptide of particular importance, is the Dl polypeptide of the photosynthetic machinery. In light the Dl polypeptide, a key molecule in the centre of the energy flow, is continuously synthesized and degraded. Degradation is more rapidly under solar UV-B radiation (Greenberg et al. 1989). Plants possess a UV-B photoreceptor which interacts with phytochrome and cryptochrome (Mohr 1986). The action spectrum of the UV-B photoreceptor shows a single prominent peak at 290 nm, and no action at wavelengths longer than 350 nm (Yatsuhasi et al. 1982). Two more key molecules in plants, which absorb in the UV-B region of the spectrum are ABA and IAA. However, it is not clear how these components determine the plant response to UV-B radiation. An increased level of ABA is associated with several types of stresses, but no increased levels of free ABA were found in leaves of Rumex patientia L. exposed to enhanced levels of UV-B radiation (Lindoo et al. 1979). In the same study, it was also shown that increased UV-B radiation had little effect on photolysis or isomerization of ABA. Degradation of IAA by UV-B radiation in sunflower seedlings has also been reported (Ros 1990). In the present study, (paper IV), however, the results suggest that UV- B radiation does not decrease the levels of free IAA.

A.5 General plant action spectrum of UV-B radiation

Biological response to UV-B radiation is wavelength dependent. The effectiveness of UV-B radiation sometimes increases exponentially with decreasing wavelength (see for example Table 1 for DNA). If 1^ is the spectral irradiance and E^ is the relative effectiveness of the irradiance at the wavelength X for a certain biological response, then the biologically effective irradiance is:

biologically effective irradiance = Jl^ E^ d\ (5.1)

where the lower and upper limits are at the wavelengths where either 1^ or

8 290 300 310 320 330 340 Wavelength, nm Fig. 2. Absolute amount of UV-B radiation reaching Lund, Sweden at noon 15 of July. Solid line refers to UV-B radiation when there is no ozone reduction, dotted line refers to 20 % ozone reduction. The values were calculated with the programme of Björn and Murphy (1985) with atmospheric pressure of 1000 mbars, relative humidity 0.5 and aerosol !evel 0.

0.0015

o.ooio • s 0.0005 -

0.0000 280 290 300 310 320 Wavelength, nm Fig 3. Biologically effective UV-B radiation, weighted with the general plant action spectrum of UV-B radiation according to Caldwell (1971), reaching Lund at noon on 15 July when there is no ozone reduction (solid line) and when there is a hypothetical 20 % ozone reduction (dotted line).

9 Ex, approach zero. Caldwell (1971) suggested a single curve for the relative photon effectiveness for plant UV-B phenomena involving nucleic acids and proteins as chromophores. Since then, this curve, normalized at 300 nm, has been extensively used as a weighting function in plant-UV-B experiments to obtain biologically effective UV-B radiation (UV-Bgg). According to Jagger (1985), "An action spectrum is the relative response of a system to different wavelengths of radiation" and "an action spectrum is a plot of the reciprocal of the number of incident photons required to produce a given effect versus wavelength". Caldwell's plant action spectrum is not based on a specific effect versus wavelength Moreover, there are studies where UV-BgE radiation has no effect on different parameters. Taking into consideration the antagonism and synergism of UV-B radiation with other wavelengths, I think it is misleading to call it a "generalized plant UV-B action spectrum". However, it is valuable for use as a weighting factor for the comparison of results between authors. A 20 % ozone reduction would result in a very small increase of solar UV radiation between 280 and 400 nm (Fig. 2). If one considers equation 5.1, and uses the "generalized plant UV-B action spectrum", a different picture arises (Fig. 3). The conclusion is that the biologically effective UV-B radiation has been increased by 3b % and the radiation amplification factor (defined as dln(UV-B effect) / din (ozone decrease)) is 1.8.

A.6 Examples of plant responses to UV-B radiation

To date, approximately 300 plant species and varieties have been studied using plants grown in growth chambers, greenhouses and in the field. UV-B radiation affects numerous biochemical/physiological processes, the development of organs as well as the composition of plant communities (Table 2). Many plants exposed to UV-B radiation accumulate UV-B absorbing pigments, i.e., flavonoids. Therefore flavonoid accumulation is considered to be one of the defence mechanisms of plants against solar UV-B radiation (Caldwell 1981, Teramura 1983, Tevini and Teramura 1989, Wellmann 1983). At the molecular level, extensive work by Hahlbrock and colleagues have shown that UV radiation induces increases in transcription, translation and * activity of chalcone synthetase, the key enzyme of the flavonoid pathway. UV • radiation also stimulates the catalytic activity of about 13 more enzymes \ specific for flavonoid synthesis (Chappell and Hahlbrock 1984, Dangl et al. » 1987, Kreuzaler et al. 1983). It was shown later that all the steps of the < sequence, e.g., de novo mRNA, enzyme and flavonoid synthesis are located in the epidermal cells where the end products accumulated (Schmelzer et al. 1988). 10 UV-B radiation may inhibit photosynthesis by causing changes to the photosynthetic apparatus including structural damage in chloroplasts r (Bornman 1989, Bornman et al. 1983). Jordan et al. (1991, 1992) have shown that UV-B radiation dramatically decreases the levels of mRNA transcripts of both the small and large subunits of Rubisco as well as the levels of mRNA transcripts of the chlorophyll a/b binding protein and the Dl protein of photosystem II. At the organ level, UV-B radiation alters leaf characteristics such as leaf area, thickness, specific leaf area, structural features of the leaf surface including epicuticular waxes, and consequently reflectance properties and light penetration into leaves (Bornman and Vogelmann 1988, 1991, Cen and Bornman 1990, Teramura 1983, Tevini and Teramura 1989). At the organism level, UV-B radiation may affect the height of plants, biomass production and carbon allocation. UV-B radiation also affects flowering (Tevini and Teramura 1989 and references therein). In a recent study, Teramura (1992) found that increased UV-B radiation resulted in earlier flowering in species from higher elevations and reduced floral biomass in plants from low elevations. At the community level, it has been shown that UV-B radiation alters the competitive balance between the crop/weed pairs of wheat and wild oats (Barnes et al. 1988). The main reason was the differential morphological response of the two species. The UV-B responsiveness of plants depends on: * the amount of photosynthetically active radiation (Cen and Bornman 1990, Mirecki and Teramura 1984, Teramura 1980, Teramura et al. 1980, Warner and Caldwell 1983), * the amount of UV-B radiation, * previous exposure to UV-B radiation (Wellmann 1983 and literature , therein), i * the microclimate and soil fertility (Murali and Teramura 1985,1986a), I * the genetic composition of plants, since interspecific differences among I species as well as intraspecific differences among cultivars have been » observed (Murali and Teramura 1986b, Teramura and Murali 1986), and 1 • the stage of plant development (Teramura and Sullivan 1987). In the present study (paper II), it is also demonstrated that in growth ,i chamber experiments, the quality of background light is very important. For example, sugar beet plants died when exposed to UV-B radiation together with ) background light depleted in the region below 450 nm, (see paper II results). | In some cases, results from experiments of one year may not be •' observed in a following year, because of modification and adaptation by plants to the above mentioned factors. Using the ozone filter technique(which uses natural UV-B radiation), Tevini et al. (1989) reported an increase of sunflower leaf area under enhanced UV-B radiation in 1987 whereas a 11 decrease was found in 1988. They explained that the difference might be due to the lower UV-B irradiance in 1987. In field experiments conducted from May to October 1986, Sullivan and Teramura (1989) reported that enhanced UV-B radiation reduced photosynthesis in Glycine max [L.] Merr. cv Essex. However, they failed to observe photosynthesis reduction in the same cultivar in a subsequent study (Teramura et al. 1990). Paradoxically, UV-B radiation also has "positive" effects on some plants, contradicting the general idea that UV-B radiation is only deleterious. UV-B radiation can, in fact, drive photosynthesis (Halldal 1964, Me Leod and Kanswisher 1962). Stimulation of photosynthetic activity may also occur. For example, this has been found for soybean (Teramura and Caldwell 1981) and the rice cultivar Kurkaruppan, where a 24 % higher maximum photosynthetic rate was found for plants grown under enhanced UV-B radiation (Teramura et al. 1991). In the present study (paper IV), higher Fv/Fmax, an index for quantum yield of photosynthesis, is also reported for sunflower seedlings exposed to UV-B radiation. Higher total and shoot dry weights for some plants were also observed. Teramura and Murali (1986) grew 23 soybean cultivars under UV-B radiation simulating a 16 % ozone reduction at College Park, Maryland, USA and reported that total dry weight for cv. Forrest was increased by 64 % in the greenhouse, and 21 % in the field. In a recent greenhouse study, Barnes et al. (1990) showed that shoot dry weight of Chenopodium album and Amaranthus retroflexus was higher for plants grown under enhanced UV-B radiation corresponding to a 20 % ozone reduction at Logan, USA. Production of a larger number of leaves has also been reported for Triticum aestivum (Barnes et al. 1990, Teramura 1980), Avena sativa, Zea mays, Avena fatua, Amaranthus retroflexus (Barnes et al. 1990), Aquilegia caerulea and A. canadensis (Larson et al. 1990) under enhanced UV-B radiation. Furthermore, increased leaf area was reported for numerous plants exposed to UV-B radiation (for a review see Teramura 1983). In the present study, increases in leaf dry weight (Beta vulgaris, paper III) and in leaf area and number (Helianthus annuus, paper IV) with UV-B radiation are reported The above discussion shows that plants respond to UV-B radiation in an unpredictable way and any prediction based on the UV-B susceptibility/tolerance of only one or two cultivars or ecotypes of a plant species is probably misleading. The prediction appears even more difficult if one takes into consideration the interaction of UV-B radiation and combined stress(es).

12 Table 2. List of recorded targets of UV-B radiation on plants, modified from Teramura (1983) and Krupa and Kickert (1989).

1. Gene expression 2. Membranes 3. Photosynthesis 4. Stomata 5. Chlorophyll/carotenoids 6. Respiration 7. UV-screening pigments 8. Soluble proteins 9. Rubisco/ PEP carboxylase/peroxidase activity 10. Lipids 11. Carbohydrates 12. Phytohormones 13. Ion transport 14. Leaf characteristics (number, area, dry weight, thickness, specific leaf area, chlorosis/bronzing/glazing, reflectance/light penetration into leaves, structural alteration of leaf surface/epicuticular waxes) 15. Pollen/flowering/reproductive potential 16. Whole plant (height, dry matter production, carbon allocation, yield) 17. Communities-alteration of competitive balance

13 B. The present study

B.I Objectives

The present study investigated the effects of ultraviolet radiation on Hibiscus rosa-sinensis L., sugar beet {Beta vulgaris L.) and sunflower (Helianthus annuus L.). In particular, the effects of UV-B radiation on two light emission phenomena, chlorophyll fluorescence and ultraweak luminescence (UL) were studied, in addition to alteration of plant growth. Parameters related to light emission were also measured, and finally the effect of UV-B radiation on hypocotyl elongation and level of free IAA were examined. The experiments in paper I were designed to test whether UV radiation affects ultraweak luminescence and chlorophyll fluorescence. The results of this paper stimulated the experiment of paper II, where the effects of UV radiation on UL and chlorophyll fluorescence as well as on growth were studied in sugar beet plants. The objective was to determine how UV radiation affects the aforementioned parameters of plants grown with light either enriched with UV-A / blue light or depleted in this region of the spectrum. The results of papers I and II provided evidence that UV radiation might act on plants via activated forms of oxygen and/or short-lived electronically excited molecules (see below). In addition, the growth of sugar beet plants exposed to both UV-B and low background irradiance was reduced. The experiments of paper III were designed to investigate how UV-B radiation affects UL and growth of sugar beet plants grown with relatively high background irradiance. Cercospora leaf spot disease was also introduced. This is one of the most widespread diseases of sugar beet, and the pathogenicity of the fungus, Cercospora beticola Sacc, is due to the production of singlet oxygen and superoxide anion via a photosensitizer produced in the fungus (Daub 1982, Daub and Hangarter 1983). Paper III also attempts to answer how the combination of these two stresses affect UL and sugar beet growth. The last part of the study (paper IV) focuses on the effects of UV-B radiation on sunflower plants. The aims of this paper were threefold: a) to examine whether the inhibition of hypocotyl elongation in sunflower by UV-B radiation is accompanied by decreased levels of IAA, b) to study how UV-B radiation otherwise affects the development of sunflower seedlings as well as the development of the individual leaves, and c) to study how UV-B radiation » affects the photosynthetic machinery of the individual leaves during their I ontogenesis. [

14 B.2 Experimental material and conditions

Detached leaves of Hibiscus rosa-sinensis were selected as experimental material in the first study (paper I) and exposed to short term UV radiation (Material and Methods, paper I). It was found that detached leaves could remain in darkness for as long as one week without any trace of visible senescence when kept moist in Petri dishes. Sugar beet (Beta vulgaris) was selected for papers II and III. Plants from an inbred genotype (in paper II) or clonally propagated from meristems of a single donor plant (paper HI) were used. The reason for this was to minimize a possible differential action of UV radiation on different genotypes. In paper II, sugar beet plants were grown in growth chambers for three weeks under low irradiance, with or without UV radiation. The background light was: i) white light (WL, which included UV-A and blue light), ii) WL together with supplementary UV-A (WL+UV-A) and iii) "yellow" light (YL, depleted in the region below 450 nm). Detailed information about the background irradiance is given in Table 1 of paper II. In paper III, sugar beet plants infected with Cercospora beticola Sacc. and non-infected plants were grown in a greenhouse under relatively high background irradiance, with or without UV-B radiation for 40 days (Materials and Methods, paper III). In paper IV, sunflower (Helianthus annuus) plants were exposed to two different background irradiances (low and high), with or without two levels of UV-B radiation (Materials and Methods, paper IV).

B.3 Parameters measured

B,3.1 Light emission

Plants not only absorb photons but they also emit them. Fluorescence, delayed light emission and ultraweak luminescence are three kinds of light emission. The first two involve re-emission of absorbed light energy from the photosynthetic machinery, whereas in ultraweak luminescence, the energy is derived from chemical reactions. All three phenomena are useful in physiological and biochemical studies of plants. Thus fluorescence and delayed light emission are used to study photosynthesis, and ultraweak luminescence is used as an index for the existence of free radicals and activated forms of oxygen. In the present study, the effects of UV radiation were studied on chlorophyll fluorescence and ultraweak luminescence.

B.3.1.1 Chlorophyll fluorescence (papers I, II and IV)

Light absorbed by chlorophyll within leaves may drive photosynthesis, be lost 15 as heat, or re-emitted as red and far-red fluorescence. When dark adapted leaves or chloroplasts are suddenly illuminated, the fluorescence follows a certain kinetic pattern known as the Kautsky curve (Papageorgiou 1975). This curve has different phases usually denoted as O, I, D, ,P, S, M, T (Krause and Weis 1984, Papageorgiou 1975). The OIDP phases, the fast induction kinetic, is mainly related to primary photochemistry of PSII, whereas the SMT, the slow kinetic, is complex and is associated with rearrangements of pigment proteins and interactions between processes in the thylakoids and in the reductive carbon cycle of the stroma (Krause and Weis 1984). At room temperature almost all fluorescence arises from Chi a molecules associated with PS II. Thus changes of chlorophyll fluorescence reflect changes in the primary processes of photosynthesis such as excitation energy transfer from PS II to PS I (Krause and Weis 1984). In the present study, the O (Fo) and P

(Fmax) phases of the Kautsky curve, and the parameters derived from them were considered. One of the most popular parameters of chlorophyll fluorescence = re erre t0 as induction is the ratio Fy/Fmax (where Pv ^p(max)"^o' f d variable fluorescence). This is proportional to the quantum yield of O2 evolution of intact leaves (Adams et al. 1990, Björkman and Demmig 1987, Demmig and Björkman 1987, Leverenz and Oquist 1987). The parameters Fo, Fy, Fy/F0, and the rate of rise to Fmax have been used in photosynthesis research and for the detection and quantification of stress effects on the photosynthetic machinery. For example, different environmental stresses increase Fo. This has been interpreted as a result of decreased efficiency of energy transfer from the antenna Chi a to the reaction centers and/or destruction of PS II reaction centers (Briantais et al. 1986). An increase in Fo accompanied by a drop in Fv may indicate photoinhibition (Björkman 1986, Baker and Horton 1988). The half-rise time (one half of the time required for fluorescence to rise from O to P) is related to the rate of the photochemical reaction and the pool size of electron acceptors on the reducing side of PS II, including the plastoquinone pool (Oquist and Wass 1988). It was shown that this parameter is a sensitive indicator for stress (Bolhar-Nordenkampf et al. 1989). The ratio F690/F735 (at room temperature) has been used as an indicator of chlorophyll content and stress conditions (Lichtenthaler and Rinderle 1988). In healthy plants, the ratio F690/F735 is between 0.85 to 1.05. Values above 1.1 may indicate lowering of chlorophyll content, and for severe stress the F690/F735 may be above 1.2. In UV research, chlorophyll fluorescence induction has been used as a tool for determining sites of damage by UV radiation (for review see Bornman 1989). In the present study, the chlorophyll fluorescence induction was measured on dark adapted leaves (papers I, II and IV). Detailed description of the instrumentation and the procedures are given in "Materials and Methods" 16 of the individual papers. In paper IV, chlorophyll fluorescence emission spectra were also determined at room temperature. From the emission spectra, the ratio of the two fluorescence maxima F690/F735 was determined at the actual maxima measured near 690 and 735 nm.

B.3.1.2 Ultraweak luminescence (papers I, II, and III)

According to Abeles (1986) ultraweak luminescence (UL; other terms are biological luminescence, biological chemiluminescence, spontaneous luminescence and biophoton emission) is "an oxidatively driven production of light, has an emission spectrum that ranges from 200 nm to 700 nm, an intensity of 10 to 1000 photons s'1 cm'2, and a quantum efficiency of 10~3 to 10"14 photons per activated molecule." There are several biological systems serving as sources of UL (Cadenas 1987) with a complex chemistry, involving interactions between activated forms of oxygen, free radicals, electronically excited species and redox transitions. Nevertheless, the principal components directly responsible for UL are: a) singlet oxygen: ^O2 , b) excited carbonyls: >C=O*, and c) fluorescent compound(s) (Campbell 1988, Cilento 1984).

a) Singlet oxygen: It can be formed in many biochemical reactions (Cadenas 1987, Kanofsky 1989, Knox and Dodge 1985). In plants the most abundant photosensitizer is chlorophyll. Energy transfer from illuminated chlorophyll to ground state molecular oxygen (3(>2) results in the production of singlet oxygen.

Chi + hv —> 3Chl 3 3 l Chl + O2 —> Chi + O2 Interactions of activated forms of oxygen, i.e superoxide anion (O2"), hydrogen peroxide (H2O2), and hydroxyl radical (*OH), or reactions via u peroxidase or lipoxygenase may also produce singlet oxygen. It is also I produced via the Russell mechanism of peroxyl radical recombination I reactions during (lipid) peroxidation.

J I 2 >CHOO —> >CHOH + O2 + >C=O 3 3 I or: 2>CHOO—> >CHOH + O2 + >C=O and 3 3 \ O2 + >C=O —-> 'O? + >C=O f When singlet oxygen decays as an individual molecule and returns to the I ground state it produces light at 1268 nm (monomole emission). When two I molecules of singlet oxygen co-operate, the light produced is at 634 and 703 I nm (dimole emission). Transitions to or from other vibrational levels can I 17 generate light of other wavelengths (Slawinski 1988). b)Triplet carbonyls ( >C=CP): The carbonyl groups of ketones and aldehydes are polar with relatively low energy levels (Slawinsk*. 1988) and thus the excited triplet state can be easily reached in biological systems (Cadenas 1984, 1987, Cilento and Adams 1988). Any unsaturated compound that can react with various activated forms of oxygen produces dioxetanes, which subsequently produce excited ketones and aldehydes. The Russell mechanism of peroxyl radical recombination reactions is an example. Triplet carbonyls have a heterogeneous emission spectrum with the maximum in biological systems around 500-560 nm (Inaba 1988), whereas a broad emission of 375- 455 nm in a model system has also been observed (Cilento 1984). c) Fluorescent compounds: The light emission is due to the energy transfer from an excited molecule (A*) to a fluorescent compound (Fluoro) e.g. chlorophyll.

A* + Fluoro —> Fluoro*—> light Fluoro*-—> Fluoro + light

Endogenous UL is the sum of the various emissions. It can provide information on the production and the levels of activated forms of oxygen and/or lipid p^roxidation in a biological object. In some cases a correlation between UL and lipid peroxidation has been observed (for discussion see Murphy and Sies 1990). It has been suggested that measurements of UL may give a more sensitive estimation of lipid peroxidation than the thiobarbituric acid assay (Cadenas et al. 1984, and references therein). Usually high levels of UL indicate high levels of activated forms of oxygen and/or elevated (lipid) peroxidation activity. However, since it is the sum of different processes, the identification of its exact source is difficult. Virtually any cell membrane can emit UL. The amount of UL can be increased by different stresses such as wounding, disease, chemicals, radiation and by electrical and thermal stress (Abeles 1986). The effects of UV radiation on ultraweak luminescence from different organs have also been studied (Table 3). With one exception (see Table 3), an increased in UL was observed from tissues exposed to UV radiation. i In the present study, UL was measured from leaves (papers I, II and * III) and storage roots (paper HI) with the apparatus described in paper I. ' Delayed fluorescence from the photosynthetic system was minimized by ;| keeping the leaves or the plants in darkness for 24 h before measurements. j

18 Table 3. Effects of UV radiation on UL form different biological objects.

Object effect References pea sprouts decrease Veselova and Veselovskii (1971) human erythrocytes increase Korchagina and Vladimirov (1972) rat liver increase Fish et al.(1965)*, Inaba (1988) rat plasma increase Inaba(1988) rat heart increase Inaba (1988) rat lung increase Inaba (1988) rat kidney increase Inaba (1988) rat skin homogenate increase Torinuki &Miura(1981) rat skin in vivo increase Torinuki&Miura(1981)

* cited by Barenboim et al. 1969.

B.3.2 Peroxidase activity and lipid peroxidation

Peroxidase and lipid peroxidation are frequently reported to be associated with UL, therefore peroxidase activity (papers I and II) and lipid peroxidation (paper III) were also measured (for procedures see "Materials and Methods" of individual papers). Abeles et al. (1979) speculated that the UL from plants was due to peroxidation of unknown endogenous substrates. Salin and Bridges (1981, 1983) also suggested that UL was linked to peroxidase activity. The exact mechanism of peroxidase-generated UL is presently unknown but singlet oxygen and excited triplet carbonyls were probably involved during peroxidase-catalyzed oxidation of various substrates such as IAA and MDA (Cadenas 1984). In systems undergoing lipid peroxidation, some of the light produced arises through a Russell-type mechanism of peroxyl radical recombination (see above) or from self reaction of alkoxyl radicals (Halliwell and Gutteridge 1989). To find out whether or not higher UL is accompanied by elevated lipid peroxidation in sugar beet leaves (paper III), lipid peroxidation was assayed with the thiobarbituric acid (TBA) test. This is perhaps the most widely used method for measuring lipid peroxidation. The lipid material is simply heated with TBA at low pH and the pink chromogen formed is measured spectrophotometrically at 532 nm. Using the extinction coefficient of the MDA-TBA product the results are expressed as nmol of MDA. This method has been critically discussed by Halliwell and Gutteridge (1989,1990). 19 In some cases it has been found that UL is accompanied by accumulation of MDA (Cadenas and Sies 1984, Murphy and Sies 1990). Inaba (1988) presented evidence that UL was associated with MDA levels for tissues exposed to UV radiation.

B.3.3 Photosynthetic pigments (papers I. II. Ill and IV^

Chlorophylls and carotenoids are pigments involved in the two light emission phenomena described above. The Fo and Fmax values in fluorescence induction curves depend to a large degree on leaf chlorophyll content (Lichtenthaler and Rinderle 1988). Chlorophyll also contributes to UL. Carotenoids, apart from transferring energy in the photosynthetic machinery, play vital roles against photo-oxidation processes by quenching singlet oxygen, and triplet state chlorophyll, as well as scavenging free radicals (Koyama 1991, Krinsky, 1979, Rau 1988). Therefore photosynthetic pigments were estimated throughout the present study. Descriptions of the methods are given in the individual papers.

B.3.4 Stomatal resistance (paper IV)

Net photosynthesis may also be limited by the diffusion of carbon dioxide entering the leaf through the stomata. Extensive work by Negash (1988) has show that UV-B radiation closes stomata and its effectiveness depends on both the quality and quantity of background light. Stomatal resistance was measured on the leaves at the first and second node of sunflower as described in "Materials and Methods" of paper IV.

B.3.5 Plant growth

Growth was measured on sugar beet and sunflower seedlings. a) Sugar beet (papers II and III) In growth chamber experiments (paper II) the effects of UV radiation combined with different qualities of background light on growth were studied. Leaf area, fresh and dry weight of storage roots were measured. In greenhouse experiments (paper III), the effects of UV radiation given alone or after and together with inoculation of Cercospora were determined by measuring fresh and dry weights of leaf laminae, petioles and storage roots. b) Sunflower (paper IV) In order to test whether inhibition of hypocotyl elongation by UV-B radiation is accompanied by lower levels of IAA, hypocotyl elongation was followed until it ceased in sunflower seedlings grown under low and high PAR, with or without UV-B radiation. Subsequently, the relative elongation rates of 20 hypocotyls were calculated. Amounts of free IAA were estimated in hypocotyls (see paper IV; materials and methods, experiment 1). Thereafter the effects of UV-B radiation on sunflower plants grown under high irradiance were studied by recording a number of parameters (see paper IV; Materials and Methods, experiment 2).

B.4 Results and discussion

B.4.1 Chlorophyll fluorescence, photosynthetic pigments and stomatal resistance

Non-irradiated detached leaves of Hibiscus had a constant Fv/F0 throughout the experiment, and this suggests that the length of the dark incubation period did not affect the photosynthetic machinery. UV treatments lowered the Fy/F0 ratio (paper I, Fig. 3). Probably PS II is the target of UV radiation, since no difference in chlorophyll content was found between control and UV treated leaves. Alternatively, structural damage of cell membranes might also be involved, since it was noted that during the time that leaves were kept in darkness after UV treatment, leakage from cell membranes occurred, leaving a brown exudate on the leaf surface. For sugar beet plants grown in growth chambers (paper II), supplementary UV-A radiation (WL+UV-A) did not have any effect on Fo,

Fmax and Fy/F0 values, but it decreased the half rise-time compared to WL (paper II, Table 5). One difficulty for the accurate interpretation of the effects of UV-A radiation on chlorophyll fluorescence was that UV-A radiation decreased Chi a content compared to control (WL) treatments (paper II, Table 4). Nevertheless, the decrease of the half-rise time might be due to change in the rate of photochemical reactions or change in the plastoquinone pool size by 2 1 UV-A radiation. A 6.17 kJ nr day UV-BBE radiation (WL+UV-B treatment) decreased Fo, Fmax and Fy/F0 and increased the half-rise time compared to WL and WL+UV-A treatments (paper II, Table 5). Lowering of Fmax by UV-B radiation has also been observed previously (Bornman et al. 1984, Iwanzik et al. 1983) and it was suggested that UV radiation increases radiationless de-excitation. Here, the lower Fo and Fmax may simply be a result of the decrease of Chi a content under WL+UV-B treatment (paper II, Table 4). However, the decrease of Fo in combination with the increase of the half-rise time indicates that the number of heat-deactivating centres is increased. In addition, a damage to the donor side would probably lower the Fmax combined with an increase of the half-rise time (for discussion see Bolhar-Nordenkampf et al. 1991). UV-B radiation has more than one target in PS II reaction centres and these are likely to be affected to varying degrees 21 (Bornman 1989, Melis et al. 1992). It is very difficult to ascertain the primary molecular target(s) of UV-B radiation. Supplementary UV-A radiation (WL+UV-A+UV-B treatment) cancelled the UV-B radiation effect for the half-rise time but no counteractive effect with UV-B was observed on the other fluorescence parameters (paper II, Table 5). For sunflower grown under 800 \unol nr2 s*1 PAR, a 6.3 2 1 kJ m" day" UV-BgE radiation did not affect Fo but increased Fmax and consequently Fv values (paper IV, Figs 7 and 8). The higher F^/Fj^^ ratios were observed in leaves from the seedlings exposed to high light together with UV-B radiation (paper IV, Figs 7 and 8) suggesting that these leaves had higher quantum yields of photosynthesis than those from plants exposed to high light only. Even though UV-B radiation can drive photosynthesis (Halldal 1964, Me Leod and Kanwisher 1962), the number of incident photons in the UV-B region was negligible (4.6 nmol nr2 s"1) compared to 800 |imol nr2 s"1 of background irradiance. Therefore, it is proposed that in the present study the higher Fv/Fmax observed with UV-B radiation is an indirect effect of the faster leaf development with UV-B radiation (paper IV, Fig. 5). The Chi a and carotenoid content, as well as the F690/F730 ratio were also higher in leaves from node 3 in sunflower plants exposed to high light together with UV-B radiation (paper IV, Table 3). It is noteworthy that the F690/F735 ratios observed in leaves from plants exposed to UV-B radiation were closer to the values observed for healthy plants (Lichtenthaler and Rinderle 1988) than those from plants grown under high light only. Concerning the effects of UV-B radiation on stomatal resistance, leaves at node 1 from plants exposed to 800 jimol m~2 s"2 PAR with 6.3 kJ m"2 day"1 UV-B^ radiation had 50 % less resistance than those from plants exposed to only 800 nmol nr2 s"1 PAR. However, there was no statistically significant difference between the treatments for leaves at node 2.

B.4.2 Ultraweak luminescence, peroxidase activity and lipid peroxidation

In both UV-C and UV-B treated Hibiscus leaves, but not controls, UL intensity first gradually increased and started to decline 72 h after the treatment (Paper I, Fig. 1). The percentage of the light emission with wavelengths exceeding 612 nm gradually increased (paper I, Fig. 2). This means that UV-induced UL has at least two emitters with different emission spectra, and that the contribution of the longer wavelength emitter to UL increases with time. The peroxidase activity rose and declined in a similar manner (paper I, Fig. 4). For sugar beet plants grown in growth chambers (paper II), the highest levels of UL occurred in leaves of plants exposed to WL+UV-B (paper II, Fig. 2). Supplementary UV-A radiation lowered the UL (UL in 22 WL+UV-A treatment was lower than that in WL treatment; UL in WL+UV-B+UV-A was lower than that in WL+UV-B treatment). In all cases, the bulk of UL exceeded 612 nm (paper II, Fig. 2). Peroxidase activity assayed in leaf extracts was approximately 10 times higher from plants grown under WL+UV-B than the activity from plants grown under WL, with the activity being about 8 times higher in plants grown under WL+UV-A+UV-B conditions (paper II, Table 3). This decrease of peroxidase activity and the lowering of UL with inclusion of UV-A together with UV-B radiation suggests that UV-A has an ameliorating effect by decreasing the photooxidative reactions. In sugar beet plants exposed to 500 nmol nr2 s"1 PAR (paper III), a 2 1 6.91 kJ nr day" UV-BgE radiation given alone increased the UL from leaves relative to control treatments (paper III, Fig. 1) but had no effect on the UL of storage roots (paper III, Fig. 2). The highest value of UL was observed with infected plants (whether exposed or not to supplementary UV-B radiation). This was also true for UL detected from the surface of storage roots, suggesting an indirect effect of the treatments on these roots. In all cases, only half of the UL was light with wavelenth above 612 nm indicating that the difference in chlorophyll content between the treatments (paper III, Table 2) did not influence the red portion of UL. UV-B radiation lowered the lipid peroxidation by 22 %, whereas Cercospora leaf spot disease increased it by 57 % compared to controls, measured with the TBA assay and expressed as concentration of MDA (paper III, Table 3). The combination of UV-B radiation and Cercospora leaf spot disease resulted in a drastic increase of lipid peroxidation (twice as much as in the control). However, levels of UL were not correlated with the amounts of MDA suggesting that lipid peroxidation might not be not the main source of UL. For example, even though leaves from infected plants exposed to UV-B radiation had much higher levels of MDA than those from infected plants under visible light, UL was about the same in both cases. However, it should be noted that although the amount of MDA in some cases correlates with UL of systems undergoing lipid peroxidation, MDA and UL emitters are formed by different pathways and at different times during the oxidative lipid deterioration process (Cadenas and Sies 1984). In addition, even though the TBA test is perhaps one of the most frequently used tests for measuring lipid peroxidation, it lacks specificity. The bulk of MDA that reacts in the TBA test is not present in the sample but is formed by decomposition of lipid peroxides during the heating stage of TBA assay (Halliwell and Gutteridge 1990, and references therein). Thus, even if peroxides do not decompose, the TBA assay can still detect them because of decomposition of peroxides during the assay itself. There are several compounds (carbohydrates, amino acids and DNA), other than MDA, that on heating with TBA give products that absorb at or close to 532 nm (Halliwell and Gutteridge 1989). The photosynthetic pigments and phenolic compounds might also interfere. Therefore measurement at 532

23 nm after a TBA assay could include contributions from these substances. The lack of specificity of the TBA assay has been shown for human plasma (Marshall et al. 1985). Using a specific enzyme method Marshall et al. (1985) estimated a mean peroxide content in human plasma of about 0.5 JIM, whereas when they expressed the results from a TBA method in terms of peroxide equivalents, they obtained a mean value of 38 \iM. Thus UV-B radiation and Cercospora leaf spot disease might decrease or increase one or more of these compounds which interfere with the measurement. Both the UL and the TBA assay lack specificity. In the case of UL the lack of specificity is not important, since endogenous UL (as the sum of various emitters) serves generally to describe the levels of peroxidation activity and activated forms of oxygen. On the other hand, the lack of specificity of TBA assay may lead to serious over- or underestimation of the lipid peroxidation occurring in the object. Thus the higher UL for leaves of non-infected sugar beets exposed to UV-B radiation compared to that for leaves of control plants indicates higher levels of activated forms of oxygen and peroxidation processes. Whether or not UV-B radiation actually decreased the levels of lipid peroxidation as it was found with the TBA assay cannot be determined with the method used.

B.4.3 Plant growth

Light below 450 nm is vital for plants exposed to UV-B radiation. Beta vulgaris grown with visible light depleted in this region of the spectrum, and exposed to UV-B light conditions, died 10 days after their transfer to experimental conditions. Blue light regulates many processes e.g. morphogenesis, cell growth and the cell cycle, stomatal opening and metabolism. The photoreactivation enzymes also have maximum efficiency in the UV-A/blue region of the spectrum. A process induced by UV-A radiation is photoprotection, a phenomenon by which near-UV administered before the inactivating UV will reduce the effect of the latter UV. Both WL+UV-A and WL+UV-B lowered the fresh and dry weight of storage roots and the leaf areas. The lowest values of these parameters were observed in plants grown under WL+UV-A+UV-B (paper II, Table 2). Non-infected sugar beet plants exposed to 500 ^imol m"2 s"1 PAR together with 6.91 kJ m"2 day'1 UV-Bgg radiation had a 19 % higher leaf dry weight than controls (non-infected plants exposed to only 500 jimol m'2 s'1 PAR). No significant difference was observed between those two treatments for any other growth parameters (paper III, Table 1). Moreover, there was no difference in growth parameters between controls and infected plants. The combined stress of UV-B radiation and Cercospora leaf spot disease decreased the dry matter of leaf laminae, petioles and storage roots by 32, 38 and 23 %, respectively, compared to the dry weight of the control plants (paper III, Table 1) showing that the two stresses together are more effective than the 24 sum of the separate stresses. To my knowledge, there is only one earlier report (Ambler et al. 1978, cited in Teramura 1983) where the effects of UV-radiation on the growth of sugar beet plants were studied. There massive UV irradiances simulating up to an 8-fold increase in ambient levels, decreased shoot dry weight and root fresh weight. In the present study, sugar beet plants were exposed to moderate UV irradiances simulating a 5 or 9 % reduction in the present amount of stratospheric ozone for Lund, Sweden on a cloudless summer day. Although different lines of sugar beet were used in papers I and II, a comparison of growth parameters from plants grown under low irradiance and exposed to UV-B radiation (paper II, Table 2) with those from plants grown under high irradiance and exposed to a similar level of UV-BBF (paper III, Table 1) reveals that sugar beet plants under high irradiance are more tolerant to UV-B radiation than those grown under low irradiance. Sunflower seedlings exposed to low irradiance (143 ^imol m~2 s"1 PAR) had the same hypocotyl elongation and the same amount of free IAA in hypocotyls as those exposed to low irradiance together with 6.3 kJ m"2 day1 UV-Bge radiation (paper IV, Figs 1 and 2; Table 1). The same was true for plants exposed to high irradiance (800 ^mol m'2 s'1 PAR), with or without 6.3 kJ m*2 day1 UV-Bgg radiation. Low irradiance gave longer hypocotyls with lower amount of free IAA than the high background irradiance. On the 2 1 other hand, 21 kJ m~ day UV-BBE radiation inhibited hypocotyl elongation (paper IV, Figs 1 and 2) but increased the amount of free IAA in hypocotyls (paper IV, Table 1). Studying the effects of UV-B radiation on hypocotyl elongation of sunflower, Ros (1990) found that UV-B radiation inhibited hypocotyl elongation. This was accompanied by decreased levels of IAA by UV-B radiation and die formation of photo-oxidation products with an inhibitory effect on plant growth (Ros 1990). His results were based on: (i) experiments ; in which sunflower seedlings were grown under low background irradiance 2 1 2 1 f (50 nmol m" s" ) and exposed to high UV-BBE radb.tion (ca 30 kJ m" day ) f and (ii) extraction of IAA from whole seedlings. The data presented here \ suggest that environmentally more relevant UV-B radiation does not inhibit hypocotyl elongation of sunflower var. Uniflorus and they are also consistent with the data presented by Tevini et al. (1989) where enhanced UV-B radiation practically does not inhibit hypocotyl elongation of sunflower cv. Polstar. In \ the present study at higher levels of UV-B radiation, the inhibition of I hypocotyl length was not accompanied by decreased levels of free IAA in ' hypocotyls. ••• Sunflower seedlings exposed to high irradiance with 6.3 kJ nr2 day1

UV-BQE radiation had more leaves (paper IV, Fig. 3) and larger leaf areas | (paper IV, Figs. 4 and 5) than those exposed to high irradiance only, although this difference gradually disappeared by the end of the experiments. At the \ 25 end of the experiment, higher specific leaf area was observed in plants exposed to high irradiance with 6.3 kJ nr2 day"1 UV-B^ radiation (paper IV, Table 2).

C. Concluding remarks

The higher levels of ultraweak luminescence observed from leaves exposed to UV radiation lead to the conclusion that part of the effect of UV radiation is due to activated forms of oxygen and free radical formation. However, the magnitude of this action depends on the experimental conditions and it is not necessarily correlated with the effects of UV-B radiation on plant growth. For example in paper III, UV-B radiation resulted in higher UL and at the same time increased leaf dry weight. The interaction of Cercospora leaf spot disease with UV-B radiation gave the highest decrease in dry matter production, whereas this interaction did not increase the UL level from infected leaves.

Ultraviolet radiation affected photosynthesis in the three plant species used in the present study. Using different parameters of chlorophyll fluorescence, the effects were either negative {Hibiscus rosa-sinensis and Beta vulgaris), or positive (Helianthus annuus) under the conditions used.

The action of UV-B radiation on sugar beet plants depended on both light quality and quantity. Under low light, UV-B radiation decreased growth, whereas under high light, it did not. It was also apparent that blue light was necessary for growth under low light conditions.

The effects of UV-B radiation on plants may be altered by other stresses. In paper III, it was also shown that while UV-B radiation increased leaf dry weight of leaf laminae and had no effect on dry weight of the other organs of sugar beet, a large reduction of dry matter was found when sugar beet plants were infected with Cercospora beticola.

Concerning the action of UV-B radiation on hypocotyl elongation and levels of free IAA, the conclusion is that environmentally more relevant UV-B radiation does not inhibit hypocotyl elongation and does not decrease the level of free IAA in sunflower var. Uniflorus. Furthermore, the higher level of UV-BQE radiation inhibited hypocotyl elongation and increased free IAA concentration in hypocotyls suggesting that another mechanism probably exists for UV-B action on hypocotyl elongation. UV-B radiation stimulated growth at the first stage of plant development and resulted in higher photosynthetic quantum efficiency as measured by the ratio Fv/Fmax- Overall, environmentally relevant levels of UV-B radiation were not harmful to the sunflower var. Uniflorus. 26 The final conclusion from the present study is that ultraviolet radiation has both harmful and enhancing effects on plants. While it is relatively easy to assume the mechanisms and explain the action for the harmful effects, it is, at present, very difficult to explain the enhancing effects of UV-B radiation.

27 Acknowledgements

I would like to thank all those who in different ways have contributed to this work.

My supervisor Professor Lars Olof Björn for your scientific and financial support. Your comments and criticism were invaluable throughout my studies. You showed me the "secrets" of UV research and taught me that any positive effect of UV radiation should not influence us negatively.

My second supervisor Dr. Janet F. Bornman for your excellent guidance and generous support.

Prof Chris H. Bornman: You were the first person who mentioned UV-B radiation research to me. Somehow this directed my interest and made me to leave the field of plant tissue culture and work with a more ecological problem.

Dr. Nils Ekelund: for your warm friendship. You were always ready to listen to me and give your suggestions.

Irina Staxén: my room-mate and friend. We consumed many cigarettes together and in this smoky atmosphere had hot discussions in the field of UV- B radiation.

Ingvar Jönsson: you showed me the practical side of plant science. You taught me about aphids and plant stresses in the greenhouse. Thanks a lot for taking care of the plants, even during weekends.

Per Westergren and Bengt Loven: Thank you for your invaluable help in setting up the light systems.

All the other friends and collegues at the Department of Plant Physiology, University of Lund.

Professor Göran Sandberg and Dr. Björn Sundberg as well as the anonymous technicians at the Department of Forest Genetics and Plant Physiology, The Swedish University of Agricultural Science, Umeå, for IAA analysis.

Thanks to my parents. In spite of being far away, your calls and long discussions were invaluable to me. Your willingness to pay thousands of drachmas to the Greek Telecommunication Organization is highly appreciated.

I would like to specially thank my dear Annika for enriching my life.

28 The tax-payers in Sweden and in the countries of The European Community are highly acknowledged. They paid my "utbildingsbidrag" (Swedish tax- payers) and a grant received from the Commission of the European Communities, Directorate General for Science, Research and Development; Joint Research Center; Div XIIE-1, Environmental Programme (the "European" tax-payers). I would like also to acknowledge Swedish Environmental Protection Agency (SNV), Swedish Natural Science Research Council (NFR) and Hilleshög AB for partial financial help.

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39