Amelioration of Ultraviolet-B-lnduced Down-Regulation of mRNA Levels for Chloroplast Proteins, by High Irradiance, is Mediated by Photosynthesis

j. Plant Physiol. \tOl. 148. pp. 100-106 (1996)

Amelioration of Ultraviolet-B-Induced Down-Regulation of mRNA Levels for Chloroplast Proteins, by High Irradiance, is Mediated by Photosynthesis SOHEILA

A.-H.

MACKERNESS,

P. JOE

BUTT, BRIAN

R.

JORDAN*,

and

BRIAN THOMAS

Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, UK Received June 24, 1995 . Accepted October 5, 1995

Summary

The mechanism by which increasing photosynthetically active radiation (PAR) reduces the sensitivity of RNA transcripts to UV-B radiation was studied in pea (Pisum sativum L.). mRNA transcript levels for rkc 5, rbc L, c~b ~nd psb A were measured. ~ver an 8 d experi~ental period in_~ea plants suppleme~ted WIth UV-B radIation under a range of conditions. Under low hght (150 /lmol m s-I), UV-B resulted In a significant decline in the levels of transcripts for all four genes which was prevented by increasing the background irradiance to 350 /lmol m -2 S-l (high light) with white light from fluorescent lamps. Increasing CO 2 levels to give photosynthesis rates equivalent to the high light treatment partially protected rbc 5 and cab transcripts and fully protected rbc L transcripts but did not prevent visible injury. Increasing light with low pressure sodium lamps, which increase photosynthesis but are not effective for activation of the DNA repair enzyme, photolyase, gave results which were not significantly different from white fluorescent high light treatments. Protection by high light was lost in the presence of the photosynthesis inhibitors CCCP and DCMU. The UV-B induced increase in the expression of chalcone synthase (chs) genes was delayed by the treatments which increased photosynthesis rates and conferred protection. The results indicate that photosynthesis plays a key role in the amelioration of UV-B induced decline in mRNA levels for proteins. The minimal role of DNA repair by photolyase indicates that reduction in photosynthesis gene transcripts in response to UV-B represents a specific regulation rather than being a consequence of DNA damage.

Key words: Pisum sativum, Photosynthesis, high PAR, RNA tramcripts, UV-B stress. Abbreviatiom: UV-B = Ultraviolet-B radiation; PAR = Photosynthetically Active Radiation; RUBISCO

1,5-bisphosphate carboxylase/oxygenase; LL = Low light; HLw = high broad spectrum light; HL Na = high light using sodium lamps; H C02 = high CO 2 (LL); LPS = low pressure sodium lamps; CCCP = carbonyl cyanide m-chlorophenylhydrazone; DeMU = 3-(3,4 dichlorophenyl) dimethylurea.

= ribulose

Introduction

Reduction in stratospheric ozone has raised concern over the increases in the levels of ultraviolet-B radiation (UV-B: 280-320 nm) reaching the earth's surface (Blumthaler and • New Zealand Institute for Crop and Food Research Limited, Levin Research Centre, Kimberley Road, Private Bag 4005, Levin, New Zealand. © 1996 by Gustav Fischer Verlag, Srullgall

Ambach, 1990). Exposure of plants to UV-B most notably leads to reduction in photosynthesis resulting in biomass reduction; anatomical changes such as decreases in root to shoot ratio and development of smaller, thicker leaves; (Teramura, 1980; Krizeck, 1975) and increases in the UV-B absorbing flavonoids (Tevini et al., 1991; Robberecht and Caldwell, 1986; Warner and Caldwell, 1983). Recently molecular studies have shown that exposure to UV-B leads to the down regulation of several key photosynthetic genes re-

Photosynthesis and UV-B induced down-regulation

suiting in the subsequent decline in the proteins. These studies have been able to explain, to some extent, the mechanism by which UV-B inhibits photosynthesis Gordan, 1995). It has bf'en recognised for some time that longer wavelength radiation (PAR: 400-700 nm) can minimise UV-B induced damage. This protection occurs at the physiological, biochemical and also molecular level (Cen and Bornman, 1990; Flint et al., 1985; Mirecki and Teramura, 1984; Jordan et al., 1992). Plants grown under high PAR have thicker leaves and increased levels of flavonoids as compared to those growth under low levels (Teramura, 1980; Cen and Bornman, 1990; Tevini et al., 1991) which tends to lessen the sensitivity of plants to UV-B (Warner and Caldwell, 1983; Murali and Teramura, 1985). These changes, however, cannot explain why plants grown under low PAR and exposed to UV-B under high PAR are less sensitive to UV-B than plants grown under low PAR and then irradiated with UV-B under low PAR (Kramer, et al., 1992; Mirecki and Teramura, 1984). The effect of high PAR, under these conditions, has been attributed to activation of photorepair mechanisms (Fernbach and Mohr, 1992; Mirecki and Teramura, 1984) which ameliorates the UV-B stress induced damage. Photorepair is thought to involve the activation of the DNA repair enzyme, photolyase (Pang and Hays, 1991). This enzyme uses energy in the range of 300 to 500 nm to reverse UV-B induced photoproducts, cyclobutane pyrimidine dimers (Sutherland, 1981). However, higher levels of visible light may also contribute to protection by providing additional substrate through increases in photosynthesis for the repair or replacement of damaged organelles or tissues (Adamse and Britz, 1992). Jordan et al. (1992) noted that increased PAR can reduce the UV-B induced down regulation of photosynthetic genes. We have used this system to study the role of photosynthesis in the protection against UV-B damage at the molecular level. In order to separate the potential contribution of photosynthesis and non-photosynthetic perception mechanisms to this protection, photosynthesis rates were manipulated by altering CO 2 levels and PAR levels using low pressure sodium lamps (LPS) which are effective for photosynthesis (Withers et al., 1991) but not for activation of photorepair enzymes. In addition, the effect of lowering of photosynthesis rates on transcript levels, under high light conditions, using inhibitors, was also studied.

101

daily. The other half of the plants (controls) were subjected to the same treatments but in the absence of UV-B radiation. For low light experiments (LL), irradiation was maintained at 150 Ilmol m- 2 s-l for the duration of the UV-B treatment. In high light experiments (HL), the illumination was increased to 350 Ilmol m- 2 S-l using either white fluorescent tubes (HLw) or low pressure sodium lamps (LPS) (HLNa ). High CO 2 (H coz ) experiments were carried out under LL conditions and at 1500 ppm CO 2 , All experiments were carried out in Weiss cabinets except for HL Na . which were carried out in Richmond cabinets as LPS lamps could be accommodated more easily in these cabinets. The spectral irradiance between 260320 nm was determined by using an Optronics 740A spectroradiometer (Optronics Laboratories, Inc., Orlando, Fl, USA). The levels ofUV-B were 4.22 and 16.7mWm- 2 nm-1 at 297nm and 313nm respectively. These UV-B levels represent 8.5 % at 297 nm and 7.5 % at 313 nm of UV-B radiation used in a comparable study Qordan et al., 1992). The levels ofUV-B in the control half of the cabinet were negligible. The third pair of leaflets was routinely used for experiments unless othetwise stated.

Photosynthesis Photosynthesis rates of the third pair of leaflets, at incident irradiation, were determined using a Combined Infrared Gas Analysis System (CIRAS-l, PP Systems, Hitchin, UK). The concentration of CO 2 in H coz experiments and irradiance levels in HLNa experiments were chosen to mimic the photosynthesis levels at the start of the HLw experiments. Values are quoted as the mean from 5 separate measurements ± standard error of the mean.

Analysis ofRNA Total RNA was extracted from leaf tissue and analysed by Northern blotting as described previously Qordan et al., 1991). Specific RNAs were detected by hybridisation with 32p labelled homologous cDNA probes. Relative amounts of hybridisation to specific bands were quantified by excising the appropriate area of the filter and determining the amount of bound radioactive probe by liquid scintillation counting in Omnisafe scintillation fluid (Life Technologies, UK). The quantitative data is presented as percentage transcripts from UV-B treated plants as compared to transcripts from control plants which were treated under the same conditions but in the absence ofUV-B irradiation. The rbc Land rbc S eDNA sequences for the large and the small subunit of RUBISCO respectively are described in Jordan et al. (1992). The psbA sequence for the D 1 polypeptide of Photosystem II is described in Jordan et al. (1991). The cab sequence for chlorophyll alb binding protein and the chs 1 sequence for chalcone synthase are described in Jordan et al. (1994).

Material and Methods Plant material and experimental conditions Pea (Pisum sativum L. cv Feltham first) seedlings were grown in a controlled environment cabinet, partitioned in two halves, with 12 h light (22°C), 12 h dark (16 0C) cycles, for 17 d. Incident radiation was provided by Philips warm white fluorescent rubes giving an irradiance of 150 Ilmol m- 2 S-l PAR. Half of the plants were then given supplementary UV-B radiation from two UV Lamps (Philips TL 40) during the 12 h photoperiod under the four experimental conditions. To exclude UV radiation below 290 nm, the UV-B lamps were wrapped in cellulose acetate sheets which were changed

Photosynthesis inhibitor studies The effect of the photosynthesis inhibitors on the transcript levels under HLw conditions were determined by using leaf discs. Ten leaf discs (1 cm 2) were floated adaxial side up in 0.1 % Tween containing either 5 11M CCCP (Sigma, UK), 25 11M DCMU (Sigma, UK) or dH 20 (control). The discs were then incubated under control or under supplemental UV-B radiation. After appropriate incubation periods, the discs were washed three times in distilled water and then frozen immediately in liquid nitrogen. The samples were prepared for Northern blot analysis as described above and quantified by liquid scintillation counting.

102

SOHElLA A.-H. MACKERNESS, P. JOE BUTT, BRIAN R. JORDAN, and BRIAN THOMAS

Statistical analysis An analysis of variance (ANOVA) was performed on quantitative data obtained from duplicate Northern analysis, for each treatment, and least significant difference (LSD) at the 5 % level determined.

Results

Visible damage In all treatments, bronzing of the leaf tissue was the first visible sign of UV-B stress and was apparent after 2 d UV-B treatment under low light (LL) conditions. This became more pronounced throughout the treatment. By 4 d the leaves had acquired a glazed appearance and by 8 d signs of chlorosis were visible. Under all other treatments, the progressive damage was less pronounced, with plants grown under high light (HLw) and high sodium light (HLNa) having reduced signs of chlorosis and tissue death after 8 d UV-B treatment. Under high CO 2 conditions (H co ) although initially plants appeared healthier than those under ambient CO 2 levels (LL) , strong bronzing became visible between day 5 and day 8 of the treatment, and their appearance was comparable to those grown under low light (LL; ambient CO 2) conditions.

Effect o/supplemental UV-B on transcript levels under LL, HL w, HL Na and H C02 conditions Total RNA was extracted from the third pair of leaflets from the base of the pea seedlings, grown in the presence and

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absence of supplemental UV-B, under all 4 experimental conditions. The integrity of the RNA was determined using Northern blot analysis. Discrete bands were observed for all probes used in hybridisations (data not shown). The steady state RNA levels were quantified by counting of the radioactive probe bound to the specific transcript after hybridisation with 32p labelled rbc 5, cab, rbc Land psb A cDNA probes and are shown in Figure 1. Under all test conditions the nuclear encoded genes, rbc 5 and cab (Fig. 1a, b) were more sensitive to UV-B treatment than chloroplast encoded genes, rbc Land psb A (Fig. 1c, d). Under LL conditions, the decline in transcript levels for all 4 genes studied in the Richmond cabinets was not significantly different from values obtained in Weiss cabinets and, therefore, the data was combined, and values represented in Figure 1 and Table 1 are average values from the 2 experiments. Photosynthetic rates of the third pair of leaflets were measured under LL conditions and were 2.5 (± 0.2) mg CO 2 dm 2 h -1 at the beginning of the experiment. Under LL conditions the nuclear encoded cab and rbc 5 transcripts were reduced dramatically by the second day of UV-B treatment, continuing to decline up to 8 d where transcripts were present only in trace amounts (Fig. 1a, b). The effect of UV-B on the chloroplast genes was not as dramatic. Transcripts for rbc L were not substantially reduced after 2 d of UV-B treatment (Fig. 1 c). The levels began to decline by the third day of treatment and continued to fall until the last sampling day. In contrast, the psb A transcript levels were not substantially affected by UV-B supplementation for up to 4 d of treatment (Fig. 1d). The percentage of RNA transcripts remaining on

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mRNA, under the four treatment conditions, after exposure to UV-B. mRNA Northern blots, probed with cDNA probes, were quantified by excides of appropriate area of the filter and counting in a liquid scintillation counter. The effect is defined as the percentage counts for UV treatments with respect to the counts from control plants, treated under the same conditions but in the absence of UV-B radiation, on equivalent days. Each point represents the mean value obtained from duplicate blots. Error bars indicate the least significant difference (LSD) taken at the 5 % level. Probes used were a rbc S, b cab, c rbc L, dpsb A. Symbols: LL (0), HLw (0), H coz (6), HLNa (+).

Photosynthesis and UV-B induced down-regulation

Table 1: Steady state levels of mRNA, under the four treatments, af-

ter exposure to UV-B for 3 d (values taken from Figure 1 and are given as the percentage countS for UV treatments with respect to the counts from control plants on equivalent days, rreated under the same conditions but in rhe absence of UV-B radiation). Values from the third day of treatment were chosen to illustrate effects of UV-B on plants during the four treatments as differences in treatments was most pronounced on this day. Means in the same row marked by are not significantly different ar the 5 % level as derermined by ANOVA. Treatments

Genes

rbcS cab rbc L psbA

LSD

LL

HLw

Heo ,

H Na

24 19 54 90-

5974 8194-

42 6091100-

60558987*

10.4 8.6 10.2 13.3

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(Fig. 1 b) where levels were slightly lower on the second to fourth day of UV-B treatment. However, the levels were still significantly higher than LL and H eo2 levels (Fig. 1 b, Table I).

Effict ofphotosynthesis inhibitors on HL w protection Increasing photosynthesis rates result in protection of the transcript levels for the 4 genes being studied (Fig. I). In order to determine whether a decrease in photosynthesis results in loss of this protection under HLw conditions, 2 photosynthesis inhibitors, DCMU and CCCP, were used in leaf disc experiments. DCMU is an inhibitor of photosynthetic electron transport and CCCP is an uncoupler of photophosphorylation. The presence of both inhibitors lead to the loss of protection under HLw conditions for all 4 genes (Fig. 2).

Changes in chs transcript levels under the four treatments the third day of each treatment from plants supplimented with UV-B as compared to control transcript levels, from plants treated under the same conditions but in the absence ofUV-B radiation, are shown for all genes in Table I. Increasing the background irradiance from 150 to 350 !Lmol m- 2 s-l resulted in an increase in photosynthetic rates from 2.5 to 8.9 (± 0.3) mg CO 2 dm 2 h -I, at the start of the experiment. The profile of RNA levels for rbc 5, cab and rbc Lunder HLw conditions was similar to LL conditions but transcript levels were not as dramatically reduced (Fig. 1a, b, c). HLw treatment had no significant effect on transcript levels for psb A. These transcripts were, however, not significantly affected by UV-B treatment under LL conditions (Fig. 1d). The protection of rbc 5 and cab transcripts was maintained throughout the 8 d UV-B treatment (Fig. 1a and b). Increased illumination under HLw conditions resulted in a significant protection of rbc L transcripts after the third day of UV-B treatment, at the point where levels sharply decline under LL levels (Fig. 1c). Table 1 clearly illustrates the extent of protection against UV-B reduction in transcript levels on the third day ofUV-B treatment. Photosynthesis rates were raised by increasing CO 2 levels, under LL conditions (HLco 2), to 8.8 (± 0.5) mg CO 2 dm 2 h -I. This photosynthesis rate was similar to that under HLw. This treatment resulted in protection of the transcripts, but only for rbc L transcripts to levels comparable to HLw (Fig. 1c). However, under H eo2 conditions, the rbc 5 and cab mRNA levels were significantly higher than under LL (ambient CO 2) conditions for the duration of the UV-B treatment (Fig. 1 a, b). The protection of rbc L transcripts under H eo2 was complete, resulting in maintenance of levels at HLw values after 3 d UV-B treatment (Fig. I c). Due to the insensitivity of psb A transcripts towards UV-B, there was little change in their levels under these conditions (Fig. I d). Low pressure sodium lights were used to increase photosynthesis to 9.0 (± 0.2) mg CO 2 dm 2 h- 1 (HL Na ), a rate again comparable to that under HLw conditions (Fig. 1). The profile of mRNA levels obtained under the HLNa • conditions was not significantly different from values obtained under HLw for all the genes (Fig. I a, c, d) with the exception of cab

UV-B irradiation leads to transient increases in chs expressions under all 4 treatments. Maximum transcript levels were reached after different time periods depending on the treatment but levels were comparable in all treatments (Fig. 3). Under LL conditions the peak value was reached on the second day in both Weiss and Richmond cabinets (Fig. 3). This peak was shifted to the third day under H eo2 conditions and to the fifth day under both HLw and HLNa conditions (Fig. 3). Discussion

In this study, by manipulating photosynthesis rates withOUt increasing the activity of photolyase, it was possible to determine the contribution of photosynthesis to the protection of transcript levels for three key photosynthetic proteins, RUBISCO, chlorophyll alb binding protein and D 1 polypeptide of PSII, under high irradiance conditions. Jordan et al. (1994) indicated that effects of UV-B on transcript levels are detectable long before any physiological differences are measurable and, therefore, this approach allows the determination of subtle effects more easily and over shorter time periods.

Increased photosynthesis protects UV-B induced decreases in tramcript levels Under all conditions, supplemental UV-B resulted in the decrease in the transcript levels for all 4 genes studied. Nuclear encoded transcripts were more sensitive to UV-B radiation than chloroplast encoded transcripts (Fig. 1). These observations are consistent with previous studies on the effect of UV-B on the mRNA levels for photosynthetic proteins Gordan et al., 1991; 1992). High irradiance during UV-B treatment reduced the UV-B induced down-regulation of the photosynthetic genes rbc 5, cab and rbc L. Similar protection for rbc 5 Gordan et al., 1992) and cab Gordan et aI., 1991) has been noted previously. Due to the relative insensitivity of psb A transcript levels to UV-B, there was little change in levels under these conditions (Fig. 1d; Table 1). The decline in transcript levels after day 4

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SOHElLA A.-H. MACKERNESS, P. JOE BUTT, BRIAN R. JORDAN, and BRIAN THOMAS

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Fig. 3: Steady state levels of leaf mRNA under the four treatments, after exposure to UV-B. Northern blots, probed with chs cDNA, were quantified by excision of appropriate area of the filter and counting in a liquid scintillation counter. The effect is defined as the percentage counts for UV treated plants with respect to the counts from control plants, treated under the same conditions but in the absence of UV-B radiation, on equivelant days. Each point represents the mean value obtained from duplicate blots. Error bars indicate the variation in duplicates. Symbols: LL (0), HLw (0), H co2 (6), HLNa (+). of UV-B treatment is most likely due to ptemature senesence of the leaves as a results of UV-B exposure.

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Fig. 2 a, b, c, d: Steady state mRNA levels of leaf discs, supplimented with UV-B under HLw conditions, after addition of water (e), CCCP (0) and DCMU (6). The effect is defined as the percentage counts for UV treatments with respect to the counts from leaf discs treated under the same conditions, but in the absence of UVB radiation, on equivalent days. Each point represents the mean value obtained from duplicate experiments. Error bars indicate the variance in the duplicate experiments. Probes used were a rbc S, b cab, c rbc L, d psbA.

Increasing photosynthesis levels to HLw levels by using high CO 2 and sodium lamps allows the separation of the potential contribution of photosynthesis and non-photophotosynthetic perception mechanisms in protection under HLw conditions. LPS lamps emit at a narrow waveband at 589 nm and do not activate DNA repair enzymes, photolyases, which require light between 300 and 500 nm (Pang and Hays, 1991). Sensitivity of transcripts to supplemental UV-B was reduced under both H eo2 and HLNa conditions for rbc S, cab and rbc L. High CO 2 levels were, however, not as effective as HLw or HLNa treatments in the protection of steady state RNA levels for the nuclear encoded genes rbc S and cab (Fig. 1 a, b) as compared to the chloroplast encoded gene, rbc L (Fig. 1c). Due to the relative insensitivity of psb A transcript levels to UV-B, again, there was little change in levels under these conditions (Fig. 1d; Table 1). Elevated CO 2 concentrations, leading to increased photosynthetic rates, resulted in a significant reduction in the decline in rbc S and cab transcripts as compared to LL, ambient CO 2 conditions, throughout the duration of the experiment (Fig. 1a, b). Previous studies have looked at the interaction of CO 2 and UV-B at the physiological level but the responses observed were inconsistent. For example, UV-B was found to reduce any CO 2 induced increases in biomass in rice and wheat (Teramura et al., 1990). In contrast, under identical conditions, no effect ofUV-B was recorded in soybean (Teramura et al., 1990). Vety few molecular studies have been carried out on plants grown in high CO 2 and none under both elevated CO 2 and UV-B conditions. In tomato, the abundance of transcripts for the nuclear-encoded chloroplast pro-

Photosynthesis and UV-B induced down-regulation teins were reduced in response to high CO 2 but chloroplastencoded transcripts were not significantly affected under the same conditions (Van Oosten et al., 1994; Van Oosten and Besford, 1994). A similar pattern was observed in the present study with pea (data not shown). Therefore, the differential effect of CO 2 on the protection of nuclear and chloroplast encoded genes towards supplemental UV-B, is likely to be due to the differences in the sensitivity of these genes to CO 2 , Transcripts of rbc L are insensitive to high levels of CO 2 and, therefore, the protective effect of higher photosynthesis under these conditions was not masked by any CO 2 effect. In contrast, rbc 5 and cab are sensitive to CO 2 and this may explain the apparently inefficient protection of transcripts. Complete protection of all genes under increased illumination by LPS lamps, a system which also does not activate photorepair mechanisms, further implies that CO 2 effects are the likely cause of the decreased protection efficiency under H eo2 conditions. These observations strongly suggest a significant role of photosynthesis in the protection of these transcripts under high irradiance and a limited contribution of photorepair mechanism. This was supported by the loss of protection when photosynthesis levels were reduced under HLw conditions using photosynthesis inhibitors, further indicating that light per se is not the major contributor to protection. The presence of photoreactivating enzyme activity has been demonstrated in many plant species, for example in water plants (Degani et al., 1980) and wheat (Taylor et al., 1995) but could not been demonstrated in several other plant species (Steinmitz and Wellman, 1986). Therefore, it is possible that in pea, photoreactivation, by photolyase, plays a negligible part in protection against short term UV-B damage or that pea does not have an active photolyase.

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day 5 for HLw and HLNa treatments, and days 2 and 3 for LL and H eo2 respectively. However, flavonoids did not accumulate to higher levels under anyone treatment (Mackerness, Jordan, Thomas, unpublished data). This observation is not surprising since flavonoid accumulation is a direct result of incident UV-B radiation (Jordan, 1993; Jordan et al., 1994) and as background lighting did not contain any UV-B, and high levels of CO 2 do not activate flavonoid synthesis (Adamse and Britz, 1992; Ziska and Teramura, 1992), the increase in flavonoids would mainly be a result of supplemental UV-B, which was constant for all treatments. In conclusion, we have shown that photosynthesis or a process linked to photosynthesis plays a key role in the amelioration of UV-B induced decline in pea mRNA levels for photosynthetic proteins. In the absence of photolyase activation, increases in photosynthesis were sufficient to account for protection of transcript levels against UV-B irradiation. Conversely, application of inhibitors of photosynthesis resulted in loss of this protection. The seemingly limited role of photolyase in this protection mechanism suggests that there is a selective and specific regulation of gene expression by UV-B radiation, corroborating previous studies (Jordan et al., 1994; Strid, 1993). Acknowledgements

This research was supported by the Biotechnology and Biological Sciences Research Council. We are grateful to Ron Pierce for running the Weiss cabinets, Rodney Edmondson for helping with the statistical analysis and to Diana Wilkins for her help with the CIRAS measurements.

References

WB stimulation ofchs expression

Chs transcripts, under all experimental conditions, were increased to a peak value in response to UV-B and then declined again. The time taken for peak values to be reached was shifted from day 2 under LL conditions to day 3 under H eo2 and day 4 under HLw and HLNa (Fig. 3). The chs genes encode the enzyme chalcone synthase, a key enzyme in the synthesis of flavonoids. Flavonoids are UV-B screening pigments produced in plants in response to UV-B radiation (Caldwell, 1981; Wellman, 1983). Increases in chs expression, in response to supplemental UV-B have been previously reported (Strid, 1993; Takeda et al., 1993). The peak shift noted in this study may indicate that stimulation of chs expression is controlled by the level of damage inflicted by UVB and that the shift represents the protection of damage by HLw , HL Na and H eo2 leading to a delay in full expression of this gene. Protection was most effective under HLw and HLNa and the delay in maximal expression of chs was longest in these treatments. However, more detailed studies will be needed to confirm this. Flavonoid concentrations under these experimental conditions have been measured and a correlation between the level of gene expression and the concentration of flavonoids during the 4 treatments has been observed (Mackerness, Jordan, Thomas, unpublished data). Maximal levels were reached at

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