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AUSTRALIAN JOURNAL OF PLANT PHYSIOLOGY Volume 24, 1997 © CSIRO Australia 1997

An international journal of plant function

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Aust. J. Plant Physiol., 1997, 24, 261–274

UV-B Irradiation Induces Differential Leaf Damage, Ultrastructural Changes and Accumulation of Specific Phenolic Compounds in Rice Cultivars Merdelyn Caasi-LitABC, Malcolm I. WhitecrossA, Murali NayuduA and Gregory J. TannerBD A

Division of Botany and Zoology, School of Life Sciences, Australian National University, Canberra, ACT 0200, Australia. B Division of Plant Industry, CSIRO, GPO Box 1600, Canberra, ACT 2601, Australia. C Institute of Plant Breeding, University of the Philippines Los Bãnos College, Laguna 4031, Philippines. D Corresponding author, email: [email protected] Abstract. Enhanced UV-B exposure of rice, Oryza sativa L., induced ultrastructural changes and visible symptoms including the formation of necrotic spots, accumulation of dark pigments, and finally desiccation of damaged leaves. Thirty-four genetically diverse cultivars were classified as either tolerant, intermediate or susceptible to UV-B irradiation on the basis of the observed damage symptoms. The development of visible symptoms was significantly reduced and delayed in highly tolerant cultivars. Highly susceptible cultivars were severely damaged by UV-B irradiation in a shorter period of time. Light and electron microscopy of these cultivars revealed intracellular and cell wall disruption in UV-B treated leaves. However, the level of disruption was significantly less in tolerant cultivars. There were significant differences in the relative levels of phenolic compounds produced in extracts of rice after UV-B exposure of the plants. Tolerant cultivars accumulated relatively higher levels of phenolics. Two prominent HPLC peaks (peak I and peak II) accumulated in response to an applied UV-B stress. There was a significant relationship between the UV-B tolerance category and the relative mean level of phenolics, and the mean area of HPLC peaks I and II. Keywords: Ultraviolet, UV-B, tolerance, susceptibility, Oryza sativa, EM, HPLC.

Introduction Depletion of stratospheric ozone caused by human activities or natural phenomena has increased the amount of radiation of wavelength 280–320 nm (UV-B) reaching the Earth’s surface. Downey (1996) showed significant stratospheric ozone losses had occurred in the two hemispheres with losses of about 4–6% per decade over the period 1979–1994. For every percentage decrease in stratospheric ozone, the amount of UV-B flux is predicted to increase by approximately double that percentage (Caldwell et al. 1989). Enhanced UV-B irradiation causes significant damage to plant ecosystems and also reduces the productivity of several economically important crop plants (Caldwell 1981; Searles et al. 1995). Increased UV-B has affected photosynthetic and growth parameters in some rice cultivars (Teramura et al. 1991; He et al. 1993; Dai et al. 1994; Hidema et al. 1996; Olszyk et al. 1996). The confirmation of a detrimental effect of UV-B would be significant because rice is a major world food crop.

Enhanced UV-B exposure leads to the formation of brown leaf spots, chlorosis and desiccation in Arabidopsis (Lois 1994) and rice (Sato et al. 1994). The accumulation of phenolic compounds appears to protect some plant tissues from direct UV-B damage. Species in which a protective effect of phenolic accumulation has been demonstrated include Arabidopsis (Li et al. 1993; Lois 1994; Ormrod et al. 1995), barley (Liu et al. 1995; Reuber et al. 1996a), beans (Beggs et al. 1985), Brassica (Alenius et al. 1995), mustard (Buchholz et al. 1995) and pea (Day and Vogelmann 1995). In rye, elevated UV-B levels increase the accumulation of flavones and isovitexin glycosides (Tevini et al. 1991) and showed the possible role of epidermisspecific flavonoids in UV-B protection (Reuber et al. 1996b). Apart from one report of synthesis of oryzalexins and momilactones (Kato et al. 1993), UV-B induced accumulation of specific compounds in rice plants has not been demonstrated. There have been three reports where the absorbance of methanol extracts has been used to estimate the amount of UV-B absorbing compounds in rice leaves 10.1071/PP96080

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Merdelyn Caasi-Lit et al.

(Teramura et al. 1991; Dai et al. 1992; He et al. 1993). These reports were unable to establish a relationship between tolerance to UV-B irradiation and the accumulation of UV absorbing compounds. Furthermore, no studies have been conducted yet on the ultrastructural changes in rice accompanying UV-B exposure. In this study, the responses of 34 rice cultivars from different provenances and maturity groups to enhanced UVB irradiation were characterised. Visible leaf damage and ultrastructural differences induced in the cultivars were examined. Leaf extracts were analysed by HLPC to determine the level and pattern of phenolic accumulation under UV-B irradiation and control conditions. The aim of this work was firstly, to determine if rice cultivars could be categorised as resistant or tolerant to enhanced UV-B irradiation; secondly, to determine if there was a common mechanism, such as the accumulation of specific phenolics, involved in tolerance of rice plants to enhanced UV-B irradiation.

Materials and Methods Plant Material Two experiments were performed, first a preliminary trial using 60-day-old plants from 21 cultivars, and subsequently, a full trial using 45-day-old plants from 34 cultivars. Both sets of results were quantitatively similar; only the results from the second trial are presented. Rice seeds were provided by the Yanco Agricultural Institute, New South Wales Department of Agriculture, Yanco, New South Wales, Australia and were chosen to represent the genetic diversity present in the rice germplasm collection (Table 1). Plant Growth Seeds were surface sterilised with 5% (w/v) sodium hypochlorite, soaked in distilled water for 2–3 days, transferred to wet tissue paper, and germinated on a mist bench at 20–25°C. When seedlings were 6–8 cm tall, they were transferred to pots (15 cm deep, 8.5 cm diameter) containing 500 g of soil. The pots were placed in a rectangular plastic tray, filled with water to the brim daily, and maintained in a glasshouse. After the first 2 weeks, the plants were fertilised with 0.2 g each of potassium chloride, ammonium sulfate and superphosphate; thereafter, they were provided with a modified nutrient solution (Cook and Evans 1983) at 2 week intervals. Ambient photosynthetic photon flux (PPF, 400–700 nm) ranged from 600 to 1200 mmol photons m–2 s–1 at midday; relative humidity was 60–70% and mean day/night air temperature was maintained at 28/18°C. Experimental Conditions and Irradiation Treatments A separate growth cabinet was used for each treatment, UV-B and control. The control cabinet had incandescent bulbs and cool white fluorescent tubes while the UV-B cabinet had cool white light, incandescent bulbs and UV-B lamps. Seedlings were transferred from the glasshouse to growth cabinets after 38 days and acclimated for 1 week prior to UV-B exposure. Each cabinet was divided into two subchambers with 34 rice cultivars arranged

randomly, two pots per cultivar and two plants per pot to make two replicates. The cabinet interiors were arranged to minimise turbulent air flow which could cause mechanical damage to leaves. The effective sampling field in the cabinet was determined using a Li-Cor light meter to designate the area with ±10% variation in UV-B dose. UV-B irradiance was measured using an IL 1700 Research Radiometer with calibrated photodetector/filters (International Light, Newburyport, USA). The visible light intensity (PPF) ranged from 300 to 350 mmol photons m–2 s–1 in the effective sampling field. UV-B irradiation in the UV-B cabinet was supplied by three UV-B lamps (Philips TL 40 W/12 UV). UV-C was removed with a sheet of 0.13 mm cellulose acetate which removed wavelengths below 290 nm. The control cabinet had a glass filter and a sheet of Mylar plastic to remove all UV-B. Both growth cabinets were maintained at a mean day/night temperature of 28/22°C with a 12 h photoperiod and 50–70% relative humidity. The height of the plant canopy was regularly adjusted to be 60 cm below the lamps to ensure uniform exposure of the plant canopy. This was done by lowering the position of the platform holding the pots. UV-B irradiated plants received biologically effective fluence (UV-BBE) of 15–16 kJ m–2 day–1 at canopy height for 10 days, which is almost twice the dose received during a Canberra summer of approximately 9 kJ m–2 day–1 (W.S. Chow, pers. comm.). This amount was weighted according to the general plant action spectrum of Caldwell (1971) and normalised to unity at 302 nm to give the same reading as the non-weighted signal at this wavelength. Assessment of Visible Symptoms The visible UV-B damage sustained by individual second and third leaves was scored in a manner similar to that used to assess insect or disease damage in crops (Painter 1951) with a non-linear scale of 1–9, where 1= no damage; 3 = spots developing on upper leaf surface (arrows); 5 = streaks developing on the veins, leaf edges and tips (arrow); 7= streaks and surrounding area starting to turn brown and dry; and 9 = whole leaves browning and drying and the plant having a stunted appearance (Fig.1A, a–e, respectively). Individual scores from at least four leaves were averaged to give the damage rating which was assessed after 2, 4, 6, 9, 10, 13 and 15 days of UV-B exposure. Each cultivar was assigned to a particular UV-B tolerance category: highly tolerant (damage rating 1–3.5), tolerant (damage rating 3.6–5.0), intermediate (damage rating 5.1–7.0), susceptible (damage rating 7.1–8.5) and highly susceptible (damage rating 8.6–9.0). Damage ratings for all cultivars in a category were averaged to give the mean damage rating. Light Microscopy, SEM and TEM Samples from the second and third leaves were collected after 20 days of UV-B exposure for TEM. Leaves were finely chopped and immediately fixed with 2.5% (w/v) glutaraldehyde in 35 mM PIPES buffer at pH 6.8, post-fixed with 1% (w/v) osmium tetroxide, dehydrated in an ethanol series, infiltrated, and polymerised with London White Resin. Sections were stained with 2% (w/v) barium permanganate and examined in a Hitachi 600 TEM. At least 50 comparable sections from each cultivar were examined and micrographs from representative sections are shown. Replicates of five leaf samples from each selected cultivar were taken from four pots for SEM. Samples were freeze-dried

UV-B Irradiation of Rice

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Table 1. Selected rice cultivars and their agronomic traits determined by Yanco Agricultural Institute, New South Wales, Australia nd, not determined; I, Indica; J, Japonica; ?, indica/japonica classification based on grain type or texture Name

Origin

Mature height (cm)

Grain weight (g/1000)

Days to maturity

Amaroo Bluebelle Padma Lido Arlesienne Milyang Doongara Koshihikari Pelde Banat 725 IR 54 M202 YRL 39 Omirt 39 Dular Fuzisaka 5 Lemont Er Bai Ai Gui Chao 2 IR 8 IR 74 Rexoro Omirt 168 China 1039 Hakkoda Goolarah Chieh Keng 46 Starbonnet Century Patna 231 Gulfrose Azucena Nato Ching Yueh 1 Rodjolele

Yanco, Australia USA India Italy France Korea Yanco, Australia Japan Yanco, Australia Romania IRRI, Philippines USA Yanco, Australia IRRI, Philippines Pakistan Japan USA China China IRRI, Philippines IRRI, Philippines USA Hungary China Japan Yanco, Australia China USA USA USA Philippines USA China Indonesia

90 98 75 110 98 80 85 105 105 110 80 90 100 110 110 90 90 90 90 85 80 115 95 115 75 110 90 115 115 110 120 110 85 120

0.02906 0.02331 0.01902 0.02370 0.04243 0.02922 0.02594 0.02844 0.02347 0.02627 0.02126 0.02838 0.02461 0.02408 0.02839 0.02532 0.02591 0.02192 0.02568 0.02835 0.02621 nd 0.02984 0.02645 0.02620 0.02615 0.02614 0.02158 0.02107 0.02702 0.03238 0.02324 0.02642 0.03314

160 150 75 110 98 80 85 105 150 145 200 150 155 145 180 140 160 200 170 200 185 190 175 130 130 170 132 180 175 170 200 160 148 200

overnight, coated with gold in a vacuum evaporator and examined with a JEOL J6400 or a Cambridge 360 Stereoscan SEM. Light microscopy samples were taken as thick sections from blocks prepared for TEM and stained with 0.2% (w/v) toluidine blue in 50 mM sodium phosphate buffer (pH 7.6). Determination of Relative Levels of Phenolics Samples of 0.3 g (fr.wt) were collected from the second and third leaves after 0, 2, 6, and 10 days of UV-B exposure. Each sample was homogenised in 3 mL of 70% (v/v) aqueous methanol containing 0.1% (w/v) ascorbic acid. The extracts were centrifuged for 5 min at 5000 gav. Phenols were estimated by the FolinCiocalteu method as modified by Singleton and Rossi (1965), using Folin-Ciocalteu reagent (Merck) diluted 10 times with MilliQ water. The A750 of the samples was calibrated against naringenin. Ascorbate was added to the extraction solvent to prevent oxidation and inhibit browning of the extracts, which otherwise takes place rapidly and quickly fouls HPLC columns during further analysis. Ascorbate reacts with the Folin-Ciocalteu reagent (Singleton and Rossi 1965) and Folin-Ciocalteu determination on

Type

J I? I J? J? J? I? J J? J I J J? J I J I J J I I I J J J J? J I? I? J? I J? J I

fresh extracts or extracts containing 0.1% (w/v) ascorbate showed that approximately 70% of the coloured product was due to added ascorbate. However, the colour produced by 0.1% (w/v) ascorbate in 70% methanol decreases with time, presumably as the ascorbate is oxidised. After 1 month, ascorbate contributes less to the colour yield of the extracts and approximately half of the colour produced in the assay is due to plant phenolics. In this study, all the assays were conducted 45–50 days after extraction and all the samples were stored in a 4°C cold room. HPLC chromatograms of rice extracts were unchanged after at least 1 year storage of rice extracts, indicating that the phenolics were stable in these solutions. The relative amounts of phenolics are given as percentage of the colour yield produced by the highest phenolic-producing cultivar, M202, after 6 days of UV-B exposure (Table 2). High Pressure Liquid Chromatography (HPLC) Duplicate 10 mL aliquots of each methanolic extract were diluted with 90 mL of MilliQ water, injected into a Beckman System Gold HPLC using either HPLC system I or system II.

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HPLC system I used a Waters Novapak C18 column (15 ´ 0.4 cm), eluted with a linear gradient from solvent A to 70% solvent B over 15 min at 1 mL/min. The void volume of the system was 1.0 mL. HPLC system II used an Activon Exsil column (3 ´ 0.46 cm), eluted with a linear gradient from solvent A to 70% solvent B over 5 min at 2mL/min. The void volume was 0.5 mL. Solvent A was 2% (v/v) acetic acid and solvent B was methanol in both systems. The absorbance of the effluent was monitored with either a single wavelength detector at 314 nm or a diode array detector. Unless mentioned, HPLC system I was used for all analyses. Statistical Analysis Data were analysed using GENSTAT (Lane et al. 1987). ANOVA was used to compare significant effects among treatments, cultivars and interactions. Means and standard errors are shown in figures. Duncan’s Multiple Range Test (DMRT) was used to compare category means in Table 3 when the cultivars were assigned to tolerance categories.

Results Development of Physical Symptoms The development of UV-B damage was scored by determining visible symptoms in the leaves and this is illustrated for the highly susceptible cultivar Dular (Fig.1A, a–e). Unirradiated leaves showed no visible signs of damage (a in Fig. 1A). Early symptoms were characterised by the formation of distinct bronze or brown spots with damage scores ranging from 2 to 3 (b in Fig.1A). Further UV-B exposure resulted in the formation of prominent bronze and brown streaks scattered along the affected leaf tissues and stretching parallel to veins on the upper surface of the leaf with damage scores ranging from 4 to 6 (c in Fig.1A). The last stage of UV-B damage was characterised by chlorosis, followed by necrosis and desiccation with damage scores ranging from 7 to 9 (refer to sections d and e in Fig.1A). The above signs and symptoms are shown in Figs 1B and C and for a number of plants after 15 days exposure to UV-B. The highly tolerant cultivars M202, Banat 725 and YRL 39 (Fig.1B, #12 and #10, Fig.1C, #13, respectively) showed few symptoms, with damage ratings below 5. Conversely, the highly susceptible cultivars Dular, Gui Chao 2 and Padma (Fig. 1C, #15, #19 and 3#, respectively) exhibited UV-B damage, showing browning and marked chlorosis with damage ratings of 9. Visible symptoms of leaf damage in tolerant cultivars were delayed and detected only after 9 days of UV-B exposure (Fig. 2a). Even after 15 days, the tolerant cultivars showed few visible symptoms and had low damage ratings from 2 to 4 (Fig. 2a). In contrast, advanced damage was

Fig. 1. UV-B damage in rice. A, the susceptible cultivar, Dular, showing damage scores of 1 (a); 3 (b); 5 (c); 7 (d); and 9 (e).



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Fig. 1. UV-B damage in rice. B,C, response of whole rice plants to enhanced UV-B irradiation after 15 days of exposure showing browning and curling of leaves of the highly susceptible cultivars Padma (C, #3), Dular (C, #15), and Gui Chao 2 (C, #19) but not the highly tolerant cultivars Banat 725 (B, #10), M202 (B, #12), or YRL 39 (C, #13). The tolerant cultivars Doongara (B, #7), IR54 (B, # 11), Lemont (B, #17), and Bluebelle (C, #2) also show limited damage. Unirradiated control plants (not shown) exhibited no damage.

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Fig 2. UV-B damage ratings of tolerant rice cultivars (a) Fuzisaka 5 (r), Rexoro (j), Banat 725 (n), Amaroo (m), M202 (s), and susceptible rice cultivars (b) Dular (r), Er Bai Ai (s), IR 8 (m), Padma (n), Gui Chao 2 (u) after 15 days of UV-B exposure.

clearly seen in susceptible cultivars as early as 2 days after UV-B treatment (Fig. 2b). Further exposure increased the symptoms dramatically. A rapid increase in visible damage occurred after 10 days of UV-B exposure in susceptible cultivars, leading to severe leaf damage with final damage ratings of 9 (Fig. 2b). On the basis of the final damage ratings taken after 15 days of UV-B exposure and the rate of development of the visible symptoms, the 34 cultivars were classified as highly tolerant, tolerant, intermediate, susceptible or highly susceptible to UV-B irradiation (Table 2). Six cultivars were classified as highly tolerant, seven as tolerant, ten as intermediate, six as susceptible and five as highly susceptible. The most tolerant cultivars were, in rank order, M202 > Amaroo > Banat 725 > Rexoro. Dular was the most sensitive cultivar, having a final damage rating of 9 after 15 days of UV-B exposure, followed by Er Bai Ai, Gui Chao 2 and Milyang 23 (Table 2). Structural Changes Cross-sections of unexposed rice leaves showed an ordered arrangement of intact cells when examined under the light microscope. UV-B irradiation of susceptible cultivars damaged the entire cell structure; in particular mesophyll and bundle sheath cells showed damage to the point of contraction and even collapse. In contrast, little if any disruption was observed in irradiated tolerant cultivars (data not shown). Under SEM, the upper surface of leaves from unirradiated control plants showed a characteristic pattern in which longitudinal vein areas were clearly delineated by parallel ridges and guard cells were turgid with a distinct

outline (Fig. 3a, b). After UV-B exposure, the leaf surface was deformed and the ridges were less prominent. The stomata in susceptible cultivars exposed to UV-B had lost the distinct outline and had become distorted as the adjoining cells deformed (Fig. 3c). In comparison, stomata of tolerant cultivars irradiated with UV-B largely maintained their shape, although some appeared to have slightly reduced turgidity (Fig. 3d). Ultrastructural damage to chloroplasts was evident in susceptible cultivars which were UV-B irradiated; the granal stacks were disrupted and the chloroplast envelope had ruptured (Fig. 4a). In the UV-B treated tolerant cultivars, the ultrastructural disruption was much less severe and was observed only after prolonged UV-B exposure (Fig. 4b). Chloroplasts from unirradiated control leaves had intact membranes and well organised granal stacks (data not shown). Accumulation of Relative Levels of Phenolic Compounds There was a significant difference in the relative amount of phenols induced by UV-B exposure among rice cultivars at all sampling times (P ² 0.001). However, treatment effects (UV-B vs. control) differed significantly (P ² 0.01) at days 2, 6, and 10. UV-B treated plants always had significantly higher relative phenolic contents than equivalent control plants. The relative amount of phenolics consistently differed among the rice cultivars from days 2 to 6 suggesting a highly significant cultivar effect (P ² 0.001). Treatment ´ cultivar interactions were shown to be significant from days 2 and 6 (P ² 0.013; P ² 0.01 respectively). Of the 34 rice cultivars screened, mean relative phenol accumulation by the highly tolerant and tolerant cultivars was 85% and 78% respectively, of the maximum level of cv.


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Table 2. Response and tolerance classification of rice cultivars to UV-B irradiation Name

Damage Relative Peak I Peak II Damage rating at phenols area at area at rating at day 6 (% of M202) day 6 day 6 day 15 at day 6

M202 Amaroo Banat 725 Rexoro Lido YRL 39

1.0 1.6 1.0 1.6 1.0 1.0

Lemont Doongara Fuzisaka 5 IR 54 Hakkoda China 1039 Bluebelle

1.4 1.0 1.3 1.4 1.2 1.0 1.0

Gulfrose Goolarah Pelde Century Patna 231 Omirt 39 Omirt 168 Rodjolele Starbonnet Arlesienne Ching Yueh 1

1.1 2.8 1.0 1.0 1.6 4.5 2.9 1.0 1.6 1.0

Chieh Keng 46 IR 8 Koshihikari Nato Azucena IR 74

1.0 5.3 3.1 3.2 1.7 3.3

Padma Milyang 23 Gui Chao 2 Er Bai Ai Dular

4.3 1.2 1.8 4.4 4.7

Highly Tolerant 100.0 70.2 80.0 92.1 74.1 98.9 Tolerant 77.5 80.2 90.9 59.7 83.7 75.8 74.7 Intermediate 81.6 83.8 88.0 68.9 83.9 51.1 60.6 75.4 67.5 85.0 Susceptible 80.7 59.1 78.7 66.6 77.3 74.3 Highly Susceptible 51.8 66.8 71.5 59.7 65.3

6.75 3.61 5.03 4.83 5.10 2.41

4.70 4.53 3.78 6.07 3.45 5.37

2.0 2.2 2.9 3.0 3.1 3.4

2.30 2.69 9.05 0.82 3.20 5.09 3.34

4.65 1.76 3.05 0.34 1.77 2.20 5.13

3.7 3.9 4.0 4.2 4.2 4.3 4.4

3.85 3.22 2.66 2.58 3.02 1.67 3.15 1.24 3.73 2.53

7.37 6.03 4.32 5.83 1.64 1.27 2.64 3.12 1.98 2.24

5.1 5.2 5.4 5.4 5.9 5.9 5.9 6.1 6.2 6.7

5.43 1.01 3.37 1.31 3.59 2.27

2.63 0.58 1.91 4.12 3.66 2.46

7.2 7.5 7.6 8.2 8.3 8.5

0.90 1.85 1.39 0.80 0

0.30 1.45 2.28 1.07 0.47

8.7 9.0 9.0 9.0 9.0

M202, after 6 days of exposure to UV-B (Table 2, column 3). Conversely, means for susceptible and highly susceptible categories were 73% and 63%, respectively, of the maximum level of cv. M202 after 6 days of UV-B exposure (Table 2). In the presence of UV-B radiation, the relative phenol level in tolerant cultivars increased by 220–250% after 6 days exposure (Fig. 5a). Further exposure led to a decline in relative phenol level between 10% and 40% of the maximum level, after 10 days exposure (Fig. 5a). In the absence of UV-B, the relative levels of phenolics in tolerant

cultivars increased slightly over the 10 days of exposure (Fig. 5b). Conversely, the relative level of phenolics in susceptible cultivars did not show any significant change under either UV-B or control conditions (Fig. 5c, d). Cultivars determined to have an intermediate tolerance to UV-B showed a pattern of relative phenol accumulation which was intermediate between those shown by the tolerant and susceptible cultivars (Table 2). HPLC Several cultivars which were clearly tolerant (M202, Banat 725, Rexoro and Fuzisaka 5) or susceptible (Dular, Padma) to UV-B irradiation were chosen for further investigation by HPLC to determine if the increase in relative phenols observed in the tolerant cultivars was due to one or more specific phenolic compounds. Chromatograms of extracts from the tolerant cultivar, M202, had two dominant peaks. Peak I eluted at 8.12 min (PI, Fig. 6a, b) and peak II at 9.73 min (PII, Fig. 6a, b), respectively. When the absorbance of the HPLC effluent was scanned using a diode array detector, all compounds which absorbed between 200 and 600 nm were detected at 314 nm, indicating that this was a suitable wavelength for detection of UV-induced compounds. A peak eluting at 1.7 min was due to unretained polar compounds such as chlorophyll (Fig. 6a–d). In extracts of cultivar M202 which had been UV-B irradiated, the level of peak I increased approximately 200% reaching a maximum after 6 days of UV-B irradiation and remaining constant for the remainder of the treatment (Fig. 6a; Fig. 7a). The level of peak I did not change significantly in control extracts of unirradiated cv. M202 (Fig. 6b; Fig. 7a). In contrast, the level of peak II was constant and did not vary significantly between UV and control treatments (Fig. 6a, b; Fig. 7a, b). Peaks I and II were either absent or present in trace amounts in extracts from the susceptible cultivar, Dular (Fig. 6c, d; Fig. 7a, b). Irradiation with UV-B had no effect on the levels of peaks I or II in extracts of Dular (Fig. 7a). The levels of these compounds in extracts of unirradiated Dular remained at a similarly low level (Fig. 7b). HPLC analysis of all cultivars with HPLC system II was conducted and results are shown in Table 2, columns 4 and 5. HPLC traces of all tolerant and highly tolerant cultivars had two prominent peaks which co-migrated with peak I and II from extracts of cv. M202. The area under peak I and II in chromatograms of extracts of tolerant and highly tolerant cultivars also increased as UV-B exposure increased, except for one tolerant cultivar, IR 54. There was a significantly reduced amount of peak I and II in all susceptible and highly susceptible cultivars except for cultivars Chieh Keng 46, Koshihikari and Azucena, where peak I and II were present in comparable levels to tolerant cultivars (Table 2).

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Fig. 3. Scanning electron micrographs of upper leaf surface showing stomatal guard cells (arrows); (a) unirradiated leaves from the susceptible cultivar, Dular, and (b) the tolerant cultivar, M202, showing a distinct outline; (c) UV-B irradiated leaf of the susceptible cultivar, Dular, showing non-turgid guard cells (arrow) becoming distorted as the adjoining cells deform; and (d) the guard cells of UV-B irradiated tolerant cultivar, M202, showing little effect after 10 days of UV-B treatment. All scale bars = 10 mm.

Relationship Between Cultivar Responses and Tolerance Category To have a clearer indication of the relationship between cultivar responses and tolerance category, responses of all cultivars in a category were pooled and the mean obtained (Table 3). Within each category, the mean damage rating

increased significantly as irradiation exposure increased except for the highly tolerant category. After 6 days UV-B exposure, the tolerant, highly tolerant and intermediate categories all had significantly lower mean damage ratings than the susceptible and highly susceptible categories (Table 3). This trend became a little clearer with increased UV-B exposure and, by day 15, a


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Fig. 4. Transmission electron micrographs of leaf mesophyll cells from unirradiated and UV-B treated plants; (a) UV-B treated leaf of susceptible cultivar, Dular, showing disruption of chloroplast grana and envelope (arrow), and (b) UV-B treated tolerant cultivar, M202, showing chloroplasts with granal stacks and intact envelope (arrow). Scale bar in both = 0.25 mm.

wider range of differential responses was observed. The highly tolerant and tolerant categories had significantly lower mean damage scores than the susceptible and highly susceptible

categories. Intermediate results were obtained after 10 days of UV-B exposure (Table 3). Similarly, there was a significant difference observed in the mean amount of peaks I and II at

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Fig. 5. Time-course of relative phenol accumulation in the tolerant cultivars M202 (m), Rexoro (d), Fuzisaka 5 (,), Banat 725 (j), and Amaroo (e), under either (a) UV-B irradiation or (b) control conditions, and time-course of relative phenol accumulation in the susceptible cultivars Gui Chao 2 (,), Dular (j), Er Bai Ai (m), IR 8 (.), Padma (e) under either (c) UV-B irradiation or (d) control conditions. For clarity, only limited numbers of cultivars are shown; for complete data, see Table 2.

day 6, between the different categories. The mean amount of peak I in the highly tolerant and tolerant categories was significantly higher than in the susceptible and highly susceptible categories (Table 3). The trends for peak II were less clear, with only the highly tolerant category having significantly higher mean levels of peak II than the highly susceptible category. However, there was no significant difference between mean levels of peak II in the tolerant and susceptible categories (Table 3). In conclusion, there was a significant inverse relationship between the mean level of peak I and peak II at day 6 (Table 3, column 5 and 6 respectively) for a tolerance category and the damage rating at day 15 (Table 3, column 4) with r2 = 0.933 and r2 = 0.91, respectively.

Evidence of the Presence of UV-B Absorbing Compounds Spectral characterisation of the minor and major chromatographic peaks of M202 extracts, obtained with a diode array monitor, indicate that all of the peaks in the chromatogram had two absorption maxima, the first at about 270 nm and the second between 320 and 350 nm. The two dominant peaks present in chromatograms of tolerant plants which had been UV-treated had similar spectral characteristics: both peaks fluoresced at 228 nm. Peak I absorbed at 271 and 324 nm while peak II absorbed at 273 and 321 nm. Preliminary work on the spectral characteristics and mobilities using TLC suggests that peak I comprises two closely migrating flavonoids. Similarly, peak II also appears to contain two closely migrating flavonoids.


271

Fig.6. HPLC chromatograms showing the time course of peak I and peak II accumulation in (a, b) the tolerant rice cultivar M202, and (c, d) the susceptible rice cultivar Dular under either (a, c) UV-B irradiation or (b, d) control conditions. The positions of peak I (PI) and peak II (PII) are shown.

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Fig. 7. The area of peak I (e) and peak II (u) in HPLC chromatograms of extracts from the tolerant rice cultivar M202, or the area of peak I (n) and peak II (s) in chromatograms of extracts from the susceptible rice cultivar Dular under either (a) UV-B irradiation or (b) control conditions.

Discussion Several studies have been conducted over the last 5 years on the responses of rice to increased UV-B. Teramura et al. (1991) examined the response of 16 rice cultivars to enhanced UV-B irradiation and reported changes in growth and photosynthesis. Dai et al. (1992) showed overall sensitivity to UV-B, derived from a response index formulated by integrating a number of agronomic characters such as plant height, leaf area, dry weight, relative growth rate and net assimilation rate, varied between four rice cultivars studied. Barnes et al. (1993) screened 22 rice cultivars and found variation in morphological responses to UV-B. Dai et al. (1994) compared responses of 188 cultivars of rice but confined comparisons to agronomic characteristics such as plant height, leaf area, plant dry weight and tiller number. In a more detailed work, He et al.

(1993) studied the accumulation of UV-B absorbing compounds in two rice cultivars, Lemont and Er Bai Ai, and found that the A280 of methanolic extracts of Lemont were lower than the A280 of methanolic extracts of Er Bai Ai. The results for the two rice cultivars were in the opposite order from results in our study. The apparent contradiction could be explained by differences in analytical techniques; for example, an increased A280 of a methanolic extract of leaves following UV-B irradiation could be due to the accumulation of peptides from protein breakdown rather than increased accumulation of phenolics. Inferences drawn about the impacts of biological pressures on crop plants require adequate replications and comparisons among a suitably large number of varieties. The results presented in our study have defined specific responses of 34 rice cultivars from a wide genetic range to enhanced UV-B irradiation.

Table 3. Comparison of the mean levels of peak I and peak II with the mean damage rating for the different tolerance categories Within any column, means having a common superscript are not significantly different at the 5% (peaks I and II) and 1% (Mean Damage Rating) levels of significance using Duncan’s Multiple Range Test (DMRT) Tolerance category Highly Tolerant Tolerant Intermediate Susceptible Highly susceptible

Mean damage rating at: Day 6 Day 10 Day 15 1.2a 1.2a 1.9a 2.9b 3.3b

1.1a 2.1 b 2.3bc 3.1d 4.7d

2.8a 4.1ab 5.8bc 7.9cd 8.9d

Mean peak area at day 6 Peak I Peak II 4.621a 3.784a 2.765b 3.396b 0.988c

4.650a 2.700c 3.644b 2.560c 1.114d


The application of a damage rating scale, similar to those used to estimate disease or insect damage on foliage, gives a more precise basis for comparison of the relative tolerance of rice cultivars to enhanced UV-B irradiation. The damage ratings showed differences in the responses of several rice cultivars to UV-B irradiation. Cultivars could be precisely rated as tolerant or susceptible on the basis of their differences in rating scores. Dular and M202 represent candidate contrasting biotypes that could be used for further studies on the mechanism(s) of UV-B response in rice. Microscopy has been used to assess damage at the tissue and ultrastructural level to provide a basis for the more macroscopic examination on which the damage rating is based. Significant anatomical and ultrastructural damage occurred in leaves of some plants irradiated with an enhanced level of UV-B. Similar ultrastructural changes have been reported previously for temperate crops like barley (Tevini et al. 1991) and pea (He et al. 1994). In 1995, Dai et al. demonstrated the reduction in dry mass of rice plants under UV-B in the glasshouse was attributed to decrease in stomatal density and opening. However, this study is the first report to show that enhanced irradiation with UV-B produces dramatic ultrastructural changes in rice leaves. Central to these results has been the finding that the 34 cultivars studied, once assigned to categories of tolerance or susceptibility, show a significant correlative relationship between the tolerance category and the relative levels of rice phenolics and of specific compounds indicated by HPLC analysis. The level of characterisation of the components in the phenolic extracts was much greater than in previous reports on rice. Two prominent HPLC peaks (peaks I and II) which accumulated in extracts of rice leaves in response to an applied UV-B stress have been identified. While the presence of these compounds appears to be strongly associated with tolerance to enhanced UV-B irradiation, phenolic accumulation may not be the only factor as there are exceptions to the high tolerance/ high phenol/ high peak area relationship. IR 54 was classified as tolerant to UV-B but had relatively low phenolics and peak I area at day 6. Conversely, cultivars Chieh Keng 46, Koshihikari and Azucena have appreciable levels of relative phenolics and peak I but were categorised as sensitive to UV-B irradiation after 15 days. However, these cultivars had damage ratings of 1.0, 3.1, and 1.7 respectively at 6 days, indicating that they were moderately tolerant in the first 6 days. Other factors such as thickness of cuticles, presence of leaf waxes and hairs and the relative erectness of habit may also be involved in tolerance to UV-B irradiation. Several of the most successful Australian cultivars such as Amaroo, YRL39, Doongara, Goolarah, and Pelde not only accumulate high levels of phenols but also possess thick cuticles, erect growth habit and greater overall leaf thickness, all of which may contribute to their tolerance to UV-B.

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A significant relationship exists between the presence of specific phenolics and tolerance to enhanced UV-B irradiation in rice cultivars. Further work is being carried out aimed at identifying the phenolic compounds present in peaks I and II and to investigate the relationship of these specific compounds with UV-B tolerance.

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Manuscript received 15 July 1996, accepted 2 January 1997