Rapid Fluence-Dependent Responses to Ultraviolet-B Radiation in Cucumber Leaves: The Role of UV-Absorbing Pigments in Damage Protection

J Plant Physiol. Vol. 148. pp. 57-62 (1996)

Rapid Fluence-Dependent Responses to Ultraviolet-B Radiation in Cucumber Leaves: The Role of UV-Absorbing Pigments in Damage Protection PAULIEN AnAMSE

and

STEVEN]. BRITZ

U.S. Dept. of Agticulture, Agricultural Research Service, Climate Suess Laboratory, Beltsville, MD 20705-2350 USA Received June 24, 1995 . Accepted October 20, 1995

Summary

The role of foliar UV-absorbing pigments (UVAP) as optical screening agents in the resistance of Cucumis sativus L. to UV-B radiation was investigated by exposing young leaves at a defined developmental stage from sensitive (cv Poinsett) and insensitive (cv Ashley) lines to brief UV-B treatments varying between 4 and 10 h. The amount of blue light (BL) or UV-A radiation during UV-B exposure was also varied. Rapid increases in UVAP immediately following UV-B were compared to damage in the same tissue (increased specific leaf weight or chlorosis) determined 72h after the start ofUV-B. Poinsett was more sensitive to both forms of UV-B damage than Ashley under conditions where the response to UV-B was not saturated. Although UVAP increased rapidly in response to UV-B, it is unlikely that optical screening by these compounds was responsible for genetic differences in sensitivity to UV-B for the following reasons: 1) the kinetics of UVAP increase were similar to that for induction of damage; 2) increases in UVAP in the UV-sensitive line (Poinsett) were similar to those in the resistant line (Ashley); and 3) BL and UV-A radiation significantly reduced damage by UV-B in cv Poinsett when given simultaneously but had relatively small stimulatory effects on rapid UVAP accumulation. These results do not rule out a general role for optical screening by UVAP nor do they exclude the possibility that qualitative differences in UVAP (e.g., as antioxidants) are the basis for cultivar differences.

Key words: Cucumis sativus L., blue light, cucumber, flavonoids, ultraviolet, chlorosis, specific leafweight. Abbreviatiom: BL = blue light 400-500 nm; cv(s) = cultivar(s); LPS = low pressure sodium; PAR = photosynthetically-active radiation 400-700 nm; UV-A = ultraviolet-A 315-400 nm; UV-B = ultravioletB 280 - 315 nm; UVAP =UV-absorbing pigments. Introduction

Genetic variability in higher plants in response to UV-B is well-documented (Murali and Teramura, 1986; Reed et aI., 1992). Determination of mechanisms underlying these differences should contribute to the development of stress tolerant crops and improve our ability to assess the impact of UV-B on plant growth. However, except for some known mutations (Li et aI., 1993; Britt et aI., 1993), the bases for most genetic differences are not well understood. Intraspecific differences in UV-B sensitivity in cucumber have been correlated with levels of UVAP (Murali and Tera© 1996 by Gustav Fischer Verlag, Stuttgarr

mura, 1986). These compounds (presumptive flavonoids; McClure, 1975) could serve as optical screens, absorbing UV while transmitting PAR (Caldwell et aI., 1983). Significantly fewer pyrimidine dimers were formed by UV-B radiation in algal cultures with higher levels of flavonoids (Takahashi et aI., 1991). UVAP increase in response to UV-B exposure in many plants, including curcurbits (Sisson, 1981). Although increased flavonoids have been suggested as an indicator of DNA damage (Beggs et aI., 1985), data from cucumber correlate accumulation of UVAP in leaves with reduced damage (Adamse et aI., 1994). UV-B exposure induced the accumulation of UVAP on a leaf area basis in two cultivars of cucum-

58

PAULIEN AnAMSE and STEVEN J. BRITZ

ber, with approximately 50 % greater response after a 3 day treatment in cv Ashley (more resistant to damage) than in cv Poinsett (less resistant). Starting values of UVAP were similar for the two lines. BL and UV-A often enhance the UV-induced synthesis of phenylpropanoid compounds (Ohl et al., 1989) and can be used to dissect the role ofUVAP levels in resistance to UV-B. Supplemental BL during UV-B treatment ameliorated damage and caused both increases in total UVAP per leaf as well as a shift in the relative amounts of different UVAP (Adamse et al., 1994). BL effects were fluence-dependent and not saturated where BL constituted up to 7 % of daily photosynthetic photon flux. Interpretation, however, was complicated by simultaneous effects of BL on leaf growth and pigment content not related to UV-B exposure. Furthermore, damage to the leaf by UV-B was probably induced during the first 24 h of exposure. UVAP on a leaf area basis increased significantly within this time, but the sensitive and resistant lines did not differ in this early pigment response and no effect of BL was detected. More detailed kinetic analysis of UV-B response during short term exposure is required to test the possibility that rapid increases in foliar UVAP content during the first hours of UV-B exposure contributed to amelioration of damage. The objectives of the current study therefore are to evaluate the role of bulk increases of UVAP in prevention of UV-B damage (e.g., through an optical screening mechanism) by comparing the kinetics of UVAP accumulation induced by UV-B in relation to the time course for induction of damage in cultivars differing in sensitivity to UV-B. Supplemental BL and UV-A were used to modifY damage and UVAP accumulation simultaneously in the same tissue. Sensitivity to UV-B is altered by background levels of PAR (Cen and Bornman, 1990), spectral interactions (Middleton and Teramura, 1994), and atmospheric CO 2 (Adamse and Britz, 1992 b). To minimize the possibility that BL or UV-A act indirectly through photosynthesis or phytochrome, background high PAR from amber LPS lamps was used to maintain high rates of photosynthesis during treatment as well as high levels of phytochrome phototransformation to the farred-absorbing form (Adamse and Britz, 1992 b; Britz and Sager,1989). Developing third leaves immediately subsequent to opening, and without prior UV-B exposure, were irradiated for 10 h or less with UV-B in the presence of high PAR from background LPS lamps ± supplemental BL or UV-A. The third leaf was selected because the prime period of UV-B sensitivity does not overlap with that of other leaves (data not shown) and because there is little direct effect ofUV-B on the first leaf which serves as a source of photosynthate (Adamse and Britz, 1992 b; Britz and Adamse, 1994). Some plants were returned to white light growth conditions without UV-B for quantification of UV-B-induced leaf damage (i.e., chlorosis or increased SLW) 72 h after the start of treatment. This approach allowed us to concentrate on responses induced rapidly by brief UV exposure, while minimizing possible differences in the kinetics of expression. In other plants, young leaves were harvested and extracted immediately after UV-B exposure to determine whether UVAP increased rapidly enough to influence damage by UV-B. Note

that increased SLW is interpreted as a sensitive indicator of UV-B damage, since the largest changes in SLW in the third leaf were associated with the greatest inhibition ofleaf growth by UV-B (Adamse et al., 1994). SLW is highly conserved and it is possible to see significant changes when differences in leaf area and dry weight are no longer discernible.

Materials and Methods Plant material Cucumber (Cucumis sativus L.) cvs Poinsett and Ashley were grown in 1.51 pots with vermiculite, flushed daily with a modified half-strength Hoagland's solution, in a growth chamber at 2TC, ca. 50 % relative humidity, and ca. 450 Ilmol mol- I CO 2 (ambient level in the building). Plants were illuminated 14 h per day with a 1: 1 mixture of 400 W high pressure sodium and metal halide lamps. PAR was stepped diurnally, starting with 1 h at 270 Ilmol m- 2 S-I, continuing with 12 h at 840 Ilmol m -2 s-I and concluding with 1h at 270 Ilmol m -2 s-I. Mter 11 days, plants were selected for uniformity and transferred to a UV exposure chamber approximately 110 min after the onsd of white light. This time corresponded to the unfolding of the third leaf.

Light treatments The UV exposure chamber was maintained at the same environmental conditions as the growth chamber except for light quality and was divided into two compartments by means of a UV-B absorbing polyester film. An adjustable bank of UV-B lamps (F40WT12-6NUVB-313, Q-panel Co., Cleveland, OH, USA) was suspended underneath the main lamp canopy. On one side, UVB313 lamps were wrapped with cellulose diacetate film (0.08 mm) to provide UV-B (+ UVB). On the other side, the lamps were wrapped with polyester film (0.13 mm) to remove UV-B while transmitting longer wavelengths (-UVB). Most PAR in the second chamber was provided by LPS lamps (SOX 180 W, Philips North America, Bloomfield, NJ) with or without supplemental BL or UV-A fluorescent lamps (respectively, F40T12/247 and F40T12/BLB GTE Sylvania, Danvers, MA). For description of light levels see Table 1. Note that the low levels of BL (Hg lines at 405 and 436 nm) and UV-A (mainly below 350 but including the Hg line at 365 nm) were emitted by the UV-B source lamps (supplemental lamps off). Wrapping the lamps in polyester substantially reduced the shorter wavelength component of UV-A. Background LPS and BL lamps produced no UV-B. UV-B sources, UV-B absorbing or transmitting filters and the resulting spectra are described in more detail elsewhere (Adamse and Britz, 1992a). Two series of experiments were conducted. In the first, plants were treated with UV-B in the presence or absence of supplemental BL (Table 1). The second series used supplemental UV-A instead of BL (Table 1). The two cultivars were treated simultaneously, but only one level of supplemental BL or UV-A could be used in a given experiment. Consequently, + BL and - BL treatments were alternated during the first series, while + UV-A and - UV-A were alternated during the second series.

Specific leaf weight and chlorosis Plants were irradiated with UV (±UVB) for 4,8 or sometimes 10 h and returned immediately to white light growth conditions. Zero time controls were subjected to a mock exposure and returned to white light. The plants were harvested 72 h after the start of the UV treatment. Specific leaf weight was determined after measuring

Rapid Effects ofUV-B

Table 1: Light conditions during UV treatments. Photon fluence

rate (Ilmol m- 2 s-l) of photosynthetically-active radiation (PAR; 400-700 nm), blue light (BL; 400-500 nm), UV-A (315-400 nm), UV-B (290-315 nm). Biologically-effective UV-B (UV-B BE normalized to 1.0 at 300 nm; Caldwell, 1971) for a maximal 10 h total exposure were 20.8 and 0.3 kJ m -2 for + UVB and - UVB, respectively.

perature) in acidified methanol (methanol: HCl : water: : 40 : 1: 59) as described previously (Adamse and Britz, 1992 b). Absorption spectra (Model 219, Caty, Varian, Palo Alto, CA, USA) were comparable to those published elsewhere (Cen and Bornman, 1990) and revealed two peaks at approximately 270 and 325 nm. Pigments were quantified at 325 nm in order to maximize sensitivity.

-----------------------

Supplement +BL -BL +UV-A -UV-A

+UVB -UVB +UVB -UVB +UVB -UVB +UVB -UVB

BL

UV-A

UV-B

PAR

59 58 6 5 6 5 5 4

9 6 9 6 52 49 9 6

2.80 0.04 2.80 0.00 2.91 0.16 2.80 0.05

826 825 778 777 531 530 530 529

Statistics Indicated values are averages of 8 to 16 samples, obtained in two or three replicate experiments. Significance of difference between means was determined based on one-sided Hests at P<0.05.

Results

UV-Absorbing Pigments

dry weight and area (LiCor Model LI-3000, Lincoln, NE, USA) of the third leaf. Chlorosis was estimated visually. Separate studies evaluating subjective scoring with a leaf scanning system indicated the two methods were comparable. The scanning system consisted of a video camera (Model 652, Dage-MTI, Michigan City, IN) and UItricon video tube (Burle Industries, Lancaster, PA), connected to a Macintosh Pc. Leaf videos were digitized (New Image Technology, Seabrook, MD) and stored as bit-mapped images (Macintosh 512 K, Apple Computer, Cupertino, CA). By adjusting the contrast, an image of the leaf was obtained either with or without chlorotic lesions visible. Pixel number for both images was compared after scanning and the percent damaged area was estimated.

UV-B treatment caused similar, rapid 20 % increases in UVAP in third leaves of cultivars Poinsett and Ashley extracted immediately following UV-B exposure (- BL and - UV-A; Fig. 1). The response appeared to be slower in the second series (± UV-A) , possibly as a result of slight differences in leaf age or background PAR. Although it is not possible to determine unambiguously whether the UV-A or BL components of the UV-B lamps affected the response to UVB, supplemental UV-A or BL did not affect UVAP accumulation in the absence of UV-B (data not shown) and had very little effect on the UV-B response in Poinsett. However, supplemental UV-A or BL stimulated rapid UVAP accumulation in Ashley, with significant UV-B effects detected after only 4h of treatment (i.e., 8.3 k] m- 2 ofUV-B BE total fluence).

UV-absorbing pigments Third leaves were harvested after 0, 4, 8 or sometime 10 hours of UV treatment and stored in liquid nitrogen. UVAP were extracted from whole leaves after incubation (24 h in darkness at room tem-

Fig.I: Effect of blue light (BL) or UV-A radiation on UV-B-induced accumulation of UV-absorbing pigments in the third leaf of cucumber cvs Poinsett and Ashley. Whitelight-grown plants were treated with UV-B (+ UVB) for up to 10 h starting 11 days after planting. Controls (- UVB) were exposed to polyester-wrapped UV-B lamps for comparable intervals and did not receive UV-B radiation. Treatment took place in a growth chamber illuminated with high PAR from LPS lamps with or without supplemental radiation (±BL or ±UV-A). Irradiances are described in Table 1. All treatments began at the same time of day (I 10 min after lights-on). Leaves were harvested immediately after treatment (± UVB), frozen in liquid nitrogen and extracted in acidic methanol. Values are the average of two (± BL) or three (± UV-A) replicate experiments and are expressed as absorbance change at 325 nm per mg dty weight (+ UVB minus - UVB) normalized with respect to - UVB controls. Bars indicate one standard error of the mean; * indicates a significant UV-B effect (P<0.05).

59

Chlorosis Induction of chlorotic lesions was observed in cv Poinsett (Fig. 2) but not in cv Ashley (data not shown), confirming

A325/mg (UV-B effect as % of -UVB) 80

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PAULIEN AoAMSE and STEVEN J. BRITZ

60

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Fig, 2: Effect of blue light (BL) or UV-A radiation on expression of UV-B-induced chlorotic lesions in the third leaf of cucumber cv Poinsett. Plants were returned to the white light chamber immediately after exposure. Damage, expressed as a percentage of the total leaf area, was assayed 72 h after the start of the UV-B treatment. Leaf three expanded from ca. 1cm 2 to 26 cm 2 in white light controls during this time. Other details are as in Fig. 1.

SLW (UV-B effect as % ol-UVB)

20

15

o

'rt--

V -BL ,. + BL

10 5

'f

POINSETI

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the differential sensitivity of the cultivars to UV-B reported previously (Adamse and Britz, 1992 b; Adamse et al., 1994). Significant chlorosis was induced by 4 h of UV-B in Poinsett in the absence of BL and approached 20 % of the leaf area for an 8 h exposure. Maximal injury was less than 10 % of leaf area during the UV-A interaction trials and longer exposures were required to see a significant effect. In spite of these differences, supplemental BL or UV-A during UV-B treatment reduced damage to a similar extent during an 8 h exposure (about 80% for BL and 70% for UV-A). Note that UV-A did not induce chlorosis when given by itself, even at much greater photon fluence rate.

6

8

10

Fig.3: Effect of blue light (BL) or UV-A radiation on UV-B-induced increase in Specific Leaf Weight (SLW) in the third leaf of cucumber cvs Poinsett and Ashley. Plants were transferred from ± UVB back to white light and leaves were harvested 72 h after the start of treatment. Values are the average of two (±BL) or three (±UV-A) replicate experiments and are expressed as the change in SLW (+ UVB minus - UVB) normalized with respect to the - UVB controls. Other details are as in Figs. 1 and 2.

Specific LeafWeight SLW in Ashley was not affected by UV-B in either series of experiments (Fig. 3). In Poinsett, UV-B exposure caused significant increases in SLW in both series of experiments, although sensitivity was greatly reduced during the second series (± UV-A) , again possibly as a result of slight differences in leaf age or background PAR during UV-B exposure. Supplemental BL dramatically reduced the UV-B-induced increase in SLW, consistent with interpretation of the changes in SLW as a damage response. Although both supplemental BL and UV-A prevented one form of UV-B damage to leaves

Rapid Effects ofUV-B

(i.e. chlorosis), UV-A did not prevent a small but significant increase in SLW following a 10 h exposure to UV-B. Note that comparable increases in SLW following 4 h of UV-B in the first series were clearly reversed by BL. These data confirm earlier results (Adamse et al., 1994) that leaf growth in Poinsett is more sensitive to UV-B than in Ashley when responses induced during the first 24 h of exposure are compared. Moreover, the differences between cultivars are not caused by a more rapid response in Poinsett. Discussion

61

concerning UVAP involvement, since results within each series are internally consistent. Comparisons between UV-A and BL are also complicated because the UV-B lamps emitted significant amounts of longer wavelength radiation (Table 1) and because plants received UV-A and BL during normal white light growth chamber conditions. Thus, it is not clear to what extent responses to UV-B were actually the result of long wavelength contamination from the UV-B lamps or interactions in the growth chamber. It is possible that all or part of the apparent differential sensitivity between cultivars is actually the result of differences in damage prevention or repair dependent on exposure to UV-A and BL provided concomitantly by the UV-B lamps. Pure sources of UV-B are required for more precise interpretation of spectral interaction. Although bulk changes in UVAP do not appear to be involved in the differential sensitivity between cultivars, it should be emphasized that Ashley and Poinsett have different complements ofUVAP as detected by HPLC (Adamse et al., 1994). BL treatment differentially affected the relative distribution of these compounds in the two cultivars. Qualitative differences in flavonoid content could be related to biochemical function. For example, flavonoids may act as antioxidants (Husain et al., 1987), protecting against oxidative stresses such as UV-B. Thus, it is possible that genetic differences are related to biochemical differences in flavonoid function. It is also possible that differences between cultivars as well as the effects of supplemental BL and UV-A are related to DNA damage and repair. The effects of BL and UV-A are consistent with rapid repair of pyrimidine dimers formed during UV irradiation (i.e., photoreactivation; Pang and Hays, 1991; Karentz et al., 1991; Quaite et al., 1992), suggesring that DNA may be a target for damaging physiological action ofUV-B exposure. It has been shown that BL increases photoreactivation (e.g., Tanada and Hendricks, 1953; Sairo and Werbin, 1969; Beggs et al., 1985, Pang and Hays, 1991). Differential responses to BL and UV-A could be explained by different photoreactivating enzymes with different spectral sensitivity (e.g., Pang and Hays, 1991).

Optical screening by UVAP may provide protection from UV-B exposure, but most studies correlating UVAP content with altered susceptibility to UV-B damage are evaluated after long-term UV exposure (days or weeks). It is therefore difficult to assess the significance of quantitative or qualitative differences in UVAP because the time of maximal sensitivity to UV-B damage is generally not known in relation to the kinetics of pigment accumulation. For example, less damaged plants may coincidently accumulate more UVAP. The current study avoids this problem by concentrating on rapid responses under conditions where UV-B effects are not saturated and clear differences between sensitive and insensitive cultivars can be demonstrated. Furthermore, differential damage is not the result of more rapid expression in the sensitive cultivar. The data obtained do not support a role for differences in UVAP accumulation as the basis for genetic differences in susceptibility to damage from UV-B radiation in cucumber. While UVAP accumulate rapidly in leaves of a sensitive cultivar (Poinsett) during UV exposure, the response curves for simultaneous induction of two forms of damage in the same tissue (chlorotic lesion and increased SLW) are similar to that for pigment accumulation. Thus, it is unlikely that UVAP provide additional screening before damage is induced. Two cultivars, Ashley and Poinsett, that differ in susceptibility to damage had similar UVAP accumulation in the absence of supplemental BL or UV-A radiation. Finally, both supplemental UV-A and BL ameliorated damage in cv Poinsett but AcknOWledgements produced only a small stimulation of UVAP accumulation This work was supported in part by U.S. Department of Agriinduced by UV-B. These results are consistent with earlier culture grant CSRS 89-37280-4799. The authots thank Drs. J. Sullistudies (Adamse et al., 1994) showing that BL stimulation of van and E. Middleton for helpful comments. large-scale UVAP accumulation was expressed between 24 and 72 h after the start of treatment. Note that UVAP were determined at 325 nm, the absorbance maximum of the extracts, References in order to optimize the discrimination of small changes. Absorbance changes, hence changes in screening of incident ra- AnAMSE, P. and S. J. BRITZ: Spectral quality of two fluorescent UV diation, would be reduced proportionately at other wavesources during long-term use. Photochem. Photobiol. 56, 641lengths (e.g., 300 nm) where extinction coefficients are lower. 644 (l992a). BL and UV-A had qualitatively similar effects on responses - - Amelioration of UV-B damage under high irradiance. I: Role of photosynthesis. Photochem. Photobiol. 56, 645-650 (1992 b). to UV-B, but quantitative comparisons are difficult to make for several reasons. The sensitivity of the third leaf to UV-B AnAMSE, P., S. J. BRITZ, and C. R. CALDWELL: Amelioration ofUVB damage under high irradiance. II: Role of blue light photoredecreased during the latter part of the study (i.e, ±UV-A) , ceptors. Photochem. Photobiol. 64, 110-115 (1994). possibly as a result of slight (ca. 12 h) increases in leaf physio- BEGGS, C. J" A. STOLZER-JEHLE, and E. WELLMANN: Isoflavonoid logical age. PAR was also lower during UV-B treatment (but formation as an indicator of UV stress in bean (Phaseolus vulgaris not during white light growth before or after) because the L.) leaves. The significance of photorepair in assessing potential UV-A lamps shaded the LPS lamps. Nonetheless, differences damage by increased solar UV-B radiation. Plant Physiol. 79, in leaf sensitivity or PAR do not affect the basic conclusions 630-634 (1985).

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