Post-recovery assessment of UV-B irradiation effects in oilseed rape plants

Tom XX Rośliny Oleiste 1999 Akademia Rolnicza w Szczecinie, Zakład Fizyki Post-recovery assessment of UV-B irradiation effects in oilseed rape plants Ocena skutków napromieniowania UV-B roślin rzepaku po okresie restytucji Key words: Słowa kluczowe: Brassica napus, chlorophyll fluorescence, recovery, ultraviolet UV-B, oilseed rape Brassica napus, fluorescencja chlorofilu, restytucja, ultrafiolet UV-B, rzepak ozimy UV-B irradiation (UV-B BE =1.4 kj m -2 d -1 ) considerably decreased the activity of primary reactions of photosynthesis in oilseed rape leaves, with stronger effect when applied with a broad-band lamp, Philips TL40/12, than with a narrow-band lamp, Philips TL100/01. The plants grown under the latter lamp showed enhanced flavonoids synthesis. After recovery, the content of ultraviolet-absorbing compounds was equal in the irradiated and the control plants. The UV-B irradiation of the plants in an early stage of development resulted in the permanent changes: significant reduction of leaf area and dry mass, which was measured after four months' recovery at the presence of white light. Zastosowane promieniowanie UV-B (UV-B BE = 1,4 kj m -2 d -1 ) znacznie obniżyło sprawność pierwotnych reakcji fotosyntezy w liściach roślin rzepaku, przy czym szerokopasmowa lampa Philips TL40/12 wykazała silniejsze oddziaływanie niż lampa wąskopasmowa Philips TL 100/01. Rośliny rosnące przy tej drugiej lampie wykazały wzmożoną syntezę flawonoidów. Po okresie restytucji poziom związków pochłaniających promieniowanie ultrafioletowe uległ wyrównaniu w roślinach napromieniowanych i kontrolnych. Napromieniowanie UV-B roślin we wczesnej fazie wzrostu wywołało trwałe skutki poprzez istotne zmniejszenie powierzchni i suchej masy liści, mierzone po 4-miesięcznym okresie restytucji. Introduction Since the beginning of the 1970s, the significant increase of UV-B radiation level reaching the earth, in the range of 280 320 nm, has been observed, which is related to the decrease in the concentration of stratospheric ozone, absorbing the bulk of the radiation in this range (Caldwell 1977, Caldwell et al. 1998). The enhanced intensity of UV-B can cause adverse changes in the anatomy and biochemistry of plants, can hinder the processes of photosynthesis, slow the growth, decrease the biomass, and as a consequence, reduce the yield of cultivated

60 plants (Bornman and Teramura 1993; Caldwell et al. 1994; Starck et al. 1995). Plants are capable to develop various protective mechanisms, of which the most important consist in enhanced synthesis of flavonoids in epidermis, the compounds which absorb radiation in this range, thus protecting the inside of leaf (Caldwell et al. 1994; 1998). It has been reported, that at low doses of UV-B, the oilseed rape, although it belongs to the group of plants which show higher sensitivity to UV-B, has the ability not only to enhance the synthesis of flavonoids but also to increase leaf thickness, which is another mean of plant s adaptation (Cen and Bornman 1993; Wilson and Greenberg 1993). The previous papers presented the effects of short-term UV-B irradiation on the primary reactions of photosynthesis (Skórska 1996, Skórska and Murkowski 1997), and described how rape plants responded to several weeks of UV-B operation. It was possible for them to develop some protective mechanisms, mainly through the enhanced synthesis of flavonoids (Skórska 1998). This paper presents the studies which were aimed at: 1) the comparison of the effects of UV-B, generated with two kinds of lamps, on the primary reactions of photosynthesis and on the concentration of flavonoids in rape plant leaves, and 2) the examination of these reactions after several months of recovery i.e. at the presence of white light without UV-B as well as the morphometric analysis of the plants. Plant material Material and methods Seeds of oilseed rape plants (Brassica napus var. oleifera L.) variety Marita were sown to the soil in pots of 500 cm 3 in the beginning of October 1998. The pots were placed in a glasshouse (temperature ca. 20 C, PAR 50-500 µmol m -2 s -1 ) on the experimental set described by Skórska et al. (1997). The plants were watered daily and fertilised twice a week with 50% Hoagland nutrient solution (KNO 3 304 mg dm -3, MgSO 4 7 H 2 O 124 mg dm -3, NH 4 H 2 PO 4 12 mg dm -3, Ca(NO 3 ) 2 4 H 2 O 471 mg dm -3, 0.001% ferric citrate, microelements). UV-B irradiation of plants The plants in the phase of second leaf were divided into three groups. One of them, marked further as U1, was subjected to UV-B irradiation using two lamps TL100W/01 (Philips, The Netherlands) with maximum emission at wavelength 312 nm, during 16 days, 4 hours daily (10.00 to 14.00), at average irradiation intensity of 1.0 W m -2. Daily dose of biologically effective UV-B BE was ca. 1.4 kj m -2 d -1. The plants in the second group (marked as U2) were irradiated by two broad-band lamps TL40W/12 (Philips, The Netherlands) emitting in the

Post-recovery assessment of UV-B irradiation effects... 61 range from 280 to 320 nm (Fig. 1), with maximum emission at 311 nm during 16 days, 4 hours daily (10.00 to 14.00) at average irradiation intensity of 0.5 W m 2. Daily average dose of biologically effective UV-B BE equalled ca. 1.4 kj m -2 d -1. The third group consisted of control plants growing in the same conditions of light and temperature as the other plants, but without the UV-B. The intensity of irradiation was measured using a radiometer IL1403 with a calibrated detector SEL 240-UVB1 (International Light Co., USA), and biologically effective dose UV-B BE was calculated on the basis of the applied lamps emission spectra and according to Caldwell model (1977). After the irradiation, the plants were left to recover they grew in the glasshouse for four months, at temperature 20 C, without UV-B, at the natural sunlight passing through glass panes. Measurements of chlorophyll fluorescence The measurements of chlorophyll a fluorescence in leaves were done by means of a fluorometer PAM-200 (Walz, Germany), using weak measuring beam (0.04 µmol m -2 s -1, 650 nm), pulse saturating light (3200 µmol m -2 s -1, 665 nm) and actinic light (120 µmol m -2 s -1 ; 665 nm). The values of the following parameters were estimated: F o, F m, F v /F m, where F o, F m, F v denote respectively intensity of the initial, maximum and variable fluorescence (F v =F m -F o ), Y photochemical rate of PSII, q P and q N - coefficients of photochemical and nonphotochemical quenching, according to generally accepted denotations (Van Kooten and Snell 1990). Determination of ultraviolet-absorbing compounds content (flavonoids) In order to determine the content of ultraviolet-absorbing pigments (mainly flavonoids), the extracts were prepared in the following way: 2.5 cm 2 of leaf tissue was placed in the solution of ethanol : water : acetate acid (79 : 20 : 1), and heated at the temperature 60 C for 30 minutes. As a coefficient of flavonoids content, the value of absorbance at the wavelength 305 nm per unit of leaf area (A 305 dm -2 ) was used. Assessment of biometric features The area of leaves was measured by means of a system DIAS (Delta T, England). The dry matter of leaves was measured using a precision balance WPS 36 (Radwag, Poland) after drying at temperature 105 C to obtain constant mass. Statistical analysis of data Taking into account the previously examined variation of the studied objects and the precision of the applied methods, the measurements were done in 5 repetitions for flavonoids content, in 6 for fluorescence, and in 8 for biometric

62 features; the results are presented in tables as average values. To determine the significance of differences between the control plants and the UV-B irradiated ones (U1 and U2), one-way analysis of variance was used, whereas the Newman-Keuls test allowed to separate the particular groups of means (marked with the same letters), on the basis of the least significant differences calculated at the significance level 0.05 (LSD 0.05 ). Results and discussion The chlorophyll fluorescence parameter, F v /F m, proportional to the maximal quantum efficiency of photosystem II (Krause et al. 1990; Schreiber et al. 1994), which is ca. 0.8 for sound leaves, decreased significantly after the UV-B irradiation (Table 1). At the same time, the decrease was much stronger in case of the broadband lamp TL40/12, emitting in the range 280 320 nm (Fig.1). The decrease of this indicator resulted from the reduction of maximal fluorescence value, F m, without a detectable change in the initial fluorescence yield, F o. Similar changes of this parameter in UV-B irradiated plants were observed by Allen et al. (1997). The UV-B emitted in narrow range > 300 nm resulted in the increase of F o without a change in F m. Such character of fluorescence changes indicates the photoinhibition of photosynthesis, similar as e.g. in thermal damage of PSII reaction centre (Krause and Weis 1984). The photochemical rate of PSII, Y, was also considerably decreased, and so was the photochemical quenching coefficient, q p, related to CO 2 assimilation rate (Schreiber et al. 1994). The decrease of Y demonstrates a disturbance in the electron transport in PSII. The UV-B irradiation did not change the value of nonphotochemical quenching coefficient, q n, which, according to the interpretation of quenching presented by Härtel and Lokstein (1995) and Eskling et al. (1997), can indicate the tolerance of the xanthophyll cycle activity in rape leaves to the applied UV-B. After the four months' recovery period, the value of the parameter F v /F m for the UV-B irradiated plants did not differ from the control, whereas both Y and q p were significantly lower than in the control plants, however did not differ from each other. The content of the ultraviolet absorbing compounds in the leaves (Table 2) increased after the irradiation with UV-B in the narrow band of emission, which indicates that the lamp had a stimulating effect on the production of such compounds. In the previous experiments, similar stimulating effect of irradiation in this range on the enhanced synthesis of flavonoids was observed (Skórska 1998). After the restitution, the content of these compounds decreased, and did not vary significantly among the three studied groups. Presumably, it was due to the ageing of the plants.

Post-recovery assessment of UV-B irradiation effects... 63 TL100W/01 (U1) TL40W/12 (U2) 1,2 1,0 I λ /I m 0,8 0,6 0,4 0,2 0,0 280 290 300 310 320 wavelength [nm] Figure 1. Emission characteristics of the lamps UV-B sources using in the experiments Charakterystyka emisyjna lamp będących źródłem promieniowania UV-B, użytych w doświadczeniu Table 1 Chlorophyll fluorescence parameters of leaves, measured directly after UV-B irradiation, and after recovery time Parametry fluorescencji chlorofilu liści, mierzone bezpośrednio po napromieniowaniu UV-B oraz po okresie restytucji Parameter F o F m F v /F m Y q P q N Measured directly after irradiation Mierzone bezpośrednio po napromieniowaniu Control 259 a 1309 a 0.80 a 0.51 a 0.66 a 0.20 a U1 470 b 1322 a 0.63 b 0.15 b 0.21 b 0.07 a U2 255 a 403 b 0.24 c 0.03 c 0.05 c 0.09 a LSD 0.05 153 399 0.28 0.21 0.28 ns Measured after recovery time after irradiation Mierzone po okresie restytucji po napromieniowaniu Control 256 a 1149 a 0.77 a 0.50 a 0.64 a 0.20 a U1 303 a 1222 a 0.75 a 0.35 b 0.49 b 0.13 a U2 280 a 1216 a 0.76 a 0.38 b 0.51 b 0.08 b LSD 0.05 ns ns ns 0.11 0.12 0.09

64 Table 2 Content of ultraviolet-absorbing compounds (flavonoids) in the rape leaves Zawartość związków pochłaniających ultrafiolet (flawonoidów) w liściach rzepaku A 305 dm -2 Control U1 U2 LSD 0.05 Measured directly after irradiation Mierzone bezpośrednio po napromieniowaniu Measured after recovery after irradiation Mierzone po okresie restytucji po napromieniowaniu 26 a 40 b 24 a 10 14 a 13 a 12 a ns The analysis of rape plants morphological features (Table 3) explicitly indicates the decrease of both leaf area and dry mass, as an effect of the applied UV-B. The radiation emitted by the narrow-band lamp also lowered the average number of leaves per plant. Similar reduction of leaf area and rape dry mass was reported by Cen and Borman (1993), immediately after the UV-B irradiation with the TL40/12 lamp, however, applied in much higher dose. Table 3 Biometric properties of rape plants after recovery time after UV-B irradiation Cechy biometryczne roślin rzepaku po restytucji po napromieniowaniu UV-B Feature Cecha Control U1 U2 LSD 0,05 Average number of leaves Średnia ilość liści Average leaf area [cm 2 ] Średnia powierzchnia liścia Average dry leaf mass [mg] Średnia sucha masa liścia 7.6 a 4.5 b 5.6 ab 2.5 51 a 25 b 22 b 12 120 a 71 b 58 b 48 The means marked with different letters differ significantly at the significance level 0.05, on the basis of the least significant difference (LSD); ns not significant difference Średnie oznaczone różnymi literami różnią się istotnie na poziomie istotności 0,05 na podstawie najmniejszej różnicy istotnej (LSD); ns brak różnicy istotnej To recapitulate, it can be stated that even the low dose of UV-B resulted in such persistent damages in the rape photosynthetic apparatus that after the four months' trial of recovery, the area and dry mass of leaves were still considerably reduced in comparison with the plants grown without the UV-B irradiation. Such changes were indicated by the values of the leaf fluorescence parameters, especially Y and q p, which, measured immediately after the irradiation, were strongly reduced, and indicated the disturbance in the photosynthetic apparatus of the irradiated plants also after the recovery period.

Post-recovery assessment of UV-B irradiation effects... 65 Acknowledgement The experiments (including the purchase of the radiometer and fluorometer) were supported by the Polish State Committee for Scientific Research as the Project 6 PO4F 07914. Bibliography Allen D.J., McKee I.F., Farage P.K., Baker N.R. 1997. Analysis of limitations to CO 2 assimilation on exposure of leaves of two Brassica napus cultivars to UV-B. Plant, Cell and Environment, 20 (5): 633-640. Bornman J.F., Teramura A.H. 1993. Effects of ultraviolet-b radiation on terrestrial plants. W: Environmental UV Photobiology, ed. Young A.R. et al., Plenum Press, New York. 14: 427-477. UV/074 Caldwell M.M. 1977. The effects of solar UV-B radiation (280-315 nm) on higher plants: implications of stratospheric ozone reduction. Research in Photobiology, 597-607. Caldwell M.M., Flint S.D., Searles P.S. 1994. Spectral balance and UV-B sensitivity of soybean: a field experiment. Plant, Cell and Environment, 17: 267-276. Caldwell M.M., Bjorn L.O., Bornman J.F., Flint S.D., Kulandaivelu G., Teramura A.H., Tevini M. 1998. Effects of increased solar ultraviolet radiation on terrestrial ecosystems. J. Photochem. Photobiol. B: Biology, 46 (1-3): 40-52. Cen Y.P., Bornman J. 1993. The effect of exposure to enhanced UV-B radiation on the penetration of monochromatic and polychromatic UV-B radiation in leaves of Brassica napus. Physiol. Plant., 87: 249-255. Eskling M., Arvidsson P.O., Akerlund H.E. 1997. The xanthophyll cycle, its regulation and components. Physiol. Plant., 100 (4): 806-816. Hartel H., Lockstein H. 1995. Relationship between quenching of maximum and dark-level chlorophyll fluorescence in vivo: dependence on photosystem II antenna size. BBA, 1228: 91-94. Krause G.H., Somersalo S., Zumbusch E., Weyers B., Laasch H. 1990. On the mechanism of photoinhibition in chloroplasts. Relationship between changes in fluorescence and activity of photosystem II. J. Plant Physiol. 136: 472-479. Schreiber U., Bilger W., Neubauer C. 1994. Chlorophyll fluorescence as a nonintrusive indicator for rapid assessment of in vivo photosynthesis. Ecophysiology of Photosynthesis (ed. Schulze E.D., Caldwell M.M.), Ecological Studies, 100: 49-70. Skórska E. 1996. Reakcja liści rzepaku na promieniowanie ultrafioletowe UV-B. Rośliny Oleiste, XVI (1): 287-292. Skórska E., Murkowski A. 1997. Wpływ napromieniowania UV-B na fotosyntezę liści rzepaku rosnących przy niedoborze niektórych składników mineralnych. Rośliny Oleiste, XVIII (1): 111-118. Skórska E., Murkowski A., Mila A. 1997. Stanowiska do uprawy roślin przy zwiększonym napromieniowaniu ultrafioletem B. Mater. IX Międzynar. Sesji Nauk. Instytutu Techniki Rolniczej AR w Szczecinie, 130-135. Skórska E. 1998. Wpływ długotrwałego napromieniowania UV-B na reakcje świetlne fotosyntezy i zawartość barwników w liściach roślin rzepaku. Rośliny Oleiste, XIX (1): 81-87.

66 Starck Z., Chołuj D., Niemyska B. 1995. Fizjologiczne reakcje roślin na niekorzystne czynniki środowiska Wyd. SGGW Warszawa. Teramura A.H., Sullivan J.H. 1994. Effect of UV-B radiation on photosynthesis and growth of terrestrial plants. Photosynthesis Research, 39 (3): 463-473. Van Kooten O., Snel J.F.H. 1990. The use of chlorophyll fluorescence nomenclature in plant stress physiology. Photosynthesis Research, 25: 147-150. Wilson M.I., Greenberg B.M. 1993. Protection of the D1 photosystem II reaction center protein from degradation in ultraviolet radiation following adaptation of Brassica napus L. to growth in ultraviolet-b. Photochem. Photobiol., 57 (3): 556-563.