Received 1 Dec. 2003 Accepted 23 Feb. 2004Supported by the State Key Basic Research and Development Plan of China (G199011707) and the National Natural Science Foundation ofChina (30170550, 30170175).

* Author for correspondence. E-mail: .

http://www.chineseplantscience.com

Acta Botanica Sinica 2004, 46 (6): 691-697

Effects of Silicon on Rice Leaves Resistance to Ultraviolet-B

LI Wen-Bin, SHI Xin-Hui , WANG He*, ZHANG Fu-Suo

(Department of Plant Nutrition, College of Resources and Environment Science, China Agricultural University,Key Laboratory of Plant-Interactions, MOE, Beijing 100094, China)

Abstract: In order to investigate the possible role of silicon in rice (Oryza sativa L.) leaves resistanceto ultraviolet-B (UV-B), experiments were conducted by using rice plants solution-cultured with or withoutsilicon supplementation. Results showed that under high UV-B irradiation the silicon-deficient leavesexhibited obvious brown spots and strips of UV damage symptoms, but the silicon-treated leaves were notaffected. A 21% and 67% increase in soluble and insoluble UV-absorbing compounds was observed in theepidermis of silicon-treated leaves, respectively. Furthermore, fluorescence microscopy revealed that agreat deal of insoluble UV-absorbing compounds was enriched in silicon bodies that were formed in the cell walls andcell lumina of epidermis of silicon-treated leaves, whereas the insoluble UV-absorbing compounds were less in theepidermis of non-silicon-treated leaves. Based on these results, it is concluded that the elevated UV resistanceof silicon-treated leaves is due to the increase of phenolic compounds in epidermis induced by silicon.Key word: rice (Oryza sativa); ultraviolet-B; phenolic compounds; silicon

The reduction in stratospheric ozone layer and the re-sulting increase in ultraviolet-B (UV-B) radiation on earthhave induced widespread public concern and become animportant scientific problem (Huang et al., 1998). In recentyears, considerable researches on UV-B resistance in plantshave been done (Janson et al., 1998; Brandt and Koch,2003). UV-B damages proteins and DNA, affects genomestability (Ishibashi et al., 2003), inhibits photosynthesis,and reduces the yield (Nayak et al., 2003). In principle, twodifferent tolerance strategies occur in vascular plants:screening of the internal tissues against the UV-B radiationand repair of inflicted damage (Janson and Gaba, 1998;Ishibashi et al., 2003). Both mechanisms complement eachother and both are apparently indispensable. Many re-searches have proved that phenolic compounds are themain substances to absorb and screen UV-B radiation inthe plant tissues. They are predominantly located in thecuticle, cell wall, and vacuole of epidermal cells which pro-tect the underlying mesophyll cells from UV-B injury (Krausset al., 1997; Hutzler et al., 1998; Schmitz-Hoerner andWeissenbo, 2003). With the elevation of UV-B radiation,the content of phenolic compounds in epidermal cells in-creased (Carlos et al., 2001), so the epidermal cells are in-dispensable barriers against ultraviolet stress. Rice is a typi-cal silicon accumulator; the level of SiO2 in the leaves is ashigh as 12% on a dry weight basis (Epstein, 1999; Rich-mond and Sussman, 2003). The insoluble SiO2 bodies, de-posited mainly in epidermal cells, are composed of 1-2

nanometer particles (Epstein, 1999). A recent report indi-cated that plastic films containing nanometer SiO2 particleshave higher capacity to absorb UV irradiation because na-nometer SiO2 particles possess super high absorptionsurface, special optic absorbing quality and quantum ef-fect (Mizutani and Nago, 1999). Another research demon-strated that UV-B radiation promoted silicon deposition inrice leaves (IRRI, 1991). So it is reasonable to ask whetherthe nanometer SiO2 particles deposited in the epidermalcells can enhance UV-B absorbing ability of rice leaves. If itdoes, whether it is due to the accumulation of UV-absorb-ing compounds induced by nanometer SiO2 particles inepidermal cells or due to the SiO2 particle itself. To answerthese questions, a series of experiments were conductedusing rice plants solution-cultured with or without siliconsupplementation. The experimental results show that sili-con can markedly enhance accumulation of phenolic com-pounds in epidermal cells of rice leaves and therefore in-crease the resistance to UV-B stress.

1 Materials and Methods1.1 Plant materials

Rice hybrid cultivar (7#)(Oryza sativa L.) fromChangsha City in Hunan Province of China was used. Seedswere germinated in quartz sand at 28 ,after 14 d, theseedlings were transplanted into 2-L plastic pots filledwith nutrient solution that was prepared according toYoshidas formula using pure water made by Millipore

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ultrapure water system (Yoshida et al., 1976). The composi-tion of nutrient solution (pH 5.6) is as follows: 4.38 mmol/LNH4NO3, 1.54 mmol/L K2SO4, 0.60 mmol/L KH2PO4, 1.0mmol/L CaCl2, 3.27 mmol/L MgSO4, 0.04 mmol/L MnCl2, 0.2mol/L (NH4)6Mo7O24, 0.6 mol/L ZnSO4, 0.6 mol/LCuSO4, 0.076 mmol/L H3BO3, 0.026 mmol/L FeSO4. The so-lution was supplemented with or without 1.5 mmol/L silicicacid. The growth chamber was 12 h light at 28 and 12 hdarkness at 25 . Air humidity was 60% R. H. (day) and80% R. H. (night) in the chamber. Ambient photosyntheticphoton flux (400-700 nm) ranged from 600-1 200mol .m-2.s-1. After growing for three months, the firstfully developed leaf was taken for experiments.1.2 Estimation of UV-B tolerance of rice leaves

According to Skaltsts method (Skaltst et al., 1994), UV-B irradiation was performed in a dark chamber at room tem-perature by using a 10 W Philips UV fluorescent lamp thatwas enveloped by a 0.13-mm cellulose acetate film to elimi-nate the light with wavelength shorter than 280 nm. BeforeUV-B treatment, the fresh leaves were cut into about 1-cm-long sections and floated on distilled water in petri dishes.The distance between the leaf sections and UV lamp was10 cm. After exposure to UV-B irradiation for 30 h, the leaveswere checked for brown spots and strips of visible UV dam-age symptoms under Olympus stereoscopic microscopeand Leica universal microscope. In order to take clearerphotographs of UV damage symptoms, the UV-B irradiatedleaves were extracted with a mixture of acetone and ethanol(1:1,V/V) to remove the chlorophyll.1.3 Histochemical localization of UV-absorbing com-pounds

In situ localization of UV-absorbing compounds in riceleaves: fresh hand-cut transverse sections of rice leaveswere sealed in 80% aqueous glycerin, and the yellow-greenautofluorescence of phenolic compounds in leaf cells wereexamined under Leica universal microscope excited by ul-traviolet light. Isolation and observation of silicon bodiesin leaf epidermis: in order to obtain epidermal peels fromrice leaves following Dayanadan et al. (1983), 1-cm-longpieces of fresh leaves were scraped on abaxial sides toremove most of the cells above the adaxial epidermis, thenthe isolated adaxial epidermis were digested in H2SO4 con-centrate for 4 d, after enough washing with distilled water,the released pure silicon bodies were collected for examina-tion of yellow-green autofluorescence of phenolic com-pounds excited by UV light.1.4 Measurement of relative levels of UV-absorbing com-pounds in epidermis

Preparation of intact epidermal tissues: according to the

procedure of Dayanadan et al. (1983), intact adaxial epider-mis was mechanically removed from fresh rice leaves bygently scraping off the abaxial side. The intact epidermalpieces free of perforations and mesophyll, determined bymicroscopic examination, were used for the followingexperiments.

Assessment of soluble UV-absorbing compounds: UV-B absorbing compounds were extracted with acid methanolmixture of MeOH-H2O-HCl (79:20:1, V/V/V) (Rozama et al.,2001). Samples of fresh adaxial epidermis were treated withabove extraction solution at 4 for 48 h, then centrifuged(10 min, 4 000 g). The UV absorption spectra of the super-natants were scanned over 200-400 nm bandwidth usingspectrophotometer (UV-2501, Shimazu Co., Japan). The larg-est absorption peak was selected for measurement of ab-sorption value of soluble UV-absorbing compounds. Therelative level of soluble UV-absorbing compounds in epi-dermis was expressed as the UV absorption value of perunit fresh sample weight. Every treatment was repeatedfive times.

Assessment of insoluble UV-absorbing compounds:according to Skaltsa et al. (1994), the above methanol ex-tracted intact epidermis was gently pressed between twoslices of filter paper and dried at room temperature. The dryepidermis was adhered to a black aluminium plate with a 4mm2 hole; the aluminium plate was then put into the sampleshelf in the UV spectrophotometer for 190-500 nm band-width scanning. For correction of the spectra and absorp-tion value, an identical control of aluminium plate withoutepidermis was inserted in the control light pathway. Ac-cording to the obtained UV absorption spectra of epidermis,the absorbance of insoluble UV-absorbing compounds inepidermis was measured at 270 nm and 320 nm and calcu-lated based on per unit leaf area. Every treatment was re-peated five times.

2 Results2.1 Effect of silicon on UV tolerance of rice leaves

Without UV-B irradiation both the silicon-treated (Fig.1b) and non-treated leaves (Fig.1a) showed no necroticspots, however, after 30 h exposure to UV-B radiation,the silicon-deficient leaves exhibited many brown spotsand strips that are typical UV damage symptoms in ac-cordance with the previous reports (Caasi-Lit et al., 1997)(Fig.1c). In contrast, there were almost no visible UVdamage symptoms in the silicon-fed leaves exposed tothe same level of UV-B radiation (Fig.1d), indicating thatsilicon can remarkably enha