Department of Biotechnology, Singhania University, India

Leaf senescence is a key developmental state in the life of plants that leads to a massive mobilization and export of nitrogen and minerals to younger tissues to prepare for the next generation and/or to allow plant survive under adverse environmental conditions (Buchanan et al., 1997). Leaf senescence serves to remobilize nutrients (especially nitrogen) from the oldest leaves to the youngest ones (Diamantoglou and Kull, 1988). The most remarkable event in leaf senescence is the disassembly of the photosynthetic apparatus within chloroplasts and thus leads to the drop off in photosynthetic activity (Grover and Mohanty, 1992). The process of senescence brings about both qualitative and quantitative changes in the composition of pigments, macromolecules, structure and organization of thylakoids, damage of electron transport system (Biswal, 1997). Senescence has been shown to modify the structural components of thylakoid complexes like PS II, PS I, Cyt b/f and ATP synthase and cause their quantitative loss (Matile, 1992; Grover, 1993). The senescence programmed is the final developmental phase of a leaf which can be influenced by several phytohormones. GA inhibits in vitro senescence of oat leaf segments (Tetley and Thimann, 1974) and Rumex leaf segments respectively (Goldthwaite and Laetsch, 1968; Whyte and Luckwill, 1966). Mutations in the F-box protein (SLY1), result in a block of GA-responsive genes (Dill et al., 2004), delay senescence when crossed to abi1 (Richards et al., 2001). Studies related to the photosynthesis in the delay of senescence by GA are scanty. Hence a study on the effect of GA on pigments, proteins and photochemical activities in Rice primary leaf segments was made during dark incubation.

Plants and treatment: Healthy seeds of Rice (Oryza sativa) were obtained from Acharya N. G. Ranga Agricultural College, Hyderabad. The seeds were surface sterilized with 0.1 % HgCl2 for 2 min and thoroughly washed with tap water and then with distilled water. The seeds were imbibed for 6 h and germinated in petridishes on filter paper for 4 days. The seedlings were randomly placed in plastic trays and watered daily with quarter strength Hoagland nutrient solution (Arnon and Hoagland, 1950) and grown in a growth chamber providing with fluorescence light (Philips, India) with a light intensity of 30-35 µ moles m-2 s-1 at 25 ±1 0C. Fully expanded 10 th day leaf segments (4-5 cm long) were cut from

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15µM GA. Studies were made on senescence induced alterations in pigments, proteins and photochemical activities by keeping the leaf segments in double distilled water in dark as control for 24-96hrs.

0.1 gm leaf segments were homogenized in a pre-chilled mortar and pestle in 10 ml of 80 % chilled acetone. The homogenate was transferred into 15 ml centrifuge tubes and centrifuged at 3000 xg for 5 min. The Chl and carotenoid concentration was measured in supernatant after dilution to a total volume of 15 ml by following the method of Arnon (1949). Carotenoid concentration was calculated according to the method of MacKinney (1941) .The protein content in the leaf segments was determined according to the method of Lowry et al. (1951). 0.1 g of leaf segments add 10 % TCA and homogenized incubated at 4 0C for 8 h. The TCA precipitates were ether-dried, dissolved in 1N NaOH. Protein content was determined using bovine serum albumin (BSA) as standard. Isolation of thylakoid membranes: Thylakoid membranes were isolated according to the procedure similar to that of Saha and Good (1970) as described in Swamy et al. (1995). One g of leaf segments were homogenized with a prechilled mortar and pestle in 10 ml of ice-cold isolation buffer containing 50 mM HEPES-NaOH (pH 7.8), 400 mM sucrose, 2 mM MgCl2 and 5 mM KCl. The homogenate was filtered through two layers of miracloth and centrifuged at 6000 xg for 5 min. The pellet was washed twice with washing buffer containing 5 mM HEPES-NaOH (pH 7.8), 100 mM sucrose, 2 mM MgCl2 and 5 mM KCl and centrifuged briefly at 2000 xg for 30 s. The supernatant was pelleted again at 6000 xg for 5 min and the pellet was finally suspended in a minimal volume of suspension buffer containing 20 mM HEPES-NaOH (pH 7.8), 100 mM sucrose, 2 mM MgCl2 and 5 mM KCl. They were immediately used for measuring the photosynthetic electron transport activities. All the operations were carried out at 4 0C in dim light. Chl was estimated according to Arnon (1949). Photosynthetic electron transport activities: Electron transport rates were measured using a Clark type oxygen electrode (Hansatech, UK) in the thylakoid membranes. The measurements were done at 25 0C under saturating intensity of white light. The 2 ml assay reaction buffer contained 50 mM HEPES-NaOH (pH 7.5), 100 mM sucrose, 2 mM MgCl2 and 5 mM KCl according to Subhan and Murthy (2000). WCE and PS I mediated activities were measured as molecular consumption. The reaction mixture for WCE contained 0.5mM MV methyl viologen (MV) while that for the PS I determination contained 5mM ascorbate, 1mM sodium azide,0.5mM MV, 0.1 mM DCPIP and 10µM DCMU. PS II mediated electron transport was studied as molecular oxygen evolution using 0.5mM p-

Total Chl was steadily declined to 30 % in control leaf segments at 96 h during dark incubation (Fig. 26). GA significantly reduced this loss to 32 % at 96 h (p< 0.01). Chl a was decreased to 27 % at 96 h during dark incubation (Fig. 27). GA significantly reduced this loss to 29 % at 96 h (p< 0.001). The Chl b degraded to 42 % at 96 h during dark incubation (Fig. 28). GA significantly reduced this loss to 51 % at 96 h (p< 0.01). Carotenoid content was steadily declined to 48 % whereas GA significantly reduced this loss to 53 % at 96 h (Fig. 29) (p< 0.05). The total protein content was decreased to 35 % in control leaf segments at 96 h during dark incubation (Fig. 30). GA significantly reduced this loss to 55 % at 96 h (p< 0.01).

The WCE activity has been measured using MV as an electron acceptor. In control thylakoid membranes WCE decreased to 42 % at 72 h, while the activity was not found at 96 h during dark incubation (Table 1). GA significantly reduced the WCE activity loss to 50 % at 72 h (p< 0.01). p-BQ supported control PS II activity decreased to 38 % during dark incubation at 96 h and this loss was significantly reduced to 45 % by GA at 96 h (Table 1) (p< 0.01). PS I activity was slightly decreased to 78 % in control thylakoid membranes at 96 h, GA significantly reduced this loss to 82 % (Table 1) (p< 0.01).

This experiment deals with the effect of GA in delaying dark incubated senescence. Chl is essential to the vital process of photosynthesis in green plants. Total Chl was steadily declined in control leaf segments at 96 h during dark incubation (Fig. 1). GA slightly reduced this loss at 96 h. Chl a degraded more than Chl b during dark incubation (Fig. 2 & 3). GA slightly reduced this loss in Chl a and Chl b at 96 h. Chls are needed for the use of light energy in photosynthesis. A biochemical relation between leaf senescence and GA was first reported by Fletcher and Osborne (1965) showing that GA retarded senescence of excised leaf tissue from Taraxacum officinale by maintaining Chl levels. According to Richmond and Lang (1957) application of cytokinin could retard leaf senescence by preventing the Chl breakdown. In barley primary leaves the number of chloroplasts per mesophyll cells was found to be particularly unchanged during the period of degradation of Chl and chloroplast proteins (Martinoia et al., 1983). Corresponding findings have been reported for senescent Festuca and Rice leaves (Camp et al., 1982; Thomas, 1983; Wardley et al., 1984). According to Matile et al., (1988)

intermediary products of Chl breakdown might be expected to accumulate in the chloroplasts. Treatment with cytokinin in permanent darkness resulted in low contents of pink pigments representing intermediary products of natural Chl breakdown (Matile et al., 1987). Carotenoid content was steadily declined during dark incubation whereas GA slightly reduced the loss in carotenoid content at 96 h (Fig. 4). During senescence, neoxanthin and β-carotene decreased concomitantly with Chl, whereas lutein and xanthophylls cycle pigments were less affected, leading to increase in lutein/ Chl and xanthophylls cycle pigments/ Chl ratios (Lu et al., 2001). The total protein content was decreased in control leaf segments during dark incubation whereas GA reduced this loss at 96 h (Fig. 5). The progressive loss in protein content throughout dark incubation is probably due to higher activity of proteases which might degrade cellular proteins during senescence. However, the protein degradation was retarded by GA which was seen as maintenance of protein content in GA treated leaf segments. The degradation of Chl and proteins in senescent leaves are biochemical expressions of chloroplasts into gerontoplasts (Matile et al., 1988). WCE decreased at 72 h, while the activity was not found at 96 h during dark incubation (Table 1). GA reduced the WCE activity loss at 72 h. p-BQ supported control PS II activity decreased at 96 h during dark incubation and this loss was reduced by GA at 96 h (Table 1). PS I activity was slightly decreased in control leaf segments at 96 h during dark incubation and GA reduced the loss in PS I activity at 96 h. (Table 1). The susceptibility of PS II may be due to alterations in oxidizing side (Choudhry and Imaseki, 1990; Swamy et al., 1995) or reducing side of PS II (Prakash et al., 1998, 1999).

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GA retarded the loss of pigments, proteins, electron transport activities. The restoration of WCE activity by GA was closely associated with the restoration of PS II activity compared to that of PS I.

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