Starch degradation during gibberellin-induced leaf sheath growth in rice

20. Starch degradation during gibberellin-induced leaf sheath growth in rice C. MATSUKURA and J. YAMAGUCHI Bioscience Center and Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa-ku, Nagoya, 464-8601 Japan Gibberellins (GAs) promote elongation of rice leaf sheath with degradation of starch granules in parenchyma cells (Matsukura et a!. 1998). Although some physiological aspects of this phenomena have been reported, regulatory mechanism(s) of the starch degradation in the leaf cells remain obscure and further analyses is needed. For this purpose we examined the expression and the activity of starch phosphorylase which catalyzes the a-1,4 bonds of starch molecules and releases glucose-l-phosphate. The plant materials were prepared by a modification of the �microdrop method�. The seedlings were pretreated with a GA-biosynthesis inhibitor, uniconazole, to reduce the endogenous GA level and to enhance the sensitivity to exogenous GAs (Matsukura et a!. 1998). Seedlings at 0 - 48 h after application of GA3 at 300 pmol/plant were excised and fixed with FAA solution (5% formaldehyde, 5% acetic acid and 45% ethanol), dehydrated through tert-butyl alcohol series, embedded in Paraplast plus tissue embedding medium (Sherwood Medical, St. Louis, Mo., USA), sectioned longitudinally at 10-12 um with a rotary microtome. The sections were stained to visualize starch granules by PAS (Periodic acid and shift) staining. After application of GA3, number of starch granules in the leaf sheath parenchyma cells decreased rapidly, whereas dense granules remained in the control plants (Fig. 1). Under these conditions the time-course change of phosphory lase activity during the leaf sheath growth was measured. Fresh rice leaf sheath were ground with a three-fold amount (w/v) of extraction buffer consisting of 100 mM HepesNaOH (pH 7.5), 1 mM EDTA, 5 mM MgCl2. The homogenate was centrifuged at 10,000 x g for 10 min at 4°C. The supernatant was assayed for starch phosphorylase directly or after boiling, and later was used as control to determine the endogenous glucose-i -phosphate level. Phosphorolytic activity was measured in coupled enzyme assay as described by Preiss et al. (1980). As shown in Fig. 2, phosphorolytic activity of GA3-applied leaf sheath distinctively increased than that of untreated control at 9 h to 12 h when the striking starch degradation occurred (Fig. 1), indicating a positive relationship between promotion of the starch degradation and increase in the phosphorolytic activity (probably an enzyme activation). We obtained and cloned two partial cDNAs encoding starch phosphorylase from rice genome project, one (accession number; D23280) is highly homologous to type L isozyme and another (D46277) to type H isozyme of potato starch phosphorylase, respectively. It has been known that type L isozyme is localized within plastids and has a low affinity to oligoglucans, whereas the type H isozyme is located within the cytosol and has a high affinity for oligoglucans (Conrads et a!. 1986). The accumulation of starch phosphorylane mRNA during the growth was detennined by northern blot experiments using above two clones as probe. No significant differences were observed in the size or accumulation levels of both mRNAs through the time-course, and also no difference of plastidic phosphorylase protein using the type L isozyme specific antibody of potato (data not shown). However we cannot rule out the possibility that total phosphorylase activity shown in Fig. 2 was due to the expression level of type H isozyme. Recently, it was reported that a redox potential generated by photosynthesis is involved in the activation of plastidic acetyl-CoA carboxylase (ACCase) which catalyzes a first step of fatty acid synthesis in chloroplasts (Sasaki eta!. 1997). It is likely that gibberellins affect the phosphorylase activities through modulation of the redox potential and cytosolic and/or plastidic pH, resulting in the striking degradation of starch granules in leaf sheath cells.

The plant materials were prepared by a modification of the �microdrop method�. The seedlings were pretreated with a GA-biosynthesis inhibitor, uniconazole, to reduce the endogenous GA level and to enhance the sensitivity to exogenous GAs (Matsukura et a!. 1998). Seedlings at 0 - 48 h after application of GA3 at 300 pmol/plant were excised and fixed with FAA solution (5% formaldehyde, 5% acetic acid and 45% ethanol), dehydrated through tert-butyl alcohol series, embedded in Paraplast plus tissue embedding medium (Sherwood Medical, St. Louis, Mo., USA), sectioned longitudinally at 10-12 um with a rotary microtome. The sections were stained to visualize starch granules by PAS (Periodic acid and shift) staining. After application of GA3, number of starch granules in the leaf sheath parenchyma cells decreased rapidly, whereas dense granules remained in the control plants (Fig. 1). Under these conditions the time-course change of phosphory lase activity during the leaf sheath growth was measured. Fresh rice leaf sheath were ground with a three-fold amount (w/v) of extraction buffer consisting of 100 mM HepesNaOH (pH 7.5), 1 mM EDTA, 5 mM MgCl2. The homogenate was centrifuged at 10,000 x g for 10 min at 4°C. The supernatant was assayed for starch phosphorylase directly or after boiling, and later was used as control to determine the endogenous glucose-i -phosphate level. Phosphorolytic activity was measured in coupled enzyme assay as described by Preiss et al. (1980). As shown in Fig. 2, phosphorolytic activity of GA3-applied leaf sheath distinctively increased than that of untreated control at 9 h to 12 h when the striking starch degradation occurred (Fig. 1), indicating a positive relationship between promotion of the starch degradation and increase in the phosphorolytic activity (probably an enzyme activation).

We obtained and cloned two partial cDNAs encoding starch phosphorylase from rice genome project, one (accession number; D23280) is highly homologous to type L isozyme and another (D46277) to type H isozyme of potato starch phosphorylase, respectively. It has been known that type L isozyme is localized within plastids and has a low affinity to oligoglucans, whereas the type H isozyme is located within the cytosol and has a high affinity for oligoglucans (Conrads et a!. 1986). The accumulation of starch phosphorylane mRNA during the growth was detennined by northern blot experiments using above two clones as probe. No significant differences were observed in the size or accumulation levels of both mRNAs through the time-course, and also no difference of plastidic phosphorylase protein using the type L isozyme specific antibody of potato (data not shown). However we cannot rule out the possibility that total phosphorylase activity shown in Fig. 2 was due to the expression level of type H isozyme. Recently, it was reported that a redox potential generated by photosynthesis is involved in the activation of plastidic acetyl-CoA carboxylase (ACCase) which catalyzes a first step of fatty acid synthesis in chloroplasts (Sasaki eta!. 1997). It is likely that gibberellins affect the phosphorylase activities through modulation of the redox potential and cytosolic and/or plastidic pH, resulting in the striking degradation of starch granules in leaf sheath cells.