Chotipa Sakulsingharoj

Subcellular compartmentation and allosteric regulation of the rice endosperm ADPglucose pyrophosphorylase

ADPglucose pyrophosphorylase (AGPase) catalyzes the first unique step in the starch biosynthetic pathway. Recent studies in barley, wheat, and maize indicate that the major endosperm enzyme exhibits different enzymatic and cellular properties than those found in leaf tissue. To determine how prevalent these properties are in other plants, the AGPase activities of rice endosperm were studied at the enzymatic and cellular levels. Unlike the barley and wheat endosperm enzymes, the rice AGPase was dependent on 3-phosphoglycerate (3-PGA) with more than 40-fold increase in catalytic activity, when assayed under near saturating 3-PGA conditions. The rice enzyme was also inhibited by Pi and this inhibition was reversed by 3-PGA. Subcellular fractionation studies indicated that the bulk (90%) of the AGPase activity was extra-plastidic, located in the cytoplasm. A smaller amount of enzyme activity (10%) was observed associated with the amyloplast fraction. The presence of two AGPase forms was supported by immunoblot analysis using anti-bodies specific for the large or small subunit of the heterotetrameric enzyme. Anti-bodies specific for the maize endosperm AGPase small subunit (BT2) recognized a major 54 kD polypeptide in the cytoplasmic fraction. A second polypeptide at 48 kD was also detected in the cytoplasmic fraction but was more abundant in the amyloplast fraction. Based on results obtained from this study, strategies for increasing starch synthesis by manipulation of AGPase activities in developing seeds and, in turn, seed yields are discussed.

Dual Regulated RNA Transport Pathways to the Cortical Region in Developing Rice Endosperm

Prolamine and glutelin RNAs are localized to two subdomains of the cortical endoplasmic reticulum (ER), the protein body ER and the cisternal ER, in developing rice seeds. The addition of nearly full-length prolamine sequences at the 3′ untranslated region of a reporter RNA redirects its localization from the cisternal ER to the protein body ER. Deletion analysis of prolamine RNA sequences indicates the presence of two partially redundant cis elements required for protein body ER targeting. The addition of glutelin 3′ untranslated region to protein body ER cis sequences, however, redirects RNA localization to the cisternal ER. These results indicate that there are at least two regulated RNA transport pathways as well as a constitutive pathway to the cortical ER.

Control of starch synthesis in cereals: metabolite analysis of transgenic rice expressing an up-regulated cytoplasmic ADP-glucose pyrophosphorylase in developing seeds.

We had previously demonstrated that expression of a cytoplasmic-localized ADPglucose pyrophosphorylase (AGPase) mutant gene from Escherichia coli in rice endosperm resulted in enhanced starch synthesis and, in turn, higher seed weights. In this study, the levels of the major primary carbon metabolites were assessed in wild type and four transgenic CS8 rice lines expressing 3- to 6-fold higher AGPase activity. Consistent with the increase in AGPase activity, all four transgenic CS8 lines showed elevated levels of ADPglucose (ADPglc) although the extent of increases in this metabolite was much higher than the extent of increases in starch as measured by seed weight. Surprisingly, the levels of several other key intermediates were significantly altered. Glucose 1-phosphate (Glc 1-P), a substrate of the AGPase reaction, as well as UDPglucose and Glc 6-P were also elevated to the same relative extent in the transgenic lines compared with the wild-type control. Analysis of metabolite ratios showed no significant differences between the wild type and transgenic lines, indicating that the reactions leading from sucrose metabolism to ADPglc formation were in near equilibrium. Moreover, glucose and fructose levels were also elevated in three transgenic lines that showed the largest differences in metabolites and seed weight over the wild type, suggesting the induction of invertase. Overall, the results indicate that the AGPase-catalyzed reaction is no longer limiting in the transgenic lines, and constraints on carbon flux into starch are downstream of ADPglc formation, resulting in an elevation of precursors upstream of ADPglc formation.

Transformation of Rice (Oryza sativa) Cultivar Taichung 65 Mediated by Agrobacterium tumefaciens

The transformation efficiency of japonica rice cv. Taichung 65 using Agrobacterium tumefaciens was investigated. Mature rice seeds were cultured on N6D medium for callus induction. Scutellum-derived calli were transformed with Agrobacterium strain AGL1, harboring binary vector pCAMBIA 1305.1 which contains gusA as a reporter gene and hptII as a selectable marker gene. After co-cultivation, it was found that calli showed transient expression of gusA gene. The transformed calli were selected on medium containing hygromycin and cefotaxime for two cycles of 2 weeks each. Then hygromycin-resistant calli were regenerated to plantlets. PCR analysis confirmed the presence of gusA and hptII genes in the genome of transgenic rice plants. Transformation efficiency of Taichung 65 in this study was 4.32 %. The results will be useful to establish transformation system for studies of gene function and genetic improvement of rice varieties.