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Plants contain at least five subfamilies of starch synthases, granule bound starch synthase (GBSS) and starch synthases I, II, III, and IV (SSI, SSII, SSIII, SSIV). In this work, two members of SSIII and SSIV, respectively, were cloned and designated OsSSIII-1/-2 and OsSSIV-1/-2 in rice. Together with six other previously reported genes, the SS gene family in rice therefore is known to be duplicated and to comprise ten SS genes distributed among the five subfamilies. The starch synthase activity of each SS was confirmed by expression and enzyme activity assay in E. coli. Expression profile analysis with reverse transcription-PCR, western blotting and zymogram, indicates that OsSSIII-2 and OsSSIV-1 are mainly expressed in endosperm, while OsSSIII-1 and OsSSIV-2 are mainly expressed in the leaves. With a similar pattern of genes encoding other enzymes for starch synthesis, (such as GBSS, SSII, ADP-glucose pyrophosphorylases, and branching enzymes), it is suggested that two divergent groups of these genes should be classified in rice. Group I genes are preferentially expressed in the endosperm and function on storage starch synthesis. Group II genes are mainly expressed in leaves and some of them in the early developing endosperm, and function on transient starch synthesis in rice.

A full-length coding domain sequence of a gene analogous to granule-bound starch synthase (GBSS; ADP-glucose-starch glucosyltransferase, EC 2.4.1.21) was cloned and defined as OsGBSSII based on a Nitrogen (N)-starvation-induced cDNA library constructed using the rapid subtraction hybridization method. The deduced amino acid sequence of OsGBSSII was 62-85% identical to those of GBSS proteins from other plant species. The exon/intron organization of OsGBSSII was similar to that of OsGBSSI. OsGBSSII was mainly expressed in leaves and its protein was exclusively bound to starch granules in rice leaves, which suggests that the amylose in rice leaves is synthesized by OsGBSSII. N-starvation-induced expression of OsGBSSII could be repressed by supplying nitrate, ammonia or amino acid (glutamic acid or glutamine), glucosamine (an inhibitor of hexokinase) or dark conditions. These results indicate that N-starvation induction was dependent on the photosynthetic product and hexokinase in rice leaves. Sugars induced the accumulation of OsGBSSII transcripts in excised leaves through glycolysis-dependent pathways. OsGBSSII gene expression is regulated by the circadian rhythm in rice leaves.

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Three starch synthase (SS) genes, OsSSII-1, OsSSII-2 and OsSSII-3, were identified in rice (Oryza sativa L.) and localized to chromosomes 10, 2 and 6, respectively. The three OsSSII full-length cDNAs were cloned, and the predicted amino acid sequences were found to share 52-73% similarity with other members of the plant SSII family. The SS activity of each OsSSII was confirmed by expression and enzyme activity assay in Escherichia coli. Expression profile analysis revealed that OsSSII-1 was expressed in endosperms, leaves and roots; OsSSII-2 was mainly expressed in leaves, while OsSSII-3 was mainly expressed in endosperms. Similar to the OsSSI proteins, the OsSSII-2 and OsSSII-3 proteins were found in the soluble as well as the starch-granule-bound fractions in rice. The roles of the OsSSII proteins in starch biosynthesis in rice and the evolutionary relationships of the genes encoding monocotyledonous and dicotyledonous class-II SS enzymes are discussed.