Identification of the ADP-glucose pyrophosphorylase isoforms essential for starch synthesis in the leaf and seed endosperm of rice (Oryza sativa L.)

Abstract

ADP-glucose pyrophosphorylase (AGP) catalyzes the first committed step of starch biosynthesis in higher plants. To identify AGP isoforms essential for this biosynthetic process in sink and source tissues of rice plants, we analyzed the rice AGP gene family which consists of two genes, OsAGPS1 and OsAGPS2, encoding small subunits (SSU) and four genes, OsAGPL1, OsAGPL2, OsAGPL3 and OsAGPL4, encoding large subunits (LSU) of this enzyme heterotetrameric complex. Subcellular localization studies using green fluorescent protein (GFP) fusion constructs indicate that OsAGPS2a, the product of the leaf-preferential transcript of OsAGPS2, and OsAGPS1, OsAGPL1, OsAGPL3, and OsAGPL4 are plastid-targeted isoforms. In contrast, two isoforms, SSU OsAGPS2b which is a product of a seed-specific transcript of OsAGPS2, and LSU OsAGPL2, are localized in the cytosol. Analysis of osagps2 and osagpl2 mutants revealed that a lesion of one of the two cytosolic isoforms, OsAGPL2 and OsAGPS2b, causes a shrunken endosperm due to a remarkable reduction in starch synthesis. In leaves, however, only the osagps2 mutant appears to severely reduce the transitory starch content. Interestingly, the osagps2 mutant was indistinguishable from wild type during vegetative plant growth. Western blot analysis of the osagp mutants and wild type plants demonstrated that OsAGPS2a is an SSU isoform mainly present in leaves, and that OsAGPS2b and OsAGPL2 are the major SSU and LSU isoforms, respectively, in the endosperm. Finally, we propose a spatiotemporal complex model of OsAGP SSU and LSU isoforms in leaves and in developing endosperm of rice plants.

Functions of Multiple Genes Encoding ADP-Glucose Pyrophosphorylase Subunits in Maize Endosperm, Embryo, and Leaf

“…Confirmation of this result for AGPLLZM and AGPSLZM is provided by a proteomics study in which both proteins were identified directly in isolated maize leaf plastids (Majeran et al, 2005). Further support comes from a proteomics analysis that identified the orthologs of AGPSLZM and AGPLEMZM in wheat amyloplasts (Balmer et al, 2006) and by fluorescence localization of GFP fusions to the AGPase SS and LS orthologs of rice (Lee et al, 2007). …”

“…Type 1 genes generate two different transcripts referred to as type 1a and type 1b (Thorbjornsen et al, 1996;Burton et al, 2002;Ohdan et al, 2005). Type 1a transcripts encode a cytosolic protein that was directly identified as the extraplastidial AGPase SS from endosperm (Burton et al, 2002;Johnson et al, 2003;Lee et al, 2007). Type 1b transcript accumulates in endosperm and is predicted to encode a plastidial protein, but the AGPase SS purified from wheat or barley amyloplasts did not match the amino acid sequence predicted by this mRNA (Burton et al, 2002;Johnson et al, 2003).…”

“…Mutational analyses have identified the genes that encode the AGPase LS in endosperm cytosol of maize (Bae et al, 1990) and rice (Lee et al, 2007). Three additional genes encoding plastidial AGPase LS exist in rice and maize Giroux et al, 1995;Lee et al, 2007;Yan et al, 2009;Huang et al, 2010), but the tissuespecific functions of this gene family have not been comprehensively established.Functional analysis of endosperm plastidial AGPase will benefit from mutations that eliminate each subunit expressed in the tissue. Classical mutations at the shrunken2 (sh2) and brittle2 (bt2) loci led to the identification and cloning of the genes encoding cytosolic AGPase LS and SS, respectively (Bae et al, 1990;Bhave et al, 1990).…”

Transcriptional regulations of the genes of starch metabolism and physiological changes in response to salt stress rice (Oryza sativa L.) seedlings

“…The large subunit (L1) and small subunit (S1 and S2a) of AGPase protein in rice crop (cv. Dongjin) has been localized in plastid organelles, while S2b small subunit protein locates in cytosol (Lee et al 2007). Also, the sensitivity and function of those subunits of AGPase protein relating to salt stress are still undiscovered.…”

“…The chloroplast organelle, especially in the endosperm of cereals, is the origin of starch biosynthesis, forming complex polysaccharides, including linear and branched molecules of glucose polymer, amylose and amylopectin, by ADP-glucose pyrophosphorylase (ADP-AGPase), starch synthase (SS) and starch branching enzymes (SBE). In starch synthesis, ADP-glucose derived from photosynthesis is synthesised by the action of ADP-GPPase, which is the case in rice (Ballicora et al 2004;Lee et al 2007). The elongation of starch proceeds via SS activity that catalyses the transfer of glycosyl moiety of ADP-glucose to the non-reducing end of pre-existing α-D-1,4-glucan primer.…”