Analysis of the rice ADPglucose transporter (OsBT1) indicates the presence of regulatory processes in the amyloplast stroma that control ADPglucose flux into starch

Abstract

Previous studies showed that efforts to further elevate starch synthesis in rice (Oryza sativa) seeds overproducing ADP-glucose (ADPglc) were prevented by processes downstream of ADPglc synthesis. Here, we identified the major ADPglc transporter by studying the shrunken3 locus of the EM1093 rice line, which harbors a mutation in the BRITTLE1 (BT1) adenylate transporter (OsBt1) gene. Despite containing elevated ADPglc levels (approximately 10-fold) compared with the wild-type, EM1093 grains are small and shriveled due to the reduction in the amounts and size of starch granules. Increases in ADPglc levels in EM1093 were due to their poor uptake of ADP-[ 14 C]glc by amyloplasts. To assess the potential role of BT1 as a rate-determining step in starch biosynthesis, the maize ZmBt1 gene was overexpressed in the wild-type and the GlgC (CS8) transgenic line expressing a bacterial glgC-TM gene. ADPglc transport assays indicated that transgenic lines expressing ZmBT1 alone or combined with GlgC exhibited higher rates of transport (approximately 2-fold), with the GlgC (CS8) and GlgC/ZmBT1 (CS8/AT5) lines showing elevated ADPglc levels in amyloplasts. These increases, however, did not lead to further enhancement in seed weights even when these plant lines were grown under elevated CO 2 . Overall, our results indicate that rice lines with enhanced ADPglc synthesis and import into amyloplasts reveal additional barriers within the stroma that restrict maximum carbon flow into starch.Cereal grains contribute a significant portion of worldwide starch production. Unlike other plant tissue, starch biosynthesis in the endosperm storage organ of cereal grains is unique in its dependence on two ADP-Glc pyrophosphorylase (AGPase) isoforms Thorbjørnsen et al., 1996;Sikka et al., 2001), a major cytosolic enzyme and a minor plastidial one, to generate ADP-glucose (ADPglc), the sugar nucleotide utilized by starch synthases in the amyloplast (Cakir et al., 2015). The majority of ADPglc in cereal endosperm is generated in the cytosol from AGPase (Tuncel and Okita, 2013) as well as by Suc synthase (Tuncel and Okita, 2013;Bahaji et al., 2014) and subsequently transported into amyloplasts by the BRITTLE-1 (BT1) protein located at the plastid envelope (Cao et al., 1995;Shannon et al., 1998).The Bt1 gene, first identified in maize (Zea mays; Mangelsdorf, 1926) and isolated by Sullivan et al. (1991), encodes a major amyloplast membrane protein ranging from 39 to 44 kD (Cao et al., 1995). The BT1 protein and its homologs belong to the mitochondrial carrier family (Sullivan et al., 1991;Haferkamp, 2007), which has a diverse range of substrates (Patron et al., 2004;Leroch et al., 2005;Kirchberger et al., 2008). The assignment of BT1 protein as the ADPglc transporter in cereal endosperms was first proposed by Sullivan et al. (1991), and then it was characterized based on the increased ADPglc levels and reduced ADPglc import rate * Address correspondence to okita@wsu.edu and hikaru.satoh.682@ m.kyushu-u.ac.jp.The authors responsible for distribution ...

Critical roles of soluble starch synthase SSIIIa and granule-bound starch synthase Waxy in synthesizing resistant starch in rice

“…4 G and E). Furthermore, the chain-length distributions in amylopectin exhibited a small increase in the abundance of branches with degrees of polymerization (DP) in the range 10-20 and a small decrease in chains with DP in the range [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50] (Fig. S5).…”

“…It is further proposed that the starch biosynthetic enzymes in the protein complex can exert a constraining effect on PPDK and AGPase to control the partitioning of ADPGlc into lipid or starch. It is known that uptake of ADPGlc by the plastid and the activity of the major SS enzymes do not limit carbon flux into starch, but that other constraints within the stroma control the flux into starch (36).…”

Gradually Decreasing Starch Branching Enzyme Expression Is Responsible for the Formation of Heterogeneous Starch Granules

“…Flipse et al (1996) suggested that, except for GBSSI, the amylose content of starch potentially correlates with the availability of ADP-Glc and the limited physical space available within the matrix of amylopectin. However, even though the BRITTLE1 (Bt1) gene was overexpressed in an up-regulated AGPase background, generating a rice line with enhanced ADP-Glc synthesis and import into amyloplasts, only a limited carbon flow into starch was observed (Cakir et al, 2016). Therefore, substrate amount is not the rate-limiting step for increasing amylose.…”

“…S2, A and C). Cakir et al (2016) reported that, when enriched ADP-Glc supply was provided, starch content could be elevated somewhat (about 10%) in the condition that the activity of starch synthase was kept unchanged. This indicated that starch content could be elevated even though the amounts and activity of starch synthase including GBSSI were not increased and only a limited amount of starch increase was detected.…”

Starch Biosynthesis in the Developing Endosperms of Grasses and Cereals

“…Genetic studies in maize, rice, and barley have shown that the predominant cytosolic isoform of AGPase is responsible for controlling the flow of carbon into storage starch in the endosperm [150][151][152][153]. ADP-Glc that is produced in the cytosol is transported into the amyloplast for starch synthesis via an ADP-Glc/ADP antiporter (termed BT1) located in the plastid inner envelope membrane [154][155][156]. The presence of extra-plastidial AGPase is not confined to domesticated cereals, but seems to be a unique feature of the endosperms of the Poaceae.…”