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

Abstract

ADP-glucose pyrophosphorylase (AGPase) provides the nucleotide sugar ADP-glucose and thus constitutes the first step in starch biosynthesis. The majority of cereal endosperm AGPase is located in the cytosol with a minor portion in amyloplasts, in contrast to its strictly plastidial location in other species and tissues. To investigate the potential functions of plastidial AGPase in maize (Zea mays) endosperm, six genes encoding AGPase large or small subunits were characterized for gene expression as well as subcellular location and biochemical activity of the encoded proteins. Seven transcripts from these genes accumulate in endosperm, including those from shrunken2 and brittle2 that encode cytosolic AGPase and five candidates that could encode subunits of the plastidial enzyme. The amino termini of these five polypeptides directed the transport of a reporter protein into chloroplasts of leaf protoplasts. All seven proteins exhibited AGPase activity when coexpressed in Escherichia coli with partner subunits. Null mutations were identified in the genes agpsemzm and agpllzm and shown to cause reduced AGPase activity in specific tissues. The functioning of these two genes was necessary for the accumulation of normal starch levels in embryo and leaf, respectively. Remnant starch was observed in both instances, indicating that additional genes encode AGPase large and small subunits in embryo and leaf. Endosperm starch was decreased by approximately 7% in agpsemzm-or agpllzm-mutants, demonstrating that plastidial AGPase activity contributes to starch production in this tissue even when the major cytosolic activity is present.Plant ADP-glucose pyrophosphorylase (AGPase) catalyzes the production of ADP-glucose (ADPGlc) and inorganic pyrophosphate (PPi) from Glc-1-P and ATP, thus generating the nucleotide sugar used by starch synthases to incorporate glucosyl units into starch. ADPGlc formation is an important metabolic control point and thus has been a genetic engineering target for crop improvement. Altering AGPase activity by transgenic means resulted in elevated yields in several starch-producing crops, including rice (Oryza sativa), maize (Zea mays), wheat (Triticum aestivum), and potato (Solanum tuberosum), although the precise nature of the metabolic and developmental changes that result remains to be elucidated (Stark et al., 1992;Greene and Hannah, 1998;Smidansky et al., 2002Smidansky et al., , 2003Smidansky et al., , 2007Hannah et al., 2012).AGPase localization appears to be strictly plastidial in most plant tissues, whereas in cereal endosperms, the majority of the enzyme is cytosolic and a minor form resides within amyloplasts (for review, see ComparotMoss and Denyer, 2009;Hannah and Greene, 2009;Geigenberger, 2011). Such an arrangement necessitates distinct modes of regulation and metabolic control of starch biosynthesis compared with other plants, including transport of ADPGlc and Glc phosphate (Glc-1-P and/or Glc-6-P) from the cytosol into the amyloplast. Subcellular fractionation revealed that 85% to 95% ...

Cloning and characterization of ADP-glucose pyrophosphorylase small subunit gene in Cyperus esculentus (yellow nutsedge)

“…Yellow nutsedge is a monocot that is more closely related to cereals than to dicots. However, previous studies have indicated that the AGPS gene in cereals is generally comprised of ten exons and nine introns (Huang et al, 2013); alternately, AGPS in cotton (Taliercio, 2010) and Arabidopsis (Hadrich et al, 2012) expressed a nine exon-eight intron structure, similar to that seen in this study.…”

“…Although the integral enzyme activity is a function of both subunits, previous studies have shown that the small subunit alone could form a homotetramer (α 4 ) and be active under saturating concentrations of substrates and 3-PGA (Ballicora et al, 1995). The ADPGlcPPase small subunit (AGPS) gene has been identified in various higher plants ( Thorbjornsen et al, 1996;Okita et al, 2001;Rosti et al, 2006;Rösti et al, 2007;Taliercio, 2010;Huang et al, 2013). In barley and rice (Okita et al, 2001), a single gene can encode two different transcripts for AGPS, whereas in maize, two paralogous genes encode two different AGPSs (Rösti et al, 2007).…”

Lineage-Specific Evolutionary Histories and Regulation of Major Starch Metabolism Genes during Banana Ripening

“…Banana AGPL genes were found only in two of the four Angiosperm AGPL sub-families identified by phylogenetic analysis (AGPLI and AGPLIII, Figure 1A ). These AGPLI and AGPLIII sub-families include known functional AGPL subunits (e.g., AtAPL1, AtAPL3, AtAPL4, agplIzm, ZmSH2, agplemzm; Crevillen et al, 2003; Huang et al, 2014) and have members in all analyzed species. In contrast, AGPLII and IV show gene loss in several lineages ( Figure 1A ).…”

Protein-Protein Interactions During Starch Biosynthesis

“…Whilst the AGPase subunits identified represent the cytosolic enzyme in maize, and therefore would be unlikely candidates to interact with starch synthases, it is possible that, in vivo, the plastidic isoforms are directly involved. We do not know which domains of the AGPase large and small subunits are involved in proteinprotein interactions, but there is a good degree of functional and structural similarity between the cytosolic and plastidic sequences (Huang et al 2014). The cytosolic enzyme accounts for approximately 99 % of the total AGPase activity (Denyer et al 1996) which may explain why the plastidic isoform was not readily detected in these protein-interaction experiments in vitro.…”