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.
Highlights
ADP-glucose pyrophosphorylase (AGPase) provides the nucleotide sugar ADP-glucose and constitutes the first step in starch biosynthesis
The convention refers to the tissue from which the transcript was first isolated, it does not imply that gene expression or function is necessarily specific to that tissue
Functional complementation of an E. coli AGPase mutation demonstrated that AGPLLZM, AGPLEMZM, AGPSLZM, AGPSEMZM, and BT2b all are capable of generating enzymatic activity when combined with at least one partner subunit, and transient expression of GFP fusion proteins showed that all five proteins are imported into plastids
Summary
ADP-glucose pyrophosphorylase (AGPase) provides the nucleotide sugar ADP-glucose and constitutes the first step in starch biosynthesis. 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. Mutations of the barley or rice type 1 gene cause major reductions in leaf enzyme activity, identifying one of the transcripts encoding leaf AGPase SS as type 1b (Rösti et al, 2006; Lee et al, 2007). A minor amount of type 2 transcript was present in barley embryo, and presumably this specifies AGPase SS in this tissue
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