A Low-Starch Barley Mutant, Risø 16, Lacking the Cytosolic Small Subunit of ADP-Glucose Pyrophosphorylase, Reveals the Importance of the Cytosolic Isoform and the Identity of the Plastidial Small Subunit

Abstract

To provide information on the roles of the different forms of ADP-glucose pyrophosphorylase (AGPase) in barley (Hordeum vulgare) endosperm and the nature of the genes encoding their subunits, a mutant of barley, Risø 16, lacking cytosolic AGPase activity in the endosperm was identified. The mutation specifically abolishes the small subunit of the cytosolic AGPase and is attributable to a large deletion within the coding region of a previously characterized small subunit gene that we have called Hv.AGP.S.1. The plastidial AGPase activity in the mutant is unaffected. This shows that the cytosolic and plastidial small subunits of AGPase are encoded by separate genes. We purified the plastidial AGPase protein and, using amino acid sequence information, we identified the novel small subunit gene that encodes this protein. Studies of the Risø 16 mutant revealed the following. First, the reduced starch content of the mutant showed that a cytosolic AGPase is required to achieve the normal rate of starch synthesis. Second, the mutant makes both A-and B-type starch granules, showing that the cytosolic AGPase is not necessary for the synthesis of these two granule types. Third, analysis of the phylogenetic relationships between the various small subunit proteins both within and between species, suggest that the cytosolic AGPase single small subunit gene probably evolved from a leaf single small subunit gene.In the endosperm of all of the species of grasses so far investigated, there are both cytosolic and plastidial forms of the enzyme of ADP-Glc pyrophosphorylase (AGPase). However, there are indications that there may be differences between species in the relative amounts of the plastidial and cytosolic activities of AGPase in the endosperm and in the nature of the genes encoding their subunits. Studies of mutants of maize (Zea mays) show that the cytosolic form accounts for Ͼ95% of the total activity of AGPase in the endosperm (Denyer et al., 1996) and that this form is essential for normal rates of starch synthesis (Tsai and Nelson, 1966;Dickinson and Preiss, 1969). These studies also show that the plastidial and cytosolic forms of AGPase are encoded by different pairs of large and small subunit genes. For example, mutations in maize at the Bt2 locus specifically affect the cytosolic single small subunit (SSU) of AGPase and eliminate the activity of AGPase in the cytosol, but they do not affect the plastidial AGPase SSU or activity Hannah et al., 2001). For barley (Hordeum vulgare), the situation is less clear, but there is evidence to suggest that barley differs from maize in the following respects.First, the plastidial activity as a proportion of the total AGPase activity is considerably higher in barley endosperm (15%; Thorbjørnsen et al., 1996b) than in maize (Ͻ5%; Denyer et al., 1996). In maize endosperm, we calculate from published measurements of the rate of starch synthesis and total AGPase activity in maize (Singletary et al., 1997) that the plastidial AGPase activity alone is not sufficient to account for t...

The Effect of the Amount of Nitrogen Fertilizer on Starch Metabolism in Leaf Sheath of Japonica and Indica Rice Varieties during the Heading Period

“…The existence of two forms of AGPase small subunits, putative cytosolic and plastidial types, has been reported in rice (Anderson et al, 1989;Sikka et al, 2001). In the present study, we investigated the transcript level of rice type 1 AGPS (AGPS1) and type 2 AGPS (AGPS2), which encode the putative cytosolic and plastidial type small subunit, respectively (Johnson et al, 2003). The heavy application of nitrogen fertilizer enhanced slightly the mRNA level of AGPS1 in all the varieties, while it decreased the expression of AGPS2 markedly in Nipponbare and Johna (Fig.…”

“…One (accession number: J04960) encodes putative cytosolic type small subunit (Anderson et al, 1989), and another (accession number: AY028315) encodes putative plastidial type small subunit (Sikka et al, 2001). Johnson et al (2003) showed that AGPase small subunit genes of grass family were classfi ed into two distinct subgroups, type 1 and type 2, which consist of cDNAs encoding the cytosolic or plastidial type small subunit, respectively. In this paper, we represented tentatively the cytosolic and plastidial type small subunit genes as AGPS1 and AGPS2, respectively.…”

Spatiotemporal Profiling of Starch Biosynthesis and Degradation in the Developing Barley Grain

“…One subunit is localized in the plastids and is the major subunit in leaves (Rö sti et al, 2006), while the other is cytosolic and contributes to starch synthesis exclusively in the endosperm (Johnson et al, 2003). A third AGP-S isoform has been described as plastidial and is localized in endosperm (Johnson et al, 2003). ADPGlc, produced by AGPase, is utilized in plastids as a Glc donor for starch synthesis by starch synthases.…”

“…Two distinct AGP-S subunits in both barley and wheat (Triticum aestivum) arise through alternative splicing of the same gene (Thorbjørnsen et al, 1996;Burton et al, 2002b). One subunit is localized in the plastids and is the major subunit in leaves (Rö sti et al, 2006), while the other is cytosolic and contributes to starch synthesis exclusively in the endosperm (Johnson et al, 2003). A third AGP-S isoform has been described as plastidial and is localized in endosperm (Johnson et al, 2003).…”

“…Multiple isoforms of each enzyme are found in graminaceous plants, and specific roles have been proposed for each isoform on the basis of either mutant or transgenic phenotypes (Morell and Myers, 2005). The first key enzyme is ADP-glucose pyrophosphorylase (AGPase), as shown in the barley (Hordeum vulgare) Risø16 mutant, in which its activity is much compromised (Johnson et al, 2003;Rö sti et al, 2006). AGPase is a heterotetramer consisting of two large (AGP-L) and two small (AGP-S) subunits, each being encoded by distinct genes (Emes et al, 2003).…”