The localization and expression of the class II starch synthases of wheat

Abstract

The starch granules of hexaploid wheat (Triticum aestivum) contain a group of three proteins known as SGP-1 (starch granule protein-1) proteins, which have apparent molecular masses of 100, 108, and 115 kD. The nature and role of these proteins has not been defined previously. We demonstrate that these polypeptides are starch synthases that are present in both the starch granule and the soluble fraction at the early stages of wheat endosperm development, but that are exclusively granule bound at mid and late endosperm development. A partial cDNA clone encoding a fragment of the 100-kD protein was obtained by screening a wheat endosperm cDNA expression library using monoclonal antibodies. Three classes of cDNA were subsequently isolated from a wheat endosperm cDNA library by nucleic acid hybridization and were shown to encode the 100-, 108-, and 115-kD proteins. The cDNA sequences are highly homologous to class II starch synthases and have the highest homology with the maize SSIIa (starch synthase IIa) gene. mRNA for the SGP-1 proteins was detected in the leaf, pre-anthesis florets, and endosperm of wheat and is highly expressed in the leaf and in the grain during the early to mid stages of development. We discuss the roles of the SGP-1 proteins in starch biosynthesis in wheat.

Figure 1

SDS-PAGE analysis of starch granule and soluble extracts from the endosperm of the wheat cv Wyuna. Each lane was loaded with the protein extracted from 5 mg of starch (starch granule extracts) or 20 μg of protein from soluble extracts. A, Lane 1, molecular mass markers; lane 2, silver-stained wheat SGPs; lane 3, activity stain of wheat SGPs. B, Lane 1, silver-stained protein standard ladder; lane 2, silver-stained wheat endosperm SGPs; lanes 3 to 14, immunoblots of SDS-PAGE gels loaded with either soluble extracts (lanes 3–8) or protease-treated starch granules (lanes 9–14). Samples were collected at different stages of endosperm development: 5 DPA (lanes 3 and 9), 7 DPA (lanes 4 and 10), 9 DPA (lanes 5 and 11), 13 DPA (lanes 6 and 12), 16 DPA (lanes 7 and 13), and 24 DPA (lanes 8 and 14). Occasional cross-reactivities of the monoclonal antibody with proteins of molecular mass lower than 100 kD were observed; however, these cross reactivities were not reproducible (unlike the consistent immunoreactivity of the 100-kD proteins) and are therefore considered to be nonspecific interactions.

Figure 2

Comparison of the deduced amino acid sequences of SSII from wheat (WSSIIA, WSSIIB, and WSSIID; this paper), maize (ZSSIIA and ZSSIIB; Harn et al., 1998), pea (PEASSII; Dry et al., 1992), and potato (POTSSII; van der Leij et al., 1991). Identical amino acid residues among each of these sequences are indicated below the sequences with an asterisk. The alignments of ZSSIIA with ZSSIIB, and PEASSII and POTSSII are essentially as described in Harn et al. (1998) and Edwards et al. (1995). All sequences are aligned to position the transit peptide cleavage site below the arrow (⇓) between residues 59 and 60 of the WSSIIA sequence. The deduced amino acid sequence from the wEL-1 insert, the sequence of SGP-B1 (peptide3), and the sequence of eight conserved regions are annotated and underlined.

Figure 2

Comparison of the deduced amino acid sequences of SSII from wheat (WSSIIA, WSSIIB, and WSSIID; this paper), maize (ZSSIIA and ZSSIIB; Harn et al., 1998), pea (PEASSII; Dry et al., 1992), and potato (POTSSII; van der Leij et al., 1991). Identical amino acid residues among each of these sequences are indicated below the sequences with an asterisk. The alignments of ZSSIIA with ZSSIIB, and PEASSII and POTSSII are essentially as described in Harn et al. (1998) and Edwards et al. (1995). All sequences are aligned to position the transit peptide cleavage site below the arrow (⇓) between residues 59 and 60 of the WSSIIA sequence. The deduced amino acid sequence from the wEL-1 insert, the sequence of SGP-B1 (peptide3), and the sequence of eight conserved regions are annotated and underlined.

Figure 3

Relationships between the primary amino acid sequences of SSs and glycogen synthase of E. coli. The dendrogram was generated using the PileUp program from Genetics Computer Group (Devereaux et al., 1984). The amino acid sequences used for the analysis are as follows (accession numbers given where reference not cited): wheat GBSS (Ainsworth et al., 1993), wheat SSI (Li et al., 1999), wheat SSII-A, wheat SSII-B, and wheat SSII-D (this paper), rice GBSS (X62134), rice SSI (D16202), maize GBSS (M24258), maize SSI (AF036891), maize SSIIa and maize SSIIb (Harn et al., 1998), maize SSII (Gao et al., 1998), pea GBSS (X88789), pea SSII (X88790), potato GBSS (X58453), potato SSI (Y10416), potato SSII (X87988), potato SSIII (X94400), and E. coli glycogen synthase (GS; J01616). Five groups of enzymes are labeled as GBSS, SSI, SSII, SSIII, and GS.

Figure 4

Alignment of conserved regions within cereal SS genes. Comparisons of cereal SSs were made based on their deduced amino acid sequences and eight conserved regions identified. Conserved regions are shown in bold and transit peptides (where defined) are shown in gray. Amino acid sequences were deduced from genes encoding; wheat GBSS (Ainsworth et al., 1993), wheat SSI (Li et al., 1999), wheat SSII-A1 (this report), maize SSIIa cDNA (Harn et al., 1998), and the maize dull-1 gene (Gao et al., 1998). The sequences of the conserved regions are defined in Figure 2.

Figure 5

Localization of cDNAs on wheat genomes by PCR. (i), Amplification with primers ssIIc and ssIIe. (ii), Amplification with primers ssIIc and ssIId. Lanes D, T. tauschii; lanes AB, N7DT7B; lanes AD, N7BT7A; lanes BD, N7AT7B; and lanes ABD, cv Chinese Spring (wild type). PCR products related to each cDNA clone are labeled. The arrow indicates a heteroduplex band that appears only when the A and D genes are amplified in the same mixture (see text).

Figure 6

Northern blot analysis of wheat wSSII mRNA in wheat. Total RNA was isolated from leaves, pre-anthesis florets, and endosperm of the wheat cv Gabo grown under defined conditions (16-h day length, 18°C day/13°C night) and probed with the wSSIIp2 DNA fragment. RNAs from leaf and pre-anthesis florets were loaded in the two left lanes. The remaining lanes contained RNA from endosperm and the extraction times (in DPA) are given above the lanes. Equivalent amounts of RNA were loaded in each lane.