Identification of the soluble starch synthase activities of maize endosperm

Abstract

This study identified the complement of soluble starch synthases (SSs) present in developing maize (Zea mays) endosperm. The product of the du1 gene, DU1, was shown to be one of the two major soluble SSs. The C-terminal 450 residues of DU1 comprise eight sequence blocks conserved in 28 known or predicted glucan synthases. This region of DU1 was expressed in Escherichia coli and shown to possess SS activity. DU1-specific antisera detected a soluble endosperm protein of more than 200 kD that was lacking in du1- mutants. These antisera eliminated 20% to 30% of the soluble SS activity from kernel extracts. Antiserum against the isozyme zSSI eliminated approximately 60% of the total soluble SS, and immunodepletion of du1- mutant extracts with this antiserum nearly eliminated SS activity. Two soluble SS activities were identified by electrophoretic fractionation, each of which correlated specifically with zSSI or DU1. Thus, DU1 and zSSI accounted for the great majority of soluble SS activity present in developing endosperm. The relative activity of the two isozymes did not change significantly during the starch biosynthetic period. DU1 and zSSI may be interdependent, because mutant extracts lacking DU1 exhibited a significant stimulation of the remaining SS activity.

Figure 1

Expression of DU1C in E. coli. Gene expression from the T7 promoter of the indicated plasmid was induced in exponential-phase E. coli cells. Total soluble lysates were fractionated by SDS-PAGE, and specific proteins containing the S-tag sequence (specified by the pET plasmid) were detected by S-protein-alkaline phosphatase conjugate. Lane 1, pET-32b; lane 2, pHC6 (DU1C in pET-32b); lane 3, pET-29b; and lane 4, pHC5 (DU1C in pET-29b). Asterisks indicate polypeptides of approximately the size predicted from the plasmid and Du1 cDNA sequences, which are present only when the DU1C coding region is contained within the plasmid.

Figure 2

Immunological detection of DU1 and SSI in kernel extracts. a, Total soluble extracts from 20-DAP kernels of the W64A genetic background homozygous for the indicated allele were fractionated by SDS-PAGE and probed with anti-DU1N or anti-SSI. An equal amount of protein was loaded in each lane. du1-M5 indicates the allele du1-R4059. The asterisk indicates full-length DU1. b, Extracts of nonmutant W64A kernels and congenic du1-Ref mutant kernels collected 20 DAP were separated into granule (i.e. 10,000g pellet) and total soluble fractions (i.e. 10,000g supernatant). An equal volume of each fraction was separated by SDS-PAGE; therefore, each pair of lanes is standardized to kernel fresh weight. The samples were probed with anti-DU1N or anti-SSI, as indicated. c, Total soluble extracts of W64A kernels harvested at various times after pollination (as indicated) were analyzed by SDS-PAGE and immunoblot analysis using anti-DU1N or anti-SSI.

Figure 3

Immunodepletion of SS activity. Total soluble extracts from kernels of the indicated genotype collected 20 DAP were treated with preimmune serum or saturating amounts of the indicated antiserum, and residual SS activity was assayed after removal of the immune complexes. The du1-Ref mutant was in the W64A genetic background. SS activity remaining after treatment with preimmune serum was defined as 100%. These values were 7.0 nmol min⁻¹ mg⁻¹ for W64A, 12.9 nmol min⁻¹ mg⁻¹ for the du1-Ref mutant, and 16.4 nmol min⁻¹ mg⁻¹ for Oh43.

Figure 4

Specific identification of SS isozymes. a, SS activity zymogram. Proteins in total soluble endosperm extracts were separated based on molecular mass by SDS-PAGE and then allowed to renature in the gel. SS substrates were provided to the entire gel, and positions of glucan synthesis were detected by staining with iodine. Two congenic strains in the W64A genetic background were analyzed, one bearing the nonmutant allele Du1 and the other containing du1-Ref (indicated as du1-). Two SS activities are evident in the nonmutant endosperm, one of which is missing from the du1-Ref extract. b, Immunoblot analysis. Proteins in duplicates of the gel shown in a were transferred to nitrocellulose paper and probed with the indicated antiserum. A polypeptide of the same mobility and genetic specificity as the larger SS activity is recognized by anti-DU1N, whereas a protein of the same mobility as the smaller SS activity is recognized by anti-SSI.

Figure 5

SS activity in total soluble kernel extracts. Total soluble extracts from kernels of the indicated genotype collected 20 DAP were assayed for SS activity in the presence or absence of exogenous primer (10 mg/mL glycogen) and 0.5 m citrate, as indicated. The du1-Ref mutant was in the W64A genetic background.