Maize starch synthases

Phytoglycogen-branching enzyme has been found in du however, no phytoglycogen was isolated by Black et al.23 Preiss and Boyer396 reported that the du mutation lowered starch synthase II activity and also lowered branching enzyme Ha activity. Gao et al.397 used a molecular approach to clone the du gene in maize endosperms and, based on amino acid sequence similarity of the predicted protein product with the soluble starch synthase III of potato,398 concluded that du most likely encodes the 180 000 molecular weight, primer-dependent soluble starch synthase described previously.399,400,401... 

Additional characterization of the two major soluble starch synthases in maize endosperm indicated distinct catalytic properties.402 Soluble starch synthase II has been reported to be encoded by the du gene.397 the initially observed reduction in BEIIa is a secondary effect and may be related to protein-to-protein interactions. 

Multiple forms of soluble starch synthases are present in barley endosperm,178 pea seeds,43 179 wheat endosperm,180482 maize endosperm,174 183-186 potato tuber,187 189 Arabidopsis,190 rice seed,191 192 Chlamydomonas reinhardtii,m sorghum seeds, teos-inte seeds and spinach leaf (the latter three are reviewed in references 2,7 and 27). 

The gene coding for full-length maize SSI (SSI-1) and genes coding for N-terminally truncated SSI (SSI-2 and SSI-3) have been individually expressed in E. coli198 and purified to homogeneity. The expressed enzymes had essentially the same properties found for other starch synthases I. In summary, with the exception of Arabidopsis,194 the function of soluble SSI in plants remains to be determined. 

The first enzymic studies done on the dul mutant were carried out in 1981, and both SSII and starch-branching enzyme Ha (SBEIIa) were found to have reduced activity in the endosperm compared to normal maize endosperm.204 SSII was shown to be different from SSI.173,205 SSII requires a primer for activity, and could not catalyze an unprimed reaction even in the presence of 0.5 M citrate, it also has less affinity for amylopectin than does SSI. However, 0.5 M citrate lowered the Km for amylopectin 17-fold. The activity with glycogen as a primer is one-half that observed with amylopectin. Therefore, glycogen is not as effective as a primer as is amylopectin, which differs from what was observed for starch synthase I. Both maize endosperm SSI and SSII had a Km for ADPGlc of 0.1 mM.196,205... 

In further experiments,185 it was found that DU1 was one of the two major soluble starch synthases, and when the C-terminal 450 residues of DU1 were expressed in E. coli, it was shown to have SS activity. Of interest was that antisera prepared against DU1 detected a soluble protein in endosperm extracts of molecular size greater than 200 kDa that was absent in dul mutants.185 The antisera reduced starch synthase activity by 20-30% in kemal extracts. In the same study, antisera prepared against SSI reduced starch synthase activity by 60%. In dul mutants, antisera prepared against SSI reduced the SS activity essentially to zero, suggesting that SSI and SSII were the only maize endosperm soluble starch synthases. Because of the high similarity in sequence of the DU1 starch synthase II to the potato SSIII, and because both are exclusively soluble, it is argued that DU1 is the evolutionary counterpart of potato SSIII.185,188 206 It is proposed that DU1 and maize SSII should be known as maize SSIII.184... 

Subsequently, the endosperm of hexaploid wheat, Triticum aestivum [L.], was shown to have a starch synthase III gene.182 A cDNA was isolated and contained an open reading frame for a 1629 amino acid polypeptide. The N-terminal region started with the transit peptide region of 67 amino acids, the N-terminal region of 656 residues, the SSSIII-specific region of 470 amino acids, and then the 436 amino acid catalytic domain in the C-terminal region. These domains were compared to those seen in starch synthases III from maize (DU1 protein),184 potato,188,206 cowpea,212 and Arabidopsis.213... 

Table 4.9 Kinetic parameters of the solubilized granule-bound starch synthase of maize endosperm175...

It is widely accepted that GBSS activity is a function of the protein coded by the waxy gene. The waxy locus gene product is a protein of molecular weight 58 KDa that is associated with starch granules and is similar to that found for the solubilized maize endosperm GBSSI.173 This protein has been extracted by heating the starch with a solution of SDS or by incubation at 37°C with 9M urea, but starch synthase activity... 

A direct relationship between the increase in the activities of starch synthase and ADPGlc PPase, and the rate of starch accumulation, has been reported for developing maize endosperm (Ozbun et al., 1973), wheat grain (Moore and Turner, 1969 Turner, 1969), and potato tubers (Sowokinos, 1976) (for a review of these, see Preiss and Levi, 1980). 

Work with a variety of plant systems has shown that multiple forms of SSS are present. Studies on barley endosperm, pea seeds, wheat endosperm, sorghum seeds, teosinte seeds, spinach leaf, maize endosperm, potato tuber, and rice seed extracts have indicated the presence of at least two major forms of SSS (reviewed in Preiss and Levi, 1980 Preiss, 1988 Preiss and Sivak, 1996), designated as types I and II. In maize leaf (Dang and Boyer, 1988) and castor bean endosperm (Goldner and Beevers, 1989), only one form of starch synthase was found, but since no extensive purification was attempted, the possibility remained that existing multiforms were not separated. Indeed, Downton and Hawker (1973) did find two forms of starch synthase in maize leaf, and thus the issue of the number of forms... 

PROPERTIES OF THE SOLUBLE STARCH SYNTHASES FROM MAIZE ENDOSPERM... 

Attempts to elute or solubilize the activity had met with little success until Macdonald and Preiss (1983, 1985) incubated ground maize starch granules with a-amylase and glucoamylase. The solubilized starch synthase activity was chromatographed on an anion exchange column and two peaks of activity were obtained, with 80% of the activity residing with starch synthase I, which eluted from the DEAE-cellulose column at a lower salt concentration than the starch synthase II fraction. 

S9, with higher starch synthase activity, had 36% of the amylose content observed in the nonmutant endosperm, whereas mutant S5, with an even lower starch synthase activity of 32%, had only 21% of the nonmutant maize amylose content. These data further support the view that the waxy protein is involved in amylose synthesis. 

The starch synthase isozymes in maize endosperm have different molecular masses. The GBSS isozyme I has a molecular mass of 60,000 GBSSII is 95,000. The SSSI has a molecular mass of 72,000, and SSSII is 95,000. Mu et al. (1994) have reported the molecular mass of maize endosperm SSSI as 76,000, which is similar to the value reported previously for SSSI (reviewed in Sivak and Preiss, 1994 Preiss and Sivak, 1996). These molecular mass values for the starch synthases are all higher than that of the E. coli glycogen synthase with a molecular weight of 52,000 (Kumar et al., 1986). 

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