Maize endosperm

Starch is formed in chloroplasts of moss, fern and green algae.18 Chlorophyceae (green algae) starch is similar to that of higher plants, and several species have been used in studies of starch biosynthesis.19,22,29 In a recent set of studies, Ball et al.22 used Chlamydomonas reinhardtii to study starch biosynthesis. They produced several Chlamydomonas mutants which produce starch with characteristics similar to starches produced by maize endosperm mutants.31-34 The various starch mutations of Chlamydomonas will be discussed in Section 3.7. Other classes of algae which produce starch are Prasinophyceae19,35 and Cryptophyceae.35,36... 

Table 3.2 Amylose content of starch granules of various size classes isolated from maize endosperm 36 days post-pollination and from intermediate size (5-6 cm) potato tubers...

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. 

Interaction of these mutants further clarifies the biosynthetic pathway. For example, the wx mutant is epistatic to all other known maize endosperm mutants and no amylose accumulates (Table 3.6). Mutants such as sh2, bt2 and sit cause major reductions in starch accumulation, but when in combination with wx, the starch produced is all amylopectin.271 In the double mutant ae wx, wx prevents the production of amylose and ae reduces the degree of branching, resulting in the accumulation of a loosely-branched polysaccharide.88 The su mutant is epistatic to du, su2 and wx relative to accumulation of phytoglycogen, but ae and sh2 are partially epistatic to su, causing a marked reduction in the su stimulated phytoglycogen accumulation (Table 3.6). The addition of du or wx to ae su partially overcomes the ae inhibitory effect, and phytoglycogen accumulates. 

In spite of these limitations to our complete knowledge of starch biosynthesis, information about the pathway of starch biosynthesis gained from studies of maize endosperm mutants can probably be generalized to other plant species because related mutants have occurred in peas, sorghum, barley, rice and Chlamydomonas, and because the same enzymes are found in starch-synthesizing tissues in other plant species. Variation in the number of isozymes and their developmental expression, and variations in cellular compartmentation, however, could result in a range of pathways with significant differences. 

Partially purified maize endosperm ADPGlc PPase (34U/mg) was found to be activated by 3PGA and Fru 6-P (25- and 17-fold, respectively) and inhibited by Pi.77 The heterotetrameric endosperm enzyme has been cloned and expressed in E. coli, and its regulatory properties were compared to an isolated allosteric mutant less sensitive to Pi inhibition.117 As indicated above, the increase of starch noted in the mutant maize endosperm ADPGlc PPase insensitive to Pi inhibition supports the importance of the allosteric effects of 3-PGA and Pi in vivo. Also as indicated above, it is believed that the major endosperm ADP-Glc PPase isoform is located in the cytosol.141... 

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 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... 

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... 

Table 4.11 Properties of maize endosperm branching isozymes 14,249...

Amylose extender mutants have been found in rice.272 The alteration of the starch structure is very similar to that reported for maize endosperm ae mutants. The defect is in the BE3 isozyme, BE3 of rice being more similar in amino acid sequence to maize BE11 than to BEl.36,272 Thus, rice BE3 may catalyze the transfer of small chains, rather than long chains. 

Amino acid sequence relationships between that of branching enzyme (BE) and amy-lolytic enzymes, such as a-amylase, pullulanase, glucosyltransferase and cyclodextrin glucanotransferase, especially at those amino acids believed to be contacts between the substrate and the amylase family enzymes, were first reported by Romeo et al.283 Baba et al.284 reported that there was a marked conservation in the amino acid sequence of the four catalytic regions of amylolytic enzymes in maize endosperm BEI. As shown in Table 4.12, four regions that putatively constitute the catalytic... 

Potato tuber BE Maize endosperm BE I Maize endosperm BE II Rice seed BE 1 Rice seed BE 3 coli glycogen BE 6. subtilis a-amylase 6. sphaericus cyclodextrinase... 

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