Fig. 1. Corn production in US (Reprinted with the permission of Genetics Society of America)
This review of starch synthesis in maize not only emphasized on those areas that have not been previously demonstrated by other authors, but also on those aspects that have advanced recently. The biochemical lesions responsible for altered starch and/or reduced quantities of starch were all identified first in maize (4-9). Although research over the past 30 years has identified the most important steps in starch synthesis in storage organs, our information is incomplete. A more complete picture will require identifying biochemical lesions in maize mutants such as sugary enhancer (se)(10), sugary2, and sugary starchy as well as finding mutants in important genes such as those encoding the soluble starch synthases, SSSI, and SSSII, so that we can establish their respective roles in starch synthesis. This deficiency in our knowledge extends also to the two branching enzymes, BEI and BEIIa, for which no null or hypomorphic mutants have been reported. We must obtain definitive evidence as to whether enzymes such as starch phosphorylase (E.C. 220.127.116.11) and the disproportionating enzyme (D-enzyme) (11) play any role in starch synthesis.
Other uncertainties concerning starch synthesis remain to be clarified: What is the mechanism of the phosphate groups linked covalently to some of the glucose molecules of starch, predominantly to C6, a process that makes phosphorylated starch. The starch synthetases require an oligosaccharide primer to which they add glucose units in a-1,4 linkages; therefore the question of the synthesis of primer for initiating starch synthesis is an open question to be elucidated. The question of what metabolite(s) enter the amyloplast as precursor(s) to the substrate(s) that will be utilized for starch synthesis has received different answers in experiments with amyloplasts isolated from several organisms. Seeds that are homozygous mutants for the most severe blocks in starch synthesis still produce 15-20% as much starch as do nonmutant controls, even when the mutant produces no protein product (12). This result raises the important question of whether the small amount of residue enzyme activity observed (in most cases encoded by another locus) is sufficient to account for the starch synthesis observed or is the mutant revealing a secondary route of synthesis?
The starch produced in nonmutant endosperms or other storage organs consists of two principle components, amylose and amylopectin. But maize sugary 1 mutant produced dominantly a soluble highly branched polysaccharide named phytoglycogen. Although amylose is usually described as a linear a-1,4 glucan, some of the molecules are sparsely branched. Takeda et al (13) found that about half the molecules in an amylose preparation, which was verified as pure amylose by gel filtration, were branched, with an average of 5.3 branches per molecule. The number average degree of polymerization, or d.p.n., indicates the average number of glucose units per polymer in starch sample. In lightly branched amylose, d.p.n. varied between 930 and 990. The variability depends on the steeping condition to which the corn kernels were subjected prior to isolation of the starch, and the maximum degree of polymerization was between 1930 and 2220. The average chain length varied between 295 and 335 glucose molecules. The iodine affinity was 20.1g/100g with an absorption maximum (lamda max) of 644 nm, following reaction with iodine. The iodine affinity or iodine-binding capacity measures the weight of iodine bound by a stated weight of starch. Under standard conditions, amylose binds approximately 20% of its own weight of iodine, whereas amylopectin binds none, so the ratio of these two components in any sample can be ascertained (14). The amount of monoacyl lipids within the starch granules is positively correlated with the amount of amylose that is present (15), and failure to remove the lipids before adding the KI/I2 agent results in an estimate of amylose that may be 3.5-7.4% lower than that of the true value (16).
The Hizukuri laboratory (17) has examined the heterogeneity of structure in corn amylose. Incubation of the amylose in 10% aqueous 1-butanol separated the sample into soluble and insoluble fractions. These fractions had linear and branched molecules in the proportions of 84:16 and 60:40, respectively. The branched molecules in the soluble fraction had a d.p.n. of 1620, about 20 branches per molecule, with short (d.p.n. -18), long (d.p.n.>230), and very long (d.p.n. not stated) chains. The large branched molecules in the soluble fraction may be immature amylopectin molecules. The branched molecules in the insoluble fraction have a d.p.n. of 2200 and a mean of 6 chains per molecule. These chains were short (d.p.n. -18) or very long (d.p.n.> 370).
The amylopectins from starch preparation discussed above (13) had an iodine affinity of 1.05 ~1.25g/100g with a lamda max 554 and 556 nm. The d.p.n. varied from 4800 to 10200 with an average chain length of 21 for all samples. The low estimate of 4800 is for starch that had been steeped in cold water and may reflect some degradation by carbohydratases. The percentages of amylopectin chains that fell into different average weights of polymerization ( d.p.n.) as measured by gel filtration for commercially prepared starch were fraction 1 (d.p.n. not stated) 10%, fraction 2 (d.p.n. 47) 20%, and fraction 3 (d.p.n. 18) 70%, so that there is a preponderance of short chains. All preparations had similar distributions of chain lengths, which together with the relative iodine affinities for amylopectins, suggest that there are long-B chains together with well-separated side chains. Manners (18) has discussed the advances in understanding of amylopectins as well as the techniques of elucidating these structures. Both the amylose and amylopectin components can be degraded completely by the combined action of fi-amylase and pullulanase, indicating that the branch points result from a-1,6 linkages. With fi-amylase alone, the amylose samples are about 82% hydrolyzed, and the amylopectin samples are 59% hydrolyzed.
Phytoglycogen is a polymer of D-glucose linked by a-1,4 and a-1,6 bonds similar to amylopectin, except that phytoglycogen is more highly branched and the products separated on gel filtration after enzymatic debranching show a chain-length distribution different from that of amylopectin. Based on the results of gel filtration, Fuwa's laboratory (19, 20) concluded that the ratio of A to B chain for phytoglycogen is low , and that the phytoglycogen apparently did not contain long B chains. Also B chains of the phytoglycogen seem comparatively uniform in chain length and are shorter in chain length than that of other maize starch. Moreover, the exterior chain length of phytoglycogen is consistently shorter than that of waxy amylopectin. Ball et al (21) suggested that phytoglycogen is the precursor of starch formation in plant starch granules.
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