Starch Synthases

The glucose moiety of ADPglucose is incorporated in the growing starch polymer by a-1,4 linkages with the help of starch synthases. Results from studies conducted thus far indicate that plants contain two types of starch synthases, namely granule bound starch synthase (GBSS) and soluble starch synthase (SS). A complex picture is emerging from studies involving plant starch synthases [8], All starches contain at least one more isoform of starch synthase other than GBSSI [27]. GBSSI is thought to be responsible for the synthesis of amylose in the matrix of starch granules found in storage tissues [28], while GBSSII is thought to synthesize amylose in non-storage tissues of many cereals studied [29], Although SS have also been detected inside the starch granule, they are mostly inactive [30]. SS are active mainly at the surface of the starch granule, in the soluble fraction of the amyloplast [8]. At least three different isoforms of SS have been identified in potato tubers. However, different plant sink organs can use different forms of SS to catalyze the glucosyl transfer from ADP-glucose to the non-reducing end of an a-1, 4-glucan.

3.1. Manipulation of GBSSI in Transgenic Plants

The activity of GBSSI has been extensively studied using amylose-free (amf) potato mutants. This amf mutation is attributed to the deletion of a single AT base pair in the region coding for the transit peptide, resulting in its improper targeting to the amyloplast [31]. In transgenic potato lines, the activity of GBSSI was reduced by 70% to 100% by expressing the cDNA in antisense orientation under the CaMV 35 S promoter [32]. In these lines, both GBSS protein and amylose were absent. The starch from the transgenic tubers resembled that from the amf potato mutant. However, in the hilum of the starch granules of the transgenic tubers, amylose was found at a percentage similar to wild-type starch [33]. The level of amylose was correlated to the GBSS protein level found during development of the starch granule.

Sense expression of gbssl cDNA in the amf potato restored the amylose levels to that found in wild-type potatoes [34]. Complementation with gbssl cDNA in wild-type background however, was found to result in co-suppression of the endogenous GBSSI gene [35]. Amylose-containing transformants of the amf potato showed no positive correlation between GBSSI activity and amylose content, indicating that GBSSI is not the sole regulating factor in amylose metabolism. There exists a linear correlation between GBSSI activity and amylose synthesis when the GBSSI activity is low and ADP-Glucose levels are not depleted. When ADP-Glucose becomes limiting, the correlation between GBSSI activity and amylose synthesis is lost. Results from the antisense and sense RNA experiments suggest that the amount of available ADP-Glucose might also contribute to the flux of amylose synthesis.

Expression of the GBSSI protein encoded by a gene from cassava in transgenic amf potato resulted in only partial complementation [36]. Complementation was greatest when a hybrid GBSSI containing the transit peptide from potato, 89 amino acids from mature potato GBSSI, and cassava GBSSI was used. This research demonstrates that there appears to be some fundamental differences in the properties of GBSSI from potato and cassava despite 80%

similarity of the mature peptides from both sources. This small difference is perhaps all that is needed to result in the sub optimal activity of the heterologous cassava GBSSI in amf potato. Another important observation made in this study was that the starch granules from cassava GBSSI-complemented amf potato had uniform size blue cores, unlike starch granules from wild-type potato expressing antisense GBSSI. This suggests that there exists a role for heterologous gene expression in tailoring the starch for various applications and processes.

3.2. Manipulation of SS in Transgenic Plants

There are at least three distinct types of SS among the plants. Repression of the SS isoforms' activity usually results in amylopectin with modified chain lengths with a concurrent increase in amylose content, suggesting distinct contributions by each isoform [8, 37-41]. It was unclear if the specific contributions were due to the intrinsic properties of the isoform per se or if they depended upon the timing of the activity at a particular stage of starch synthesis. From antisense RNA and immunoprecipitation experiments in potato, it was inferred that SSIII and SSII contribute approximately 80% and 10-15% of the activity, respectively [37, 38], SSI was shown to be highly active in source tissues and barely active in the sink tissue of potato [39].

Reduction in the SSIII activity by over 80% results in cracked starch granules [37, 38] with amylopectin enriched in chains of 6 glucose units, i.e. degree of polymerization of 6 glucose units (dp 6) [40]. Reduction in the activity of SSII resulted in amylopectin that was enriched in chains of dp 8-12 and depleted in chains of dpl5-25 [40]. Cracked starch granules were also visible when total SS (mainly SSII and SSIII) activity was reduced by 60% [41]. In addition, the granules also appeared to be deeply sunken with holes through the center [40], Finally, the amylopectin from the transgenic plants with repressed SSII and SSIII had shorter branches and a reduction in the phosphorylation of the starch.

Based on the results from these experiments it is evident that there is an interaction, possibly in more ways than a synergistic manner, between SSII and SSIII during the production of amylopectin [40, 41], A reduction in either SSI or SSII activity has no effect on the net flux of carbon into starch in the sink tissues, but results in 50% decrease in phosphorylation, especially when SSII activity is repressed [39].

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