Starch branching enzymes (SBE) catalyze the formation of a-1,6 linkages in amylopectin. Multiple forms of SBE also exist and may be classified into two classes, A and B . Although there are known mutants for genes encoding for SBEA isoforms, none have been isolated so far in genes encoding for SBEB. Based on this fact, it has even been suggested that SBEB probably does not contribute much to starch synthesis , Although this line of argument has not been ruled out, other alternate propositions such as: i) that a mutation in this gene could be lethal, ii) that it might be encoded by multiple genes, and iii) that screening techniques are not sensitive enough, are emerging as other valid reasons [43, 44],
Although six different SBE isoforms are encountered, SBEA in potato is touted to play only a minor role in starch synthesis and was not even isolated and characterized until recently.
When endogenous expression was suppressed using antisense RNA technology, SBEA activity in leaves was reduced considerably, but not in tubers. However, differences in tuber starch properties were noticed . Average chain lengths increased even though there were no increases in large chain lengths, and the phosphorus content and apparent amylose levels were elevated. In barley, two isoforms of SBEA were characterized, and their genes were found to diverge only in their 5' end due to the presence of an intron containing a retro-transposon like element , This larger isoform was shown to be endosperm specific. Due to redundancy combined with tissue specificity of SBE expression by different isoforms, it is possible to account for the differences in starch structure observed in transgenic potato despite the lack of SBEA activity.
In potato, the major form of BE is of the SBEB type . When endogenous SBEB gene expression was repressed using antisense RNA approach, the physico-chemical properties of the starch were altered even though the amylose and amylopectin ratio and branch length of the amylopectin were largely unchanged . Phosphorous levels were elevated due to increases in 3- and 6-linked glucose phosphates. When SBEB was inactivated in amf potato by antisense techniques, again there was no change in the amylose and amylopectin ratios . There was, however, a reduction in the number of starch granules.
The role of SBEB in monocot endosperms is little understood. The situation becomes complex due to presence of multiple isoforms for SBEB and a lack of good screening techniques. In hexaploid wheat, it is suggested that there are up to ten different SBEB genes alone, although all of these may not encode functional SBE . Transgenic wheat lines with sbeb RNA expressed in antisense orientation driven by the rice actin promoter showed different levels of repression in the SBE activity . In one line where the activity of SBE was ten fold lower, the starch was less crystalline, and had a slight reduction in the high molecular weight component. However, the gel assays used could not differentiate in the screening of the various isoforms of SBE. Thus it was unclear how much activity of the SBE in the transgenic line was contributed by each isoform.
4.3. Sense expression of a glycogen branching enzyme (GBE) gene in potato
Glycogen is the non-phytic equivalent of starch in living organisms and branch chains in glycogen are made possible by the action of GBE. When a gene for glycogen branching enzyme from Eschericia coli was expressed in wild type and amf potato as a chimeric protein behind the GBSS transit peptide sequence and two N-terminal amino acids of the mature GBSS, the resulting transgenic potato exhibited starch containing increased branching degree [52, 53]. The branches were mostly short chains. However, neither the size nor the morphology of the starch granule was altered. This demonstrates that GBE is more akin to SBEA than SBEB in its function and can be utilized to alter starch physico-chemical properties.
5. DEBRANCfflNG ENZYME AND DISPROPORTIONATING ENZYMES
There is a greater degree of acceptance that starch debranching enzyme (DBE) activity contributes to the final starch structure , A model describing the role for DBE stemmed from studies in Chlamydomonas [54, 55], and research is being carried out in plants to resolve the roles for various isoforms of DBEs. Inconsistent reports have implicated both isoamylase (debranches amylopectin but not pullulan) and pullulanase/limit-dextrinase/R-enzyme (debranches pullulan) in amylopectin formation based on mutant analyses in maize [56, 57]. In barley, wheat and potato, isoamylase was found to be abundantly active in sink tissues , On the contrary, it was inactive during most of the early part of seed germination, and was found to gain activity only in the late seed germination stage. Based on this, it was suggested that in developing barley endosperm isoamylase plays a critical role in starch synthesis, and that it probably assists pullulanases and a-amylases in germinating seed. However, the question as to whether pullulanase contributes to starch biosynthesis in these plants was not addressed. Cloning of the genes for these enzymes should lead the way for the production of transgenic plants, which could be employed in furthering our understanding on the regulation of starch synthesis.
Although believed to be part of the starch degradation pathway a-1,4 glucanotransferases (disproportionating enzymes, or D-enzymes) have been reported to be present at the time of starch synthesis in plants . In Chlamydomonas D-enzyme was found to be active on the outer chains of amylopectin and glycogen . From this study, it was evident that D-enzyme is involved in the direct transfer of glucans onto the outer chains of amylopectin. In Chlamydomonas absence of D-enzyme resulted in an increase of unbranched malto-oligosaccharides, a lowering of starch content, amylopectin with modified branch chain pattern, and alterations in starch granule size and crystallinity. However, modification in plant starch metabolism by engineering the expression of D-enzyme has not been reported so far.
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