**4. Altering the composition and amount of starch by biotechnological manipulation of enzymes**

The alteration of starch quantity and quality can be achieved through the overexpression of some enzymes involved in starch synthesis [60], by mutations or RNAi technology, such as the inhibition of potato SSII, SSIII and GBSS [61], or the decrease in the expression of wheat BEIIa and BEIIb [62,63]. In this way, by affecting the catalytic activity of enzymes involved in the synthesis of amylose or amylopectin, it could be possible to obtain starches for different purposes. Table 3 presents a summary of some of the varieties of plants (transgenic, mutant or silenced by RNAi) that exhibit altered levels of amylose.

The production of high amylose starch is of particular interest because its amount is correlated with the amount of RS in food. Foods with higher content of RS have the potential to improve human health and lower the risk of serious noninfectious diseases. As described above, the amylose content can be increased by the inactivation of the enzymes involved in amylopectin synthesis. In this way, RNAi was used to down-regulate the two different isoforms of starch-branching enzyme (BE) II (BEIIa and BEIIb) in wheat endosperm. Whereas the inhibition of BEIIb expression alone had no effect on amylose content; the decrease of both, BEIIa and BEIIb expression, resulted in the accumulation of starch containing more than 70% of amylose. When this high amylose starch was used to feed rats as a whole meal, it was observed that short-chain fatty acids such as butyrate, propionate and acetate increased with respect to controls. Short chain fatty acids are derived from the anaerobic fermentation of polysaccharides in the large intestine and are important in improving colonic health. These results indicate that this high-amylose wheat has a significant potential to improve human health through its RS content [62].

The decrease of BEIIb enzyme activity in rice is also traditionally associated with elevated amylose content, increased gelatinization temperature, and a decreased proportion of short amylopectin branches. To further elucidate the structural and functional role of this enzyme, the phenotypic effects of down-regulating BEIIb expression in rice endosperm were characterized by Buttardo and coworkers [64] by artificial microRNA (amiRNA) and hairpin RNA (hp-RNA) gene silencing. The results showed that RNA silencing of BEIIb expression in rice grains did not affect the expression of the other major isoforms of BE or SS proteins. The increase in about 2-fold of amylose content was not due to an increase in the relative proportion of amylose chains but instead was due to significantly elevated levels of long and intermediate chains of amylopectin. Rice altered by the amiRNA technique produced a more extreme starch phenotype than those modified using the hp-RNA technique, with a greater increase in the proportion of long and intermediate chains of amylopectin. The major structural modifications of starch produced in the amiRNA lines led to more severe alterations in starch granule morphology and crystallinity as well as digestibility of freshly cooked grains [64].


Ref: NA, not applicable

498 The Complex World of Polysaccharides

**manipulation of enzymes** 

disproportionating enzyme [85]. In particular, the initial steps of starch degradation at the granule surface are regulated mainly by phosphorylation [50,51]. Furthermore, a starch biosynthetic enzyme, the starch synthase III (SSIII) from *Arabidopsis thaliana* (AtSSIII) has been reported by our group to have a regulatory role in the synthesis of transient starch [33]. This enzyme contains 1025 amino acid residues and has an N-terminal transit peptide for chloroplast localization followed by three in tandem starch-binding domains (SBD D1, D2 and D3, residues 22-591), which bind to raw starch and its individual components, amylose or amylopectin [53,54,58,59]. The adsorption experiments show that the SBD123 region binds preferentially to amylose, and that the D1 domain is mainly responsible for this selective binding. The D2 domain contains two binding sites including amino acid residues Y394 (binding site 1) and W366 (binding site 2) which act in cooperation with the D1 domain in the binding activity while G335 and W340 have a minor role [54]. It is worth mentioning that our work was the first report on the existence of an SBD in a synthesizing enzyme

(AtSSIII) and the first experimental evidence of its starch binding capacity.

(transgenic, mutant or silenced by RNAi) that exhibit altered levels of amylose.

significant potential to improve human health through its RS content [62].

**4. Altering the composition and amount of starch by biotechnological** 

The alteration of starch quantity and quality can be achieved through the overexpression of some enzymes involved in starch synthesis [60], by mutations or RNAi technology, such as the inhibition of potato SSII, SSIII and GBSS [61], or the decrease in the expression of wheat BEIIa and BEIIb [62,63]. In this way, by affecting the catalytic activity of enzymes involved in the synthesis of amylose or amylopectin, it could be possible to obtain starches for different purposes. Table 3 presents a summary of some of the varieties of plants

The production of high amylose starch is of particular interest because its amount is correlated with the amount of RS in food. Foods with higher content of RS have the potential to improve human health and lower the risk of serious noninfectious diseases. As described above, the amylose content can be increased by the inactivation of the enzymes involved in amylopectin synthesis. In this way, RNAi was used to down-regulate the two different isoforms of starch-branching enzyme (BE) II (BEIIa and BEIIb) in wheat endosperm. Whereas the inhibition of BEIIb expression alone had no effect on amylose content; the decrease of both, BEIIa and BEIIb expression, resulted in the accumulation of starch containing more than 70% of amylose. When this high amylose starch was used to feed rats as a whole meal, it was observed that short-chain fatty acids such as butyrate, propionate and acetate increased with respect to controls. Short chain fatty acids are derived from the anaerobic fermentation of polysaccharides in the large intestine and are important in improving colonic health. These results indicate that this high-amylose wheat has a

The decrease of BEIIb enzyme activity in rice is also traditionally associated with elevated amylose content, increased gelatinization temperature, and a decreased proportion of short amylopectin branches. To further elucidate the structural and functional role of this enzyme, **Table 3.** Amylose content of different lines (wt, mutant and/or transgenic). Adapted from [10].

The roles of BEIIa and BEIIb in defining the structure of amylose and amylopectin were also examined in barley (*Hordeum vulgare*) endosperm. Barley lines with low expression of either BEIIa, BE IIb or both isoforms were generated through RNA-mediated silencing technology. These lines enabled the study of the role of each of these proteins in determining the amylose content, the distribution of chain lengths, and the frequency of branching in both amylose and amylopectin. A high amylose phenotype (> 70%) was observed in lines expressing lower levels of BEIIa and BEIIb, while a reduction in the expression of either of these isoforms alone had minor impact on amylose content. The structure and properties of the barley high amylose starch resulting from the decrease in the expression of both BEII isoforms were found to be similar to those observed in amylose mutants of maize, which result from mutations that decrease the expression of the BEIIb gene. The analysis of amylopectin chain length distribution indicated that both BEIIa and BEIIb isoforms have distinct roles in determining the fine structure of amylopectin. A significant reduction in the frequency of branches in amylopectin was observed only when both BEIIa and BEIIb were reduced, whereas there was a significant increase in the branching frequency of amylose when BEIIb alone was reduced [61,65].

Other way of modifying amylase content is by SS expression. Amylose and amylopectin of rice mutants deficient in endosperm SS isoforms, either SSI (ΔSSI) or SSIIIa (ΔSSIIIa), were found to have an altered structure respect to to their parent (cv. *Nipponbare*, Np). The amylose content was higher in the mutants (Np, 15.5%; ΔSSI, 18.2%; ΔSSIIIa, 23.6%), and the molar ratio of branched amylose and its side chains was increased. In addition, the chainlength distribution of the β-amylase limit dextrins of amylopectin showed high regularity, which is consistent with the reported cluster structure. The mole % of the B(1)-B(3) fractions was changed slightly in ΔSSI, which is consistent with the proposed role of SSI in elongating the external part of clusters. In ΔSSIIIa, it has been observed a significant increase in the B(1) fraction and a decrease in both, the B(2) and B(3) fractions. The internal chain length of the B(2) and B(3) fractions appeared to be slightly altered, suggesting that the deficiency in SS affected the actions of branching enzyme(s) [66].

In another approach, SSIIIa null mutants of rice (*Oryza sativa*) were generated using retrotransposon insertion and chemical mutagenesis. The amylopectin B(2) to B(4) chains with degree of polymerization (DP) >/= 30 and the M(r) of amylopectin were reduced to about 60% and 70% in the mutants, suggesting that SSIIIa plays an important role in the elongation of amylopectin B(2) to B(4) chains. Chains with DP 6 to 9 and DP 16 to 19 decreased while chains with DP 10 to 15 and DP 20 to 25 increased in the amylopectin mutants. These changes in the SSIIIa mutants are almost opposite images of those of SSIdeficient rice mutant and were caused by 1.3- to 1.7-fold increase of the amount of SSI in the mutant endosperm. Furthermore, the amylose content and the extralong chains (DP >/= 500) of amylopectin were increased by 1.3- and 12-fold, respectively. These changes of starch composition of the mentioned mutants are due to the increase in about 1.7-fold of GBSSI activity. The starch granules of the mutants were found to be smaller with round shape and less crystalline. Thus, SSIIIa deficiency, the second major SS isoforrm in developing rice endosperm, affected either the structure of amylopectin, amylase content, and also the physicochemical properties of starch granules in two ways: directly by the SSIIIa deficiency itself and indirectly by up-regulation of both SSI and GBSSI mRNA [67].

By a different approach Safford et al [68] reported no effect on the amylose content of potato starch after the downregulation of the expression of the major branching enzyme isozyme (BE). However, a notable increase (50 – 100%) of the phosphorous content was detected. Although the almost complete suppression of the branching enzyme activity (less than 5% respect to wt levels) in transgenic potato tubers, no changes in amylose content of the starches derived from these transgenic lines were detected. Differences in the gelatinization properties (an increase of up to 5°C in the peak temperature and viscosity onset temperature) are reported, suggesting that these changes correlated with the branching pattern of the starch that result in changes of the double helix length. It is also possible that the increased phosphate content observed in the transgenic starches resulted in the elevation of the gelatinization temperature [68].

500 The Complex World of Polysaccharides

when BEIIb alone was reduced [61,65].

affected the actions of branching enzyme(s) [66].

expressing lower levels of BEIIa and BEIIb, while a reduction in the expression of either of these isoforms alone had minor impact on amylose content. The structure and properties of the barley high amylose starch resulting from the decrease in the expression of both BEII isoforms were found to be similar to those observed in amylose mutants of maize, which result from mutations that decrease the expression of the BEIIb gene. The analysis of amylopectin chain length distribution indicated that both BEIIa and BEIIb isoforms have distinct roles in determining the fine structure of amylopectin. A significant reduction in the frequency of branches in amylopectin was observed only when both BEIIa and BEIIb were reduced, whereas there was a significant increase in the branching frequency of amylose

Other way of modifying amylase content is by SS expression. Amylose and amylopectin of rice mutants deficient in endosperm SS isoforms, either SSI (ΔSSI) or SSIIIa (ΔSSIIIa), were found to have an altered structure respect to to their parent (cv. *Nipponbare*, Np). The amylose content was higher in the mutants (Np, 15.5%; ΔSSI, 18.2%; ΔSSIIIa, 23.6%), and the molar ratio of branched amylose and its side chains was increased. In addition, the chainlength distribution of the β-amylase limit dextrins of amylopectin showed high regularity, which is consistent with the reported cluster structure. The mole % of the B(1)-B(3) fractions was changed slightly in ΔSSI, which is consistent with the proposed role of SSI in elongating the external part of clusters. In ΔSSIIIa, it has been observed a significant increase in the B(1) fraction and a decrease in both, the B(2) and B(3) fractions. The internal chain length of the B(2) and B(3) fractions appeared to be slightly altered, suggesting that the deficiency in SS

In another approach, SSIIIa null mutants of rice (*Oryza sativa*) were generated using retrotransposon insertion and chemical mutagenesis. The amylopectin B(2) to B(4) chains with degree of polymerization (DP) >/= 30 and the M(r) of amylopectin were reduced to about 60% and 70% in the mutants, suggesting that SSIIIa plays an important role in the elongation of amylopectin B(2) to B(4) chains. Chains with DP 6 to 9 and DP 16 to 19 decreased while chains with DP 10 to 15 and DP 20 to 25 increased in the amylopectin mutants. These changes in the SSIIIa mutants are almost opposite images of those of SSIdeficient rice mutant and were caused by 1.3- to 1.7-fold increase of the amount of SSI in the mutant endosperm. Furthermore, the amylose content and the extralong chains (DP >/= 500) of amylopectin were increased by 1.3- and 12-fold, respectively. These changes of starch composition of the mentioned mutants are due to the increase in about 1.7-fold of GBSSI activity. The starch granules of the mutants were found to be smaller with round shape and less crystalline. Thus, SSIIIa deficiency, the second major SS isoforrm in developing rice endosperm, affected either the structure of amylopectin, amylase content, and also the physicochemical properties of starch granules in two ways: directly by the SSIIIa deficiency

By a different approach Safford et al [68] reported no effect on the amylose content of potato starch after the downregulation of the expression of the major branching enzyme isozyme (BE). However, a notable increase (50 – 100%) of the phosphorous content was detected.

itself and indirectly by up-regulation of both SSI and GBSSI mRNA [67].

Other strategy to obtain high amylose starches was carried out by Itoh et al [60]. The Waxy (Wx) gene encodes a granule-bound starch synthase (GBSS) that plays a key role in the amylose synthesis of rice and other plant species. In rice, it has been described two functional Wx alleles: Wx(a), which produces higher amounts of amylose, and Wx(b), which produces low amounts of this polymer due to a mutation in the 5' splicing site of intron 1. When the Wx(a) cDNA was introduced into null-mutant Japonica rice (wx) the amylose content were 6-11% higher than that of the original cultivar, Labelle, which carries the Wx(a) allele, although the levels of the Wx protein in the transgenic rice were equal to those of cv. Labelle [60].

Finally, using *A. thaliana* null mutant lines for the SSIII locus, it has been postulated that SSIII has a regulatory role in the starch synthesis process [33]. These mutant lines show a higher accumulation of leaf starch during the day due to an apparent increase in biosynthetic rate. Besides, starch granules show physical alterations and higher phosphate content [33]. These data suggest that SSIII might have a negative regulatory role in starch synthesis. Previously, SSIII had been associated to a starch-excess phenotype, although indirectly through its association with regulatory proteins such as 14-3-3 [69]. In addition other SS isoform, SSIV, has been described to be essential for the initiation process of starch granule synthesis since *A. thaliana* SSIV mutant plants show just one large starch granule per plastid. The role of this isoform in the formation of the starch granule could be replaced in part by the SSIII isoform since the concomitant elimination of both enzymes in Arabidopsis block the starch synthesis. These data suggests that the remaining synthase activities are unable to start the synthesis of the starch granule. Recently, SSIV has been postulated to be also involved in the regulation of starch accumulation since its overexpression increases the starch levels in Arabidopsis leaves by 30%–40%. In addition, SSIV-overexpressing lines display a higher growth rate. The increase in starch content as a consequence of enhanced SSIV expression is also observed in long-term storage starch organs such as potato tubers [70].
