Control of starch branching in barley defined through differential RNAi suppression of starch branching enzyme IIa and IIb

Abstract

The roles of starch branching enzyme (SBE, EC 2.4.1.18) IIa and SBE IIb in defining the structure of amylose and amylopectin in barley (Hordeum vulgare) endosperm were examined. Barley lines with low expression of SBE IIa or SBE IIb, and with the low expression of both isoforms were generated through RNA-mediated silencing technology. These lines enabled the study of the role of each of these isoforms in determining the amylose content, the distribution of chain lengths, and the frequency of branching in both amylose and amylopectin. In lines where both SBE IIa and SBE IIb expression were reduced by >80%, a high amylose phenotype (>70%) was observed, while a reduction in the expression of either of these isoforms alone had minor impact on amylose content. The structure and properties of the high amylose starch resulting from the concomitant reduction in the expression of both isoforms of SBE II in barley were found to approximate changes seen in amylose extender mutants of maize, which result from lesions eliminating expression of the SBE IIb gene. Amylopectin chain length distribution analysis indicated that both SBE IIa and SBE IIb isoforms play distinct roles in determining the fine structure of amylopectin. A significant reduction in the frequency of branches in amylopectin was noticed only when both SBE IIa and SBE IIb were reduced, whereas there was a significant increase in the branching frequency of amylose when SBE IIb alone was reduced. Functional interactions between SBE isoforms are suggested, and a possible inhibitory role of SBE IIb on other SBE isoforms is discussed.

Gradually Decreasing Starch Branching Enzyme Expression Is Responsible for the Formation of Heterogeneous Starch Granules

“…The high accumulation of amylose in crystalline lamellae disorders the packing of lamellar structure and destroys the granule layered organization (Blazek and Gilbert, 2011). The very high amylose in wheat and barley starches alters starch morphology from disk-shaped to sickle-shaped granules with a significant loss of birefringence (Regina et al, 2006(Regina et al, , 2010Carciofi et al, 2012;Slade et al, 2012).…”

Molecular genetic analysis of glucan branching enzymes from plants and bacteria in Arabidopsis reveals marked differences in their functions and capacity to mediate starch granule formation

“…S6). It would be interesting to see whether MOSs are present in other situations where starch made predominantly by BE1 was found to have high amylose (Bhattacharyya et al, 1990;Regina et al, 2006Regina et al, , 2010.…”

“…In contrast, loss of class II BE results in significant changes, such as decreased starch content and a high apparent amylose content. This has been observed in several species, including maize (Stinard et al, 1993), potato , pea (Pisum sativum; Bhattacharyya et al, 1990), rice (Mizuno et al, 1993), barley (Hordeum vulgare; Regina et al, 2010), and wheat (Triticum aestivum; Regina et al, 2006). The high apparent amylose content was caused at least in part by the accumulation of less-frequently branched amylopectin that stains with a higher wavelength of maximal absorption (l max ) than that of the wild type (Boyer et al, 1976).…”

New insight in cereal starch degradation: identification and structural characterization of four α-amylases in bread wheat

“…α-1,4-Glucanotransferases or starch synthases have exhibited partial redundancies between isoforms allowing to partial complementation of mutations and therefore maintaining starch crystal integrity to some extents [65]. In barley, similar partial redundancy was described for the starch branching enzyme class 2, SBEIIb mutation being remained asymptomatic due to partial complementation by SBEIIa [66].…”