Mutations Affecting Starch Synthase III in Arabidopsis Alter Leaf Starch Structure and Increase the Rate of Starch Synthesis

Abstract

The role of starch synthase (SS) III (SSIII) in the synthesis of transient starch in Arabidopsis (Arabidopsis thaliana) was investigated by characterizing the effects of two insertion mutations at the AtSS3 gene locus. Both mutations, termed Atss3-1 and Atss3-2, condition complete loss of SSIII activity and prevent normal gene expression at both the mRNA and protein levels. The mutations cause a starch excess phenotype in leaves during the light period of the growth cycle due to an apparent increase in the rate of starch synthesis. In addition, both mutations alter the physical structure of leaf starch. Significant increases were noted in the mutants in the frequency of linear chains in amylopectin with a degree of polymerization greater than approximately 60, and relatively small changes were observed in chains of degree of polymerization 4 to 50. Furthermore, starch in the Atss3-1 and Atss3-2 mutants has a higher phosphate content, approximately two times that of wild-type leaf starch. Total SS activity is increased in both Atss3 mutants and a specific SS activity appears to be up-regulated. The data indicate that, in addition to its expected direct role in starch assembly, SSIII also has a negative regulatory function in the biosynthesis of transient starch in Arabidopsis.Starch serves a fundamental role in the life cycle of plants as the primary carbohydrate storage form for chemical energy. Transient starch is produced and degraded over short periods of time to satisfy the ongoing energy requirements of plant development (e.g. over the course of the diurnal cycle in the leaf). In contrast, storage starch accumulates and persists in heterotrophic tissues in anticipation of future plant energy needs, such as germination or sprouting. The anabolism and catabolism of both storage and transient starch forms requires precise control of the expression and/or activity of many different enzymes.Starch consists of two homopolymers of a-D-glucosyl units joined in linear arrays by a-(1!4) glycosidic bonds, amylose (Am) and amylopectin (Ap). Ap is the more abundant polymer, and it contains a-(1!6) branch linkages at a frequency of approximately 5%. The organized architectural arrangement of linear and branched chains in Ap enables efficient packaging of large amounts of Glc into insoluble starch granules. The basic enzymatic steps responsible for Ap structure begin with the formation of the activated glucosyl donor ADP-Glc (ADPG), catalyzed by ADPG pyrophosphorylase. Reactions utilizing ADPG to build a-(1!4)-linked linear glucosyl chains are catalyzed by the starch synthases (SSs). Branch linkages are introduced by branching enzymes (BEs), which catalyze cleavage of an internal a-(1!4) linkage and creation of a new a-(1!6) linkage by transfer of the released reducing end to a C6 hydroxyl. Starch-debranching enzymes (DBEs) hydrolyze branch linkages and are thought to be involved in starch biosynthesis, based on evidence that DBE mutants produce less starch than normal and contain a highly branched polysaccharide not obse...

Leaf Starch Turnover Occurs in Long Days and in Falling Light at the End of the Day

“…However, the extent and timing of plateauing of starch content in continuous light varies considerably between studies. For example, plateauing of starch content occurred in 16-h days in some previous studies of Arabidopsis (Carrari et al, 2005;Li et al, 2009) but was not observed in others (Lu et al, 2005;Zhang et al, 2005;Szydlowski et al, 2009).…”

A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana

“…Whole plant starch staining was carried out as described (35). In brief, plants were first boiled in 80% (v/v) ethanol to remove pigments and then stained with fresh made iodine solution (10 g liter Ϫ1 KI, 1 g liter Ϫ1 I 2 ).…”

Function and Characterization of Starch Synthase I Using Mutants in Rice

“…Mutants defective in GBSSI in several species (waxy rice [Sano, 1984]; waxy maize [Tsai, 1974]; waxy barley [Hordeum vulgare ;Eriksson, 1962]; waxy wheat [Triticum aestivum; Nakamura et al, 1995;Triticum monococcum;Fujita et al, 2001]; amf potato [Solanum tuberosum; Hovenkamp-Hermelink et al, 1987]; lam pea [Pisum sativum; Denyer et al, 1995]), SSIII of maize (dull-1; Gao et al, 1998), mutants deficient in SSIIa in rice (most japonica rice varieties are mutants of indica rice varieties [Umemoto et al, 2002;Nakamura et al, 2005]), in barley (sex6; Morell et al, 2003), in wheat (GSP-1 null; Yamamori et al, 2000) and in maize (sugary2; Zhang et al, 2004), and mutants for SSII in pea (rugosus5; Craig et al, 1998) have been identified and analyzed. Most recently, SSI (Delvalle et al, 2005) and SSIII (Zhang et al, 2005) mutants in Arabidopsis (Arabidopsis thaliana) leaves have been isolated. However, mutants for the remaining SS isozymes are yet to be found.…”