Starch Synthesis in Arabidopsis. Granule Synthesis, Composition, and Structure

Abstract

The aim of this work was to characterize starch synthesis, composition, and granule structure in Arabidopsis leaves. First, the potential role of starch-degrading enzymes during starch accumulation was investigated. To discover whether simultaneous synthesis and degradation of starch occurred during net accumulation, starch was labeled by supplying 14 CO 2 to intact, photosynthesizing plants. Release of this label from starch was monitored during a chase period in air, using different light intensities to vary the net rate of starch synthesis. No release of label was detected unless there was net degradation of starch during the chase. Similar experiments were performed on a mutant line (dbe1) that accumulates the soluble polysaccharide, phytoglycogen. Label was not released from phytoglycogen during the chase indicating that, even when in a soluble form, glucan is not appreciably degraded during accumulation. Second, the effect on starch composition of growth conditions and mutations causing starch accumulation was studied. An increase in starch content correlated with an increased amylose content of the starch and with an increase in the ratio of granule-bound starch synthase to soluble starch synthase activity. Third, the structural organization and morphology of Arabidopsis starch granules was studied. The starch granules were birefringent, indicating a radial organization of the polymers, and x-ray scatter analyses revealed that granules contained alternating crystalline and amorphous lamellae with a periodicity of 9 nm. Granules from the wild type and the high-starch mutant sex1 were flattened and discoid, whereas those of the high-starch mutant sex4 were larger and more rounded. These larger granules contained "growth rings" with a periodicity of 200 to 300 nm. We conclude that leaf starch is synthesized without appreciable turnover and comprises similar polymers and contains similar levels of molecular organization to storage starches, making Arabidopsis an excellent model system for studying granule biosynthesis.The Arabidopsis leaf is an excellent system in which to study starch granule biosynthesis for several reasons. First, starch accumulates in large amounts over a short period; up to one-half of the carbon assimilated through photosynthesis is stored as starch during the light period. As a consequence, it is possible to analyze the composition and structure of starch made over a period of a few hours by a defined set of enzymes. In contrast, starch synthesis in storage organs occurs over a long developmental period, during which there are usually considerable changes in the complement of starch-synthesizing enzymes (Smith and Martin, 1993; Burton et al., 1995) and in overall cellular conditions. Second, the rate of starch synthesis in leaves can be controlled by altering the irradiance and measured accurately by supplying 14 CO 2 . Third, our knowledge of the complete genome sequence of Arabidopsis and the availability of transposon and T-DNA-tagged populations enables specific knockout mutation...

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

“…They suggested that an unknown mechanism increasingly diverted newly assimilated C toward Suc, leading to a reduced flux of C into starch synthesis rather than the onset of degradation after prolonged periods in the light (Li et al, 1992). It is possible that this pulse-chase experiment failed to detect starch degradation because 14 C starch was buried within the starch granule beneath newly synthesized 12 C starch during the chase and thus protected from degradation occurring at the granule surface (Zeeman et al, 2002). However, it is also possible that the cause of the commonly observed cessation of starch accumulation in continuous light differs between species.…”

“…The simplifying assumption that leaf starch degradation is low or absent during the light period is supported by pulse-chase experiments with 14 CO 2 , which failed to detect significant starch degradation in standard light periods in pea (Pisum sativum; Kruger et al, 1983), pepper (Capsicum annuum; Grange, 1984), wild-type Arabidopsis (Arabidopsis thaliana; Walters et al, 2004;Zeeman et al, 2002), and tobacco (Nicotiana tabacum; Häusler et al, 1998) leaves (limitations of this approach are discussed in Supplemental Text S1). However, starch degradation may occur during long periods in continuous light, in some mutants with chloroplast export deficiencies, and in response to acute abiotic stress.…”

Starch Serves as Carbohydrate Storage in Nematode-Induced Syncytia

“…PSH1 appears to be dispensable for starch degradation. Zeeman et al (2002) suggested that PHS1 is important for tolerance against abiotic stress because Atphs1 mutants were susceptible for transient water deficiency. Both PHS isoforms, however, were highly regulated during the diurnal cycle and PHS2 is of special interest because it is cytosolic and might play a role in the conversion from starch via maltose into Glc .…”

“…It is synthesized mainly during photosynthesis in the light period buffering CO 2 fixation or in storage organs like tubers, bulbs, or seeds (Dennis and Blakeley, 2000). In recent years, numerous studies have been performed to increase knowledge of starch metabolism, including in the model plant Arabidopsis (Arabidopsis thaliana; Zeeman et al, 2002Zeeman et al, , 2004Siedlecka et al, 2003;Smith et al, 2004;Delvallé et al, 2005;Lloyd et al, 2005;Dumez et al, 2006). In most of these studies, plant leaf material of the wild type and mutant lines was used to investigate starch metabolism under different light regimes or altered sugar concentrations.…”

Phosphorylation of Transitory Starch Is Increased during Degradation

“…Amylose comprises predominantly linear chains of a-1,4-linked Glc residues, whereas amylopectin exists as a branched a-1,4:a-1,6 D-glucan polymer. Amylopectin accounts for about 75% of the starch weight in reserve starch (Ball et al, 1998) and typically more than 90% in transitory starch (Zeeman et al, 2002).…”