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...

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Reductions in activity of SSIII, the major isoform of starch synthase responsible for amylopectin synthesis in the potato tuber, result in fissuring of the starch granules. To discover the causes of the fissuring, and thus to shed light on factors that influence starch granule morphology in general, SSIII antisense lines were compared with lines with reductions in the major granule-bound isoform of starch synthase (GBSS) and lines with reductions in activity of both SSIII and GBSS (SSIII/GBSS antisense lines). This revealed that fissuring resulted from the activity of GBSS in the SSIII antisense background. Control (untransformed) lines and GBSS and SSIII/GBSS antisense lines had unfissured granules. Starch analyses showed that granules from SSIII antisense tubers had a greater number of long glucan chains than did granules from the other lines, in the form of larger amylose molecules and a unique fraction of very long amylopectin chains. These are likely to result from increased flux through GBSS in SSIII antisense tubers, in response to the elevated content of ADP-glucose in these tubers. It is proposed that the long glucan chains disrupt organization of the semi-crystalline parts of the matrix, setting up stresses in the matrix that lead to fissuring.Little is known about the processes that determine the morphology of starch granules, but important clues have been gained from studies of mutant plants with altered granule morphology. Many such plants carry mutations in genes encoding isoforms of starch synthase and starch-branching enzyme responsible for the synthesis of amylopectin, the branched ␣1,4, ␣1,6 glucan, which forms the semi-crystalline matrix of the granule. For example, mutations in pea affecting starch-branching enzyme A (at the r locus (1)) and starch synthase II (SSII, 1 at the rug5 locus (2)) convert the normally ovoid granules of the embryo into deeply fissured, multilobed structures and highly twisted, contorted structures, respectively (2, 3). Mutations in maize affecting starch-branching enzyme IIb (at the amylose-extender locus (4)) convert the normally polyhedral granules of the endosperm into irregular, elongated structures (5).The altered granule morphology of the mutants presumably results from alterations in the structures and relative amounts of the glucan polymers of which the granule is comprised. Most mutations affecting isoforms of starch synthase and starch branching enzyme affect both of these parameters. The r and rug5 mutations of pea and the amylose-extender mutation of maize affect the proportion of short and long chains in amylopectin, the chain-length distribution within the short-chain population, and the ratio within the granule of amylopectin to amylose, the essentially linear glucan that makes up 20 -30% of storage starches (2, 6 -9). However, it is not known which of these changes in granule composition and polymer structure leads to the changes in granule morphology.Transgenic potato plants with reduced activity of isoforms of starch synthase provide a good system ...

Starch granules from higher plants contain alternating zones of semicrystalline and amorphous material known as growth rings. The regulation of growth ring formation is not understood. We provide several independent lines of evidence that growth ring formation in the starch granules of potato (Solanum tuberosum) tubers is not under diurnal control. Ring formation is not abolished by growth in constant conditions, and ring periodicity and appearance are relatively unaffected by a change from a 24-h to a 40-h photoperiod, and by alterations in substrate supply to the tuber that are known to affect the diurnal pattern of tuber starch synthesis. Some, but not all, of the features of ring formation are consistent with the involvement of a circadian rhythm. Such a rhythm might operate by changing the relative activities of starch-synthesizing enzymes: Growth ring formation is disrupted in tubers with reduced activity of a major isoform of starch synthase. We suggest that physical as well as biological mechanisms may contribute to the control of ring formation, and that a complex interplay of several factors may by involved.Starch granules from every higher plant species studied so far contain alternating regions of semicrystalline and amorphous material commonly known as growth rings. Growth rings can be observed by light microscopy, by atomic force microscopy, and by scanning and transmission electron microscopy (SEM and TEM) after treatment of granules with acid or degradative enzymes. These methods reveal that the rings represent alternating concentric layers of high/low refractive index, density, crystallinity, and resistance to chemical and enzymatic attack (Badenhuizen, 1939; Badenhuizen, 1959; Buttrose, 1960;Gallant and Guilbot, 1969;Hall and Sayre, 1973; Baker et al., 2001).The origin of growth rings remains obscure. Previous studies have suggested that one of two biological mechanisms could regulate their formation. First, their formation could be under the control of a diurnal rhythm that is dependent on day/night variations in the environment, such as a light/dark regime or alternating temperature cycles. Meyer (1895) hypothesized that growth of the granule follows a diurnal rhythm and that one growth ring is laid down per day. Support for this comes from studies of growth rings in the starch of developing cereal endosperm. Granules from barley (Hordeum vulgare) endosperm were claimed to have one growth ring for each day after their initiation (Buttrose, 1960), and growth rings were not visible in granules from the endosperm of wheat (Triticum aestivum) and barley plants grown in constant light and temperature (Van de Sande-Bakhuyzen, 1925; Buttrose, 1960 Buttrose, , 1962. Rings reappeared in the peripheral regions of granules when plants grown initially in constant conditions were transferred to a day-night regime during the course of granule development (Buttrose, 1962). These observations lead Buttrose (1962) to propose that growth ring formation is controlled by a diurnal rhythm that is dependent on ...

Summary• We have developed methods, based on confocal microscopy and three-dimensional (3D) modelling, for the analysis of complex tissues and individual nuclei. These methods were used to study the development of early wheat ( Triticum aestivum ) endosperm as a whole and of endosperm nuclei undergoing polyploidization.• Fixed sections of immature caryopses were either stained with SYTOX Green or used for fluorescence in situ hybridization (FISH) to visualize centromeres, telomeres and a rye chromosome arm substitution. Each section was imaged as a confocal image stack. By using A MIRA 3.0 for computer image processing, rendered models were produced of the whole endosperm and of individual nuclei.• We followed endosperm development up to the formation of a complete syncytium, which develops via a dorsal and a ventral plate of nuclei in the central cell. Modelling of nuclei showed that wheat chromosomes are not anchored to the nuclear membrane and become more randomly positioned in endoreduplicated nuclei.• This analysis produced a precise description of the positioning of nuclei throughout the developing endosperm and of chromosomal domains in single nuclei.