The aim of this work was to investigate starch granule numbers in Arabidopsis (Arabidopsis thaliana) leaves. Lack of quantitative information on the extent of genetic, temporal, developmental, and environmental variation in granule numbers is an important limitation in understanding control of starch degradation and the mechanism of granule initiation. Two methods were developed for reliable estimation of numbers of granules per chloroplast. First, direct measurements were made on large series of consecutive sections of mesophyll tissue obtained by focused ion beam-scanning electron microscopy. Second, average numbers were calculated from the starch contents of leaves and chloroplasts and estimates of granule mass based on granule dimensions. Examination of wild-type plants and accumulation and regulation of chloroplast (arc) mutants with few, large chloroplasts provided the following new insights. There is wide variation in chloroplast volumes in cells of wild-type leaves. Granule numbers per chloroplast are correlated with chloroplast volume, i.e. large chloroplasts have more granules than small chloroplasts. Mature leaves of wild-type plants and arc mutants have approximately the same number of granules per unit volume of stroma, regardless of the size and number of chloroplasts per cell. Granule numbers per unit volume of stroma are also relatively constant in immature leaves but are greater than in mature leaves. Granule initiation occurs as chloroplasts divide in immature leaves, but relatively little initiation occurs in mature leaves. Changes in leaf starch content over the diurnal cycle are largely brought about by changes in the volume of a fixed number of granules.Chloroplasts in Arabidopsis (Arabidopsis thaliana) mesophyll cells are generally stated to contain about five starch granules at the end of the light period (Zeeman et al., 2002(Zeeman et al., , 2007, but nothing is known about how this number is determined or the extent of genetic, temporal, developmental, and environmental variation in the number. This is an important limitation in understanding control of starch degradation at night, a process essential for the normal growth of the plant (Gibon et al., 2006;Smith and Stitt, 2007;Stitt et al., 2007;Usadel et al., 2008). The surface area and volume of starch granules in the chloroplast are relevant to the control of starch degradation in two ways. First, surface area can potentially limit the rate of degradation during the night. This limitation is not a major determinant of the rate of degradation in wild-type Arabidopsis leaves in controlled conditions; degradation is near linear through most of the night and consumes almost all of the starch reserves by dawn. If degradation were limited by surface area, the rate would decline with time through the night. However, reductions in starch granule numbers may result in limitation of starch degradation. The starch synthase4 (ss4) mutant of Arabidopsis has only one starch granule per chloroplast. It has a low rate of starch degradation and a low...

Small-angle X-ray scattering (SAXS) methods are extensively used for characterizing macromolecular structure and dynamics in solution. The computation of theoretical scattering profiles from three-dimensional models is crucial in order to test structural hypotheses. Here, a new approach is presented to efficiently compute SAXS profiles that are based on three-dimensional Zernike polynomial expansions. Comparison with existing methods and experimental data shows that the Zernike method can be used to effectively validate three-dimensional models against experimental data. For molecules with large cavities or complicated surfaces, the Zernike method more accurately accounts for the solvent contributions. The program is available as open-source software at http://sastbx.als.lbl.gov.