Starch synthase 4 is essential for coordination of starch granule formation with chloroplast division during Arabidopsis leaf expansion.

Arabidopsis thaliana mutants lacking the SS4 isoform of starch synthase have strongly reduced numbers of starch granules per chloroplast, suggesting that SS4 is necessary for the normal generation of starch granules. To establish whether it plays a direct role in this process, we investigated the circumstances in which granules are formed in ss4 mutants. Starch granule numbers and distribution and the accumulation of starch synthase substrates and products were investigated during ss4 leaf development, and in ss4 mutants carrying mutations or transgenes that affect starch turnover or chloroplast volume. We found that immature ss4 leaves have no starch granules, but accumulate high concentrations of the starch synthase substrate ADPglucose. Granule numbers are partially restored by elevating the capacity for glucan synthesis (via expression of bacterial glycogen synthase) or by increasing the volumes of individual chloroplasts (via introduction of arc mutations). However, these granules are abnormal in distribution, size and shape. SS4 is an essential component of a mechanism that coordinates granule formation with chloroplast division during leaf expansion and determines the abundance and the flattened, discoid shape of leaf starch granules.

Control of starch granule numbers in Arabidopsis chloroplasts.

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.

Step-by-step acquisition of the gibberellin-DELLA growth-regulatory mechanism during land-plant evolution.

Angiosperms (flowering plants) evolved relatively recently and are substantially diverged from early land plants (bryophytes, lycophytes, and others [1]). The phytohormone gibberellin (GA) adaptively regulates angiosperm growth via the GA-DELLA signaling mechanism [2-7]. GA binds to GA receptors (GID1s), thus stimulating interactions between GID1s and the growth-repressing DELLAs [8-12]. Subsequent 26S proteasome-mediated destruction of the DELLAs promotes growth [13-17]. Here we outline the evolution of the GA-DELLA mechanism. We show that the interaction between GID1 and DELLA components from Selaginella kraussiana (a lycophyte) is GA stimulated. In contrast, GID1-like (GLP1) and DELLA components from Physcomitrella patens (a bryophyte) do not interact, suggesting that GA-stimulated GID1-DELLA interactions arose in the land-plant lineage after the bryophyte divergence ( approximately 430 million years ago [1]). We further show that a DELLA-deficient P. patens mutant strain lacks the derepressed growth characteristic of DELLA-deficient angiosperms, and that both S. kraussiana and P. patens lack detectable growth responses to GA. These observations indicate that early land-plant DELLAs do not repress growth in situ. However, S. kraussiana and P. patens DELLAs function as growth-repressors when expressed in the angiosperm Arabidopsis thaliana. We conclude that the GA-DELLA growth-regulatory mechanism arose during land-plant evolution and via independent stepwise recruitment of GA-stimulated GID1-DELLA interaction and DELLA growth-repression functions.