RESUMEN
A putative phosphatase, LSF1 (for LIKE SEX4; previously PTPKIS2), is closely related in sequence and structure to STARCH-EXCESS4 (SEX4), an enzyme necessary for the removal of phosphate groups from starch polymers during starch degradation in Arabidopsis (Arabidopsis thaliana) leaves at night. We show that LSF1 is also required for starch degradation: lsf1 mutants, like sex4 mutants, have substantially more starch in their leaves than wild-type plants throughout the diurnal cycle. LSF1 is chloroplastic and is located on the surface of starch granules. lsf1 and sex4 mutants show similar, extensive changes relative to wild-type plants in the expression of sugar-sensitive genes. However, although LSF1 and SEX4 are probably both involved in the early stages of starch degradation, we show that LSF1 neither catalyzes the same reaction as SEX4 nor mediates a sequential step in the pathway. Evidence includes the contents and metabolism of phosphorylated glucans in the single mutants. The sex4 mutant accumulates soluble phospho-oligosaccharides undetectable in wild-type plants and is deficient in a starch granule-dephosphorylating activity present in wild-type plants. The lsf1 mutant displays neither of these phenotypes. The phenotype of the lsf1/sex4 double mutant also differs from that of both single mutants in several respects. We discuss the possible role of the LSF1 protein in starch degradation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Hojas de la Planta/metabolismo , Almidón/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cloroplastos/enzimología , ADN Bacteriano/genética , Glucanos/metabolismo , Mutagénesis Insercional , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Hojas de la Planta/genética , ARN de Planta/genéticaRESUMEN
In most species, the precursor for starch synthesis, ADPglucose, is made exclusively in the plastids by the enzyme ADPglucose pyrophosphorylase (AGPase). However, in the endosperm of grasses, including the economically important cereals, ADPglucose is also made in the cytosol via a cytosolic form of AGPase. Cytosolic ADPglucose is imported into plastids for starch synthesis via an ADPglucose/ADP antiporter (ADPglucose transporter) in the plastid envelope. The genes encoding the two subunits of cytosolic AGPase and the ADPglucose transporter are unique to grasses. In this review, the evolutionary origins of this unique endosperm pathway of ADPglucose synthesis and its functional significance are discussed. It is proposed that the genes encoding the pathway originated from a whole-genome-duplication event in an early ancestor of the grasses.
Asunto(s)
Evolución Biológica , Vías Biosintéticas , Grano Comestible/metabolismo , Poaceae/metabolismo , Almidón/biosíntesis , Grano Comestible/enzimología , Grano Comestible/genética , Glucosa/metabolismo , Glucosa-1-Fosfato Adenililtransferasa/genética , Glucosa-1-Fosfato Adenililtransferasa/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Poaceae/enzimología , Poaceae/genética , Almidón/genéticaRESUMEN
Starch is the major storage carbohydrate in plants. It is comprised of glucans that form semicrystalline granules. Glucan phosphorylation is a prerequisite for normal starch breakdown, but phosphoglucan metabolism is not understood. A putative protein phosphatase encoded at the Starch Excess 4 (SEX4) locus of Arabidopsis thaliana was recently shown to be required for normal starch breakdown. Here, we show that SEX4 is a phosphoglucan phosphatase in vivo and define its role within the starch degradation pathway. SEX4 dephosphorylates both the starch granule surface and soluble phosphoglucans in vitro, and sex4 null mutants accumulate phosphorylated intermediates of starch breakdown. These compounds are linear alpha-1,4-glucans esterified with one or two phosphate groups. They are released from starch granules by the glucan hydrolases alpha-amylase and isoamylase. In vitro experiments show that the rate of starch granule degradation is increased upon simultaneous phosphorylation and dephosphorylation of starch. We propose that glucan phosphorylating enzymes and phosphoglucan phosphatases work in synergy with glucan hydrolases to mediate efficient starch catabolism.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Proteínas Tirosina Fosfatasas/metabolismo , Almidón/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Metabolismo de los Hidratos de Carbono , ADN Bacteriano/genética , Glucanos/metabolismo , Mutagénesis Insercional , Fosforilación , Proteínas Tirosina Fosfatasas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
We report that protein phosphorylation is involved in the control of starch metabolism in Arabidopsis leaves at night. sex4 (starch excess 4) mutants, which have strongly reduced rates of starch metabolism, lack a protein predicted to be a dual specificity protein phosphatase. We have shown that this protein is chloroplastic and can bind to glucans and have presented evidence that it acts to regulate the initial steps of starch degradation at the granule surface. Remarkably, the most closely related protein to SEX4 outside the plant kingdom is laforin, a glucan-binding protein phosphatase required for the metabolism of the mammalian storage carbohydrate glycogen and implicated in a severe form of epilepsy (Lafora disease) in humans.