Blocking the Metabolism of Starch Breakdown Products in Arabidopsis Leaves Triggers Chloroplast Degradation

In most plants, a large fraction of photo-assimilated carbon is stored in the chloroplasts during the day as starch and remobilized during the subsequent night to support metabolism. Mutations blocking either starch synthesis or starch breakdown in Arabidopsis thaliana reduce plant growth. Maltose is the major product of starch breakdown exported from the chloroplast at night. The maltose excess 1 mutant （mex1）, which lacks the chloroplast envelope maltose transporter, accumulates high levels of maltose and starch in chloroplasts and develops a distinctive but previously unexplained chlorotic phenotype as leaves mature. The introduction of additional mutations that prevent starch synthesis, or that block maltose production from starch, also prevent chlorosis of mex1. In contrast, introduction of mutations in disproportionating enzyme （DPE1） results in the accumulation of maltotriose in addition to maltose, and greatly increases chlorosis. These data suggest a link between maltose accumulation and chloroplast homeostasis. Microscopic analyses show that the mesophyll cells in chlorotic mex1 leaves have fewer than half the number of chloroplasts than wild-type cells. Transmission electron microscopy reveals autophagy-like chloroplast degradation in both mex1 and the dpe1/mex1 double mutant. Microarray analyses reveal substantial reprogramming of metabolic and cellular processes, suggesting that organellar protein turnover is increased in mex1, though leaf senescence and senescence-related chlorophyll catabolism are not induced. We propose that the accumulation of maltose and malto-oligosaccharides causes chloroplast dysfunction, which may by signaled via a form of retrograde signaling and trigger chloroplast degradation.     

植物海藻糖代谢及海藻糖-6-磷酸信号研究进展

海藻糖代谢和海藻糖-6-磷酸（T6P）信号途径在植物生长和发育过程中具有重要的调控作用。T6P是海藻糖的代谢前体,是植物响应碳元素可用性、调控生长发育的关键信号分子。植物体中除了自身的海藻糖合成途径外,由病原菌产生的海藻糖或T6P能够导致植物代谢和发育的重新编程。植物不同阶段的生长发育,包括胚胎发育、幼苗生长、成花诱导及叶片衰老等,都受T6P的调控。T6P信号的一个关键互作因子是蔗糖非发酵相关激酶1（SnRKl）,T6P能够抑制SnRK1的催化活性,进而调控植物的生长和发育过程。     

大豆叶片淀粉的降解及淀粉降解酶

在90μmol m~(-2)s~(-1)光强以下可见大豆叶片淀粉的降解,降解速率为0.8～3.8mg淀粉dm~(-2)h~(-1)。淀粉降解通过水解及磷酸解两条途径,α,β—淀粉酶的最适pH5～6,磷酸化酶pH7～8。α—淀粉酶活力随叶片的成长显著增强,β—淀粉酶则有所减弱。叶片淀粉积累或消耗时此三酶活力无显著变化。 黄化小麦叶片照光转绿过程中此三酶活力变化不大。黄化玉米叶片照光转绿过程中磷酸化酶活力降低,β—淀粉酶活力增强。     

Overproduction of Trehalose: Heterologous Expression of Escherichia coli Trehalose-6-Phosphate Synthase and Trehalose-6-Phosphate Phosphatase in Corynebacterium glutamicum

Trehalose is a disaccharide with potential applications in the biotechnology and food industries. We propose a method for industrial production of trehalose, based on improved strains of Corynebacterium glutamicum. This paper describes the heterologous expression of Escherichia coli trehalose-synthesizing enzymes trehalose-6-phosphate synthase (OtsA) and trehalose-6-phosphate phosphatase (OtsB) in C. glutamicum, as well as its impact on the trehalose biosynthetic rate and metabolic-flux distributions, during growth in a defined culture medium. The new recombinant strain showed a five- to sixfold increase in the activity of OtsAB pathway enzymes, compared to a control strain, as well as an almost fourfold increase in the trehalose excretion rate during the exponential growth phase and a twofold increase in the final titer of trehalose. The heterologous expression described resulted in a reduced specific glucose uptake rate and Krebs cycle flux, as well as reduced pentose pathway flux, a consequence of downregulated glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The results proved the suitability of using the heterologous expression of Ots proteins in C. glutamicum to increase the trehalose biosynthetic rate and yield and suggest critical points for further improvement of trehalose overproduction in C. glutamicum.     

Feedback Inhibition of Ammonium Uptake by a Phospho-Dependent Allosteric Mechanism in Arabidopsis

The acquisition of nutrients requires tight regulation to ensure optimal supply while preventing accumulation to toxic levels. Ammonium transporter/methylamine permease/rhesus (AMT/Mep/Rh) transporters are responsible for ammonium acquisition in bacteria, fungi, and plants. The ammonium transporter AMT1;1 from Arabidopsis thaliana uses a novel regulatory mechanism requiring the productive interaction between a trimer of subunits for function. Allosteric regulation is mediated by a cytosolic C-terminal trans-activation domain, which carries a conserved Thr (T460) in a critical position in the hinge region of the C terminus. When expressed in yeast, mutation of T460 leads to inactivation of the trimeric complex. This study shows that phosphorylation of T460 is triggered by ammonium in a time- and concentration-dependent manner. Neither Gln nor l-methionine sulfoximine–induced ammonium accumulation were effective in inducing phosphorylation, suggesting that roots use either the ammonium transporter itself or another extracellular sensor to measure ammonium concentrations in the rhizosphere. Phosphorylation of T460 in response to an increase in external ammonium correlates with inhibition of ammonium uptake into Arabidopsis roots. Thus, phosphorylation appears to function in a feedback loop restricting ammonium uptake. This novel autoregulatory mechanism is capable of tuning uptake capacity over a wide range of supply levels using an extracellular sensory system, potentially mediated by a transceptor (i.e., transporter and receptor).     

植物叶片暂时淀粉分解途径研究进展

植物叶片暂时淀粉主要分解途径包括如下过程:叶绿体中半结晶状淀粉粒在葡聚糖-水双激酶(GWD)和磷酸葡聚糖-水双激酶(PWD)作用下磷酸化,使淀粉粒结构松散;异淀粉酶(ISA3)作用于松散淀粉粒而释放出磷酸葡聚糖,再经磷酸葡聚糖磷酸酶(SEX4)水解去除磷酸而生成可溶性线性葡聚糖;葡聚糖在β-淀粉酶(BAM3)催化下水解生成麦芽糖后,再通过麦芽糖载体(MEX1)转运至细胞质.该文主要综述了以上转化过程中涉及的底物、生成物和催化酶类的研究进展情况,同时简述了植物叶片暂时淀粉分解的次要途径和抗逆性相关途径,并提出了该领域目前存在的问题和今后研究方向.     

Inhibition by Catalase of Dark-mediated Glucose-6-Phosphate Dehydrogenase Activation in Pea Chloroplasts

Dark activation of light-inactivated glucose-6-phosphate dehydrogenase was inhibited by catalase in a broken pea chloroplast system. Partially purified glucose-6-phosphate dehydrogenase from pea leaf chloroplasts can be inactivated in vitro by dithiothreitol and thioredoxin and reactivated by H₂O₂. The in vitro activation by H₂O₂ was not enhanced by horseradish peroxidase, and dark activation in the broken chloroplast system was only slightly inhibited by NaCN. These results indicate that the dark activation of glucose-6-phosphate dehydrogenase may involve oxidation by H₂O₂ of SH groups on the enzyme which were reduced in the light by the light effect mediator system.     

Starch Gel Electrophoresis of Fructose-6-Phosphate Phosphoketolase in the Genus Bifidobacterium

Three groups of nomenspecies of the genus Bifidobacterium were distinguished by the different mobility of their fructose-6-phosphate phosphoketolase in starchgel electrophoresis; there is apparently a close relatedness between electrophoretic type of phosphoketolase and habitat.     

Feedback Inhibition of Deoxy-d-xylulose-5-phosphate Synthase Regulates the Methylerythritol 4-Phosphate Pathway

The 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway leads to the biosynthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the precursors for isoprene and higher isoprenoids. Isoprene has significant effects on atmospheric chemistry, whereas other isoprenoids have diverse roles ranging from various biological processes to applications in commercial uses. Understanding the metabolic regulation of the MEP pathway is important considering the numerous applications of this pathway. The 1-deoxy-d-xylulose-5-phosphate synthase (DXS) enzyme was cloned from Populus trichocarpa, and the recombinant protein (PtDXS) was purified from Escherichia coli. The steady-state kinetic parameters were measured by a coupled enzyme assay. An LC-MS/MS-based assay involving the direct quantification of the end product of the enzymatic reaction, 1-deoxy-d-xylulose 5-phosphate (DXP), was developed. The effect of different metabolites of the MEP pathway on PtDXS activity was tested. PtDXS was inhibited by IDP and DMADP. Both of these metabolites compete with thiamine pyrophosphate for binding with the enzyme. An atomic structural model of PtDXS in complex with thiamine pyrophosphate and Mg²⁺ was built by homology modeling and refined by molecular dynamics simulations. The refined structure was used to model the binding of IDP and DMADP and indicated that IDP and DMADP might bind with the enzyme in a manner very similar to the binding of thiamine pyrophosphate. The feedback inhibition of PtDXS by IDP and DMADP constitutes an important mechanism of metabolic regulation of the MEP pathway and indicates that thiamine pyrophosphate-dependent enzymes may often be affected by IDP and DMADP.     

Trehalose and trehalase in Arabidopsis

Trehalase is ubiquitous in higher plants. So far, indications concerning its function are scarce, although it has been implicated in the detoxification of exogenous trehalose. A putative trehalase gene, T19F6.15, has been identified in the genome sequencing effort in Arabidopsis. Here we show that this gene encodes a functional trehalase when its cDNA is expressed in yeast, and that it is expressed in various plant organs. Furthermore, we present results on the distribution and activity of trehalase in Arabidopsis and we describe how inhibition of trehalase by validamycin A affects the plants response to exogenous trehalose (alpha-D-glucopyranosyl-[1, 1]-alpha-D-glucopyranoside). Trehalase activity was highest in floral organs, particularly in the anthers (approximately 700 nkat g(-1) protein) and maturing siliques (approximately 250 nkat g(-1) protein) and much lower in leaves, stems, and roots (less than 50 nkat g(-1) protein). Inhibition of trehalase in vivo by validamycin A led to the accumulation of an endogenous substance that had all the properties of trehalose, and to a strong reduction in sucrose and starch contents in flowers, leaves, and stems. Thus, trehalose appears to be an endogenous substance in Arabidopsis, and trehalose and trehalase may play a role in regulating the carbohydrate allocation in plants.     

Cooperative D1 Degradation in the Photosystem II Repair Mediated by Chloroplastic Proteases in Arabidopsis

Light energy constantly damages photosynthetic apparatuses, ultimately causing impaired growth. Particularly, the sessile nature of higher plants has allowed chloroplasts to develop unique mechanisms to alleviate the irreversible inactivation of photosynthesis. Photosystem II (PSII) is known as a primary target of photodamage. Photosynthetic organisms have evolved the so-called PSII repair cycle, in which a reaction center protein, D1, is degraded rapidly in a specific manner. Two proteases that perform processive or endopeptidic degradation, FtsH and Deg, respectively, participate in this cycle. To examine the cooperative D1 degradation by these proteases, we engaged Arabidopsis (Arabidopsis thaliana) mutants lacking FtsH2 (yellow variegated2 [var2]) and Deg5/Deg8 (deg5 deg8) in detecting D1 cleaved fragments. We detected several D1 fragments only under the var2 background, using amino-terminal or carboxyl-terminal specific antibodies of D1. The appearance of these D1 fragments was inhibited by a serine protease inhibitor and by deg5 deg8 mutations. Given the localization of Deg5/Deg8 on the luminal side of thylakoid membranes, we inferred that Deg5/Deg8 cleaves D1 at its luminal loop connecting the transmembrane helices C and D and that the cleaved products of D1 are the substrate for FtsH. These D1 fragments detected in var2 were associated with the PSII monomer, dimer, and partial disassembly complex but not with PSII supercomplexes. It is particularly interesting that another processive protease, Clp, was up-regulated and appeared to be recruited from stroma to the thylakoid membrane in var2, suggesting compensation for FtsH deficiency. Together, our data demonstrate in vivo cooperative degradation of D1, in which Deg cleavage assists FtsH processive degradation under photoinhibitory conditions.     

Degradation of Glucan Primers in the Absence of Starch Synthase 4 Disrupts Starch Granule Initiation in Arabidopsis

Arabidopsis leaf chloroplasts typically contain five to seven semicrystalline starch granules. It is not understood how the synthesis of each granule is initiated or how starch granule number is determined within each chloroplast. An Arabidopsis mutant lacking the glucosyl-transferase, STARCH SYNTHASE 4 (SS4) is impaired in its ability to initiate starch granules; its chloroplasts rarely contain more than one large granule, and the plants have a pale appearance and reduced growth. Here we report that the chloroplastic α-amylase AMY3, a starch-degrading enzyme, interferes with granule initiation in the ss4 mutant background. The amy3 single mutant is similar in phenotype to the wild type under normal growth conditions, with comparable numbers of starch granules per chloroplast. Interestingly, the ss4 mutant displays a pleiotropic reduction in the activity of AMY3. Remarkably, complete abolition of AMY3 (in the amy3 ss4 double mutant) increases the number of starch granules produced in each chloroplast, suppresses the pale phenotype of ss4, and nearly restores normal growth. The amy3 mutation also restores starch synthesis in the ss3 ss4 double mutant, which lacks STARCH SYNTHASE 3 (SS3) in addition to SS4. The ss3 ss4 line is unable to initiate any starch granules and is thus starchless. We suggest that SS4 plays a key role in granule initiation, allowing it to proceed in a way that avoids premature degradation of primers by starch hydrolases, such as AMY3.     

d-Glucose 6-Phosphate Cycloaldolase: Inhibition Studies and Aldolase Function

d-Glucose 6-phosphate cycloaldolase is inhibited 83% by 0.66 mm EDTA and stimulated 1.7-fold by 0.6 mm KCl. Dihydroxyacetone phosphate, an analog of the last three carbons in the proposed intermediate, d-xylo-5-hexulose 6-phosphate, acts as a partially competitive inhibitor. Treatment with NaBH₄ in the presence of dihydroxyacetone phosphate does not cause permanent inactivation as would be expected if a Schiff base were being formed. In these properties it resembles a type II, metal-containing aldolase. Photooxidation in the presence of Rose Bengal inactivates this enzyme. NAD⁺ partially protects against this photooxidation. Cells grown on medium lacking myoinositol had four times as much enzyme activity as cells grown on medium containing 100 mg of myoinositol per liter.     

Stress-Induced GSK3 Regulates the Redox Stress Response by Phosphorylating Glucose-6-Phosphate Dehydrogenase in Arabidopsis

Diverse stresses such as high salt conditions cause an increase in reactive oxygen species (ROS), necessitating a redox stress response. However, little is known about the signaling pathways that regulate the antioxidant system to counteract oxidative stress. Here, we show that a Glycogen Synthase Kinase3 from Arabidopsis thaliana (ASKα) regulates stress tolerance by activating Glc-6-phosphate dehydrogenase (G6PD), which is essential for maintaining the cellular redox balance. Loss of stress-activated ASKα leads to reduced G6PD activity, elevated levels of ROS, and enhanced sensitivity to salt stress. Conversely, plants overexpressing ASKα have increased G6PD activity and low levels of ROS in response to stress and are more tolerant to salt stress. ASKα stimulates the activity of a specific cytosolic G6PD isoform by phosphorylating the evolutionarily conserved Thr-467, which is implicated in cosubstrate binding. Our results reveal a novel mechanism of G6PD adaptive regulation that is critical for the cellular stress response.     

The Trehalose 6-Phosphate/SnRK1 Signaling Pathway Primes Growth Recovery following Relief of Sink Limitation

Trehalose 6-P (T6P) is a sugar signal in plants that inhibits SNF1-related protein kinase, SnRK1, thereby altering gene expression and promoting growth processes. This provides a model for the regulation of growth by sugar. However, it is not known how this model operates under sink-limited conditions when tissue sugar content is uncoupled from growth. To test the physiological importance of this model, T6P, SnRK1 activities, sugars, gene expression, and growth were measured in Arabidopsis (Arabidopsis thaliana) seedlings after transfer to cold or zero nitrogen compared with sugar feeding under optimal conditions. Maximum in vitro activities of SnRK1 changed little, but T6P accumulated up to 55-fold, correlating with tissue Suc content in all treatments. SnRK1-induced and -repressed marker gene expression strongly related to T6P above and below a threshold of 0.3 to 0.5 nmol T6P g⁻¹ fresh weight close to the dissociation constant (4 µm) of the T6P/ SnRK1 complex. This occurred irrespective of the growth response to Suc. This implies that T6P is not a growth signal per se, but through SnRK1, T6P primes gene expression for growth in response to Suc accumulation under sink-limited conditions. To test this hypothesis, plants with genetically decreased T6P content and SnRK1 overexpression were transferred from cold to warm to analyze the role of T6P/SnRK1 in relief of growth restriction. Compared with the wild type, these plants were impaired in immediate growth recovery. It is concluded that the T6P/SnRK1 signaling pathway responds to Suc induced by sink restriction that enables growth recovery following relief of limitations such as low temperature.The nonreducing Glc disaccharide, trehalose [α-d-glucopyranosyl-(1→1)-α-d-glucopyranoside] is widespread in nature. In resurrection plants, fungi, bacteria, and nonvertebrate animals, it performs a role as a carbon source and stress protection compound (Elbein et al., 2003; Paul et al., 2008). In the majority of plants, however, amounts of trehalose are too low to perform this function. Instead, the pathway has developed into a specialized system that regulates and integrates metabolism with growth and development (Schluepmann et al., 2003; Lunn et al., 2006; Ramon and Rolland, 2007; Gómez et al., 2010). This system is indispensible throughout seed and vegetative development (Eastmond et al., 2002; van Dijken et al., 2004; Gómez et al., 2010), and evidence suggests that the critical function is performed by the precursor of trehalose, trehalose 6-P (T6P). There is one known trehalose biosynthesis pathway in plants from the intermediates Glc 6-P and UDP-Glc catalyzed by trehalose phosphate synthase (TPS), which synthesizes T6P. T6P is then converted to trehalose by trehalose phosphate phosphatase (TPP). The regulation of T6P content in plants by TPSs and TPPs is not well understood. TPS1 is thought to account for most TPS catalytic activity in plants (Vandesteene et al., 2010). All 10 TPPs are now known to be catalytically active (Vandesteene et al., 2012); however, their specific contribution to T6P homeostasis is not known. Evidence suggests that T6P is a sugar signal in plants. T6P responds strongly to Suc supply when Suc is fed to seedlings grown in culture and in response to an increase in Suc in illuminated leaves (Lunn et al., 2006). Biosynthetic pathways for cell wall (Gómez et al., 2006) and starch synthesis (Kolbe et al., 2005) are regulated by T6P, supporting the observation that T6P promotes carbon utilization and growth of seedlings at high sugar levels when its content is increased through expression of otsA, a TPS-encoding gene from Escherichia coli (Schluepmann et al., 2003; Paul et al., 2010). In contrast, expression of otsB, a corresponding TPP-encoding gene from E. coli, decreases T6P content and inhibits growth in the presence of high sugar (Schluepmann et al., 2003; Paul et al., 2010). Given the importance of T6P in the regulation of growth and end-product synthesis, targets for its interaction have been eagerly sought.Recently, it was found that T6P inhibits the protein kinase SnRK1 in growing tissues of plants (Zhang et al., 2009; Debast et al., 2011; Delatte et al., 2011; Martínez-Barajas et al., 2011) through an intermediary factor. SnRK1 (AKIN10/AKIN11) is a member of the SNF1-related AMPK group of protein kinases that perform central functions in the regulation of responses of cells to endogenous energy and carbon status (Hardie, 2007). Baena-González et al. (2007) established that over 1000 genes are regulated by SnRK1 involved in biosynthetic, growth, and stress responses. It was observed that, in addition to cell wall and starch synthesis, T6P could regulate amino acid metabolism, protein, and nucleotide synthesis (Zhang et al., 2009) and is most likely connected to hormone signaling (Zhang et al., 2009; Paul et al., 2010). A model is proposed where SnRK1 inhibits growth processes when sugar and energy supplies are scarce, thus enabling survival under starvation stress conditions. When sugar supply is plentiful, T6P accumulates and inhibits SnRK1 blocking expression of genes involved in the stress survival response and inducing genes involved in the feast response, including growth processes. Interestingly, plants with altered SnRK1 activity display similar phenotypes to plants with altered T6P in both growth and developmental processes such that plants with genetically decreased T6P content resemble those with overexpressed SnRK1 and vice versa (Schluepmann et al., 2003; Baena-González et al., 2007; Wingler et al., 2012).Sugars fluctuate widely in plants in response to changes in photosynthesis and in response to environmental variables. Sugar starvation conditions, such as those induced by deep shade, limit growth through lack of sugar availability; SnRK1 would be active under such conditions. High sugar availability, however, does not necessarily indicate good conditions for growth and high growth rates. For example, under low-temperature and limiting nutrient supply, growth is limited in spite of abundant sugar availability (Paul and Stitt, 1993; Usadel et al., 2008). This is termed sink-limited growth, when growth is limited by capacity of sinks, i.e. growing regions to use assimilate. It departs from the famine model of growth regulation by SnRK1. The interrelationship between T6P, SnRK1, and growth is not known under such conditions. Here, we vary growth conditions by temperature and nutrient supply to induce sink-limited growth and feed Suc and Glc at physiological levels (15 mm). We show a strong specific interrelationship between T6P and Suc and SnRK1-regulated gene expression under all conditions irrespective of growth rate. This implies that T6P is not a growth signal per se, but through SnRK1, T6P primes gene expression for growth. By priming, we mean being in a prepared state with an advanced capacity to activate growth following relief of a growth limitation, such as low temperature. To test that T6P/SnRK1 enable growth recovery following relief from sink limitation, plants with genetically decreased T6P content and SnRK1 overexpression were transferred from cold to warm. Compared with the wild type, these plants were impaired in immediate growth recovery. It is concluded that T6P responds to Suc induced by growth restriction. This enables growth recovery following relief of limitations downstream of T6P/SnRK1, such as low temperature. Our findings are included in a model for the regulation of growth by the T6P/SnRK1 signaling pathway.     

Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors

Clathrin plays important roles in intracellular membrane traffic including endocytosis of plasma membrane proteins and receptors and protein sorting between the trans-Golgi network (TGN) and endosomes. Whether clathrin serves additional roles in receptor recycling, degradative sorting, or constitutive secretion has remained somewhat controversial. Here we have used acute pharmacological perturbation of clathrin terminal domain (TD) function to dissect the role of clathrin in intracellular membrane traffic. We report that internalization of major histocompatibility complex I (MHCI) is inhibited in cells depleted of clathrin or its major clathrin adaptor complex 2 (AP-2), a phenotype mimicked by application of Pitstop® inhibitors of clathrin TD function. Hence, MHCI endocytosis occurs via a clathrin/AP-2-dependent pathway. Acute perturbation of clathrin also impairs the dynamics of intracellular clathrin/adaptor complex 1 (AP-1)- or GGA (Golgi-localized, γ-ear-containing, Arf-binding protein)-coated structures at the TGN/endosomal interface, resulting in the peripheral dispersion of mannose 6-phosphate receptors. By contrast, secretory traffic of vesicular stomatitis virus G protein, recycling of internalized transferrin from endosomes, or degradation of EGF receptor proceeds unperturbed in cells with impaired clathrin TD function. These data indicate that clathrin is required for the function of AP-1- and GGA-coated carriers at the TGN but may be dispensable for outward traffic en route to the plasma membrane.     

Regulation of Starch Biosynthesis in Plant Leaves: Activation and Inhibition of ADPglucose Pyrophosphorylase

The ADPglucose pyrophosphorylases of 7 plant-leaf tissues were partially purified and characterized. In all cases the enzymes showed stability to heat treatment at 65 degrees for 5 minutes in the presence of 0.02 m phosphate buffer, pH 7.0. The leaf ADPglucose pyrophosphorylases were activated 5 to 15-fold by 3-phosphoglycerate. Fructose-6-phosphate and fructose 1, 6-diphosphate stimulated ADPglucose pyrophosphorylase to lesser extents. The A(0.5) (conc of activator required to give 50% of the observed maximal activation) of 3-phosphoglycerate for the barley enzyme was 7 x 10(-6)m while for the sorghum enzyme it was 3.7 x 10(-4)m. Inorganic phosphate proved to be an effective inhibitor of ADPglucose synthesis. The I(0.5) (conc of inhibitor that gave 50% inhibition of activity for the various leaf enzymes varied from 2 x 10(-5)m (barley) to 1.9 x 10(-4)m (sorghum). This inhibition was reversed or antagonized by the activator 3-phosphoglycerate. These results form the basis for an hypothesis of the regulation of leaf starch biosynthesis.     

Purification and Some Catalytic Properties of Glucose-6-Phosphate Dehydrogenase Isoforms from Barley Leaves

The cytosolic and chloroplastic isoforms of glucose-6-phosphate dehydrogenase (G₆PDH) were separated and purified from barley leaves (Hordeum vulgare L.). In etiolated leaves, only the cytosolic isoform was expressed. The molecular mass of the cytosolic enzyme, G₆PDH₁, was 112±8 kDa and that of the chloroplast enzyme, G₆PDH₂, was 136±7 kDa. The K_m values for glucose-6-phosphate and NADP were 0.133 and 0.041 mM for G₆PDH₁, and 0.275 and 0.062 mM for G₆PDH₂, respectively. The pH optimum was 8.2 for G₆PDH₁ and 7.8 for G₆PDH₂. The enzyme is absolutely specific for NADP. NADPH is a competitive inhibitor of the G₆PDH₁ in respect to glucose-6-phosphate (G₆P) and NADP (K_i = 0.050 and 0.025 mM, respectively). NADPH is a competitive inhibitor of the G₆PDH₂ in respect to NADP (K_i = 0.010 mM), but a non-competitive inhibitor in respect to the G₆P. ADP, AMP, UTP, NAD, and NADH had no effect on the activity of G₆PDH. ATP inhibited the G₆PDH₂ activity.     

STARCH-EXCESS4 Is a Laforin-Like Phosphoglucan Phosphatase Required for Starch Degradation in Arabidopsis thaliana

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 α-1,4-glucans esterified with one or two phosphate groups. They are released from starch granules by the glucan hydrolases α-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.