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.     

Osh proteins regulate phosphoinositide metabolism at ER-plasma membrane contact sites

Sac1 phosphoinositide (PI) phosphatases are essential regulators of PI-signaling networks. Yeast Sac1, an integral endoplasmic reticulum (ER) membrane protein, controls PI4P levels at the ER, Golgi, and plasma membrane (PM). Whether Sac1 can act in trans and turn over PI4P at the Golgi and PM from the ER remains a paradox. We find that Sac1-mediated PI4P metabolism requires the oxysterol-binding homology (Osh) proteins. The PH domain-containing family member, Osh3, localizes to PM/ER membrane contact sites dependent upon PM PI4P levels. We reconstitute Osh protein-stimulated Sac1 PI phosphatase activity in vitro. We also show that the ER membrane VAP proteins, Scs2/Scs22, control PM PI4P levels and Sac1 activity in vitro. We propose that Osh3 functions at ER/PM contact sites as both a sensor of PM PI4P and an activator of the ER Sac1 phosphatase. Our findings further suggest that the conserved Osh proteins control PI metabolism at additional membrane contact sites.     

Enzymes of starch and sucrose metabolism in Zea mays leaves

Mesophyll and bundle sheath cells of maize leaves were separated and enzymes of starch and sucrose metabolism assayed. The starch content and activities of ADPglucose (ADPG) starch synthetase and phosphorylase expressed both on a chlorophyll and a protein basis were much lower in mesophyll cells compared to bundle sheath preparations. Exposure of the leaves to continuous illumination for 2·5 days caused the starch content of mesophyll cells to rise greatly and led to considerable increases in ADPG starch synthetase and phosphorylase activity. In glasshouse grown leaves the bulk of invertase, sucrose phosphate synthetase, sucrose phosphatase, UDPglucose pyrophosphorylase and amylase was situated in the mesophyll layer. Sucrose synthetase, ADPG starch synthetase and phosphorylase were largely confined to the bundle sheath. No enzyme could be completely assigned to one particular cell layer. Upon continuous illumination both ADPG starch synthetase and phosphorylase increased in the mesophyll bythe same relative amount. The mesophyll is likely to be a major site for sucrose synthesis in maize leaves.     

A plastid-localized glycogen synthase kinase 3 modulates stress tolerance and carbohydrate metabolism

Glycogen synthase kinase 3 (GSK-3) was originally identified as a regulator of glycogen synthesis in mammals. Like starch in plants, glycogen is a polymer of glucose, and serves as an energy and carbon store. Starch is the main carbohydrate store in plants. Regulation of starch metabolism, in particular in response to environmental cues, is of primary importance for carbon and energy flow in plants but is still obscure. Here, we provide evidence that MsK4, a novel Medicago sativa GSK-3-like kinase, connects stress signalling with carbon metabolism. MsK4 was found to be a plastid-localized protein kinase that is associated with starch granules. High-salt stress rapidly induced the in vivo kinase activity of MsK4. Metabolic profiling of MsK4 over-expressor lines revealed changes in sugar metabolism, including increased amounts of maltose, the main degradation product of starch in leaves. Plants over-expressing MsK4 showed improved tolerance to salt stress. Moreover, under high-salinity conditions, MsK4-over-expressing plants accumulated significantly more starch and showed modified carbohydrate content compared with wild-type plants. Overall, these data indicate that MsK4 is an important regulator that adjusts carbohydrate metabolism to environmental stress.     

A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism

In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs) and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190) were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.     

Identification of mutants in phosphorus metabolism

Phosphorus availability is often limiting for plant growth. However, little is known of the pathways and mechanisms that regulate phosphorus (P) uptake and distribution in plants. We have developed a screen based on the induction of secreted root acid phosphatase activity by low‐P stress to identify mutants of Arabidopsis thaliana with defects in P metabolism. Acid phosphatase activity was detected visually in the roots of A. thaliana seedlings grown in vitro on low‐P medium, using the chromogenic substrate, 5‐bromo‐4‐chloro‐3‐indolyl‐phosphate (BCIP). In low‐P stress conditions the roots of wild‐type plants stained blue, as the induced root acid phosphatase cleaved BCIP to release the coloured product. Potential mutants were identified as having white, or pale blue, roots under these conditions. Out of approximately 79 000 T‐DNA mutagenised seedlings screened, two mutants with reduced acid phosphatase staining were further characterised. Both exhibited reduced growth and differences in their P contents when compared to wild‐type A. thaliana. The mutant with the most severe phenotype, pho3, accumulated high levels of anthocyanins and starch in a distinctive visual pattern within the leaves. The phenotypes of these mutants are distinct from two previously identified phosphorus mutants (phol and pho2) and from an acid phosphatase deficient mutant (pupl) of A. thaliana. This suggested that the screening method was robust and might lead to the identification of further mutants with the potential for increasing our understanding of P nutrition.     

Rice debranching enzyme isoamylase3 facilitates starch metabolism and affects plastid morphogenesis

Debranching enzymes, which hydrolyze α-1 and 6-glucosidic linkages in α-polyglucans, play a dual role in the synthesis and degradation of starch in plants. A transposon-inserted rice mutant of isoamylase3 (isa3) contained an increased amount of starch in the leaf blade at the end of the night, indicating that ISA3 plays a role in the degradation of transitory starch during the night. An epitope-tagged ISA3 expressed in Escherichia coli exhibited hydrolytic activity on β-limit dextrin and amylopectin. We investigated whether ISA3 plays a role in amyloplast development and starch metabolism in the developing endosperm. ISA3-green fluorescent protein (GFP) fusion protein expressed under the control of the rice ISA3 promoter was targeted to the amyloplast stroma in the endosperm. Overexpression of ISA3 in the sugary1 mutant, which is deficient in ISA1 activity, did not convert water-soluble phytoglycogen to starch granules, indicating that ISA1 and ISA3 are not functionally redundant. Both overexpression and loss of function of ISA3 in the endosperm generated pleomorphic amyloplasts and starch granules. Furthermore, chloroplasts in the leaf blade of isa3 seedlings were large and pleomorphic. These results suggest that ISA3 facilitates starch metabolism and affects morphological characteristics of plastids in rice.     

Mechanistic Insights into Glucan Phosphatase Activity against Polyglucan Substrates

Glucan phosphatases are central to the regulation of starch and glycogen metabolism. Plants contain two known glucan phosphatases, Starch EXcess4 (SEX4) and Like Sex Four2 (LSF2), which dephosphorylate starch. Starch is water-insoluble and reversible phosphorylation solubilizes its outer surface allowing processive degradation. Vertebrates contain a single known glucan phosphatase, laforin, that dephosphorylates glycogen. In the absence of laforin, water-soluble glycogen becomes insoluble, leading to the neurodegenerative disorder Lafora Disease. Because of their essential role in starch and glycogen metabolism glucan phosphatases are of significant interest, yet a comparative analysis of their activities against diverse glucan substrates has not been established. We identify active site residues required for specific glucan dephosphorylation, defining a glucan phosphatase signature motif (CζAGΨGR) in the active site loop. We further explore the basis for phosphate position-specific activity of these enzymes and determine that their diverse phosphate position-specific activity is governed by the phosphatase domain. In addition, we find key differences in glucan phosphatase activity toward soluble and insoluble polyglucan substrates, resulting from the participation of ancillary glucan-binding domains. Together, these data provide fundamental insights into the specific activity of glucan phosphatases against diverse polyglucan substrates.     

The phosphoglucan phosphatase like sex Four2 dephosphorylates starch at the C3-position in Arabidopsis

Starch contains phosphate covalently bound to the C6-position (70 to 80% of total bound phosphate) and the C3-position (20 to 30%) of the glucosyl residues of the amylopectin fraction. In plants, the transient phosphorylation of starch renders the granule surface more accessible to glucan hydrolyzing enzymes and is required for proper starch degradation. Phosphate also confers desired properties to starch-derived pastes for industrial applications. In Arabidopsis thaliana, the removal of phosphate by the glucan phosphatase Starch Excess4 (SEX4) is essential for starch breakdown. We identified a homolog of SEX4, LSF2 (Like Sex Four2), as a novel enzyme involved in starch metabolism in Arabidopsis chloroplasts. Unlike SEX4, LSF2 does not have a carbohydrate binding module. Nevertheless, it binds to starch and specifically hydrolyzes phosphate from the C3-position. As a consequence, lsf2 mutant starch has elevated levels of C3-bound phosphate. SEX4 can release phosphate from both the C6- and the C3-positions, resulting in partial functional overlap with LSF2. However, compared with sex4 single mutants, the lsf2 sex4 double mutants have a more severe starch-excess phenotype, impaired growth, and a further change in the proportion of C3- and C6-bound phosphate. These findings significantly advance our understanding of the metabolism of phosphate in starch and provide innovative options for tailoring novel starches with improved functionality for industry.     

Carbohydrate metabolism in Musa paradisiaca

Considerable variations exist in the content of glucose, fructose, sucrose, starch and protein and in the activities of enzymes involved in carbohydrate metabolism between different parts of the banana plant (Musa paradisiaca). Sucrose synthetase is present in the highest concentration in rootstock and fruit pulp, and sucrose phosphate synthetase in the pseudostem. The highest ratio of the activity of sucrose phosphate synthetase to sucrose synthetase is found in leaves. Acid invertase is present in leaves, leaf-sheath and fruit pulp and is not demonstrable in rootstock and pseudostem. Neutral invertase activity is high in pseudostem and leaf-sheath. Starch phosphorylase is largely concentrated in fruit pulp and rootstock. The maximum activity of ATP:d-phosphoglucose (ADPG) pyrophosphorylase is found in rootstock. β-Amylase is not demonstrable in rootstock and is largely concentrated in leaf-sheath. Hexokinase is most active in rootstock and the lowest in leaves. Acid phosphatase and alkaline phosphatase activity is highest in fruit pulp and pseudostem. Glucosephosphate isomerase is most active in the rootstock and lowest in the leaves.     

Starch metabolism in developing strawberry (Fragaria × ananassa) fruits

Fruit starch reserves can be an important contributor to the sugar content of some ripe fruit, and despite the relatively high financial premiums (compared to other fruit) commanded by ripe strawberries, neither their starch or sugar biochemistry has been examined in detail. This study assessed the rate of starch biosynthesis and breakdown in developing strawberry and sought to determine the temporal changes in the activities of selected enzymes known to be involved in sucrose-starch interconversions. Scanning electron microscopy revealed that starch levels appeared greatest in immature strawberry ( Fragaria × ananassa , cv. Elsanta) at 7 days postanthesis, as evidenced by a decrease in the number of cells containing starch granules as ripening progressed. Levels of key enzymes of starch and sugar metabolism estimated using Western blotting and enzyme activity analysis showed that activities did not correlate with antigen levels. In particular, enzyme activity recovery experiments indicated that losses were due to non-proteinaceous inhibitors, and in particular protein binding: highlighting the potential for misinterpretation of enzyme activity data gathered from ripening (strawberry) fruit tissue extracts. Consequently, in vitro experiments using [U-¹⁴C] glucose revealed that incorporation to starch is low (11%) at the earliest developmental stages when starch content is greatest. Starch synthesis rate then declines to non-detectable levels as fruit expand and ripen. These results show that starch accumulates extremely early in the fruit formation process and that starch degradation predominates during fruit growth and development. We estimate that breakdown of transient starch can contribute up to 3% of the sugar accumulated in ripe fruit.     

The role of transitory starch in C(3), CAM, and C(4) metabolism and opportunities for engineering leaf starch accumulation

Essentially all plants store starch in their leaves during the day and break it down the following night. This transitory starch accumulation acts as an overflow mechanism when the sucrose synthesis capacity is limiting, and transitory starch also acts as a carbon store to provide sugar at night. Transitory starch breakdown can occur by either of two pathways; significant progress has been made in understanding these pathways in C(3) plants. The hydrolytic (amylolytic) pathway generating maltose appears to be the primary source of sugar for export from C(3) chloroplasts at night, whereas the phosphorolytic pathway supplies carbon for chloroplast reactions, in particular in the light. In crassulacean acid metabolism (CAM) plants, the hydrolytic pathway predominates when plants operate in C(3) mode, but the phosphorolytic pathway predominates when they operate in CAM mode. Information on transitory starch metabolism in C(4) plants has now become available as a result of combined microscopy and proteome studies. Starch accumulates in all cell types in immature maize leaf tissue, but in mature leaf tissues starch accumulation ceases in mesophyll cells except when sugar export from leaves is blocked. Proper regulation of the amount of carbon that goes into starch, the pathway of starch breakdown, and the location of starch accumulation could help ensure that engineering of C(4) metabolism is coordinated with the downstream reactions required for efficient photosynthesis.     

Adjustment of growth, starch turnover, protein content and central metabolism to a decrease of the carbon supply when Arabidopsis is grown in very short photoperiods

Arabidopsis was grown in a 12, 8, 4 or 3 h photoperiod to investigate how metabolism and growth adjust to a decreased carbon supply. There was a progressive increase in the rate of starch synthesis, decrease in the rate of starch degradation, decrease of malate and fumarate, decrease of the protein content and decrease of the relative growth rate. Carbohydrate and amino acids levels at the end of the night did not change. Activities of enzymes involved in photosynthesis, starch and sucrose synthesis and inorganic nitrogen assimilation remained high, whereas five of eight enzymes from glycolysis and organic acid metabolism showed a significant decrease of activity on a protein basis. Glutamate dehydrogenase activity increased. In a 2 h photoperiod, the total protein content and most enzyme activities decreased strongly, starch synthesis was inhibited, and sugars and amino acids levels rose at the end of the night and growth was completely inhibited. The rate of starch degradation correlated with the protein content and the relative growth rate across all the photoperiod treatments. It is discussed how a close coordination of starch turnover, the protein content and growth allows Arabidopsis to avoid carbon starvation, even in very short photoperiods.     

Starch metabolism in leaves

Starch is the most abundant storage carbohydrate produced in plants. The initiation of transitory starch synthesis and degradation in plastids depends mainly on diurnal cycle, post-translational regulation of enzyme activity and starch phosphorylation. For the proper structure of starch granule the activities of all starch synthase isoenzymes, branching enzymes and debranching enzymes are needed. The intensity of starch biosynthesis depends mainly on the activity of AGPase (adenosine 5'-diphosphate glucose pyrophosphorylase). The key enzymes in starch degradation are beta-amylase, isoamylase 3 and disproportionating enzyme. However, it should be underlined that there are some crucial differences in starch metabolism between heterotrophic and autotrophic tissues, e.g. is the ability to build multiprotein complexes responsible for biosynthesis and degradation of starch granules in chloroplasts. The observed huge progress in understanding of starch metabolism was possible mainly due to analyses of the complete Arabidopsis and rice genomes and of numerous mutants with altered starch metabolism in leaves. The aim of this paper is to review current knowledge on transient starch metabolism in higher plants.     

Mannitol metabolism in cultured plant cells

Non-structural storage carbohydrates were measured in 9-day-old barley ( Hordeum vulgare L. cv. Brant) primary leaves. Accumulation rates of starch, sucrose and total non-structural carbohydrates (TNC) were approximately linear when measured between 2- and 12-h of light. Progressively higher TNC accumulation rates were observed at higher irradiance levels (i.e., comparing 250, 550 and 1050 ·mol m⁻² s⁻¹). Synthesis of a low-molecular-weight fructan also was enhanced by high irradiances. Low irradiance treatments decreased leaf sucrose levels and there was a corresponding increase in the lag period preceding starch synthesis in the light. Increased starch accumulation rates were usually observed when sucrose concentrations were high. These and other results suggested that cytosolic sucrose concentrations affected starch metabolism in the chloroplast. However, sucrose accumulation rates increased and starch storage decreased when barley seedlings were transferred from 20 to 10°C during the light period. Lowering the night temperature from 20 to 10°C for a single dark period 8-days after planting increased the TNC content of barley primary leaves at the beginning of day nine. In this experiment, TNC accumulation rates of treated and untreated leaves were similar. Changes in the accumulation rate of TNC were usually observed within 2- to 4-h after barley seedlings were exposed to altered environmental conditions. Monitoring rapid changes in leaf carbohydrate levels is a sensitive method for assessing the effects of environmental treatments on photosynthetic metabolism.     

Protein, nucleic acid and starch metabolism in the duckweed Spirodela oligorrhiza, treated with cytokinins

Bacteria-free cultures of Spirodela oligorrhiza continue to increase in frond number for 2 to 3 days after transfer to darkness. There is then no further increase in frond number for 3 to 4 weeks, although DNA, RNA and protein synthesis continue at decreased rates and starch accumulates in the plants. We refer to such ;non-growing' plants in darkness as dormant. Adding kinetin to dormant Spirodela initiated increased DNA, RNA and protein synthesis within 1h, although new fronds were not detected until 24h after the addition of kinetin. The frond number then continued to increase. Starch accumulated in dormant plants. Accumulation of starch appeared to be a consequence of inhibition of growth rather than the converse. No evidence was obtained for a block in [(14)C]glucose metabolism that might explain the lack of growth in darkness in the absence of kinetin. In darkness, more ribosomes were membrane-bound in dormant Spirodela than in Spirodela growing with kinetin. Similarities between the response of Spirodela to darkness, stringent control in bacteria and pleiotypic controls in animal cells are discussed. It is suggested that all three processes are ultimately controlled by specific protein kinases that are individually sensitive to different effectors.