Pathway of starch breakdown in photosynthetic tissues of Pisum sativum.

1. The aim of this work was to discover the pathway of starch breakdown in the photosynthetic tissues of Pisum sativum. 2. Measurements of the starch in the leaves of plants grown in photoperiods of 12 or 18 h showed that starch, synthesized in the light, was rapidly metabolized in the dark at rates of 0.04--0.06 mumol glucose/min per g fresh weight. 3. The maximum catalytic activities of alpha-amylase, beta-amylase, hexokinase, alpha-glucan phosphorylase and phosphoglucomutase in extracts of leaves showed no diurnal variation in either photoperiod, and exceeded estimates of the rate of net starch breakdown in the dark. 4. Studies with intact chloroplasts, isolated from young shoots and from leaves, indicated that pea chloroplasts do not contain significant activities of alpha-amylase, beta-amylase and hexokinase, although some of the latter may be attached to the outside of the chloroplast envelope. These studies also showed that pea chloroplasts contained sufficient alpha-glucan phosphorylase and phosphoglucomutase to mediate the observed rates of starch breakdown. 5. It is proposed that starch breakdown in pea chloroplasts is phosphorolytic.     

Properties of α-glucan phosphorylase from pea chloroplasts

A partially purified preparation of α-glucan phosphorylase was obtained from chloroplasts of Pisum sativum by ion-exchange chromatography and gel filtration. The preparation, in which no other enzyme that metabolized starch or glucose 1 -phosphate could be detected, was characterized. The optimum for phosphorolysis was pH 7.2; at pH 8.0 the activity was reduced by 50%. The preparation showed normal hyperbolic kinetics with the substrates, and catalysed the formation of [¹⁴C]glucose 1-phosphate from ¹⁴C-labelled starch grains from pea chloroplasts. None of the following, generally at 5 and 10 mM, significantly altered the rate of phosphorolysis: glucose, fructose, sucrose, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate, ATP, ADP, AMP, 6-phosphogluconate, 2-phosphoglycollate, Mg²⁺, dithiothreitol. However, phosphorolysis was inhibited by ADPglucose. Measurements of ADPglucose in leaves and in isolated chloroplasts showed that none could be detected in the dark and suggested that the concentration in the light was high enough to cause a modest inhibition of the phosphorylase. The control of the breakdown of chloroplast starch is discussed.     

Maltose is the major form of carbon exported from the chloroplast at night

Transitory starch is formed in chloroplasts during the day and broken down at night. We investigated carbon export from chloroplasts resulting from transitory-starch breakdown. Starch-filled chloroplasts from spinach (Spinacia oleracea L. cv. Nordic IV) were isolated 1 h after the beginning of the dark period and incubated for 2.5 h, followed by centrifugation through silicone oil. Exported products were measured in the incubation medium to avoid measuring compounds retained inside the chloroplasts. Maltose and glucose made up 85% of the total exported products and were exported at rates of 626 and 309 nmol C mg^–1 chlorophyll h^–1, respectively. Net export of phosphorylated products was less than 5% and higher maltodextrins were not detected. Maltose levels in leaves of bean (Phaseolus vulgaris L. cv. Linden), spinach, and Arabidopsis thaliana (L.) Heynh. were low in the light and high in the dark. Maltose levels remained low and unchanged during the light/dark cycle in two starch-deficient Arabidopsis mutants, stf1, deficient in plastid phosphoglucomutase, and pgi, deficient in plastid phosphoglucoisomerase. Through the use of nonaqueous fractionation, we determined that maltose was distributed equally between the chloroplast and cytosolic fractions during darkness. In the light there was approximately 24% more maltose in the cytosol than the chloroplast. Taken together these data indicate that maltose is the major form of carbon exported from the chloroplast at night as a result of starch breakdown. We hypothesize that the hydrolytic pathway for transitory-starch degradation is the primary pathway used when starch is being converted to sucrose and that the phosphorolytic pathway provides carbon for other purposes.Abbreviations CAM crassulacean acid metabolism - Chl chlorophyll - DHAP dihydroxyacetone phosphate - FBPase fructose bisphosphatase - GAP glyceraldehyde-3-phosphate - G6P glucose 6-phosphate - PGA 3-phosphoglycerate - TPT triose phosphate translocator - WT wild type     

Capacities of pea chloroplasts to catalyse the oxidative pentose phosphate pathway and glycolysis

The aim of this work was to measure the capacities of pea (Pisum sativum) shoot chloroplasts to catalyse the oxidative pentose phosphate pathway and glycolysis. Of the total activities in the unfractionated homogenates, appreciable proportions of those of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and phosphofructokinase, and smaller but significant proportions of those of phosphopyruvate hydratase and pyruvate kinase were recovered in crude preparations of chloroplasts, and co-purified with intact chloroplasts on sucrose gradients. The activities in the chloroplasts showed considerable latency that was closely correlated with chloroplast integrity. Phosphoglyceromutase activity in the above preparations of chloroplasts did not exceed that expected from cytoplasmic contamination. The mass-action ratio for phosphoglyceromutase in illuminated isolated chloroplasts differed markedly from the enzyme's equilibrium constant. Isolated chloroplasts converted 2-phosphoglycerate to pyruvate. The enzyme activities of the chloroplasts were compared with the rates of respiration and starch breakdown in pea leaves in the dark. It is concluded that in the dark chloroplasts could metabolize all the products of starch breakdown and catalyse much of the respiration of pea shoots via the oxidative pentose phosphate pathway and/or glycolysis as far as 3-phosphoglycerate. It is suggested that pea shoot chloroplasts lack phosphoglyceromutase but contain some phosphopyruvate hydratase and pyruvate kinase.     

Diurnal oscillation of amylolytic activity in spinach chloroplasts

Chloroplasts isolated from spinach (Spinacia oleracea L., cv. vital^R) plants grown under controlled light/dark and temperature regimes, contained the phosphorolytic and amylolytic pathways for starch breakdown. The latter consists at least of α- and β-amylase and maltase. Only low amylolytic activity was observed in chloroplasts isolated during the light phase. In chloroplasts prepared during the dark phase, this activity was almost twice as high. These diurnal oscillations of the amylolytic activity were maintained when the plants were kept in prolonged darkness or continuous light. The amylolytic system exhibited a sharp pH optimum between 5.8 and 6.0. Phosphorylase activity, when assayed with saturating concentrations of inorganic phosphate, did not show diurnal fluctuations.     

A starch-accumulating mutant of Arabidopsis thaliana deficient in a chloroplastic starch-hydrolysing enzyme

The aim of this work was to identify enzymes that participate in the degradation of transitory starch in Arabidopsis . A mutant line was isolated by screening leaves at the end of the night for the presence of starch. The mutant had a higher starch content than the wild-type throughout the diurnal cycle. This accumulation was due to a reduction in starch breakdown, leading to an imbalance between the rates of synthesis and degradation. No reduction in the activity of endo-amylase (α-amylase), β-amylase, starch phosphorylase, maltase, pullulanase or D-enzyme could be detected in crude extracts of leaves of the mutant. However, native PAGE in gels containing amylopectin revealed that a starch-hydrolysing activity, putatively identified as an endo-amylase and present in wild-type chloroplasts, was absent or appreciably reduced in the mutant. This is the first time that a specific enzyme required for starch degradation has been identified in leaves.     

Starch degradation in isolated spinach chloroplasts

A method for loading isolated intact spinach (Spinacia oleracea L.) chloroplasts with ¹⁴C-starch is described. These intact chloroplasts were incubated aerobically in the dark for 30 minutes. Radioactivity in starch declined and glyceric acid 3-phosphate and maltose were the major radioactive products. It is proposed that starch is degraded within the chloroplast to glyceric acid 3-phosphate and to maltose.     

Carbon balance and circadian regulation of hydrolytic and phosphorolytic breakdown of transitory starch

Transitory starch is formed in chloroplasts during the day and broken down at night. Transitory starch degradation could be regulated by light, circadian rhythms, or carbon balance. To test the role of these potential regulators, starch breakdown rates and metabolites were measured in bean (Phaseolus vulgaris) and Arabidopsis (Arabidopsis thaliana) plants. In continuous light, starch and maltose levels oscillated in a circadian manner. Under photorespiratory conditions, transitory starch breakdown occurred in the light faster than at night and glucose-6-P (G6P) was elevated. Nonaqueous fractionation showed that the increase in G6P occurred in the chloroplast. When Arabidopsis plants lacking the plastidic starch phosphorylase enzyme were placed under photorespiratory conditions, G6P levels remained constant, indicating that the increased chloroplastic G6P resulted from phosphorolytic starch degradation. Maltose was increased under photorespiratory conditions in both wild type and plants lacking starch phosphorylase, indicating that regulation of starch breakdown may occur at a point preceding the division of the hydrolytic and phosphorolytic pathways. When bean leaves were held in N2 to suppress photosynthesis and Suc synthesis without increasing photorespiration, starch breakdown did not occur and maltose and G6P levels remained constant. The redox status of the chloroplasts was found to be oxidized under conditions favoring starch degradation.     

Characterization of starch breakdown in the intact spinach chloroplast

Starch degradation with a rate of 1 to 2 microgram-atom carbon per milligram chlorophyll per hour was monitored in the isolated intact spinach (Spinacia oleracea) chloroplast which had been preloaded with ¹⁴C-starch photosynthetically from ¹⁴CO₂. Starch breakdown was dependent upon inorganic phosphate and the ¹⁴C-labeled intermediates formed were principally those of the Embden-Meyerhof pathway from glucose phosphate to glycerate 3-phosphate. In addition, isotope was found in ribose 5-phosphate and in maltose and glucose. The appearance of isotope in the intermediates of the Embden-Meyerhof pathway but not in the free sugars was dependent upon the inorganic phosphate concentration. Dithiothreitol shifted the flow of ¹⁴C from triose-phosphate to glycerate 3-phosphate. Iodoacetic acid inhibited starch breakdown and caused an accumulation of triose-phosphate. This inhibition of starch breakdown was overcome by ATP. The inhibitory effect of ionophore A 23187 on starch breakdown was reversed by the addition of magnesium ions. The formation of maltose but not glucose was impaired by the ionophore. The inhibition of starch breakdown by glycerate 3-phosphate was overcome by inorganic phosphate. Fructose 1,6-bisphosphate and ribose 5-phosphate did not affect the rate of polysaccharide metabolism but increased the flow of isotope into maltose. Starch breakdown was unaffected by the uncoupler (trifluoromethoxyphenylhydrazone), electron transport inhibitors (rotenone, cyanide, salicylhydroxamic acid), or anaerobiosis. Hexokinase and the dehydrogenases of glucose 6-phosphate and gluconate 6-phosphate were detected in the chloroplast preparations. It was concluded (a) that chloroplastic starch was degraded principally by the Embden-Meyerhof pathway and by a pathway involving amylolytic cleavage; (b) ATP required in the Embden-Meyerhof pathway is generated by substrate phosphorylation in the oxidation of glyceraldehyde 3-phosphate to glycerate 3-phosphate; and (c) the oxidative pentose phosphate pathway is the probable source of ribose 5-phosphate.     

Physiological rates of starch breakdown in isolated intact spinach chloroplasts

Starch breakdown with rates above 10 μatom carbon per mg chlorophyll per hour has been monitored in spinach chloroplasts and compares favorably with the rates in whole leaves. Intact starch-loaded chloroplasts were prepared from protoplasts to avoid rupture during mechanical homogenization and rapid centrifugation. Particular attention was paid to the identification of all the products of starch degradation and to measuring the actual rates of their accumulation. The products of starch breakdown included triose phosphate, 3-phosphoglycerate, CO₂, glucose, and some maltose. Comparison of the rates of metabolism of added glucose and of the conversion of starch to phosphorylated intermediates showed that starch phosphorolysis was the major pathway leading to phosphorylated endproducts. From the results, the relative contribution of phosphorolysis and hydrolysis to starch breakdown and the contribution of glycolysis and the oxidative pentose phosphate cycle can be estimated. Phosphate has a large influence on the metabolism of the chloroplast in the dark.     

Regulation of adenylate levels in intact spinach chloroplasts

Starch phosphorylase activity in extracts of spinach or pea leaves and of isolated chloroplasts was determined and separated by electrophoresis in polyacrylamide gels. In spinach leaf extracts, a specific activity of 16 nmol glucose 1-phosphate formed per min per mg protein was found, whereas a lower value (6 nmol per min per mg protein) was observed in preparations of isolated chloroplasts which were about 75% intact. In the spinach leaf extracts two forms of phosphorylase were found; chloroplast preparations almost exclusively contained one of these. In pea leaf extracts the specific activity was 10 nmol glucose 1-phosphate formed per min per mg protein. Three forms of phosphorylase contributed to this activity. Preparations of isolated chloroplasts with an intactness of about 85% exhibited a lower specific activity (5nmol per min per mg protein) and contained two of these three phosphorylase forms.Abbreviations G1P Glucose 1-phosphate - P_i orthophosphate - Tris Tris (hydroxymethyl)aminomethane - MES 2(N-morpholino)ethane sulphonic acid - EDTA ethylenediamine tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid     

Autophagy Contributes to Leaf Starch Degradation

Transitory starch, a major photosynthetic product in the leaves of land plants, accumulates in chloroplasts during the day and is hydrolyzed to maltose and Glc at night to support respiration and metabolism. Previous studies in Arabidopsis thaliana indicated that the degradation of transitory starch only occurs in the chloroplasts. Here, we report that autophagy, a nonplastidial process, participates in leaf starch degradation. Excessive starch accumulation was observed in Nicotiana benthamiana seedlings treated with an autophagy inhibitor and in autophagy-related (ATG) gene-silenced N. benthamiana and in Arabidopsis atg mutants. Autophagic activity in the leaves responded to the dynamic starch contents during the night. Microscopy showed that a type of small starch granule-like structure (SSGL) was localized outside the chloroplast and was sequestered by autophagic bodies. Moreover, an increased number of SSGLs was observed during starch depletion, and disruption of autophagy reduced the number of vacuole-localized SSGLs. These data suggest that autophagy contributes to transitory starch degradation by sequestering SSGLs to the vacuole for their subsequent breakdown.     

High-Affinity Maltose Binding and Transport by the Thermophilic Anaerobe Thermoanaerobacter ethanolicus 39E

Thermoanaerobacter ethanolicus is a gram-positive thermophile that produces considerable amounts of ethanol from soluble sugars and polymeric substrates, including starch. Growth on maltose, a product of starch hydrolysis, was associated with the production of a prominent membrane-associated protein that had an apparent molecular weight of 43,800 and was not detected in cells grown on xylose or glucose. Filter-binding assays revealed that cell membranes bound maltose with high affinity. Metabolic labeling of T. ethanolicus maltose-grown cells with [¹⁴C]palmitic acid showed that this protein was posttranslationally acylated. A maltose-binding protein was purified by using an amylose resin affinity column, and the binding constant was 270 nM. Since maltase activity was found only in the cytosol of fractionated cells and unlabeled glucose did not compete with radiolabeled maltose for uptake in whole cells, it appeared that maltose was transported intact. In whole-cell transport assays, the affinity for maltose was approximately 40 nM. Maltotriose and α-trehalose competitively inhibited maltose uptake in transport assays, whereas glucose, cellobiose, and a range of disaccharides had little effect. Based on these results, it appears that T. ethanolicus possesses a high-affinity, ABC type transport system that is specific for maltose, maltotriose, and α-trehalose.     

Starch depletion in germinating wheat,wrinkled-seeded peas and senescing tobacco leaves

The residual starches of germinating wheat and barley grains show similar structural changes. Germinating wheat grains produce malto-oligosaccharides and dextrins. The starch of wrinkled-seeded peas showed some structural changes during germination, but the starch from senescing tobacco leaves showed none. Neither peas nor tobacco produced malto-oligosaccharides or dextrins at any stage. Wrinkled-seeded peas showed some differences to smooth-seeded peas in enzyme content, and starch was probably degraded by phosphorylase initially with α-amylase acting after 3 days. In senescing tobacco leaves the only significant enzyme activities were α-amylase and maltase. Wheat closely resembled barley in showing amylolytic breakdown.     

Ectopic maltase alleviates dwarf phenotype and improves plant frost tolerance of maltose transporter mutants

Maltose, the major product of starch breakdown in Arabidopsis (Arabidopsis thaliana) leaves, exits the chloroplast via the maltose exporter1 MEX1. Consequently, mex1 loss-of-function plants exhibit substantial maltose accumulation, a starch-excess phenotype and a specific chlorotic phenotype during leaf development. Here, we investigated whether the introduction of an alternative metabolic route could suppress the marked developmental defects typical for mex1 loss-of-function mutants. To this end, we ectopically expressed in mex1  chloroplasts a functional maltase (MAL) from baker’s yeast (Saccharomyces cerevisiae, chloroplastidial MAL [cpMAL] mutants). Remarkably, the stromal MAL activity substantially alleviates most phenotypic peculiarities typical for mex1 plants. However, the cpMAL lines contained only slightly less maltose than parental mex1 plants and their starch levels were, surprisingly, even higher. These findings point to a threshold level of maltose responsible for the marked developmental defects in mex1. While growth and flowering time were only slightly retarded, cpMAL lines exhibited a substantially improved frost tolerance, when compared to wild-types. In summary, these results demonstrate the possibility to bypass the MEX1 transporter, allow us to differentiate between possible starch-excess and maltose-excess responses, and demonstrate that stromal maltose accumulation prevents frost defects. The latter insight may be instrumental for the development of crop plants with improved frost tolerance.

Expressing a yeast maltase in chloroplasts of the Arabidopsis maltose transporter mutant mex1 prevents the marked developmental defects typical for that mutant and enhances plant frost tolerance.     

A cytosolic glucosyltransferase is required for conversion of starch to sucrose in Arabidopsis leaves at night

Maltose is exported from the Arabidopsis chloroplast as the main product of starch degradation at night. To investigate its fate in the cytosol, we characterised plants with mutations in a gene encoding a putative glucanotransferase (disproportionating enzyme; DPE2), a protein similar to the maltase Q (MalQ) gene product involved in maltose metabolism in bacteria. Use of a DPE2 antiserum revealed that the DPE2 protein is cytosolic. Four independent mutant lines lacked this protein and displayed a decreased capacity for both starch synthesis and starch degradation in leaves. They contained exceptionally high levels of maltose, and elevated levels of glucose, fructose and other malto-oligosaccharides. Sucrose levels were lower than those in wild-type plants, especially at the start of the dark period. A glucosyltransferase activity, capable of transferring one of the glucosyl units of maltose to glycogen or amylopectin and releasing the other, was identified in leaves of wild-type plants. Its activity was sufficient to account for the rate of starch degradation. This activity was absent from dpe2 mutant plants. Based on these results, we suggest that DPE2 is an essential component of the pathway from starch to sucrose and cellular metabolism in leaves at night. Its role is probably to metabolise maltose exported from the chloroplast. We propose a pathway for the conversion of starch to sucrose in an Arabidopsis leaf.