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Controlled conversion of leaf starch to sucrose at night is essential for the normal growth of Arabidopsis. The conversion involves the cytosolic metabolism of maltose to hexose phosphates via an unusual, multidomain protein with 4-glucanotransferase activity, DPE2, believed to transfer glucosyl moieties to a complex heteroglycan prior to their conversion to hexose phosphate via a cytosolic phosphorylase. The significance of this complex pathway is unclear; conversion of maltose to hexose phosphate in bacteria proceeds via a more typical 4-glucanotransferase that does not require a heteroglycan acceptor. It has recently been suggested that DPE2 generates a heterogeneous series of terminal glucan chains on the heteroglycan that acts as a glucosyl buffer to ensure a constant rate of sucrose synthesis in the leaf at night. Alternatively, DPE2 and/or the heteroglycan may have specific properties important for their function in the plant. To distinguish between these ideas, we compared the properties of DPE2 with those of the Escherichia coli glucanotransferase MalQ. We found that MalQ cannot use the plant heteroglycan as an acceptor for glucosyl transfer. However, experimental and modeling approaches suggested that it can potentially generate a glucosyl buffer between maltose and hexose phosphate because, unlike DPE2, it can generate polydisperse malto-oligosaccharides from maltose. Consistent with this suggestion, MalQ is capable of restoring an essentially wild-type phenotype when expressed in mutant Arabidopsis plants lacking DPE2. In light of these findings, we discuss the possible evolutionary origins of the complex DPE2-heteroglycan pathway.
Die nun begonnene Reihe „studieren++“ resultiert aus einer von der Universität Potsdam angebotenen Vorlesungsreihe. Das Besondere an dieser Vorlesungsreihe ist der multidisziplinäre Anspruch und die konsequent umgesetzte Zusammenarbeit über Disziplingrenzen hinweg. Die nicht nur über Instituts-, sondern über Fakultätsgrenzen praktizierte Interdisziplinarität erlaubt die Betrachtung eines Problems oder Sachverhalts aus unterschiedlichen Blickwinkeln. Wissenschaftliche Fragestellungen sind komplex und nicht immer auf eine Disziplin beschränkt. Sie in ihrer Gänze erfassen und nachhaltige Lösungsstrategien oder Konzepte entwickeln zu können gelingt oft nur durch eine multidisziplinäre Kooperation. Eine Lehrveranstaltung wie die vorliegende ist nicht nur für die Studierenden einer Universität eine hervorragende Möglichkeit, um über die Grenzen der eigenen Disziplin hinaus zu blicken und die Zusammenarbeit mit Wissenschaftlerinnen und Wissenschaftlern aus anderen Bereichen zu pflegen. So lernt man, sich in andere Sichtweisen hineinzuversetzen und sich zwischen den Disziplinen zu bewegen – eine Kompetenz, die in der hochkomplexen Arbeitswelt von heute von hohem Nutzen ist.
Der vorliegende erste Band der Reihe hat „Raum und Zahl“ zum Thema und ist aus einer Ringvorlesung aus dem Wintersemester 2013/2014 entstanden. Drei der fünf Fakultäten, insgesamt neun Institute der Universität Potsdam, haben sich an der Vorlesung beteiligt und sich dieses spannenden Themas angenommen. Als jemand, der sich jahrelang wissenschaftlich mit algorithmischer Geometrie sowie mit raumbezogenen Datenbanken und Navigationssystemen beschäftigt hat, kann ich nur bekräftigen, dass die Bezüge zwischen Raum und Zahl, zwischen Räumen und Zahlen, noch viel stärker im öffentlichen Bewusstsein verankert gehören. Räume auch quantitativ zu erfassen und zu verstehen ist eine Kulturtechnik, die an Wichtigkeit eher noch zunimmt, vor allem vor dem Hintergrund, dass wir genetisch nicht allzu gut auf derartige Herausforderungen vorbereitet sind. Denn viele unserer einschlägigen Gene entstammen noch aus der Zeit der Savanne, einer Zeit, zu der das Raumkonzept sich fast ausschließlich auf die unmittelbare räumliche Umgebung bezog und Zahlen jenseits von 10 nur wenig Relevanz für das eigene Überleben hatten.
Als Präsident der Universität Potsdam freut es mich ganz besonders, dass sich die hier vertretenen Wissenschaftler bereit erklärt haben, ihre Überlegungen mit den Studierenden und ihren Kolleginnen und Kollegen zu teilen. Herrn Kollegen Hans-Joachim Petsche möchte ich für sein Engagement danken und ihm zu dieser gelungenen Reihe gratulieren. Der Geist der Wissenschaft, der nicht nur einsam im Büro oder Labor gelebt wird, sondern gerade an einer Universität auch aktiv nach außen getragen werden sollte, wird hier in besonderer Weise sichtbar. Ich wünsche Ihnen viel Freude bei der Lektüre des Bandes und freue mich auf weitere Veröffentlichungen in dieser Reihe.
A technique has been developed to measure absolute intracellular oxygen concentrations in green plants. Oxygen-sensitive phosphorescent microbeads were injected into the cells and an optical multifrequency phase-modulation technique was used to discriminate the sensor signal from the strong autofluorescence of the plant tissue. The method was established using photosynthesis-competent cells of the giant algae Chara corallina L., and was validated by application to various cell types of other plant species.
Water-soluble heteroglycans (SHG) were isolated from leaves of wild-type Arabidopsis thaliana L. and from two starch-deficient mutants. Major constituents of the SHG are arabinose, galactose, rhamnose, and glucose. SHG was separated into low (< 10 kDa; SHG(S)) and high (> 10 kDa; SHG(L)) molecular weight compounds. SHG(S) was resolved into approximately 25 distinct oligoglycans by ion exchange chromatography. SHG(L) was further separated into two subfractions, designated as subfraction I and II, by field flow fractionation. For the intracellular localization of the various SHG compounds several approaches were chosen: first, leaf material was subjected to non-aqueous fractionation. The apolar gradient fractions were characterized by monitoring markers and were used as starting material for the SHG isolation. Subfraction I and SHG(S) exhibited a distribution similar to that of cytosolic markers whereas subfraction II cofractionated with crystalline cellulose. Secondly, intact organelles were isolated and used for SHG isolation. Preparations of intact organelles (mitochondria plus peroxisomes) contained no significant amount of any heteroglycan. In isolated intact microsomes a series of oligoglycans was recovered but neither subfraction I nor II. In in vitro assays using glucose 1-phosphate and recombinant cytosolic (Pho 2) phosphorylase both SHG(S) and subfraction I acted as glucosyl acceptor whereas subfraction II was essentially inactive. Rabbit muscle phosphorylase a did not utilize any of the plant glycans indicating a specific Pho 2-glycan interaction. As revealed by in vivo labeling experiments using (CO2)-C-14 carbon fluxes into subfraction I and II differed. Furthermore, in leaves the pool size of subfraction I varied during the light-dark regime
Reversible binding of the starch-related R1 protein to the surface of transitory starch granules
(2000)
Biomarkers are used to predict phenotypical properties before these features become apparent and, therefore, are valuable tools for both fundamental and applied research. Diagnostic biomarkers have been discovered in medicine many decades ago and are now commonly applied. While this is routine in the field of medicine, it is of surprise that in agriculture this approach has never been investigated. Up to now, the prediction of phenotypes in plants was based on growing plants and assaying the organs of interest in a time intensive process. For the first time, we demonstrate in this study the application of metabolomics to predict agronomic important phenotypes of a crop plant that was grown in different environments. Our procedure consists of established techniques to screen untargeted for a large amount of metabolites in parallel, in combination with machine learning methods. By using this combination of metabolomics and biomathematical tools metabolites were identified that can be used as biomarkers to improve the prediction of traits. The predictive metabolites can be selected and used subsequently to develop fast, targeted and low-cost diagnostic biomarker assays that can be implemented in breeding programs or quality assessment analysis. The identified metabolic biomarkers allow for the prediction of crop product quality. Furthermore, marker-assisted selection can benefit from the discovery of metabolic biomarkers when other molecular markers come to its limitation. The described marker selection method was developed for potato tubers, but is generally applicable to any crop and trait as it functions independently of genomic information.
Feedback inhibition of starch degradation in arabidopsis leaves mediated by trehalose 6-phosphate
(2013)
Many plants accumulate substantial starch reserves in their leaves during the day and remobilize them at night to provide carbon and energy for maintenance and growth. In this paper, we explore the role of a sugar-signaling metabolite, trehalose-6-phosphate (Tre6P), in regulating the accumulation and turnover of transitory starch in Arabidopsis (Arabidopsis thaliana) leaves. Ethanol-induced overexpression of trehalose-phosphate synthase during the day increased Tre6P levels up to 11-fold. There was a transient increase in the rate of starch accumulation in the middle of the day, but this was not linked to reductive activation of ADP-glucose pyrophosphorylase. A 2- to 3-fold increase in Tre6P during the night led to significant inhibition of starch degradation. Maltose and maltotriose did not accumulate, suggesting that Tre6P affects an early step in the pathway of starch degradation in the chloroplasts. Starch granules isolated from induced plants had a higher orthophosphate content than granules from noninduced control plants, consistent either with disruption of the phosphorylation-dephosphorylation cycle that is essential for efficient starch breakdown or with inhibition of starch hydrolysis by beta-amylase. Nonaqueous fractionation of leaves showed that Tre6P is predominantly located in the cytosol, with estimated in vivo Tre6P concentrations of 4 to 7 mu M in the cytosol, 0.2 to 0.5 mu M in the chloroplasts, and 0.05 mu M in the vacuole. It is proposed that Tre6P is a component in a signaling pathway that mediates the feedback regulation of starch breakdown by sucrose, potentially linking starch turnover to demand for sucrose by growing sink organs at night.
The biochemical function of the Laforin-like dual-specific phosphatase AtSEX4 (EC 3.1.3.48) has been studied. Crystalline maltodextrins representing the A- or the B-type allomorph were prephosphorylated using recombinant glucan, water dikinase (StGWD) or the successive action of both plastidial dikinases (StGWD and AtPWD). AtSEX4 hydrolyzed carbon 6-phosphate esters from both the prephosphorylated A- and B-type allomorphs and the kinetic constants are similar. The phosphatase also acted on prelabeled carbon-3 esters from both crystalline maltodextrins. Similarly, native starch granules prelabeled in either the carbon-6 or carbon-3 position were also dephosphorylated by AtSEX4. The phosphatase did also hydrolyze phosphate esters of both prephosphorylated maltodextrins when the (phospho)glucans had been solubilized by heat treatment. Submillimolar concentrations of nonphosphorylated maltodextrins inhibited AtSEX4 provided they possessed a minimum of length and had been solubilized. As opposed to the soluble phosphomaltodextrins, the AtSEX4- mediated dephosphorylation of the insoluble substrates was incomplete and at least 50% of the phosphate esters were retained in the pelletable (phospho) glucans. The partial dephosphorylation of the insoluble glucans also strongly reduced the release of nonphosphorylated chains into solution. Presumably, this effect reflects fast structural changes that following dephosphorylation occur near the surface of the maltodextrin particles. A model is proposed defining distinct stages within the phosphorylation/dephosphorylation-dependent transition of alpha-glucans from the insoluble to the soluble state.
Reserve starch is an important plant product but the actual biosynthetic process is not yet fully understood. Potato (Solanum tuberosum) tuber discs from various transgenic plants were used to analyse the conversion of external sugars or sugar derivatives to starch. By using in vitro assays, a direct glucosyl transfer from glucose 1-phosphate to native starch granules as mediated by recombinant plastidial phosphorylase was analysed. Compared with labelled glucose, glucose 6-phosphate or sucrose, tuber discs converted externally supplied [C-14] glucose 1-phosphate into starch at a much higher rate. Likewise, tuber discs from transgenic lines with a strongly reduced expression of cytosolic phosphoglucomutase, phosphorylase or transglucosidase converted glucose 1-phosphate to starch with the same or even an increased rate compared with the wild-type. Similar results were obtained with transgenic potato lines possessing a strongly reduced activity of both the cytosolic and the plastidial phosphoglucomutase. Starch labelling was, however, significantly diminished in transgenic lines, with a reduced concentration of the plastidial phosphorylase isozymes. Two distinct paths of reserve starch biosynthesis are proposed that explain, at a biochemical level, the phenotype of several transgenic plant lines.
We applied a top-down systems biology approach to understand how Chlamydomonas reinhardtii acclimates to long-term heat stress (HS) and recovers from it. For this, we shifted cells from 25 to 42 degrees C for 24 h and back to 25 degrees C for >= 8 h and monitored abundances of 1856 proteins/protein groups, 99 polar and 185 lipophilic metabolites, and cytological and photosynthesis parameters. Our data indicate that acclimation of Chlamydomonas to long-term HS consists of a temporally ordered, orchestrated implementation of response elements at various system levels. These comprise (1) cell cycle arrest; (2) catabolism of larger molecules to generate compounds with roles in stress protection; (3) accumulation of molecular chaperones to restore protein homeostasis together with compatible solutes; (4) redirection of photosynthetic energy and reducing power from the Calvin cycle to the de novo synthesis of saturated fatty acids to replace polyunsaturated ones in membrane lipids, which are deposited in lipid bodies; and (5) when sinks for photosynthetic energy and reducing power are depleted, resumption of Calvin cycle activity associated with increased photorespiration, accumulation of reactive oxygen species scavengers, and throttling of linear electron flow by antenna uncoupling. During recovery from HS, cells appear to focus on processes allowing rapid resumption of growth rather than restoring pre-HS conditions.
Parenchyma cells from tubers of Solanum tuberosum L. convert several externally supplied sugars to starch but the rates vary largely. Conversion of glucose 1-phosphate to starch is exceptionally efficient. In this communication, tuber slices were incubated with either of four solutions containing equimolar [U-C-14]glucose 1-phosphate, [U-C-14]sucrose, [U-C-14]glucose 1-phosphate plus unlabelled equimolar sucrose or [U-C-14]sucrose plus unlabelled equimolar glucose 1-phosphate. C-14-incorporation into starch was monitored. In slices from freshly harvested tubers each unlabelled compound strongly enhanced C-14 incorporation into starch indicating closely interacting paths of starch biosynthesis. However, enhancement disappeared when the tubers were stored. The two paths (and, consequently, the mutual enhancement effect) differ in temperature dependence. At lower temperatures, the glucose 1-phosphate-dependent path is functional, reaching maximal activity at approximately 20 degrees C but the flux of the sucrose-dependent route strongly increases above 20 degrees C. Results are confirmed by in vitro experiments using [U-C-14]glucose 1-phosphate or adenosine-[U-C-14]glucose and by quantitative zymograms of starch synthase or phosphorylase activity. In mutants almost completely lacking the plastidial phosphorylase isozyme(s), the glucose 1-phosphate-dependent path is largely impeded. Irrespective of the size of the granules, glucose 1-phosphate-dependent incorporation per granule surface area is essentially equal. Furthermore, within the granules no preference of distinct glucosyl acceptor sites was detectable. Thus, the path is integrated into the entire granule biosynthesis. In vitro C-14-incorporation into starch granules mediated by the recombinant plastidial phosphorylase isozyme clearly differed from the in situ results. Taken together, the data clearly demonstrate that two closely but flexibly interacting general paths of starch biosynthesis are functional in potato tuber cells.
Both photoautotrophic and heterotrophic plant cells are capable of accumulating starch inside the plastid. However, depending on the metabolic state of the respective cell the starch-related carbon fluxes are different. The vast majority of the transitory starch biosynthesis relies on the hexose phosphate pools derived from the reductive pentose phosphate cycle and, therefore, is restricted to ongoing photosynthesis. Transitory starch is usually degraded in the subsequent dark period and mainly results in the formation of neutral sugars, such as glucose and maltose, that both are exported into the cytosol. The cytosolic metabolism of the two carbohydrates includes reversible glucosyl transfer reactions to a heteroglycan that are mediated by two glucosyl transferases. DPE2 and PHS2 (or, in all other species, Pho2).
In heterotrophic cells, accumulation of starch mostly depends on the long distance transport of reduced carbon compounds from source to sink organs and, therefore, includes as an essential step the import of carbohydrates from the cytosol into the starch forming plastids.
In this communication, we focus on starch metabolism in heterotrophic tissues from Arabidopsis thaliana wild type plants (and in various starch-related mutants as well). By using hydroponically grown A. thaliana plants, we were able to analyse starch-related biochemical processes in leaves and roots from the same plants. Within the roots we determined starch levels and the morphology of native starch granules. Cytosolic and apoplastic heteroglycans were analysed in roots and compared with those from leaves of the same plants. A. thaliana mutants lacking functional enzymes either inside the plastid (such as phosphoglucomutase) or in the cytosol (disproportionating isoenzyme 2 or the phosphorylase isozyme, PHS2) were included in this study. In roots and leaves from the three mutants (and from the respective wild type organ as well), starch and heteroglycans as well as enzyme patterns were analysed.
Almost all glucosyl transfer reactions rely on glucose-1-phosphate (Glc-1-P) that either immediately acts as glucosyl donor or as substrate for the synthesis of the more widely used Glc dinucleotides, ADPglucose or UDPglucose. In this communication, we have analyzed two Glc-1-P-related processes: the carbon flux from externally supplied Glc-1-P to starch by either mesophyll protoplasts or intact chloroplasts from Arabidopsis (Arabidopsis thaliana). When intact protoplasts or chloroplasts are incubated with [U-C-14]Glc-1-P, starch is rapidly labeled. Incorporation into starch is unaffected by the addition of unlabeled Glc-6-P or Glc, indicating a selective flux from Glc-1-P to starch. However, illuminated protoplasts incorporate less C-14 into starch when unlabeled bicarbonate is supplied in addition to the C-14-labeled Glc-1-P. Mesophyll protoplasts incubated with [U-C-14] Glc-1-P incorporate C-14 into the plastidial pool of adenosine diphosphoglucose. Protoplasts prepared from leaves of mutants of Arabidopsis that lack either the plastidial phosphorylase or the phosphoglucomutase isozyme incorporate C-14 derived from external Glc-1-P into starch, but incorporation into starch is insignificant when protoplasts from a mutant possessing a highly reduced ADPglucose pyrophosphorylase activity are studied. Thus, the path of assimilatory starch biosynthesis initiated by extraplastidial Glc-1-P leads to the plastidial pool of adenosine diphosphoglucose, and at this intermediate it is fused with the Calvin cycle-driven route. Mutants lacking the plastidial phosphoglucomutase contain a small yet significant amount of transitory starch.
The origin of second harmonic generation (SHG) in starch granules was investigated using ab initio quantum mechanical modeling and experimentally examined using polarization-in, polarization-out (PIPO) second harmonic generation microscopy. Ab initio calculations revealed that the largest contribution to the SHG signal from A- and B-type allomorphs of starch originates from the anisotropic organization of hydroxide and hydrogen bonds mediated by aligned water found in the polymers. The hypothesis was experimentally tested by imaging maize starch granules under various hydration and heat treatment conditions that alter the hydrogen bond network. The highest SHG intensity was found in fully hydrated starch granules, and heat treatment diminished the SHG intensity. The PIPO SHG imaging showed that dried starch granules have a much higher nonlinear optical susceptibility component ratio than fully hydrated granules. In contrast, deuterated starch granules showed a smaller susceptibility component ratio demonstrating that SHG is highly sensitive to the organization of the hydroxyl and hydrogen bond network. The polarization SHG imaging results of potato starch granules, representing starch allomorph B, were compared to those of maize starch granules representing allomorph A. The results showed that the amount of aligned water was higher in the maize granules. Nonlinear microscopy of starch granules provides evidence that varying hydration conditions leads to significant changes in the nonlinear susceptibility ratio as well as the SHG intensity, supporting the hypothesis from ab initio calculations that the dominant contribution to SHG is due to the ordered hydroxide and hydrogen bond network.
The present study established the way in which plastidial alpha-glucan phosphorylase (Pho1) synthesizes maltodextrin (MD) which can be the primer for starch biosynthesis in rice endosperm. The synthesis of MD by Pho1 was markedly accelerated by branching enzyme (BE) isozymes, although the greatest effect was exhibited by the presence of branching isozyme I (BEI) rather than by isozyme IIa (BEIIa) or isozyme IIb (BEIIb). The enhancement of the activity of Pho1 by BE was not merely due to the supply of a non-reducing ends. At the same time, Pho1 greatly enhanced the BE activity, possibly by generating a branched carbohydrate substrate which is used by BE with a higher affinity. The addition of isoamylase to the reaction mixture did not prevent the concerted action of Pho1 and BEI. Furthermore, in the product, the branched structure was, at least to some extent, maintained. Based on these results we propose that the interaction between Pho1 and BE is not merely due to chain-elongating and chain-branching reactions, but occurs in a physically and catalytically synergistic manner by each activating the mutual capacity of the other, presumably forming a physical association of Pho1, BEI and branched MDs. This close interaction might play a crucial role in the synthesis of branched MDs and the branched MDs can act as a primer for the biosynthesis of amylopectin molecules.
Plastidial phosphorylase is required for normal starch synthesis in Chlamydomonas reinhardtii
(2006)
Among the three distinct starch phosphorylase activities detected in Chlamydomonas reinhardtii, two distinct plastidial enzymes (PhoA and PhoB) are documented while a single extraplastidial form (PhoC) displays a higher affinity for glycogen as in vascular plants. The two plastidial phosphorylases are shown to function as homodimers containing two 91-kDa (PhoA) subunits and two 110-kDa (PhoB) subunits. Both lack the typical 80-amino-acid insertion found in the higher plant plastidial forms. PhoB is exquisitely sensitive to inhibition by ADP-glucose and has a low affinity for malto-oligosaccharides. PhoA is more similar to the higher plant plastidial phosphorylases: it is moderately sensitive to ADP-glucose inhibition and has a high affinity for unbranched malto-oligosaccharides. Molecular analysis establishes that STA4 encodes PhoB. Chlamydomonas reinhardtii strains carrying mutations at the STA4 locus display a significant decrease in amounts of starch during storage that correlates with the accumulation of abnormally shaped granules containing a modified amylopectin structure and a high amylose content. The wild-type phenotype could be rescued by reintroduction of the cloned wild-type genomic DNA, thereby demonstrating the involvement of phosphorylase in storage starch synthesis.
Metabolites and lipids are the final products of enzymatic processes, distinguishing the different cellular functions and activities of single cells or whole tissues. Understanding these cellular functions within a well-established model system requires a systemic collection of molecular and physiological information. In the current report, the green alga Chlamydomonas reinhardtii was selected to establish a comprehensive workflow for the detailed multi-omics analysis of a synchronously growing cell culture system. After implementation and benchmarking of the synchronous cell culture, a two-phase extraction method was adopted for the analysis of proteins, lipids, metabolites and starch from a single sample aliquot of as little as 10-15million Chlamydomonas cells. In a proof of concept study, primary metabolites and lipids were sampled throughout the diurnal cell cycle. The results of these time-resolved measurements showed that single compounds were not only coordinated with each other in different pathways, but that these complex metabolic signatures have the potential to be used as biomarkers of various cellular processes. Taken together, the developed workflow, including the synchronized growth of the photoautotrophic cell culture, in combination with comprehensive extraction methods and detailed metabolic phenotyping has the potential for use in in-depth analysis of complex cellular processes, providing essential information for the understanding of complex biological systems.
Glucan, water dikinase (GWD) and phosphoglucan, water dikinase (PWD) are required for normal starch metabolism. We analysed starch phosphorylation in Arabidopsis wildtype plants and mutants lacking either GWD or PWD using P-31 NMR. Phosphorylation at both C6- and C3-positions of glucose moieties in starch was drastically decreased in GWD-deficient mutants. In starch from PWD-deficient plants C3-bound phosphate was reduced to levels close to the detection limit. The latter result contrasts with previous reports according to which GWD phosphorylates both C6- and C3-positions. In these studies, phosphorylation had been analysed by HPLC of acid-hydrolysed glucans. We now show that maltose-6-phosphate, a product of incomplete starch hydrolysis, co-eluted with glucose-3-phosphate under the chromatographic conditions applied. Re-examination of the specificity of the dikinases using an improved method demonstrates that C6- and C3-phosphorylation is selectively catalysed by GWD and PWD, respectively.
Functional interactions of plastidial phosphorylase (Phol) and starch branching enzymes (BEs) from the developing rice endosperm are the focus of this study. In the presence of both Phol and BE, the same branched primer molecule is elongated and further branched almost simultaneously even at very low glucan concentrations present in the purified enzyme preparations. By contrast, in the absence of any BE, glucans are not, to any significant extent, elongated by Phol. Based on our in vitro data, in the developing rice endosperm, Phol appears to be weakly associated with any of the BE isozymes. By using fluorophore-labeled malto-oligosaccharides, we identified maltose as the smallest possible primer for elongation by Phol. Linear dextrins act as carbohydrate substrates for BEs. By functionally interacting with a BE, Phol performs two essential functions during the initiation of starch biosynthesis in the rice endosperm: First, it elongates maltodextrins up to a degree of polymerization of at least 60. Second, by closely interacting with BEs, Phol is able to elongate branched glucans efficiently and thereby synthesizes branched carbohydrates essential for the initiation of amylopectin biosynthesis.
The subcellular distribution of starch-related enzymes and the phenotype of Arabidopsis mutants defective in starch degradation suggest that the plastidial starch turnover is linked to a cytosolic glycan metabolism. In this communication, a soluble heteroglycan (SHG) from leaves of Pisum sativum L. has been studied. Major constituents of the SHG are galactose, arabinose and glucose. For subcellular location, the SHG was prepared from isolated protoplasts and chloroplasts. On a chlorophyll basis, protoplasts and chloroplasts yielded approximately 70% and less than 5%, respectively, of the amount of the leaf-derived SHG preparation. Thus, most of SHG resides inside the cell but outside the chloroplast. SHG is soluble and not membrane-associated. Using membrane filtration, the SHG was separated into a <10 kDa and a >10 kDa fraction. The latter was resolved into two subfractions (I and II) by field-flow fractionation. In the protoplast-derived >10 kDa SHG preparation the subfraction I was by far the most dominant compound. beta-Glucosyl Yariv reagent was reactive with subfraction II, but not with subfraction I. In in vitro assays the latter acted as glucosyl acceptor for the cytosolic (Pho 2) phosphorylase but not for rabbit muscle phosphorylase. Glycosidic linkage analyses of subfractions I and II and of the Yariv reagent reactive glycans revealed that all three glycans contain a high percentage of arabinogalactan-like linkages. However, SHG possesses a higher content of minor compounds, namely glucosyl, mannosyl, rhamnosyl and fucosyl residues. Based on glycosyl residues and glycosidic linkages, subfraction I possesses a more complex structure than subfraction II
The phosphorylation of amylopectin by the glucan, water dikinase (GWD; EC 2.7.9.4) is an essential step within starch metabolism. This is indicated by the starch excess phenotype of GWD-deficient plants, such as the sex1-3 mutant of Arabidopsis (Arabidopsis thaliana). To identify starch-related enzymes that rely on glucan-bound phosphate, we studied the binding of proteins extracted from Arabidopsis wild-type leaves to either phosphorylated or nonphosphorylated starch granules. Granules prepared from the sex1-3 mutant were prephosphorylated in vitro using recombinant potato (Solanum tuberosum) GWD. As a control, the unmodified, phosphate free granules were used. An as-yet uncharacterized protein was identified that preferentially binds to the phosphorylated starch. The C-terminal part of this protein exhibits similarity to that of GWD. The novel protein phosphorylates starch granules, but only following prephosphorylation with GWD. The enzyme transfers the beta-P of ATP to the phosphoglucan, whereas the gamma-P is released as orthophosphate. Therefore, the novel protein is designated as phosphoglucan, water dikinase (PWD). Unlike GWD that phosphorylates preferentially the C6 position of the glucose units, PWD phosphorylates predominantly (or exclusively) the C3 position. Western-blot analysis of protoplast and chloroplast fractions from Arabidopsis leaves reveals a plastidic location of PWD. Binding of PWD to starch granules strongly increases during net starch breakdown. Transgenic Arabidopsis plants in which the expression of PWD was reduced by either RNAi or a T-DNA insertion exhibit a starch excess phenotype. Thus, in Arabidopsis leaves starch turnover requires a close collaboration of PWD and GWD
The starch excess phenotype of Arabidopsis mutants defective in the starch phosphorylating enzyme glucan, water dikinase (EC 2.7.9.4) indicates that phosphorylation of starch is required for its degradation. However, the underlying mechanism has not yet been elucidated. In this study, two in vivo systems have been established that allow the analysis of phosphorylation of transitory starch during both biosynthesis in the light and degradation in darkness. First, a photoautotrophic culture of the unicellular green alga Chlamydomonas reinhardtii was used to monitor the incorporation of exogenously supplied P-32 orthophosphate into starch. Illuminated cells incorporated P-32 into starch with a constant rate during 2 h. By contrast, starch phosphorylation in darkened cells exceeded that in illuminated cells within the first 30 min, but subsequently phosphate incorporation declined. Pulse-chase experiments performed with P-32/P-31 orthophosphate revealed a high turnover of the starch-bound phosphate esters in darkened cells but no detectable turnover in illuminated cells. Secondly, leaf starch granules were isolated from potato (Solanum tuberosum) plants grown under controlled conditions and glucan chains from the outer granule layer were released by isoamylase. Phosphorylated chains were purified and analyzed using high performance anion-exchange chromatography and matrix-assisted laser desorption/ionization mass spectrometry. Glucans released from the surface of starch granules that had been isolated from darkened leaves possessed a considerably higher degree of phosphorylation than those prepared from leaves harvested during the light period. Thus, in the unicellular alga as well as in potato leaves, net starch degradation is accompanied with an increased phosphorylation of starch
During starch degradation, chloroplasts export neutral sugars into the cytosol where they appear to enter a complex glycan metabolism. Interactions between glycans and glucosyl transferases residing in the cytosol were studied by analyzing transgenic potato (Solanum tuberosum L.) plants that possess either decreased or elevated levels of the cytosolic (Pho 2) phosphorylase isoform. Water-soluble heteroglycans (SHGs) were isolated from these plants and were characterized. SHG contains, as major constituents, arabinose, rhamnose, galactose and glucose. Non-aqueous fractionation combined with other separation techniques revealed a distinct pool of the SHG that is located in the cytosol. Under in vitro conditions, the cytosolic heteroglycans act as glucosyl acceptor selectively for Pho 2. Acceptor sites were characterized by a specific hydrolytic degradation following the Pho 2-catalyzed glucosyl transfer. The size distribution of the cytosolic SHG increased during the dark period, indicating a distinct metabolic activity related to net starch degradation. Antisense inhibition of Pho 2 resulted in increased glucosyl and rhamnosyl contents of the glycans. Overexpression of Pho 2 decreased the content of both residues. Compared with the wild type, in both types of transgenic plants the size of the cytosolic glycans was increased
A technique has been developed to measure absolute intracellular oxygen concentrations in green plants. Oxygen- sensitive phosphorescent microbeads were injected into the cells and an optical multifrequency phase-modulation technique was used to discriminate the sensor signal from the strong auto fluorescence of the plant tissue. The method was established using photosynthesis- competent cells of the giant algae Chara corallina L., and was validated by application to various cell types of other plant species
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.