The N-terminal Part of Arabidopsis thaliana Starch Synthase 4 Determines the Localization and Activity of the Enzyme

Starch synthase 4 (SS4) plays a specific role in starch synthesis because it controls the number of starch granules synthesized in the chloroplast and is involved in the initiation of the starch granule. We showed previously that SS4 interacts with fibrillins 1 and is associated with plastoglobules, suborganelle compartments physically attached to the thylakoid membrane in chloroplasts. Both SS4 localization and its interaction with fibrillins 1 were mediated by the N-terminal part of SS4. Here we show that the coiled-coil region within the N-terminal portion of SS4 is involved in both processes. Elimination of this region prevents SS4 from binding to fibrillins 1 and alters SS4 localization in the chloroplast. We also show that SS4 forms dimers, which depends on a region located between the coiled-coil region and the glycosyltransferase domain of SS4. This region is highly conserved between all SS4 enzymes sequenced to date.Weshow that the dimerization seems to be necessary for the activity of the enzyme. Both dimerization and the functionality of the coiled-coil region are conserved among SS4 proteins from phylogenetically distant species, such as Arabidopsis and Brachypodium. This finding suggests that the mechanism of action of SS4 is conserved among different plant species.España Secretaría de Estado de Investigación, Desarrollo e Innovación BIO2012–35043España, Consejería de Economía BIO118

Regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 by the nitrogen source: Effect of ammonium

Glutamine synthetase activity from Synechocystis sp. strain PCC 6803 is regulated as a function of the nitrogen source available in the medium. Addition of 0.25 mM NH4Cl to nitrate-grown cells promotes a clear short-term inactivation of glutamine synthetase, whose enzyme activity decreases to 5 to 10% of the initial value in 25 min. The intracellular levels of glutamine, determined under various conditions, taken together with the results obtained with azaserine (an inhibitor of transamidases), rule out the possibility that glutamine per se is responsible for glutamine synthetase inactivation. Nitrogen starvation attenuates the ammonium-mediated glutamine synthetase inactivation, indicating that glutamine synthetase regulation is modulated through the internal balance between carbon-nitrogen compounds and carbon compounds. The parallelism observed between the glutamine synthetase activity and the internal concentration of α-ketoglutarate suggests that this metabolite could play a role as a positive effector of glutamine synthetase activity in Synechocystis sp. Despite the similarities of this physiological system to that described for enterobacteria, the lack of in vivo 32P labeling of glutamine synthetase during the inactivation process excludes the existence of an adenylylation-deadenylylation system in this cyanobacterium.Dirección General de Investigación Científica y Técnica PB 88-002

Purification and Properties of Glutamine Synthetases from the Cyanobacteria Synechocystis sp. Strain PCC 6803 and Calothrix sp. Strain PCC 7601

Glutamine synthetases (GSs) from two cyanobacteria, one unicellular (Synechocystis sp. strain PCC 6803) and the other filamentous (Calothrix sp. strain PCC 7601 [Fremyella diplosiphon]), were purified to homogeneity. The biosynthetic activities of both enzymes were strongly inhibited by ADP, indicating that the energy charge of the cell might regulate the GS activity. Both cyanobacteria exhibited an ammonium-mediated repression of GS synthesis. In addition, the Synechocystis sp. showed an inactivation of GS promoted by ammonium that had not been demonstrated previously in cyanobacteria.Comisión Asesora de Investigación Científica y Técnica 45/85 85-047

Integrated functions among multiple starch synthases determine both amylopectin chain length and branch linkage location in Arabidopsis leaf starch

This study assessed the impact on starch metabolism in Arabidopsis leaves of simultaneously eliminating multiple soluble starch synthases (SS) from among SS1, SS2, and SS3. Double mutant ss1- ss2- or ss1- ss3- lines were generated using confirmed null mutations. These were compared to the wild type, each single mutant, and ss1- ss2- ss3- triple mutant lines grown in standardized environments. Double mutant plants developed similarly to the wild type, although they accumulated less leaf starch in both short-day and long-day diurnal cycles. Despite the reduced levels in the double mutants, lines containing only SS2 and SS4, or SS3 and SS4, are able to produce substantial amounts of starch granules. In both double mutants the residual starch was structurally modified including higher ratios of amylose:amylopectin, altered glucan chain length distribution within amylopectin, abnormal granule morphology, and altered placement of α(1→6) branch linkages relative to the reducing end of each linear chain. The data demonstrate that SS activity affects not only chain elongation but also the net result of branch placement accomplished by the balanced activities of starch branching enzymes and starch debranching enzymes. SS3 was shown partially to overlap in function with SS1 for the generation of short glucan chains within amylopectin. Compensatory functions that, in some instances, allow continued residual starch production in the absence of specific SS classes were identified, probaby accomplished by the granule bound starch synthase GBSS1.ANR Génoplante GPLA0611GEuropean Union-FEDER, Région Nord Pas de Calais ARCir PlantTEQ5National Science Foundation DBI-0209789Comisión Interministerial de Ciencia y Tecnología BIO2009-07040Junta de Andalucía P09-CVI-470

Enhancing the expression of starch synthase class IV results in increased levels of both transitory and long-term storage starch

Starch is an important renewable raw material with an increasing number of applications.Several attempts have been made to obtain plants that produce modified versions of starchor higher starch yield. Most of the approaches designed to increase the levels of starch havefocused on the increment of the amount of ADP-glucose or ATP available for starch biosyn-thesis. In this work, we show that the overexpression of starch synthase class IV (SSIV)increases the levels of starch accumulated in the leaves ofArabidopsisby 30%–40%. In addi-tion,SSIV-overexpressing lines display a higher rate of growth. The increase in starch contentas a consequence of enhancedSSIVexpression is also observed in long-term storage starchorgans such as potato tubers. Overexpression ofSSIVin potato leads to increased tuber starchcontent on a dry weight basis and to increased yield of starch production in terms of tons ofstarch⁄hectare. These results identify SSIV as one of the regulatory steps involved in the con-trol of the amount of starch accumulated in plastids.Comisión Interministerial de Ciencia y Tecnología de España y Fondo Europeo de Desarrollo Regional BIO2009-07040, BIO2007-63915 y PET2008-0106Junta de Andalucía P09-CVI-470

Disruption of both chloroplastic and cytosolic FBPase genes results in a dwarf phenotype and important starch and metabolite changes in Arabidopsis thaliana

In this study, evidence is provided for the role of fructose-1,6-bisphosphatases (FBPases) in plant development and carbohydrate synthesis and distribution by analysing two Arabidopsis thaliana T-DNA knockout mutant lines, cyfbp and cfbp1, and one double mutant cyfbp cfbp1 which affect each FBPase isoform, cytosolic and chloroplastic, respectively. cyFBP is involved in sucrose synthesis, whilst cFBP1 is a key enzyme in the Calvin–Benson cycle. In addition to the smaller rosette size and lower rate of photosynthesis, the lack of cFBP1 in the mutants cfbp1 and cyfbp cfbp1 leads to a lower content of soluble sugars, less starch accumulation, and a greater superoxide dismutase (SOD) activity. The mutants also had some developmental alterations, including stomatal opening defects and increased numbers of root vascular layers. Complementation also confirmed that the mutant phenotypes were caused by disruption of the cFBP1 gene. cyfbp mutant plants without cyFBP showed a higher starch content in the chloroplasts, but this did not greatly affect the phenotype. Notably, the sucrose content in cyfbp was close to that found in the wild type. The cyfbp cfbp1 double mutant displayed features of both parental lines but had the cfbp1 phenotype. All the mutants accumulated fructose-1,6-bisphosphate and triose-phosphate during the light period. These results prove that while the lack of cFBP1 induces important changes in a wide range of metabolites such as amino acids, sugars, and organic acids, the lack of cyFBP activity in Arabidopsis essentially provokes a carbon metabolism imbalance which does not compromise the viability of the double mutant cyfbp cfbp1.España, Ministerio de Economía y Competitividad BIO2009-07297España, Ministerio de Economía y Competitividad BIO2012-33292Junta de Andalucía P07-CVI-279

Glutamina sintetasa de la cianobacteria unicelular synechocystis sp. PCC 6803: Purificación, estudios de su regulación por la fuente de nitrógeno y clonación del gen glnA

Las cianobacterias, organismos procariotas fotosintéticos, asimilan el amonio principalmente por la acción secuencial de las enzimas Glutamina Sintetasa y Glutamato Sintasa (ruta GS-GOGAT), de las cuales es la Glutamina Sintetasa (GS) la que experimenta una regulación de su actividad. Se ha purificado a homogeneidad la GS de Synechocystis sp. PCC 6803. La enzima pura presenta una actividad específica de 143 u/mg proteína y está compuesta por un solo tipo de subunidad de 52 KDA. La actividad biosintética de la enzima es dependiente de MG2+ y se ve inhibida por ADP y AMP principalmente. Los estudios inmunológicos de las GSS de Synechocystis PCC 6803, Synechococcus PCC 6301 y Calothrix PCC 7601 demuestran el alto grado de conservación de esta enzima dentro de las cianobacterias. La actividad GS de Synechocystis está regulada por la fuente de nitrógeno, encontrándose la enzima activa cuando la fuente es nitrato e inactiva en un 90% cuando dicha fuente es amonio. El control de esta regulación parece estar ejercido por el balance en la concentración intracelular de compuestos carbono- nitrogenados frente a compuestos carbonados. Proponemos que este control puede ser ejercido por el balance intracelular de Glutamina y 2-Oxo-Glutarato. La regulación de la Glutamina Sintetasa de Synechocystis se lleva a cabo también a nivel de síntesis de la proteína, siendo los niveles de proteína GS cuando la fuente de nitrógeno es nitrato el doble que cuando es amonio. La perdida de actividad GS no parece ser debida a la modificación covalente de la enzima. Proponemos que dicha inactivación está provocada por la unión no covalente de un compuesto fosforilado. Se ha clonado el gen estructural de la GS, glnA, de Synechocystis mediante hibridación heteróloga con un fragmento del gen glnA de la Cianobacteria Anabaena PCC 7120. El gen complementa la auxotrófia de Glutamina de un mutante de Escherichia coli carente de actividad GS. En este trabajo presentamos la caracterización de la Glutamina Sintetasa de la cianobacteria unicelular Synechocystis PCC 6803, describiendo su purificación, principales características cinéticas y respuesta a la presencia de distintos compuestos relacionados con el metabolismo del nitrógeno. Asimismo presentamos un estudio sobre la regulación de la actividad y síntesis de dicha enzima en respuesta al tipo de fuente de nitrógeno disponible para el organismo. Por otro lado, presentamos la clonación del gen glnA de esta cianobacteria y describimos la obtención de una estirpe, AM 6, derivada de Synechocystis que presenta integrado en su cromosoma el gen glnA de Anabaena PCC 7120. La clonación del gen permite el estudio de una seria de interesantes cuestiones acerca de la estructura y regulación de esta enzima y junto con la estirpe AM 6, puede servir en el estudio de las diferencias existentes en la regulación de la GS de Anabaena PCC 7120 y Synechocystis PCC 6803

The Different Large Subunit Isoforms of Arabidopsis thaliana ADP-glucose Pyrophosphorylase Confer Distinct Kinetic and Regulatory Properties to the Heterotetrameric Enzyme

ADP-glucose pyrophosphorylase catalyzes the first and limiting step in starch biosynthesis and is allosterically regulated by the levels of 3-phosphoglycerate and phosphate in plants. ADP-glucose pyrophosphorylases from plants are heterotetramers composed of two types of subunits (small and large). In this study, the six Arabidopsis thaliana genes coding for ADP-glucose pyrophosphorylase isoforms (two small and four large subunits) have been cloned and expressed in an Escherichia coli mutant deficient in ADP-glucose pyrophosphorylase activity. The co-expression of the small subunit APS1 with the different Arabidopsis large subunits (APL1, APL2, APL3, and APL4) resulted in heterotetramers with different regulatory and kinetic properties. Heterotetramers composed of APS1 and APL1 showed the highest sensitivity to the allosteric effectors as well as the highest apparent affinity for the substrates (glucose-1-phosphate and ATP), whereas heterotetramers formed by APS1 and APL2 showed the lower response to allosteric effectors and the lower affinity for the substrates. No activity was detected for the second gene coding for a small subunit isoform (APS2) annotated in the Arabidopsis genome. This lack of activity is possibly due to the absence of essential amino acids involved in catalysis and/or in the binding of glucose-1-phosphate and 3-phosphoglycerate. Kinetic and regulatory properties of the different heterotetramers, together with sequence analysis has allowed us to make a distinction between sink and source enzymes, because the combination of different large subunits would provide a high plasticity to ADP-glucose pyrophosphorylase activity and regulation. This is the first experimental data concerning the role that all the ADP-glucose pyrophosphorylase isoforms play in a single plant species. This phenomenon could have an important role in vivo, because different large subunits would confer distinct regulatory properties to ADP-glucose pyrophosphorylase according to the necessities for starch synthesis in a given tissue.Ministerio de Ciencia y Tecnología PB98-1122 and BMC2002-00984Junta de Andalucía CVI-281United States Department of Energy DOE-FGD2-93ER20121Northern Regional United States Department of Agriculture NC-14

General distribution of the nitrogen control gene ntcA in cyanobacteria

The ntcA gene from Synechococcus sp. strain PCC 7942 encodes a regulatory protein which is required for the expression of all of the genes known to be subject to repression by ammonium in that cyanobacterium. Homologs to ntcA have now been cloned by hybridization from the cyanobacteria Synechocystis sp. strain PCC 6803 and Anabaena sp. strain PCC 7120. Sequence analysis has shown that these ntcA genes would encode polypeptides strongly similar (77 to 79% identity) to the Synechococcus NtcA protein. Sequences hybridizing to ntcA have been detected in the genomes of nine other cyanobacteria that were tested, including strains of the genera Anabaena, Calothrix, Fischerella, Nostoc, Pseudoanabaena, Synechococcus, and Synechocystis.This work was supported by grant B1089-0527 from Comisión Interministerial de Ciencia y Tecnología, by grant PB90-0114 from Dirección General de Investigación Científica y Técnica, and by Junta de Andalucía, Spain. J.E.F. was the recipient of a fellowship from Fundación Cámara Urzaiz-Universidad de Sevilla, Seville, Spain