Goizeder Almagro

Sucrose synthase activity in the sus1/sus2/sus3/sus4 Arabidopsis mutant is sufficient to support normal cellulose and starch production

Sucrose synthase (SUS) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate-glucose and fructose. In Arabidopsis, a multigene family encodes six SUS (SUS1-6) isoforms. The involvement of SUS in the synthesis of UDP-glucose and ADP-glucose linked to Arabidopsis cellulose and starch biosynthesis, respectively, has been questioned by Barratt et al. [(2009) Proc Natl Acad Sci USA 106:13124–13129], who showed that (i) SUS activity in wild type (WT) leaves is too low to account for normal rate of starch accumulation in Arabidopsis, and (ii) different organs of the sus1/sus2/sus3/sus4 SUS mutant impaired in SUS activity accumulate WT levels of ADP-glucose, UDP-glucose, cellulose and starch. However, these authors assayed SUS activity under unfavorable pH conditions for the reaction. By using favorable pH conditions for assaying SUS activity, in this work we show that SUS activity in the cleavage direction is sufficient to support normal rate of starch accumulation in WT leaves. We also demonstrate that sus1/sus2/sus3/sus4 leaves display WT SUS5 and SUS6 expression levels, whereas leaves of the sus5/sus6 mutant display WT SUS1–4 expression levels. Furthermore, we show that SUS activity in leaves and stems of the sus1/sus2/sus3/sus4 and sus5/sus6 plants is ∼85% of that of WT leaves, which can support normal cellulose and starch biosynthesis. The overall data disprove Barratt et al. (2009) claims, and are consistent with the possible involvement of SUS in cellulose and starch biosynthesis in Arabidopsis.

Starch biosynthesis, its regulation and biotechnological approaches to improve crop yields

Structurally composed of the glucose homopolymers amylose and amylopectin, starch is the main storage carbohydrate in vascular plants, and is synthesized in the plastids of both photosynthetic and non-photosynthetic cells. Its abundance as a naturally occurring organic compound is surpassed only by cellulose, and represents both a cornerstone for human and animal nutrition and a feedstock for many non-food industrial applications including production of adhesives, biodegradable materials, and first-generation bioethanol. This review provides an update on the different proposed pathways of starch biosynthesis occurring in both autotrophic and heterotrophic organs, and provides emerging information about the networks regulating them and their interactions with the environment. Special emphasis is given to recent findings showing that volatile compounds emitted by microorganisms promote both growth and the accumulation of exceptionally high levels of starch in mono- and dicotyledonous plants. We also review how plant biotechnologists have attempted to use basic knowledge on starch metabolism for the rational design of genetic engineering traits aimed at increasing starch in annual crop species. Finally we present some potential biotechnological strategies for enhancing starch content.

Enhancing sucrose synthase activity results in increased levels of starch and ADP-glucose in maize (Zea mays L.) seed endosperms.

Sucrose synthase (SuSy) is a highly regulated cytosolic enzyme that catalyzes the conversion of sucrose and a nucleoside diphosphate into the corresponding nucleoside diphosphate glucose and fructose. In cereal endosperms, it is widely assumed that the stepwise reactions of SuSy, UDPglucose pyrophosphorylase and ADPglucose (ADPG) pyrophosphorylase (AGP) take place in the cytosol to convert sucrose into ADPG necessary for starch biosynthesis, although it has also been suggested that SuSy may participate in the direct conversion of sucrose into ADPG. In this study, the levels of the major primary carbon metabolites, and the activities of starch metabolism-related enzymes were assessed in endosperms of transgenic maize plants ectopically expressing StSUS4, which encodes a potato SuSy isoform. A total of 29 fertile lines transformed with StSUS4 were obtained, five of them containing a single copy of the transgene that was still functional after five generations. The number of seeds per ear of the five transgenic lines containing a single StSUS4 copy was comparable with that of wild-type (WT) control seeds. However, transgenic seeds accumulated 10-15% more starch at the mature stage, and contained a higher amylose/amylopectin balance than WT seeds. Endosperms of developing StSUS4-expressing seeds exhibited a significant increase in SuSy activity, and in starch and ADPG contents when compared with WT endosperms. No significant changes could be detected in the transgenic seeds in the content of soluble sugars, and in activities of starch metabolism-related enzymes when compared with WT seeds. A suggested metabolic model is presented wherein both AGP and SuSy are involved in the production of ADPG linked to starch biosynthesis in maize endosperm cells.

Arabidopsis thaliana Mutants Lacking ADP-Glucose Pyrophosphorylase Accumulate Starch and Wild-type ADP-Glucose Content: Further Evidence for the Occurrence of Important Sources, other than ADP-Glucose Pyrophosphorylase, of ADP-Glucose Linked to Leaf Starch Biosynthesis

It is widely considered that ADP-glucose pyrophosphorylase (AGP) is the sole source of ADP-glucose linked to bacterial glycogen and plant starch biosynthesis. Genetic evidence that bacterial glycogen biosynthesis occurs solely by the AGP pathway has been obtained with glgC⁻ AGP mutants. However, recent studies have shown that (i) these mutants can accumulate high levels of ADP-glucose and glycogen, and (ii) there are sources other than GlgC, of ADP-glucose linked to glycogen biosynthesis. In Arabidopsis, evidence showing that starch biosynthesis occurs solely by the AGP pathway has been obtained with the starchless adg1-1 and aps1 AGP mutants. However, mounting evidence has been compiled previewing the occurrence of more than one important ADP-glucose source in plants. In attempting to solve this 20-year-old controversy, in this work we carried out a judicious characterization of both adg1-1 and aps1. Both mutants accumulated wild-type (WT) ADP-glucose and approximately 2% of WT starch, as further confirmed by confocal fluorescence microscopic observation of iodine-stained leaves and of leaves expressing granule-bound starch synthase fused with GFP. Introduction of the sex1 mutation affecting starch breakdown into adg1-1 and aps1 increased the starch content to 8-10% of the WT starch. Furthermore, aps1 leaves exposed to microbial volatiles for 10 h accumulated approximately 60% of the WT starch. aps1 plants expressing the bacterial ADP-glucose hydrolase EcASPP in the plastid accumulated normal ADP-glucose and reduced starch when compared with aps1 plants, whereas aps1 plants expressing EcASPP in the cytosol showed reduced ADP-glucose and starch. Moreover, aps1 plants expressing bacterial AGP in the plastid accumulated WT starch and ADP-glucose. The overall data show that (i) there occur important source(s), other than AGP, of ADP-glucose linked to starch biosynthesis, and (ii) AGP is a major determinant of starch accumulation but not of intracellular ADP-glucose content in Arabidopsis.

Post-Translational Redox Modification of ADP-Glucose Pyrophosphorylase in Response to Light is Not a Major Determinant of Fine Regulation of Transitory Starch Accumulation in Arabidopsis Leaves

ADP-glucose pyrophosphorylase (AGP) is a heterotetrameric enzyme comprising two small and two large subunits that catalyze the production of ADP-glucose linked to starch biosynthesis. The current paradigm on leaf starch metabolism assumes that post-translational redox modification of AGP in response to light is a major determinant of fine regulation of transitory starch accumulation. According to this view, under oxidizing conditions occurring during the night the two AGP small subunits (APS1) are covalently linked via an intermolecular disulfide bridge that inactivates the protein, whereas under reducing conditions occurring during the day NADP-thioredoxin reductase C (NTRC)-dependent reductive monomerization of APS1 activates the enzyme. In this work we have analyzed changes in the redox status of APS1 during dark-light transition in leaves of plants cultured under different light intensities. Furthermore, we have carried out time-course analyses of starch content in ntrc mutants, and in aps1 mutants expressing the Escherichia coli redox-insensitive AGP (GlgC) in the chloroplast. We also characterized aps1 plants expressing a redox-insensitive, mutated APS1 (APS1mut) form in which the highly conserved Cys81 residue involved in the formation of the intermolecular disulfide bridge has been replaced by serine. We found that a very moderate, NTRC-dependent APS1 monomerization process in response to light occurred only when plants were cultured under photo-oxidative conditions. We also found that starch accumulation rates during the light in leaves of both ntrc mutants and GlgC-expressing aps1 mutants were similar to those of wild-type leaves. Furthermore, the pattern of starch accumulation during illumination in leaves of APS1mut-expressing aps1 mutants was similar to that of APS1-expressing aps1 mutants at any light intensity. The overall data demonstrate that post-translational redox modification of AGP in response to light is not a major determinant of fine regulation of transitory starch accumulation in Arabidopsis.

Escherichia coli glycogen metabolism is controlled by the PhoP-PhoQ regulatory system at submillimolar environmental Mg2+ concentrations, and is highly interconnected with a wide variety of cellular processes

Using the Keio collection of gene-disrupted mutants of Escherichia coli, we have recently carried out a genome-wide screening of the genes affecting glycogen metabolism. Among the mutants identified in the study, Delta mgtA, Delta phoP and Delta phoQ cells, all lacking genes that are induced under low extracellular Mg2+ conditions, displayed glycogen-deficient phenotypes. In this work we show that these mutants accumulated normal glycogen levels when the culture medium was supplemented with submillimolar Mg2+ concentrations. Expression analyses conducted in wild-type, Delta phoP and Delta phoQ cells showed that the glgCAP operon is under PhoP-PhoQ control in the submillimolar Mg2+ concentration range. Subsequent screening of the Keio collection under non-limiting Mg2+ allowed the identification of 183 knock-out mutants with altered glycogen levels. The stringent and general stress responses, end-turnover of tRNA, intracellular AMP levels, and metabolism of amino acids, iron, carbon and sulfur were major determinants of glycogen levels. glgC::lacZY expression analyses using mutants representing different functional categories revealed that the glgCAP operon belongs to the RelA regulon. We propose an integrated metabolic model wherein glycogen metabolism is (a) tightly controlled by the energy and nutritional status of the cell and (b) finely regulated by changes in environmental Mg2+ occurring at the submillimolar concentration range.

Microbial Volatile-Induced Accumulation of Exceptionally High Levels of Starch in Arabidopsis Leaves Is a Process Involving NTRC and Starch Synthase Classes III and IV

Microbial volatiles promote the accumulation of exceptionally high levels of starch in leaves. Time-course analyses of starch accumulation in Arabidopsis leaves exposed to fungal volatiles (FV) emitted by Alternaria alternata revealed that a microbial volatile-induced starch accumulation process (MIVOISAP) is due to stimulation of starch biosynthesis during illumination. The increase of starch content in illuminated leaves of FV-treated hy1/cry1, hy1/cry2, and hy1/cry1/cry2 Arabidopsis mutants was many-fold lower than that of wild-type (WT) leaves, indicating that MIVOISAP is subjected to photoreceptor-mediated control. This phenomenon was inhibited by cordycepin and accompanied by drastic changes in the Arabidopsis transcriptome. MIVOISAP was also accompanied by enhancement of the total 3-phosphoglycerate/Pi ratio, and a two- to threefold increase of the levels of the reduced form of ADP-glucose pyrophosphorylase. Using different Arabidopsis knockout mutants, we investigated the impact in MIVOISAP of downregulation of genes directly or indirectly related to starch metabolism. These analyses revealed that the magnitude of the FV-induced starch accumulation was low in mutants impaired in starch synthase (SS) classes III and IV and plastidial NADP-thioredoxin reductase C (NTRC). Thus, the overall data showed that Arabidopsis MIVOISAP involves a photocontrolled, transcriptionally and post-translationally regulated network wherein photoreceptor-, SSIII-, SSIV-, and NTRC-mediated changes in redox status of plastidial enzymes play important roles.

Genome-Wide Screening of Genes Whose Enhanced Expression Affects Glycogen Accumulation in Escherichia coli

Using a systematic and comprehensive gene expression library (the ASKA library), we have carried out a genome-wide screening of the genes whose increased plasmid-directed expression affected glycogen metabolism in Escherichia coli. Of the 4123 clones of the collection, 28 displayed a glycogen-excess phenotype, whereas 58 displayed a glycogen-deficient phenotype. The genes whose enhanced expression affected glycogen accumulation were classified into various functional categories including carbon sensing, transport and metabolism, general stress and stringent responses, factors determining intercellular communication, aggregative and social behaviour, nitrogen metabolism and energy status. Noteworthy, one-third of them were genes about which little or nothing is known. We propose an integrated metabolic model wherein E. coli glycogen metabolism is highly interconnected with a wide variety of cellular processes and is tightly adjusted to the nutritional and energetic status of the cell. Furthermore, we provide clues about possible biological roles of genes of still unknown functions.

Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding β-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic β-amylase encoding genes in pgi1 leaves, which was accompanied by increased β-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P)H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.

Volatile compounds emitted by diverse phytopathogenic microorganisms promote plant growth and flowering through cytokinin action

It is known that volatile emissions from some beneficial rhizosphere microorganisms promote plant growth. Here we show that volatile compounds (VCs) emitted by phylogenetically diverse rhizosphere and non-rhizhosphere bacteria and fungi (including plant pathogens and microbes that do not normally interact mutualistically with plants) promote growth and flowering of various plant species, including crops. In Arabidopsis plants exposed to VCs emitted by the phytopathogen Alternaria alternata, changes included enhancement of photosynthesis and accumulation of high levels of cytokinins (CKs) and sugars. Evidence obtained using transgenic Arabidopsis plants with altered CK status show that CKs play essential roles in this phenomenon, because growth and flowering responses to the VCs were reduced in mutants with CK-deficiency (35S:AtCKX1) or low receptor sensitivity (ahk2/3). Further, we demonstrate that the plant responses to fungal VCs are light-dependent. Transcriptomic analyses of Arabidopsis leaves exposed to A. alternata VCs revealed changes in the expression of light- and CK-responsive genes involved in photosynthesis, growth and flowering. Notably, many genes differentially expressed in plants treated with fungal VCs were also differentially expressed in plants exposed to VCs emitted by the plant growth promoting rhizobacterium Bacillus subtilis GB03, suggesting that plants react to microbial VCs through highly conserved regulatory mechanisms.