Ignacio Ezquer

Enhancing Sucrose Synthase Activity in Transgenic Potato ( Solanum tuberosum L.) Tubers Results in Increased Levels of Starch, ADPglucose and UDPglucose and Total Yield

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. To determine the impact of SuSy activity in starch metabolism and yield in potato (Solanum tuberosum L.) tubers we measured sugar levels and enzyme activities in tubers of SuSy-overexpressing potato plants grown in greenhouse and open field conditions. We also transcriptionally characterized tubers of SuSy-overexpressing and -antisensed potato plants. SuSy-overexpressing tubers exhibited a substantial increase in starch, UDPglucose and ADPglucose content when compared with controls. Tuber dry weight, starch content per plant and total yield of SuSy-overexpressing tubers increased significantly over those of control plants. In contrast, activities of enzymes directly involved in starch metabolism in SuSy-overexpressing tubers were normal when compared with controls. Transcriptomic analyses using POCI arrays and the MapMan software revealed that changes in SuSy activity affect the expression of genes involved in multiple biological processes, but not that of genes directly involved in starch metabolism. These analyses also revealed a reverse correlation between the expressions of acid invertase and SuSy-encoding genes, indicating that the balance between SuSy- and acid invertase-mediated sucrolytic pathways is a major determinant of starch accumulation in potato tubers. Results presented in this work show that SuSy strongly determines the intracellular levels of UDPglucose, ADPglucose and starch, and total yield in potato tubers. We also show that enhancement of SuSy activity represents a useful strategy for increasing starch accumulation and yield in potato tubers.

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.

Microbial Volatile Emissions Promote Accumulation of Exceptionally High Levels of Starch in Leaves in Mono-and Dicotyledonous Plants

Microbes emit volatile compounds that affect plant growth and development. However, little or nothing is known about how microbial emissions may affect primary carbohydrate metabolism in plants. In this work we explored the effect on leaf starch metabolism of volatiles released from different microbial species ranging from Gram-negative and Gram-positive bacteria to fungi. Surprisingly, we found that all microbial species tested (including plant pathogens and species not normally interacting with plants) emitted volatiles that strongly promoted starch accumulation in leaves of both mono- and dicotyledonous plants. Starch content in leaves of plants treated for 2 d with microbial volatiles was comparable with or even higher than that of reserve organs such as potato tubers. Transcriptome and enzyme activity analyses of potato leaves exposed to volatiles emitted by Alternaria alternata revealed that starch overaccumulation was accompanied by up-regulation of sucrose synthase, invertase inhibitors, starch synthase class III and IV, starch branching enzyme and glucose-6-phosphate transporter. This phenomenon, designated as MIVOISAP (microbial volatiles-induced starch accumulation process), was also accompanied by down-regulation of acid invertase, plastidial thioredoxins, starch breakdown enzymes, proteins involved in internal amino acid provision and less well defined mechanisms involving a bacterial- type stringent response. Treatment of potato leaves with fungal volatiles also resulted in enhanced levels of sucrose, ADPglucose, UDPglucose and 3-phosphoglycerate. MIVOISAP is independent of the presence of sucrose in the culture medium and is strongly repressed by cysteine supplementation. The discovery that microbial volatiles trigger starch accumulation enhancement in leaves constitutes an unreported mechanism for the elicidation of plant carbohydrate metabolism by microbes.

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.

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.

A suggested model for potato MIVOISAP involving functions of central carbohydrate and amino acid metabolism, as well as actin cytoskeleton and endocytosis

We have recently found that microbial species ranging from Gram-negative and Gram-positive bacteria to different fungi emit volatiles that strongly promote starch accumulation in leaves of both mono- and di-cotyledonous plants. Transcriptome and enzyme activity analyses of potato leaves exposed to volatiles emitted by Alternaria alternata revealed that starch over-accumulation was accompanied by enhanced 3-phosphoglycerate to Pi ratio, and changes in functions involved in both central carbohydrate and amino acid metabolism. Exposure to microbial volatiles also promoted changes in the expression of genes that code for enzymes involved in endocytic uptake and traffic of solutes. With the overall data we propose a metabolic model wherein important determinants of accumulation of exceptionally high levels of starch include (a) upregulation of ADPglucose-producing SuSy, starch synthase III and IV, proteins involved in the endocytic uptake and traffic of sucrose, (b) down-regulation of acid invertase, starch breakdown enzymes and proteins involved in internal amino acid provision, and (c) 3-phosphoglycerate-mediated allosteric activation of ADPglucose pyrophosphorylase.