The effect of microbial volatiles on plant starch metabolism

Starch is a branched homopolisaccharide of ¿-1,4-linked glucose subunits with ¿-1,6- linked glucose subunits with ¿-1,6-linked glucose at the branching points. It represents the major storage carbohydrate in most higher plants. In addition, this polysaccharide represents the most common carbohydrate in the human diet. Starch is abundant in storage organs such as cereal seeds and tubers, and has multiple applications in the paper, textiles, cosmetics, pharmaceutical and construction industries. Plants are able to perceive biotic stimuli by recognizing a multitude of different signaling compounds originating from the interacting organisms. Contact with these substances not only adjusts plant defense reactions, but very often leads to decrease in photosynthesis, and to a transition from source to sink status in photosynthetic tissues. Some of these compounds are volatiles with molecular masses less than 300 Da and a high vapor pressure. Given the lack of knowledge regarding how microbial volatiles may affect plant primary carbohydrate metabolism, in this study I explored the effect of volatiles released from different microbial species on starch metabolism.In the First chapter of the work I explored the effect on leaf starch metabolism of volatiles released from different microbial species ranging from Gram-negative and Gram-positive bacteria, and fungi. All microbial species tested (including plant pathogens and species normally not interacting with plants) emitted volatiles that strongly promoted starch accumulation in leaves of both mono- and di-cotyledonous plants. Starch content in leaves of plants treated during 2 days with microbial volatiles was comparable or even higher than that of reserve organs such as potato tubers. Microbial volatiles promoting starch accumulation are sensed in leaves. Starch structure and amylose content were severely affected during MIcrobial VOlatiles Induced Starch Accumulation Process (MIVOISAP). Treatment of leaves with fungal volatiles (FVs) emitted by Alternaria alternata resulted in enhanced levels of sucrose, ADP-glucose (ADPG), UDP-glucose and 3-phosphoglycerate (3PGA). MIVOISAP occurs independently of the presence of sucrose in the culture medium and is strongly repressed by cysteine supplementation. Transcriptome and enzyme activity analyses of potato leaves exposed to FVs emitted by A. alternata showed that starch over-accumulation was accompanied by up-regulation of sucrose synthase (SuSy), invertase inhibitors, starch synthases (SS) class III and IV, starch branching enzyme, and glucose-6-phosphate transporter. This phenomenon, was also accompanied by down-regulation of acid invertase, plastidial thioredoxins, starch breakdown enzymes, proteins involved in internal amino acid provision, and less well defi ned mechanisms involving bacteria-type stringent response. In chapter 2, time-course analyses of starch accumulation in Arabidopsis leaves exposed to FVs emitted by A. alternata revealed that MIVOISAP is due to stimulation of starch biosynthesis during illumination. The increase of starch content in illuminated leaves of FVs-treated hy1/cry1, hy1/cry2 and hy1/cry1/cry2 Arabidopsis mutants was many-fold lower than that of WT leaves when plants were illuminated with white and red light. Thus the overall data indicated that MIVOISAP is subjected to photoreceptor-mediated control. This phenomenon was inhibited by cycloheximide and cordycepin, and accompanied by drastic changes in the Arabidopsis transcriptome, providing evidence that MIVOISAP is partially subjected to transcriptional control. MIVOISAP was also accompanied by enhancement of the total 3-PGA/ inorganic phosphate (Pi) ratio and a 2-3 fold increase of the levels of the reduced form of ADP- glucose pyrophosphorylase (AGP). Using different Arabidopsis knockout mutants I investigated the impact in MIVOISAP of down-regulation of genes directly or indirectly related with starch metabolism. These analyses revealed that the magnitude of the FVs-induced starch accumulation was low in mutants impaired in SS class III and IV, and plastidial NADP-thioredoxin reductase C (NTRC). The overall data thus showed that MIVOISAP involves a photocontrolled, transcriptionally and post-translationally regulated network wherein photoreceptors, SSIII, SSIV and NTRC-mediated changes in redox status of plastidial enzyme(s) play important roles.In attempting to solve a 20 years-old controversy on the possible occurrence of starch biosynthetic pathway(s) additional/alternative to the "classic" AGP pathway, in chapter 3 I carried out a judicious characterization of the starch-less adg1-1 and aps1 mutants impaired in AGP. Quantitative measurement analyses revealed that both mutants accumulated WT ADPG and ca. 2% of WT starch, as further confi rmed by confocal fl uorescence microscopic observation of iodine stained leaves and of leaves expressing granule bound starch synthase fused with green fl uorescent protein. Introduction of the sex1 mutation affecting starch breakdown into adg1-1 and aps1 increased the starch content to 8-10% of the WT starch. Importantly, aps1 leaves exposed to FVs emitted by A. alternata for 10 h accumulated ca. 60% of the WT starch. aps1 plants expressing the bacterial ADP-glucose hydrolase (EcASPP) in the plastid accumulated normal ADPG and reduced starch when compared with aps1 plants, whereas aps1 plants expressing EcASPP in the cytosol showed reduced ADPG and starch. Moreover, aps1 plants expressing a bacterial AGP in the plastid accumulated WT starch and ADPG. The overall data showed that (a) there occur important source(s), other than AGP, of ADPG linked to starch biosynthesis, and (b) AGP is a major determinant of starch accumulation but not of intracellular ADPG content in Arabidopsis.Peer Reviewe