The mechanisms underlying starch granule initiation remain unknown. We have recently reported that mutation of soluble starch synthase IV (SSIV) in Arabidopsis thaliana results in restriction of the number of starch granules to a single, large, particle per plastid, thereby defining an important component of the starch priming machinery. In this work, we provide further evidence for the function of SSIV in the priming process of starch granule formation and show that SSIV is necessary and sufficient to establish the correct number of starch granules observed in wild-type chloroplasts. The role of SSIV in granule seeding can be replaced, in part, by the phylogenetically related SSIII. Indeed, the simultaneous elimination of both proteins prevents Arabidopsis from synthesizing starch, thus demonstrating that other starch synthases cannot support starch synthesis despite remaining enzymatically active. Herein, we describe the substrate specificity and kinetic properties of SSIV and its subchloroplastic localization in specific regions associated with the edges of starch granules. The data presented in this work point to a complex mechanism for starch granule formation and to the different abilities of SSIV and SSIII to support this process in Arabidopsis leaves.

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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.

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 .

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.

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.

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.

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