Fabrice Wattebled

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.

Distinct functional properties of isoamylase-type starch debranching enzymes in monocot and dicot leaves

Maize and Arabidopsis starch debranching enzymes have evolved separately so that the monocot protein possesses enzymatic activity by itself whereas the dicot protein requires a partner for activity. Isoamylase-type starch debranching enzymes (ISA) play important roles in starch biosynthesis in chloroplast-containing organisms, as shown by the strict conservation of both catalytically active ISA1 and the noncatalytic homolog ISA2. Functional distinctions exist between species, although they are not understood yet. Numerous plant tissues require both ISA1 and ISA2 for normal starch biosynthesis, whereas monocot endosperm and leaf exhibit nearly normal starch metabolism without ISA2. This study took in vivo and in vitro approaches to determine whether organism-specific physiology or evolutionary divergence between monocots and dicots is responsible for distinctions in ISA function. Maize (Zea mays) ISA1 was expressed in Arabidopsis (Arabidopsis thaliana) lacking endogenous ISA1 or lacking both native ISA1 and ISA2. The maize protein functioned in Arabidopsis leaves to support nearly normal starch metabolism in the absence of any native ISA1 or ISA2. Analysis of recombinant enzymes showed that Arabidopsis ISA1 requires ISA2 as a partner for enzymatic function, whereas maize ISA1 was active by itself. The electrophoretic mobility of recombinant and native maize ISA differed, suggestive of posttranslational modifications in vivo. Sedimentation equilibrium measurements showed recombinant maize ISA1 to be a dimer, in contrast to previous gel permeation data that estimated the molecular mass as a tetramer. These data demonstrate that evolutionary divergence between monocots and dicots is responsible for the distinctions in ISA1 function.

Function of isoamylase-type starch debranching enzymes ISA1 and ISA2 in the Zea mays leaf

Conserved isoamylase-type starch debranching enzymes (ISAs), including the catalytic ISA1 and noncatalytic ISA2, are major starch biosynthesis determinants. Arabidopsis thaliana leaves require ISA1 and ISA2 for physiological function, whereas endosperm starch is near normal with only ISA1. ISA functions were characterized in maize (Zea mays) leaves to determine whether species-specific distinctions in ISA1 primary structure, or metabolic differences in tissues, are responsible for the differing ISA2 requirement. Genetic methods provided lines lacking ISA1 or ISA2. Biochemical analyses characterized ISA activities in mutant tissues. Starch content, granule morphology, and amylopectin fine structure were determined. Three ISA activity forms were observed in leaves, two ISA1/ISA2 heteromultimers and one ISA1 homomultimer. ISA1 homomultimer activity existed in mutants lacking ISA2. Mutants without ISA2 differed in leaf starch content, granule morphology, and amylopectin structure compared with nonmutants or lines lacking both ISA1 and ISA2. The data imply that both the ISA1 homomultimer and ISA1/ISA2 heteromultimer function in the maize leaf. The ISA1 homomultimer is present and functions in the maize leaf. Evolutionary divergence between monocots and dicots probably explains the ability of ISA1 to function as a homomultimer in maize leaves, in contrast to other species where the ISA1/ISA2 heteromultimer is the only active form.

Functions of maize genes encoding pyruvate phosphate dikinase in developing endosperm

Significance Mutations affecting the transcription factor encoded by the gene o2 are important in maize agriculture because they result in improved grain nutritional quality. The mutations also cause detrimental effects by reducing kernel hardness and diminishing agronomic quality and food applications. The undesirable characteristics are not fully understood because the o2 product regulates multiple targets that could contribute to the phenotype. This study investigated one target that had not been previously mutated, pyruvate phosphate dikinase (PPDK), and showed that PPDK deficiency in isolation causes the negative phenotype associated with reduced kernel hardness. Thus, maize improvement may be better accomplished by targeting individual metabolic pathways determining protein and amino acid balance rather than pleiotropic regulators such as the o2 transcription factor. Maize opaque2 (o2) mutations are beneficial for endosperm nutritional quality but cause negative pleiotropic effects for reasons that are not fully understood. Direct targets of the bZIP transcriptional regulator encoded by o2 include pdk1 and pdk2 that specify pyruvate phosphate dikinase (PPDK). This enzyme reversibly converts AMP, pyrophosphate, and phosphoenolpyruvate to ATP, orthophosphate, and pyruvate and provides diverse functions in plants. This study addressed PPDK function in maize starchy endosperm where it is highly abundant during grain fill. pdk1 and pdk2 were inactivated individually by transposon insertions, and both genes were simultaneously targeted by endosperm-specific RNAi. pdk2 accounts for the large majority of endosperm PPDK, whereas pdk1 specifies the abundant mesophyll form. The pdk1- mutation is seedling-lethal, indicating that C4 photosynthesis is essential in maize. RNAi expression in transgenic endosperm eliminated detectable PPDK protein and enzyme activity. Transgenic kernels weighed the same on average as nontransgenic siblings, with normal endosperm starch and total N contents, indicating that PPDK is not required for net storage compound synthesis. An opaque phenotype resulted from complete PPDK knockout, including loss of vitreous endosperm character similar to the phenotype conditioned by o2-. Concentrations of multiple glycolytic intermediates were elevated in transgenic endosperm, energy charge was altered, and starch granules were more numerous but smaller on average than normal. The data indicate that PPDK modulates endosperm metabolism, potentially through reversible adjustments to energy charge, and reveal that o2- mutations can affect the opaque phenotype through regulation of PPDK in addition to their previously demonstrated effects on storage protein gene expression.

Starch Biosynthesis in Leaves and Its Regulation

Plants assimilate carbon during photosynthesis using light energy to reduce atmospheric CO2 and to produce sugars and chemical energy (ATP). Sugars are partly incorporated directly into starch granules in leaf chloroplasts for short-term storage or are exported to non-photosynthetic organs for long-term storage. Indeed, starch accumulation in photosynthetic tissues is transient since it undergoes recurrent cycles of synthesis and degradation following day/night oscillation. Transient starch is synthesized during the day while photosynthesis is active and is degraded at night to provide carbon and energy to the plant when photosynthesis is inactive. Conversely, storage starch accumulates over long periods in storage organs such as seeds or tubers where it is degraded to sustain germination before photosynthesis becomes effective. Transient and storage starch syntheses occur in plastid stroma and involve dedicated enzymatic activities typically supported by several genetically independent isoforms. Although highly similar, both processes hold specific features regarding synthesis and regulation. In this chapter, we describe the mechanism of starch synthesis in photosynthetic tissues (mostly leaves) and its regulation. Several aspects are specifically highlighted here such as: (1) the function of starch synthases for the initiation of starch synthesis and the elongation of the amylopectin- and amylose-forming glucans, (2) the implication of branching enzymes and debranching enzymes for the formation of branch points and the control of their distribution within the polysaccharides, and (3) the regulation of the pathway in leaves especially by the circadian clock , the redox state of the cell and the influence of physiological factors, and the formation of protein-protein complexes.

From dusk till dawn: the Arabidopsis thaliana sugar starving responsive network

Plant growth and development are tightly controlled by photosynthetic carbon availability. The understanding of mechanisms governing carbon partitioning in plants will be a valuable tool in order to satisfy the rising global demand for food and biofuel. The goal of this study was to determine if sugar starvation responses were transcriptionally coordinated in Arabidopsis thaliana. A set of sugar-starvation responsive (SSR) genes was selected to perform a co-expression network analysis. Posteriorly, a guided-gene approach was used to identify the SSR-network from public data and to discover candidate regulators of this network. In order to validate the SSR network, a global transcriptome analysis was realized on three A. thaliana starch-deficient mutants. The starch-deficient phenotype in leaves induces sugar starvation syndrome at the end of the night due to the absence of photosynthesis. Promoter sequences of genes belonging to the SSR-network were analyzed in silico reveling over-represented motifs implicated in light, abscisic acid, and sugar responses. A small cluster of protein encoding genes belonging to different metabolic pathways, including three regulatory proteins, a protein kinase, a transcription factor, and a blue light receptor, were identified as the cornerstones of the SSR co-expression network. In summary, a large transcriptionally coordinated SSR network was identified and was validated with transcriptional data from three starch-deficient mutant lines. Candidate master regulators of this network were point out.

The Chlamydomonas mex1 mutant shows impaired starch mobilization without maltose accumulation

The MEX1 locus of Chlamydomonas reinhardtii was identified in a genetic screen as a factor that affects starch metabolism. Mutation of MEX1 causes a slow-down in the mobilization of storage polysaccharide. Cosegregation and functional complementation analyses were used to assess the involvement of the Mex1 protein in starch degradation. Heterologous expression experiments performed in Escherichia coli and Arabidopsis thaliana allowed us to test the capacity of the algal protein in maltose export. In contrast to the A. thaliana mex1 mutant, the mutation in C. reinhardtii does not lead to maltose accumulation and growth impairment. Although localized in the plastid envelope, the algal protein does not transport maltose efficiently across the envelope, but partly complements the higher plant mutant. Both Mex orthologs restore the growth of the E. coli ptsG mutant strain on glucose-containing medium, revealing the capacity of these proteins to transport this hexose. These findings suggest that Mex1 is essential for starch mobilization in both Chlamydomonas and Arabidopsis, and that this protein family may support several functions and not only be restricted to maltose export across the plastidial envelope.

E. coli glycogen branching enzyme restores synthesis of starch-like polyglucans in an Arabidopsis mutant devoid of endogenous starch branching enzymes

Starch synthesis requires several enzymatic activities including branching enzymes (BEs) responsible for the formation of α(1→6) linkages. Distribution and number of these linkages are further controlled by debranching enzymes (DBEs) that cleave some of them, rendering the polyglucan water-insoluble and semi-crystalline. Although the activity of BEs and DBEs is mandatory to sustain normal starch synthesis, the relative importance of each in the establishment of the plant storage polyglucan (i.e. water-insolubility, crystallinity, presence of amylose) is still debated. Here, we have substituted the activity of BEs in Arabidopsis with that of the Escherichia coli glycogen branching enzyme (GlgB). The latter is the BE counterpart in the metabolism of glycogen, a highly branched water-soluble and amorphous storage polyglucan. GlgB was expressed in the be2 be3 double mutant of Arabidopsis that is devoid of BE activity and consequently free of starch. The synthesis of a water-insoluble, partly crystalline, amylose-containing starch-like polyglucan was restored in GlgB-expressing plants, suggesting that BEs origin only have a limited impact on establishing essential characteristics of starch. Moreover, the balance between branching and debranching is crucial for the synthesis of starch, as an excess of branching activity results in the formation of highly branched, water-soluble, poorly crystalline polyglucan.Starch synthesis requires several enzymatic activities including branching enzymes (BEs) responsible for the formation of α(1→6) linkages. Distribution and number of these linkages are further controlled by debranching enzymes (DBEs) that cleave some of them, rendering the polyglucan water-insoluble and semi-crystalline. Although the activity of BEs and DBEs is mandatory to sustain normal starch synthesis, the relative importance of each in the establishment of the plant storage polyglucan (i.e. water-insolubility, crystallinity, presence of amylose) is still debated. Here, we have substituted the activity of BEs in Arabidopsis with that of the Escherichia coli glycogen branching enzyme (GlgB). The latter is the BE counterpart in the metabolism of glycogen, a highly branched water-soluble and amorphous storage polyglucan. GlgB was expressed in the be2 be3 double mutant of Arabidopsis that is devoid of BE activity and consequently free of starch. The synthesis of a water-insoluble, partly crystalline, amylose-containing starch-like polyglucan was restored in GlgB-expressing plants, suggesting that BEs only have a limited impact on establishing essential characteristics of starch. Moreover, the balance between branching and debranching is crucial for the synthesis of starch, as an excess of branching activity results in the formation of highly branched, water-soluble, poorly crystalline polyglucan.