Enzymatic fingerprinting reveals specific xyloglucan and pectin signatures in the cell wall purified with primary plasmodesmata.

Plasmodesmata (PD) pores connect neighbouring plant cells and enable direct transport across the cell wall. Understanding the molecular composition of these structures is essential to address their formation and later dynamic regulation. Here we provide a biochemical characterisation of the cell wall co-purified with primary PD of Arabidopsis thaliana cell cultures. To achieve this result we combined subcellular fractionation, polysaccharide analyses and enzymatic fingerprinting approaches. Relative to the rest of the cell wall, specific patterns were observed in the PD fraction. Most xyloglucans, although possibly not abundant as a group, were fucosylated. Homogalacturonans displayed short methylated stretches while rhamnogalacturonan I species were remarkably abundant. Full rhamnogalacturonan II forms, highly methyl-acetylated, were also present. We additionally showed that these domains, compared to the broad wall, are less affected by wall modifying activities during a time interval of days. Overall, the protocol and the data presented here open new opportunities for the study of wall polysaccharides associated with PD.

TRANSPARENT TESTA 16 and 15 act through different mechanisms to control proanthocyanidin accumulation in Arabidopsis testa.

Flavonoids are secondary metabolites that fulfil a multitude of functions during the plant life cycle. In Arabidopsis proanthocyanidins (PAs) are flavonoids that specifically accumulate in the innermost integuments of the seed testa (i.e. endothelium), as well as in the chalaza and micropyle areas, and play a vital role in protecting the embryo against various biotic and abiotic stresses. PAs accumulation in the endothelium requires the activity of the MADS box transcription factor TRANSPARENT TESTA (TT) 16 (ARABIDOPSIS B-SISTER/AGAMOUS-LIKE 32) and the UDP-glycosyltransferase TT15 (UGT80B1). Interestingly tt16 and tt15 mutants display a very similar flavonoid profiles and patterns of PA accumulation. By using a combination of genetic, molecular, biochemical, and histochemical methods, we showed that both TT16 and TT15 act upstream the PA biosynthetic pathway, but through two distinct genetic routes. We also demonstrated that the activity of TT16 in regulating cell fate determination and PA accumulation in the endothelium is required in the chalaza prior to the globular stage of embryo development. Finally this study provides new insight showing that TT16 and TT15 functions extend beyond PA biosynthesis in the inner integuments of the Arabidopsis seed coat.

Two loci control phytoglycogen production in the monocellular green alga Chlamydomonas reinhardtii.

The STA8 locus of Chlamydomonas reinhardtii was identified in a genetic screen as a factor that controls starch biosynthesis. Mutations of STA8 cause a significant reduction in the amount of granular starch produced during nutrient limitation and accumulate phytoglycogen. The granules remaining in sta8 mutants are misshapen, and the abundance of amylose and long chains in amylopectin is altered. Mutations of the STA7 locus, which completely lack isoamylase activity, also cause accumulation of phytoglycogen, although sta8 and sta7 mutants differ in that there is a complete loss of granular starch in the latter. This is the first instance in which mutations of two different genetic elements in one plant species have been shown to cause phytoglycogen accumulation. An analytical procedure that allows assay of isoamylase in total extracts was developed and used to show that sta8 mutations cause a 65% reduction in the level of this activity. All other enzymes known to be involved in starch biosynthesis were shown to be unaffected in sta8 mutants. The same amount of total isoamylase activity (approximately) as that present in sta8 mutants was observed in heterozygous triploids containing two sta7 mutant alleles and one wild-type allele. This strain, however, accumulates normal levels of starch granules and lacks phytoglycogen. The total level of isoamylase activity, therefore, is not the major determinant of whether granule production is reduced and phytoglycogen accumulates. Instead, a qualitative property of the isoamylase that is affected by the sta8 mutation is likely to be the critical factor in phytoglycogen production.

Biochemical characterization of wild-type and mutant isoamylases of Chlamydomonas reinhardtii supports a function of the multimeric enzyme organization in amylopectin maturation.

Chlamydomonas reinhardtii mutants of the STA8 gene produce reduced amounts of high amylose starch and phytoglycogen. In contrast to the previously described phytoglycogen-producing mutants of C. reinhardtii that contain no residual isoamylase activity, the sta8 mutants still contained 35% of the normal amount of enzyme activity. We have purified this residual isoamylase and compared it with the wild-type C. reinhardtii enzyme. We have found that the high-mass multimeric enzyme has reduced its average mass at least by one-half. This coincides with the disappearance of two out of the three activity bands that can be seen on zymogram gels. Wild-type and mutant enzymes are shown to be located within the plastid. In addition, they both act by cleaving off the outer branches of polysaccharides with no consistent difference in enzyme specificity. Because the mutant enzyme was demonstrated to digest phytoglycogen to completion in vitro, we propose that its inability to do so in vivo supports a function of the enzyme complex architecture in the processing of pre-amylopectin chains.

The debranching enzyme complex missing in glycogen accumulating mutants of Chlamydomonas reinhardtii displays an isoamylase-type specificity.

To investigate the functions of debranching enzymes in starch biosynthesis, we have partially purified and characterized these activities from wild type and mutant sta7 Chlamydomonas reinhardtii. Mutants of the STA7 locus substitute synthesis of insoluble granular starch by that of small amounts of glycogen-like material. The mutants were previously shown to lack an 88 kDa debranching enzyme. Two distinct debranching activities were detected in wild-type strains. The 88 kDa debranching enzyme subunit missing in glycogen-producing mutants (CIS1) is shown to be part of a multimeric enzyme complex. A monomeric 95 kDa debranching enzyme (CLD1) cleaved alpha-1,6 linkages separated by as few as three glucose residues while the multimeric complex was unable to do so. Both enzymes were able to debranch amylopectin while the alpha-1,6 linkages of glycogen were completely debranched by the multimeric complex only. Therefore CLD1 and the multimeric debranching enzyme display respectively the limit-dextrinase (pullulanase) and isoamylase-type specificities. Various mutations in the STA7 locus caused the loss of both CIS1 and of the multimeric isoamylase complex. In contrast to rice and maize mutants that accumulate phytoglycogen owing to mutation of an isoamylase-type DBE, isoamylase depletion in Chlamydomonas did not result in any qualitative or quantitative difference in pullulanase activity.

The structure and expression of the wheat starch synthase III gene. Motifs in the expressed gene define the lineage of the starch synthase III gene family.

The endosperm of hexaploid wheat (Triticum aestivum [L.]) was shown to contain a high molecular weight starch synthase (SS) analogous to the product of the maize du1 gene, starch synthase III (SSIII; DU1). cDNA and genomic DNA sequences encoding wheat SSIII were isolated and characterized. The wheat SSIII cDNA is 5,346 bp long and contains an open reading frame that encodes a 1,628-amino acid polypeptide. A putative N-terminal transit peptide, a 436-amino acid C-terminal catalytic domain, and a central 470-amino acid SSIII-specific domain containing three regions of repeated amino acid similarity were identified in the wheat gene. A fourth region between the transit peptide and the SSIII-specific domain contains repeat motifs that are variable with respect to motif sequence and repeat number between wheat and maize. In dicots, this N-terminal region does not contain repeat motifs and is truncated. The gene encoding wheat SSIII, designated ss3, consists of 16 exons extending over 10 kb, and is located on wheat chromosome I. Expression of ss3 mRNA in wheat was detected in leaves, pre-anthesis florets, and from very early to middle stage of endosperm development. The entire N-terminal variable repeat region and the majority of the SSIII-specific domain are encoded on a single 2,703-bp exon. A gene encoding a class III SS from the Arabidopsis genome sequencing project shows a strongly conserved exon structure to the wheat ss3 gene, with the exception of the N-terminal region. The evolutionary relationships of the genes encoding monocot and dicot class III SSs are discussed.

PROCUSTE1 encodes a cellulose synthase required for normal cell elongation specifically in roots and dark-grown hypocotyls of Arabidopsis.

Mutants at the PROCUSTE1 (PRC1) locus show decreased cell elongation, specifically in roots and dark-grown hypocotyls. Cell elongation defects are correlated with a cellulose deficiency and the presence of gapped walls. Map-based cloning of PRC1 reveals that it encodes a member (CesA6) of the cellulose synthase catalytic subunit family, of which at least nine other members exist in Arabidopsis. Mutations in another family member, RSW1 (CesA1), cause similar cell wall defects in all cell types, including those in hypocotyls and roots, suggesting that cellulose synthesis in these organs requires the coordinated expression of at least two distinct cellulose synthase isoforms.

Genetic and biochemical evidence for the involvement of alpha-1,4 glucanotransferases in amylopectin synthesis

We describe a novel mutation in the Chlamydomonas reinhardtii STA11 gene, which results in significantly reduced granular starch deposition and major modifications in amylopectin structure and granule shape. This defect simultaneously leads to the accumulation of linear malto-oligosaccharides. The sta11-1 mutation causes the absence of an alpha-1,4 glucanotransferase known as disproportionating enzyme (D-enzyme). D-enzyme activity was found to be correlated with the amount of wild-type allele doses in gene dosage experiments. All other enzymes involved in starch biosynthesis, including ADP-glucose pyrophosphorylase, debranching enzymes, soluble and granule-bound starch synthases, branching enzymes, phosphorylases, alpha-glucosidases (maltases), and amylases, were unaffected by the mutation. These data indicate that the D-enzyme is required for normal starch granule biogenesis in the monocellular alga C. reinhardtii.

Biochemical characterization of the chlamydomonas reinhardtii alpha-1,4 glucanotransferase supports a direct function in amylopectin biosynthesis

Plant alpha-1,4 glucanotransferases (disproportionating enzymes, or D-enzymes) transfer glucan chains among oligosaccharides with the concomitant release of glucose (Glc). Analysis of Chlamydomonas reinhardtii sta11-1 mutants revealed a correlation between a D-enzyme deficiency and specific alterations in amylopectin structure and starch biosynthesis, thereby suggesting previously unknown biosynthetic functions. This study characterized the biochemical activities of the alpha-1,4 glucanotransferase that is deficient in sta11-1 mutants. The enzyme exhibited the glucan transfer and Glc production activities that define D-enzymes. D-enzyme also transferred glucans among the outer chains of amylopectin (using the polysaccharide chains as both donor and acceptor) and from malto-oligosaccharides into the outer chains of either amylopectin or glycogen. In contrast to transfer among oligosaccharides, which occurs readily with maltotriose, transfer into polysaccharide required longer donor molecules. All three enzymatic activities, evolution of Glc from oligosaccharides, glucan transfer from oligosaccharides into polysaccharides, and transfer among polysaccharide outer chains, were evident in a single 62-kD band. Absence of all three activities co-segregated with the sta11-1 mutation, which is known to cause abnormal accumulation of oligosaccharides at the expense of starch. To explain these data we propose that D-enzymes function directly in building the amylopectin structure.

The localization and expression of the class II starch synthases of wheat.

The starch granules of hexaploid wheat (Triticum aestivum) contain a group of three proteins known as SGP-1 (starch granule protein-1) proteins, which have apparent molecular masses of 100, 108, and 115 kD. The nature and role of these proteins has not been defined previously. We demonstrate that these polypeptides are starch synthases that are present in both the starch granule and the soluble fraction at the early stages of wheat endosperm development, but that are exclusively granule bound at mid and late endosperm development. A partial cDNA clone encoding a fragment of the 100-kD protein was obtained by screening a wheat endosperm cDNA expression library using monoclonal antibodies. Three classes of cDNA were subsequently isolated from a wheat endosperm cDNA library by nucleic acid hybridization and were shown to encode the 100-, 108-, and 115-kD proteins. The cDNA sequences are highly homologous to class II starch synthases and have the highest homology with the maize SSIIa (starch synthase IIa) gene. mRNA for the SGP-1 proteins was detected in the leaf, pre-anthesis florets, and endosperm of wheat and is highly expressed in the leaf and in the grain during the early to mid stages of development. We discuss the roles of the SGP-1 proteins in starch biosynthesis in wheat.

Novel, starch-like polysaccharides are synthesized by an unbound form of granule-bound starch synthase in glycogen-accumulating mutants of Chlamydomonas reinhardtii.

In vascular plants, mutations leading to a defect in debranching enzyme lead to the simultaneous synthesis of glycogen-like material and normal starch. In Chlamydomonas reinhardtii comparable defects lead to the replacement of starch by phytoglycogen. Therefore, debranching was proposed to define a mandatory step for starch biosynthesis. We now report the characterization of small amounts of an insoluble, amylose-like material found in the mutant algae. This novel, starch-like material was shown to be entirely dependent on the presence of granule-bound starch synthase (GBSSI), the enzyme responsible for amylose synthesis in plants. However, enzyme activity assays, solubilization of proteins from the granule, and western blots all failed to detect GBSSI within the insoluble polysaccharide matrix. The glycogen-like polysaccharides produced in the absence of GBSSI were proved to be qualitatively and quantitatively identical to those produced in its presence. Therefore, we propose that GBSSI requires the presence of crystalline amylopectin for granule binding and that the synthesis of amylose-like material can proceed at low levels without the binding of GBSSI to the polysaccharide matrix. Our results confirm that amylopectin synthesis is completely blocked in debranching-enzyme-defective mutants of C. reinhardtii.

Starches from A to C. Chlamydomonas reinhardtii as a model microbial system to investigate the biosynthesis of the plant amylopectin crystal.

Wide-angle powder x-ray diffraction analysis was carried out on starch extracted from wild-type and mutant Chlamydomonas reinhardtii cells. Strains containing no defective starch synthases as well as mutants carrying a disrupted granule-bound starch synthase structural gene displayed the A type of diffraction pattern with a high degree of crystallinity. Mutants carrying a defect for the major soluble starch synthase (SSS), SSS II, were characterized by a switch to the B type of diffraction pattern with very low crystallinity. Mutant strains carrying SSS I as the only glucan elongation enzyme regained some of their crystallinity but switched to the C type of diffraction pattern. Differential scanning calorimetry analysis correlated tightly with the x-ray diffraction results. Together with the electron microscopy analyses, these results establish C. reinhardtii as a microbial model system displaying all aspects of cereal starch synthesis and structure. We further show that SSS II is the major enzyme involved in the synthesis of crystalline structures in starch and demonstrate that SSS I alone builds a new type of amylopectin structure.

Preamylopectin Processing: A Mandatory Step for Starch Biosynthesis in Plants.

It has been generally assumed that the [alpha]-(1->4)-linked and [alpha]-(1->6)-branched glucans of starch are generated by the coordinated action of elongation (starch synthases) and branching enzymes. We have identified a novel Chlamydomonas locus (STA7) that when defective leads to a wipeout of starch and its replacement by a small amount of glycogen-like material. Our efforts to understand the enzymological basis of this phenotype have led us to determine the selective disappearance of an 88-kD starch hydrolytic activity. We further demonstrate that this enzyme is a debranching enzyme. Cleavage of the [alpha]-(1->6) linkage in a branched precursor of amylopectin (preamylopectin) has provided us with the ground rules for understanding starch biosynthesis in plants. Therefore, we propose that amylopectin clusters are synthesized by a discontinuous mechanism involving a highly specific glucan trimming mechanism.

Storage, Photosynthesis, and Growth: The Conditional Nature of Mutations Affecting Starch Synthesis and Structure in Chlamydomonas.

Growth-arrested Chlamydomonas cells accumulate a storage polysaccharide that bears strong structural and functional resemblance to higher plant storage starch. It is synthesized by similar enzymes and responds in an identical fashion to the presence of mutations affecting these activities. We found that log-phase photosynthetically active algae accumulate granular [alpha](1->4)-linked, [alpha](1->6)-branched glucans whose shape, cellular location, and structure differ markedly from those of storage starch. That synthesis of these two types of polysaccharides is controlled by both a common and a specific set of genes was evidenced by the identification of a new Chlamydomonas (STA4) locus specifically involved in the biosynthesis of storage starch. Mutants defective in STA4 accumulated a new type of high-amylose storage starch displaying an altered amylopectin chain size distribution. It is expected that the dual nature and functions of starch synthesis in unicellular green algae will yield new insights into the biological reasons for the emergence of starch in the eukaryotic plant cell.