Alison M. Smith

The wrinkled-seed character of pea described by Mendel is caused by a transposon-like insertion in a gene encoding starch-branching enzyme

We describe the cloning of the r (rugosus) locus of pea (Pisum sativum L.), which determines whether the seed is round or wrinkled. Wrinkled (rr) seeds lack one isoform of starch-branching enzyme (SBEI), present in round (RR or Rr) seeds. A major polymorphism in the SBEI gene between near-isogenic RR and rr lines shows 100% cosegregation with the r locus, establishing that the SBEI gene is at the r locus. An aberrant transcript for SBEI is produced in rr embryos. In rr lines the SBEI gene is interrupted by a 0.8 kb insertion that is very similar to the Ac/Ds family of transposable elements from maize. Failure to produce SBEI has complex metabolic consequences on starch, lipid, and protein biosynthesis in the seed.

A Mutant of Arabidopsis Lacking a Chloroplastic Isoamylase Accumulates Both Starch and Phytoglycogen

In this study, our goal was to evaluate the role of starch debranching enzymes in the determination of the structure of amylopectin. We screened mutant populations of Arabidopsis for plants with alterations in the structure of leaf starch by using iodine staining. The leaves of two mutant lines stained reddish brown, whereas wild-type leaves stained brownish black, indicating that a more highly branched polyglucan than amylopectin was present. The mutants were allelic, and the mutation mapped to position 18.8 on chromosome 1. One mutant line lacked the transcript for a gene with sequence similarity to higher plant debranching enzymes, and both mutants lacked a chloroplastic starch-hydrolyzing enzyme. This enzyme was identified as a debranching enzyme of the isoamylase type. The loss of this isoamylase resulted in a 90% reduction in the accumulation of starch in this mutant line when compared with the wild type and in the accumulation of the highly branched water-soluble polysaccharide phytoglycogen. Both normal starch and phytoglycogen accumulated simultaneously in the same chloroplasts in the mutant lines, suggesting that isoamylase has an indirect rather than a direct role in determining amylopectin structure.

β-AMYLASE4, a Noncatalytic Protein Required for Starch Breakdown, Acts Upstream of Three Active β-Amylases in Arabidopsis Chloroplasts

This work investigated the roles of β-amylases in the breakdown of leaf starch. Of the nine β-amylase (BAM)–like proteins encoded in the Arabidopsis thaliana genome, at least four (BAM1, -2, -3, and -4) are chloroplastic. When expressed as recombinant proteins in Escherichia coli, BAM1, BAM2, and BAM3 had measurable β-amylase activity but BAM4 did not. BAM4 has multiple amino acid substitutions relative to characterized β-amylases, including one of the two catalytic residues. Modeling predicts major differences between the glucan binding site of BAM4 and those of active β-amylases. Thus, BAM4 probably lost its catalytic capacity during evolution. Total β-amylase activity was reduced in leaves of bam1 and bam3 mutants but not in bam2 and bam4 mutants. The bam3 mutant had elevated starch levels and lower nighttime maltose levels than the wild type, whereas bam1 did not. However, the bam1 bam3 double mutant had a more severe phenotype than bam3, suggesting functional overlap between the two proteins. Surprisingly, bam4 mutants had elevated starch levels. Introduction of the bam4 mutation into the bam3 and bam1 bam3 backgrounds further elevated the starch levels in both cases. These data suggest that BAM4 facilitates or regulates starch breakdown and operates independently of BAM1 and BAM3. Together, our findings are consistent with the proposal that β-amylase is a major enzyme of starch breakdown in leaves, but they reveal unexpected complexity in terms of the specialization of protein function.

Major differences in isoforms of starch-branching enzyme between developing embryos of round- and wrinkled-seeded peas (Pisum sativum L.)

In order to determine whether round-and wrinkled-seeded peas (Pisum sativum L.) differ in the activity and properties of starch-branching enzyme (1,4-α-D-glucan, 1,4-α-D-glucan-6-glycosyl transferase; EC 2.4.1.18) in their developing embryos, essentially isogenic lines of peas, differing only at the r (rugosus) locus that confers the round (RR, Rr) or wrinkled (rr) phenotype, were studied. Activity of the enzyme rises rapidly from an early stage of development in embryos of round peas, but only at later stages in embryos of wrinkled peas. The purified enzyme from mature embryos of round peas can be resolved into two isoforms that differ in molecular weight and in their ability to branch amylose. The purified enzyme from mature embryos of wrinkled peas is a single protein with the same molecular weight and branching properties as one of the isoforms from embryos of round peas. The difference in activity of starch-branching enzyme between embryos of round and wrinkled peas is likely to be due to the absence from embryos of wrinkled peas of one of the isoforms occurring in embryos of round peas.

Glycine decarboxylase is confined to the bundle-sheath cells of leaves of C3−C4 intermediate species

Immunogold labelling has been used to determine the cellular distribution of glycine decarboxylase in leaves of C3, C3−C4 intermediate and C4 species in the genera Moricandia, Panicum, Flaveria and Mollugo. In the C3 species Moricandia foleyi and Panicum laxum, glycine decarboxylase was present in the mitochondria of both mesophyll and bundle-sheath cells. However, in all the C3−C4 intermediate (M. arvensis var. garamatum, M. nitens, M. sinaica, M. spinosa, M. suffruticosa, P. milioides, Flaveria floridana, F. linearis, Mollugo verticillata) and C4 (P. prionitis, F. trinervia) species studied glycine decarboxylase was present in the mitochondria of only the bundle-sheath cells. The bundle-sheath cells of all the C3−C4 intermediate species have on their centripetal faces numerous mitochondria which are larger in profile area than those in mesophyll cells and are in close association with chloroplasts and peroxisomes. Confinement of glycine decarboxylase to the bundle-sheath cells is likely to improve the potential for recapture of photorespired CO2 via the Calvin cycle and could account for the low rate of photorespiration in all C3−C4 intermediate species.

At least three different RNA polymerase holoenzymes direct transcription of the agarase gene (dagA) of streptomyces coelicolor A3(2)

Using a combination of gel filtration and anion exchange FPLC, three different RNA polymerase holoenzymes from Streptomyces coelicolor A3(2) have been separated. Each holoenzyme transcribes from only one of the four promoters of the S. coelicolor A3(2) dagA gene. Holoenzyme reconstitution experiments identified the sigma factors responsible for recognition of two of the promoters. The previously identified E sigma 49 transcribes from the dagA p3 promoter, whereas a novel species, E sigma 28, recognizes the dagA p2 promoter. Circumstantial evidence suggests that the third holoenzyme, which transcribes from the dagA p4 promoter, is the previously identified E sigma 35. This level of transcriptional complexity supports the idea that RNA polymerase heterogeneity may play a central role in the regulation and coordination of gene expression in this biochemically and morphologically complex bacterium.

Evidence that glucose 6-phosphate is imported as the substrate for starch synthesis by the plastids of developing pea embryos.

The aim of this work was to determine in what form carbon destined for starch synthesis crosses the membranes of plastids in developing pea (Pisum sativum L.) embryos. Plastids were isolated mechanically and incubated in the presence of ATP with the following 14C-labelled substrates: glucose, fructose, glucose 6-phosphate, glucose 1-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate. Glucose 6-phosphate was the only substrate that supported physiologically relevant rates of starch synthesis. Incorporation of label from glucose 6-phosphate into starch was dependent upon the integrity of the plastids and the presence of ATP. The rate of incorporation approached saturation at a glucose 6-phosphate concentration of less than 1 mM. It is argued that glucose 6-phosphate is likely to enter the plastid as the source of carbon for starch synthesis in vivo.

Photorespiratory metabolism and immunogold localization of photorespiratory enzymes in leaves of C3 and C3-C4 intermediate species of Moricandia

Photorespiratory metabolism of the C3-C4 intermediate species Moricandia arvensis (L.) DC has been compared with that of the C3 species, Moricandia moricandioides (Boiss.) Heywood. Assays of glycollate oxidase (EC 1.1.3.1), glyoxylate aminotransferases (EC 2.6.1.4, EC 2.6.1.45) and hydroxypyruvate reductase (EC 1.1.1.29) indicate that the capacity for flux through the photorespiratory cycle is similar in both species. Immunogold labelling with monospecific antibodies was used to investigate the cellular locations of ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), glycollate oxidase, and glycine decarboxylase (EC 2.1.2.10) in leaves of the two species. Ribulose 1,5-bisphosphate carboxylase/oxygenase was confined to the stroma of chloroplasts and glycollate oxidase to the peroxisomes of all photosynthetic cells in leaves of both species. However, whereas glycine decarboxylase was present in the mitochondria of all photosynthetic cells in M. moricandioides, it was only found in the mitochondria of bundle-sheath cells in M. arvensis. We suggest that localized decarboxylation of glycine in the leaves of M. arvensis will lead to improved recapture of photorespired CO2 and hence a lower rate of photorespiration.

Evidence that the “waxy” protein of pea (Pisum sativum L.) is not the major starch-granule-bound starch synthase

The aim of this work was to identify the starch-granule-bound starch synthase of developing pea embryos. When starch-granule-bound proteins were solubilised by digestion of granules with α-amylase and fractionated on a Mono Q anion-exchange column, activity of starch synthase eluted as three peaks. The distribution of activity in fractions from the column coincided with that of a 77-kDa protein. An antibody to this protein inhibited starch-synthase activity both in solubilised, starch-granule-bound protein and on intact starch granules. Recoveries of activity through extraction, solubilisation and chromatography indicate that this protein is the major, if not the only, form of starch synthase on the starch granule. The major, 59-kDa protein of the pea starch granule is antigenically related to the product of thewaxy locus of potato, which has previously been identified as the starch-granule-bound starch synthase of the tuber. However, the distribution of the 59-kDa protein did not coincide with that of starch-synthase activity in fractions from the Mono Q column. An antibody to the 59-kDa protein did not inhibit starch-synthase activity. The results raise questions about the relationship between “waxy” proteins and starch-granule-bound starch synthases generally.

Distribution of photorespiratory enzymes between bundle-sheath and mesophyll cells in leaves of the C3-C4 intermediate species Moricandia arvensis (L.) DC

In order to study the location of enzymes of photorespiration in leaves of the C3−C4 intermediate species Moricandia arvensis (L.). DC, protoplast fractions enriched in mesophyll or bundlesheath cells have been prepared by a combination of mechanical and enzymic techniques. The activities of the mitochondrial enzymes fumarase (EC 4.2.1.2) and glycine decarboxylase (EC 2.1.2.10) were enriched by 3.0- and 7.5-fold, respectively, in the bundle-sheath relative to the mesophyll fraction. Enrichment of fumarase is consistent with the larger number of mitochondria in bundle-sheath cells relative to mesophyll cells. The greater enrichment of glycine decarboxylase indicates that the activity is considerably higher on a mitochondrial basis in bundle-sheath than in mesophyll cells. Serine hydroxymethyltransferase (EC 2.1.2.1) activity was enriched by 5.3-fold and glutamate-dependent glyoxylate-aminotransferase (EC 2.6.1.4) activity by 2.6-fold in the bundle-sheath relative to the mesophyll fraction. Activities of serine- and alanine-dependent glyoxylate aminotransferase (EC 2.6.1.45 and EC 2.6.1.4), glycollate oxidase (EC 1.1.3.1), hydroxypyruvate reductase (EC 1.1.1.81), glutamine synthetase (EC 6.3.1.2) and phosphoribulokinase (EC 2.7.1.19) were not significantly different in the two fractions. These data provide further independent evidence to complement earlier immunocytochemical studies of the distribution of photorespiratory enzymes in the leaves of this species, and indicate that while mesophyll cells of M. arvensis have the capacity to synthesize glycine during photorespiration, they have only a low capacity to metabolize it. We suggest that glycine produced by photorespiratory metabolism in the mesophyll is decarboxylated predominantly by the mitochondria in the bundle sheath.