Plastidial phosphorylase is required for normal starch synthesis in Chlamydomonas reinhardtii

Abstract

SummaryAmong the three distinct starch phosphorylase activities detected in Chlamydomonas reinhardtii, two distinct plastidial enzymes (PhoA and PhoB) are documented while a single extraplastidial form (PhoC) displays a higher affinity for glycogen as in vascular plants. The two plastidial phosphorylases are shown to function as homodimers containing two 91-kDa (PhoA) subunits and two 110-kDa (PhoB) subunits. Both lack the typical 80-amino-acid insertion found in the higher plant plastidial forms. PhoB is exquisitely sensitive to inhibition by ADP-glucose and has a low affinity for malto-oligosaccharides. PhoA is more similar to the higher plant plastidial phosphorylases: it is moderately sensitive to ADP-glucose inhibition and has a high affinity for unbranched malto-oligosaccharides. Molecular analysis establishes that STA4 encodes PhoB. Chlamydomonas reinhardtii strains carrying mutations at the STA4 locus display a significant decrease in amounts of starch during storage that correlates with the accumulation of abnormally shaped granules containing a modified amylopectin structure and a high amylose content. The wild-type phenotype could be rescued by reintroduction of the cloned wild-type genomic DNA, thereby demonstrating the involvement of phosphorylase in storage starch synthesis.

Crystal Structure of the Chlamydomonas Starch Debranching Enzyme Isoamylase ISA1 Reveals Insights into the Mechanism of Branch Trimming and Complex Assembly

“…Flanking restriction sites were introduced into the 5Ј end of the first 3.5-kb PCR product by using EcoRI Isa2F (GGA ATT CAT GAT ACA AGG ACA CGT CCA G) and IntIsa2-5 (GCC GAG TAC ACG TAC GTA CCC AG) primers, whereas a XbaI restriction site was introduced in the second 5-kb PCR product covering the end of the genomic DNA using IntIsa2-3 (GGT TCT GGC GGT TTG GTT GCG G) and XbaIsa2R (GTC TAG ATC AGT GCT TCC GGG CCG CCG C) primers. The fulllength ISA2 genomic sequence was then assembled from PCR products using a unique KpnI restriction site found in the gene and cloned into the pSL18 plasmid (21). This plasmid was called pSL-Isa2 and was used to transform Baf06 and BafV13 mutant strains with the glass beads method (22).…”

Transcriptome for Photobiological Hydrogen Production Induced by Sulfur Deprivation in the Green Alga Chlamydomonas reinhardtii

“…7,2008 TRANSCRIPTOME FOR PHOTOBIOLOGICAL HYDROGEN PRODUCTION 1973 during PBHP. Genes involved in protein degradation that showed an increase in transcript abundance encode a 26S proteasome subunit S3, a putative serine carboxypeptidase, a putative ubiquitin, an E2 ubiquitin-conjugating enzyme and a ubiquitin carboxyl-terminal hydrolase (Table 1, proteolysis).…”

Mutation of the Plastidial α-Glucan Phosphorylase Gene in Rice Affects the Synthesis and Structure of Starch in the Endosperm

“…Furthermore, there is evidence that disproportionating enzyme (Colleoni et al, 1999;Ball and Morell, 2003) and a-glucan phosphorylase (Schupp and Ziegler, 2004;Dauvillé e et al, 2006) are also involved in this process.…”

“…These results indicate that PhoB is required for normal starch biosynthesis in the alga. Interestingly, PhoB has a low affinity for MOS, whereas PhoA, the other plastidial Pho in C. reinhardtii, has a high affinity for MOS as does the higher plant Pho1 (Dauvillé e et al, 2006).…”