PDIL1-2 can indirectly and negatively regulate expression of the AGPL1 gene in bread wheat

BACKGROUND: ADP-glucose pyrophosphorylase (AGPase), the key enzyme in plant starch biosynthesis, is a heterotetramer composed of two identical large subunits and two identical small subunits. AGPase has plastidial and cytosolic isoforms in higher plants, whereas it is mainly detected in the cytosol of grain endosperms in cereal crops. Our previous results have shown that the expression of the TaAGPL1 gene, encoding the cytosolic large subunit of wheat AGPase, temporally coincides with the rate of starch accumulation and that its overexpression dramatically increases wheat AGPase activity and the rate of starch accumulation, suggesting an important role. METHODS: In this study, we performed yeast one-hybrid screening using the promoter of the TaAGPL1 gene as bait and a wheat grain cDNA library as prey to screen out the upstream regulators of TaAGPL1 gene. And the barley stripe mosaic virus-induced gene-silencing (BSMV-VIGS) method was used to verify the functional characterization of the identified regulators in starch biosynthesis. RESULTS: Disulfide isomerase 1-2 protein (TaPDIL1-2) was screened out, and its binding to the TaAGPL1-1D promoter was further verified using another yeast one-hybrid screen. Transiently silenced wheat plants of the TaPDIL1-2 gene were obtained by using BSMV-VIGS method under field conditions. In grains of BSMV-VIGS-TaPDIL1-2-silenced wheat plants, the TaAGPL1 gene transcription levels, grain starch contents, and 1000-kernel weight also significantly increased. CONCLUSIONS: As important chaperones involved in oxidative protein folding, PDIL proteins have been reported to form hetero-dimers with some transcription factors, and thus, our results suggested that TaPDIL1-2 protein could indirectly and negatively regulate the expression of the TaAGPL1 gene and function in starch biosynthesis.

Single nucleotide polymorphisms in partial sequences of the gene encoding the large sub-units of ADP-glucose pyrophosphorylase within a representative collection of 10 Musa genotypes

Background ADP-glucose pyrophosphorylase (AGPase) is a rate-limiting enzyme catalyzing the first step in the starch biosynthesis pathway in higher plants. To date, there are no reported variants or isoforms of the AGPase enzyme in bananas (Musa spp. family Musaceae) as is the case of other plants. In this study, genomic DNA sequences homologous to the gene encoding one of the large subunits of the enzyme were amplified from 10 accessions of the genus Musa, including representatives of wild ancestors (AA and BB genomes), dessert bananas (AA, AAA, AB and AAB genomes), plantains (AAB genome) and cooking bananas (ABB and AAA genomes), and studied in order to find single nucleotide polymorphisms (SNP) base variations in Musa accessions. Results In the 810-base pair amplicons of the AGPase large sub-unit (LSU) gene analyzed in ten Musa accessions, a total of 36 SNPs and insertions/deletions (indels) were found. The phylogenetic analysis revealed fifteen distinct haplotypes, which were grouped into four variants. Deep examination of SNPs in the 2nd exon in the LSU of AGPase showed that at seven locations, five SNPs altered their amino acid sequence. Conclusions This work reveals the possible number of AGPase enzyme isoforms and their molecular levels in banana. Molecular markers could be designed from SNPs present in these banana accessions. This information could be useful for the development of SNP-based molecular markers for Musa germplasm, and alteration of the allosteric properties of AGPase to increase the starch content and manipulate the starch quality of banana fruits.

Genome Mapping of an Extreme Thermophile, Thermus caldophilus GK24

Genome of an extreme thermophile, Thermus caldophilus GK24 has been analyzed to construct the genomic map. The genomic DNAs encapsulated in agarose gel were digested with SspI, EcoRI, SpeI, and HpaI restriction endonucleases, and then the resulting genomic DNA fragments were analyzed by pulsed-field gel electrophoresis. Its restriction map has been constructed by analyzing sizes of the restriction fragments obtained from both complete and partial digestions. The circular form of its genome was composed of about 1.98 Mbp and a megaplasmid. The genomic loci for the genes of xylose isomerase, thioredoxin, tRNA-16S rRNA, 23S rRNA, L5 ribosomal protein, ADP-glucose pyrophosphorylase, DNA-ligase, and Tca DNA polymerase were determined by both Southern hybridization and PCR.

Enzymes of glycolytic and pentose phosphate pathways in cytosolic and leucoplastic fractions of developing seeds of Brassica campestris.

Distribution of the enzymes of glycolytic and pentose phosphate pathways were studied in cytosolic and leucoplastic fractions of the developing seeds of Brassica. Leucoplasts were isolated using a discontinuous percoll gradient. Intactness of leucoplasts was checked by ADP-glucose pyrophosphorylase assay in presence and absence of triton X-100. No contamination by microbodies, mitochondria and cytosol was observed as assessed by measuring the activities of marker enzymes. The recovery, latency and specific activity of each enzyme in different fractions were compared. The leucoplastic fraction contained complete set of the enzymes of glycolytic and pentose phosphate pathways, indicating that the two subcellular compartments metabolize carbon independently by these pathways. However, the enzymes showed higher activities in cytosolic fraction as compared to those in the leucoplasts, suggesting the need for exchange of metabolites in the two compartments through various translocators, for acting in cooperation to produce energy, reducing power and carbon skeletons for different biosynthetic activities in the non-photosynthetic plastids. Based on these compartmentation studies, a model for carbon flow for fatty acid synthesis in leucoplasts of developing Brassica seeds has been proposed.