Root transcripts associated with arsenic accumulation in hyperaccumulator Pteris vittata.

Hyperaccumulation of arsenic (As) by brake fern Pteris vittata has been described as an important genetic trait that provides an option for development of a sustainable phytoremediation process for As mitigation. Accumulation of very high concentration of arsenic in above-ground tissues may be the result of arsenic vacuole compartmentalization, but the mechanism(s) of arsenic uptake and transport by underground tissues are largely unknown. In this study, we made an attempt towards understanding the molecular mechanism of As hyperaccumulation in this plant. A time-dependent As accumulation study indicates an exponential accumulation of As from 7 to 30 days of arsenic exposure in fronds, and day 3-7 in roots. Root transcriptome analysis identified 554,973 transcripts. Further, subsets of 824 transcripts were differentially expressed between treated and control samples. Many of the genes of critical As-stress response, transcription factors and metal transporters, biosynthesis of chelating compounds involved in uptake and accumulation mechanisms were identified. The genes that were highly expressed such as cysteine-rich RLK, and ABC transporter G family member 26 needs further studies along with arsenite transmembrane transporter. The analysis of generated transcriptome dataset has provided valuable information and platform for further functional studies.

Characterization of UDP-glucosyltransferase from Indigofera tinctoria.

Indican is a secondary metabolite in Indigofera tinctoria; its synthesis from indoxyl and UDP-glucose is catalyzed by a UDP-glucosyltransferase (UGT). In this study, we partially purified UGT extracted from I. tinctoria leaves and analyzed the protein by peptide mass fingerprinting. We identified two fragments that were homologous to UGT after comparison with the transcriptomic data of I. tinctoria leaves. The fragments were named itUgt1 and itUgt2 and were amplified using rapid amplification of cDNA ends polymerase chain reaction to obtain full-length cDNAs. The resultant nucleotide sequences of itUgt1 and itUgt2 encoded peptides of 477 and 475 amino acids, respectively. The primary structure of itUGT1 was 89% identical to that of itUGT2 and contained an important plant secondary product glycosyltransferase (PSPG) box sequence and a UGT motif. The recombinant proteins expressed in Escherichia coli were found to possess high indican synthesis activity. Although the properties of the two proteins itUGT1 and itUGT2 were very similar, itUGT2 was more stable at high temperatures than itUGT1. Expression levels of itUGT mRNA and protein in plant tissues were examined by UGT assay, immunoblotting, and semi-quantitative reverse transcription polymerase chain reaction. So far, we presume that itUGT1, but not itUGT2, primarily catalyzes indican synthesis in I. tinctoria leaves.

RNA-Seq analysis for indigo biosynthesis pathway genes in Indigofera tinctoria and Polygonum tinctorium.

Natural indigo is the most important blue dye for textile dyeing and valuable secondary metabolite biosynthesized in Indigofera tinctoria and Polygonum tinctorium plants. Present investigation is made to generation of gene resource for pathway enrichment and to understand possible gene expression involved in indigo biosynthesis. The data about raw reads and the transcriptome assembly project has been deposited at GenBank under the accessions SRA180766 and SRX692542 for I. tinctoria and P. tinctorium, respectively.

Cloning, in silico characterization and prediction of three dimensional structure of SbDof1, SbDof19, SbDof23 and SbDof24 proteins from Sorghum [Sorghum bicolor (L.) Moench].

In the present study, four full-length Dof (DNA-binding with one finger) genes from Sorghum bicolor namely SbDof1, SbDof19, SbDof23, and SbDof24 were PCR amplified, gel eluted, cloned, and sequenced (accession number HQ540084, HQ540085, HQ540086, and HQ540087, respectively). These sequences were further characterized in silico by subjecting them to homology search, multiple sequence alignment, phylogenetic tree construction, and protein functional analysis, revealing their identity to Dof like proteins. Phylogenetic analysis of cloned SbDof genes along with other reported Dof proteins revealed existence of two major groups A and B, while group A was further bifurcated into two sub-groups (viz., I and II). Motif scan analysis of SbDof proteins revealed the presence of glycine- and alanine-rich profiles in SbDof1, while proline-rich profile was observed in SbDof23. Asparagines, methionine, and serine-rich profiles were common in case of both SbDof19 and SbDof24 proteins. The three dimensional structures of SbDof proteins were predicted by I-TASSER server based on multiple threading method. The modeled structures were refined by energy minimization and their stereo chemical qualities were validated by PROCHECK and QMEAN server indicating the acceptability of the predicted models. The final models were submitted to PMDB database with assigned PMDB IDs, i.e., PM0077395, PM0077396, PM0077397, PM0077398, and PM0076448 for SbDof1, SbDof19, SbDof23, SbDof24, and Dof domain, respectively. Based on gene ontology (GO) terms in I-TASSER server putative functions of modeled SbDof proteins were also predicted.