The hnRNP F/H homologue of Trypanosoma brucei is differentially expressed in the two life cycle stages of the parasite and regulates splicing and mRNA stability.

Trypanosomes are protozoan parasites that cycle between a mammalian host (bloodstream form) and an insect host, the Tsetse fly (procyclic stage). In trypanosomes, all mRNAs are trans-spliced as part of their maturation. Genome-wide analysis of trans-splicing indicates the existence of alternative trans-splicing, but little is known regarding RNA-binding proteins that participate in such regulation. In this study, we performed functional analysis of the Trypanosoma brucei heterogeneous nuclear ribonucleoproteins (hnRNP) F/H homologue, a protein known to regulate alternative splicing in metazoa. The hnRNP F/H is highly expressed in the bloodstream form of the parasite, but is also functional in the procyclic form. Transcriptome analyses of RNAi-silenced cells were used to deduce the RNA motif recognized by this protein. A purine rich motif, AAGAA, was enriched in both the regulatory regions flanking the 3' splice site and poly (A) sites of the regulated genes. The motif was further validated using mini-genes carrying wild-type and mutated sequences in the 3' and 5' UTRs, demonstrating the role of hnRNP F/H in mRNA stability and splicing. Biochemical studies confirmed the binding of the protein to this proposed site. The differential expression of the protein and its inverse effects on mRNA level in the two lifecycle stages demonstrate the role of hnRNP F/H in developmental regulation.

Genome-wide identification, structure analysis and expression profiling of phospholipases D under hormone and abiotic stress treatment in chickpea (Cicer arietinum).

Phospholipases D (PLDs) are phospholipid hydrolyzing enzymes and crucial components of lipid signaling in plants. PLDs are implicated in stress responses in different plants however, characterization of PLDs in chickpea is missing. Here, we identify 13 PLD genes in the chickpea genome. PLD family could be divided into α, ß, δ, ε and ζ isoforms based on sequence and structure. Protein remodeling described that chickpea PLDs are composed of defined arrangements of α-helix, ß-sheets and short loops. Phylogenetic analysis suggested evolutionary conservation of chickpea PLD family with dicots. In-planta subcellular localization showed the plasma membrane localization of chickpea PLDs. All PLD promoters had hormone and stress related cis-regulatory elements, which suggested overlapping function of PLDs in hormone and abiotic stress signaling. The qRT-PCR expression analysis revealed that most PLD genes are differentially expressed in multiple abiotic stresses (drought, salt and cold stress). Moreover, several PLD genes had overlapping expression in abiotic stress and ABA and JA treatment. These observations indicate the involvement of PLD gene family in cross-talk of phytohormone and abiotic stress signaling in chickpea. Thus, present study opens new avenues of utilizing PLD related information for understanding hormone-regulated abiotic stress signaling in legume crops.

Genomic and expression analysis indicate the involvement of phospholipase C family in abiotic stress signaling in chickpea (Cicer arietinum).

Phospholipase C proteins are phospholipid hydrolysing enzymes and crucial components of abiotic stress triggered lipid signaling in plants. PLCs are implicated in plant reaction to drought, salinity, and cold stress responses, however, characterization of the PLC family in the legume crop chickpea is missing. Here, we identify and describe nine PLC encoding genes in the chickpea genome. Phylogenetic analysis showed that the chickpea PLC family has evolved through a common path in dicots. Subcellular localization of fluorescence tagged proteins confirmed cytoplasmic and plasma membrane bound forms of PLCs in chickpea. The promoters of all the PLC genes are comprised of several hormone response related, development and abiotic stress related cis-regulatory elements. Expression analysis in five developmental stages (germination, seedling, vegetative, reproductive and senescence) showed significant expression of multiple PLCs in germination, vegetative and reproductive stages, suggesting their diverse role in various developmental processes. qRT-PCR expression analysis of the entire PLC gene family under drought, salt and cold stresses revealed that most PLC genes are differentially expressed in multiple abiotic stresses. These observations indicate the involvement of PLC gene family in abiotic stress signaling and responses in important legume crop. The present study opens new avenues for utilizing PLC- related information in biotechnological programs for abiotic stress tolerance and legume crop improvement.

Expressed sequence tags and molecular cloning and characterization of gene encoding pinoresinol/lariciresinol reductase from Podophyllum hexandrum.

Podophyllotoxin, an aryltetralin lignan, is the source of important anticancer drugs etoposide, teniposide, and etopophos. Roots/rhizome of Podophyllum hexandrum form one of the most important sources of podophyllotoxin. In order to understand genes involved in podophyllotoxin biosynthesis, two suppression subtractive hybridization libraries were synthesized, one each from root/rhizome and leaves using high and low podophyllotoxin-producing plants of P. hexandrum. Sequencing of clones identified a total of 1,141 Expressed Sequence Tags (ESTs) resulting in 354 unique ESTs. Several unique ESTs showed sequence similarity to the genes involved in metabolism, stress/defense responses, and signalling pathways. A few ESTs also showed high sequence similarity with genes which were shown to be involved in podophyllotoxin biosynthesis in other plant species such as pinoresinol/lariciresinol reductase. A full length coding sequence of pinoresinol/lariciresinol reductase (PLR) has been cloned from P. hexandrum which was found to encode protein with 311 amino acids and show sequence similarity with PLR from Forsythia intermedia and Linum spp. Spatial and stress-inducible expression pattern of PhPLR and other known genes of podophyllotoxin biosynthesis, secoisolariciresinol dehydrogenase (PhSDH), and dirigent protein oxidase (PhDPO) have been studied. All the three genes showed wounding and methyl jasmonate-inducible expression pattern. The present work would form a basis for further studies to understand genomics of podophyllotoxin biosynthesis in P. hexandrum.

Rice WNK1 is regulated by abiotic stress and involved in internal circadian rhythm.

In Mammalian system the WNK (with no lysine kinase) serine-threonine protein kinase gene family is suggested to be involved in regulating ion homeostasis and other pathophysiological processes including cancer, hypertension and renal ion transport. In plant system the information about WNK genes is very poor. However, WNK-like genes have also been identified in plants, including ten in Arabidopsis, designated AtWNK1-AtWNK10. Here we report the cloning and characterization of a homologue of AtWNK1 gene from Oryza sativa indica cultivar Pusa Basmati-1 rice and designated as OsWNK1. The specific feature of this gene is lysine residue in kinase subdomain II, which is essential for the coordination of ATP in the active center and conserved among all other kinases, is absent. OsWNK1 was found to respond differentially under various abiotic stresses like cold, heat, salt, drought. OsWNK1 gene showed rhythmic expression profile under diurnal and circadian conditions at the transcription level. Our data indicates that OsWNK1 in rice might play a role in abiotic stress tolerance and that it is involved in internal rhythm.