Vajinder Kumar

Characterization of Upstream Sequences of the LOJ Gene Leads to Identification of a Novel Enhancer Element Conferring Lateral Organ Junction-Specific Expression in Arabidopsis thaliana

Isolation and characterization of promoters are important in understanding gene regulation and genetic engineering of crop plants. Earlier, a pentatricopeptide repeat protein (PPR) encoding gene (At2g39230), designated as Lateral Organ Junction (LOJ) gene, was identified through T-DNA promoter trapping in Arabidopsis thaliana. The upstream sequence of the LOJ gene conferred on the reporter gene a novel LOJ-specific expression. The present study was aimed at identifying and characterizing the cis-regulatory motifs responsible for tissue-specific expression in the −673 and +90 bases upstream of the LOJ gene recognized as LOJ promoter. In silico analysis of the LOJ promoter revealed the presence of a few relevant regulatory motifs and a unique feature like AT-rich inverted repeat. Deletion analysis of the LOJ promoter confirmed the presence of an enhancer-like element in the distal region (−673/−214), which stimulates a minimal promoter-like sequence in the −424/−214 region in a position and orientation autonomous manner. The −136/+90 region of the LOJ promoter was efficient in driving reporter gene expression in tissues like developing anthers and seeds of Arabidopsis. A positive regulation for the seed- and anther-specific expression module was contemplated within the 5′ untranslated region of the LOJ gene. However, this function was repressed in the native context by the lateral organ junction-specific expression. The present study has led to the identification of a novel lateral organ junction-specific element and an enhancer sequence in Arabidopsis with potential applications in plant genetic engineering.

Effect of overexpression of Arabidopsis thaliana SHB1 and KLUH genes on seed weight and yield contributing traits in Indian mustard (Brassica juncea L. (Czern.))

Seed size is a highly heritable trait in mustard and increasing seed size could improve crop productivity. Here we report the effect of overexpression of two Arabidopsis genes SHB1 and KLUH on seed weight and other yield contributing traits in Brassica juncea. KLUH gene was expressed under its own promoter, whereas the constitutive HPL promoter drove SHB1. Transgenic status was confirmed through PCR, RT-PCR and Southern analyses. The three SHB1 events contained single copy of transgene, whereas 12 KLUH events carried 1–4 copies of transgenes. All transgenics were fertile and comparable to non-transgenic counterpart for most of the agronomic traits in both T0 and T1 generations. Seed size and seed weight in SHB1 transgenics were 30–40% higher than the control, whereas one KLUH event showed significant increase in seed weight. Constitutive overexpression of SHB1 in B. juncea had undesirable pleiotropic effects that were comparable to the Arabidopsis lines overexpressing IKU2.

Expression analysis of a MATE-type transporter gene of Arabidopsis and its orthologues in rice and chickpea under salt stress

Salinity is one of the major abiotic stresses that adversely affect the crop growth and productivity. Salt responsive genes belonging to MATE efflux proteins reportedly play a significant role imparting salt tolerance to plants. In the present study, AT5G52050, a putative salt responsive gene from Arabidopsis thaliana encoding MATE efflux family protein which functions as an antiporter and its orthologues in rice (LOC_Os02g45380) and chickpea (LOC101489496) have been identified. The expression pattern of these genes was validated by quantitative reverse transcription polymerase chain reaction. In silico analysis of the upstream promoter regions of these genes revealed the presence of several conserved sequence motifs related to salt response either in single or in multiple copies. A phylogenetic analysis to understand the evolutionary relationship of MATE family genes in Arabidopsis, rice and chickpea revealed conservation of MATE family genes between dicots and monocots. The genes identified in this study may serve as promising candidates for further elucidation of the salt tolerance mechanism in plants.

Transmembrane START domain proteins: in silico identification, characterization and expression analysis under stress conditions in chickpea (Cicer arietinum L.)

Steroidogenic acute regulatory related transfer (StART) proteins that are involved in transport of lipid molecules, play a myriad of functions in insects, mammals and plants. These proteins consist of a modular START domain of approximately 200 amino acids which binds and transfers the lipids. In the present study we have performed a genome-wide search for all START domain proteins in chickpea. The search identified 36 chickpea genes belonging to the START domain family. Through a phylogenetic tree reconstructed with Arabidopsis, rice, chickpea, and soybean START proteins, we were able to identify four transmembrane START (TM-START) proteins in chickpea. These four proteins are homologous to the highly conserved mammalian phosphatidylcholine transfer proteins. Multiple sequence alignment of all the transmembrane containing START proteins from Arabidopsis, rice, chickpea, and soybean revealed that the amino acid residues to which phosphatidylcholine binds in mammals, is also conserved in all these plant species, implying an important functional role and a very similar mode of action of all these proteins across dicots and monocots. This study characterizes a few of the not so well studied transmembrane START superfamily genes that may be involved in stress signaling. Expression analysis in various tissues showed that these genes are predominantly expressed in flowers and roots of chickpea. Three of the chickpea TM-START genes showed induced expression in response to drought, salt, wound and heat stress, suggesting their role in stress response.

A Simple PCR Cloning Step to Utilize a Binary Vector for Identification and Characterization of Enhancer Element in Plant Promoters

Identification and characterization of regulatory elements, such as enhancers, require designing of specialized plant transformation vector to testify its unique features of position and orientation-independent core-promoter activation. Very few plant transformation vectors are available till date for identification of enhancer elements in plant promoters. Hence, we need to utilize the existing binary plant transformation vectors, such as pBI101, through tedious and time-consuming restriction enzyme-based cloning steps. We describe here a simple one-step PCR-based cloning strategy to introduce a 35S CaMV minimal promoter in a binary plant transformation vector pBI101, and utilize the unique features to identify a lateral organ junction tissue-specific enhancer element in a plant promoter from Arabidopsis thaliana.