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Plant and Cell Physiology Advance Access published online on June 15, 2005
Plant and Cell Physiology, doi:10.1093/pcp/pci154
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Plant and Cell Physiology 2005 © The Japanese Society of Plant Physiologists (JSPP); all rights reserved.
Received January 31, 2005
Accepted June 9, 2005

Regular Paper

Cloning and Regulation of a Stress Regulated Pennisetum glaucum Vacuolar ATPase c Gene and Characterization of Its Promoter that Expresses in Shoot Hairs and Floral Organs

Wricha Tyagi 1, Divya Rajagopal 1, Sneh Lata Singla-Pareek 1, M. K. Reddy 1, and S. K. Sopory 1*

1 Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi-110067, India

* To whom correspondence should be addressed.
S. K. Sopory, E-mail: sopory{at}icgeb.res.in


  Abstract

We have cloned and characterised the cDNA, genomic clone and upstream promoter region of a vacuolar ATPase c subunit (PgVHA-c1) from Pennisetum glaucum. The deduced amino acid sequence shows 98-71% sequence identity with V-ATPase from rice and Arabidopsis and is a highly hydrophobic protein with four trans-membrane regions. PgVHA-c1-GFP fusion protein is expressed in BY2 cells on the endo-membranes surrounding vacuoles, however, PgVHA-c1 could not functionally complement V-ATPase-c deletion mutants of yeast. The sequence analysis of genomic clone revealed the presence of two introns in coding region and the splice junctions followed the typical canonical GU-AG consensus sequence. The transcript analysis showed the expression of PgVHA-c1 was stimulated more in response to salinity stress and very marginally in response to drought and low temperature stress. Exogenous application of abscisic acid, salicylic acid and calcium stimulated the transcript level in the absence of stress. We have cloned the 5' flanking regions of the PgVHA-c1 and mapped its transcript start site at 78 bp upstream of ATG. Transgenic tobacco with promoter::GUS constructs showed that the region -288/+78 was sufficient for GUS expression. The expression of reporter gene even with the full-length promoter was limited to shoot hairs and to male and female reproductive organs. The DRE- and ABRE- like elements in the promoter did not show consensus flanking regions, however, gel mobility shift assays showed that Pennisetum has specific transacting factors that showed binding to the core DRE, ABRE and TCA elements.

Keywords: ABA; calcium; promoter; Pennisetum glaucum; salicylic acid-stress.
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