Cloning and regulation of a stress-regulated Pennisetum glaucum vacuolar ATPase c gene and characterization of its promoter that is expressed in shoot hairs and floral organs.
We have cloned and characterized the cDNA, genomic clone and upstream promoter region of a vacuolar ATPase (V-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 transmembrane 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 the genomic clone revealed the presence of two introns in the coding region, and the splice junctions followed the typical canonical GU-AG consensus sequence. The transcript analysis showed that 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 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 the reporter gene even with the full-length promoter was limited to shoot hairs and to male and female reproductive organs. The dehydration-responsive element (DRE) and ABA-responsive element (ABRE) 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.
Tyagi
Wricha
W
Plant Molecular Biology, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi-110067, India.
Rajagopal
Divya
D
Singla-Pareek
Sneh Lata
SL
Reddy
Malireddy K
MK
Sopory
Sudhir K
SK
D001483:Base Sequence; D003001:Cloning, Molecular; D018744:DNA, Plant; D024202:Electrophoretic Mobility Shift Assay; D035264:Flowers / Q000201:enzymology*; D005816:Genetic Complementation Test; D005966:Glucuronidase / Q000235:genetics; D008969:Molecular Sequence Data; D031782:Pennisetum / Q000201:enzymology* / Q000235:genetics; D018520:Plant Shoots / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D020156:Salicylic Acid / Q000494:pharmacology; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics*
D018744:DNA, Plant; D005966:Glucuronidase; D025262:Vacuolar Proton-Translocating ATPases; D020156:Salicylic Acid