Molecular Cloning of Tomato Plasma Membrane H⁺-ATPase

Abstract

Two cDNA clones (LHA1 and LHA2) from tomato (Lycopersicon esculentum) which likely encode isoforms of the plasma membrane H+-ATPase were isolated. The longest cDNA (3229 base pairs), LHAI, comprises an open reading frame that encodes a 956 amino acid, 105 kilodalton polypeptide with several potential transmembrane domains. In vitro transcription and translation of LHA1 yields a major translation product of approximately 100 kilodaltons that is immunoprecipitable with antiserum to the com root plasma membrane H -ATPase. LHA2 encodes a portion of a coding sequence that is 96% identical to LHA1, suggesting that LHA2 encodes an isoform of the H+-ATPase. Genomic DNA gel blot analysis indicates that both LHA1 and LHA2 hybridize to a common set of six to eight restriction fragments at moderate stringency and to single distinct fragments at high stringency. LHA1 and LHA2 map to distinct sites on chromosomes three and six, respectively. RNA gel blot analysis indicates that both LHA1 and LHA2 hybridize to 3.4 kilobase pair transcripts present in both leaves and roots, although the LHA2 transcript is relatively more abundant in leaves than in roots. These results indicate that in tomato as many as six to eight genes may encode the plasma membrane H+-ATPase, two of which are expressed at the level of mRNA in both roots and leaves.The plant plasma membrane H+ translocating ATPase (EC 3.6.1.35) plays a central role in the physiology and bioenergetics of the plant cell. It is the primary active transport enzyme associated with the plasma membrane and its activity is responsible for generating the membrane potential and concomitant electrochemical gradient which, in turn, drives the translocation of a number of solutes including cations, anions, amino acids, sugars, and hormones across the plasma membrane by secondary transport systems (23,25,30). The activity of the H+ translocating ATPase also contributes to the maintenance of intracellular and extracellular pH (25) and regulation of the activity of the plasma membrane H+-ATPase has been proposed to mediate a broad range of physiological responses which play a central role in the growth and development of plants (25). One such response is auxininduced growth for which it has been proposed that activation

Assessment of the Number and Expression of P-Type H+-ATPase Genes in Tomato

“…Approximately 5 X lo5 clones from a tomato (Lycopersicon esculentum) X Charon 35 genomic library (Deikman and Fisher, 1988) were screened with an EcoRI-EcoRV restriction fragment encompassing bp 1304 to 2201 of the cDNA clone LHA1 (Ewing et al, 1990) by plaque hybridization (Sambrook et al, 1989). Prior to hybridization, filters were prewashed for 1 h at 42OC in 5X SSC, 0.5% SDS (w/v), and 1 m EDTA (pH 8.0) and prehybridized at 42OC for 8 h in 50% formamide (v/v), 5X SSPE, 5X Denhardt's solution, 0.2% SDS (w/v), and 100 pg/mL sheared and heat-denatured herring sperm DNA.…”

“…Tomato genomic DNA (5 pg) was digested with the indicated restriction enzymes, fractionated on a 0.8% agarose gel, and blotted to nylon membranes as described previously (Southern, 1975;Ewing et al, 1990). Membranes were hybridized with individual restriction fragments of the cDNAs LHA1 and LHA2 (which include those portion:; amplified by the F1 and F2 primers) as described previously (Ewing et al, 1990) and the PCR-amplified Fl-F2 region of LHA3 through LHA7 genomic clones, labeled with [32P]dA'IP by random priming (Feinberg and Vogelstein, 1983).…”

“…Membranes were hybridized with individual restriction fragments of the cDNAs LHA1 and LHA2 (which include those portion:; amplified by the F1 and F2 primers) as described previously (Ewing et al, 1990) and the PCR-amplified Fl-F2 region of LHA3 through LHA7 genomic clones, labeled with [32P]dA'IP by random priming (Feinberg and Vogelstein, 1983). Hybridization was carried out at 65OC in 1% SDS (w/v), 1 M sodium chloride, 10% dextran sulfate (w/v), and 100 pg/mL denatured salmon sperm DNA with labeled probe added at 5 >I: lo6 cpm/mL (10 ng/mL).…”

“…To generate intemal standards, the cDNA clone LHA2 in the vector pCGNl703 (Ewing et al, 1990) was modified by the insertion of a 705-bp fragment of heterologous DNA (a Dra1,HincII fragment from bp 35 to 740 of the full-length ascidian cytoskeletal actin cDNA) within the region amplified by the Fl-F2 primer pair. RNA was transcribed from this modified LHA2 cDNA in vitro using T7 RNA polymerase and treated with RNase-free DNase (Stratagene) to remove template DNA.…”

“…Previously, genomic DNA gel-blot analysis had demonstrated the presence of multiple cross-hybridizing sequences related to the tomato H+-ATPase cDNAs LHA1 and LHA2 (Ewing et al, 1990). To identify additional genes that might encode H+-ATPases in tomato, genomic clones crosshybridizing to the previously characterized LHA1 cDNA were isolated.…”