Calcium-dependent regulatory mechanisms participate in diverse developmentally, hormonally, and environmentally regulated processes, with the precise control of cytosolic Ca2+ concentration being critical to such mechanisms. In plant cells, P-type Ca2+-ATPases localized in the plasma membrane and the endoplasmic reticulum are thought to play a central role in regulating cytoplasmic Ca2+ concentrations. Ca2+-ATPase activity has been identified in isolated plant cell membranes, but the protein has not been characterized at the molecular level. We have isolated a partial-length cDNA (LCA1) and a complete genomic clone (gLCA13) encoding a putative endoplasmic reticulum-localized Ca2+-ATPase in tomato. The deduced amino acid sequence specifies a protein (Lycopersicon Ca2+-ATPase) of 1048 amino acids with a molecular mass of 116 kDa, eight probable transmembrane domains, and all of the highly conserved functional domains common to P-type cation-translocating ATPases. In addition, the protein shares -50% amino acid sequence identity with animal sarcoplasmic/endoplasmic reticulum Ca2+-ATPases but <30% identity with other P-type ATPases. Genomic DNA blot hybridization analysis indicates that the Lycopersicon Ca2+-ATPase is encoded by a single gene. RNA blot hybridization analysis indicates the presence of three transcript sizes in root tissue and a single, much less abundant, transcript in leaves. Lycopersicon Ca2+-ATPase mRNA levels increase dramatically upon a 1-day exposure to 50 mM NaCl. Thus this report describes the primary structure of a higher-plant Ca2+-ATPase and the regulation of its mRNA abundance by salt stress.

Seven genomic fragments encoding isoforms of tomato (Lycopersicon esculentum) plasma membrane H+-ATPase were cloned and characterized. Genomic DNA gel-blot analysis indicated that probes corresponding to LHAl through LHA7 hybridized to a common set of seven to nine restriction fragments at moderate stringency and to single, distinct fragments at high stringency. RNA gel-blot and polymerase chain reaction (PCR)-based RNA analyses indicated that LHA1, LHA2, and LHA4 transcripts were present in all organs examined (roots, hypocotyls, stems, immature leaves, mature leaves, green fruit, and red ripe fruit). LHAl mRNA was present at similar abundance in all organs, LHA2 mRNA was most abundant in hypocotyls and leaves, and LHA4 mRNA was most abundant in roots and hypocotyls. RNA gel-blot and RNAbased PCR assays indicated that lHA3, lHA.5, LHA6, and LHA7 mRNA was present at very low or nondetectable levels in all organs, suggesting that these genes are either expressed at very low levels or in organs not examined or that they are regulated by hormonal or environmental cues that were not tested. Indoleacetic acid (IAA) treatment of tomato hypocotyl segments resulted in modest changes in abundance of LHA1, LHA2, and LHA4 transcripts, but these changes were not correlated with the time course of IAAinduced growth. In addition, constitutively silent LHA genes were not activated by IAA. These results indicate that at least seven genomic sequences are present in tomato that may encode plasma membrane H+-ATPases, at least three of which are expressed relatively abundantly at the mRNA level.The enzyme primarily responsible for active transport in plants is the plasma membrane H+-ATPase. It is a member of an evolutionarily related family of cation-translocating ATPases that includes the Ca2+-ATPases of plants and animals, the plasma membrane H+-ATPase of fungi, and the Na+,K+-ATPase of animals. This family of P-type ATPases is characterized by formation of a phosphorylated intermediate, inhibition by vanadate, and structural similarity in several conserved domains (Serrano, 1989). The activity of the H+-ATPase generates an electrochemical gradient across the plant cell plasma membrane that drives a number of secondary transport systems, includmg those responsible for the translocation of cations, anions, amino acids, sugars, and hormones (Poole, 1978;Reinhold and Kaplan, 1984). The activity of the H+-ATPase also contributes to the maintenance of intracellular and extracellular pH (Smith and Raven, 1979).

Transient expression following agroinfiltration of plant tissue was investigated as a system for producing recombinant protein. As a model system, Agrobacterium tumefaciens containing the beta-glucuronidase (GUS) gene was vacuum infiltrated into lettuce leaf disks. Infiltration with a suspension of 10(9) colony forming units/mL followed by incubation for 72 h at 22 degrees C in continuous darkness produced a maximum of 0.16% GUS protein based on dry tissue or 1.1% GUS protein based on total soluble protein. This compares favorably to expression levels for commercially manufactured GUS protein from transgenic corn seeds. A. tumefaciens culture medium pH between 5.6 and 7.0 and surfactant concentrations < or = 100 ppm in the vacuum infiltration did not affect GUS expression, while infiltration with an A. tumefaciens density of 10(7) and 10(8) colony forming units/mL, incubation at 29 degrees C, and a surfactant concentration of 1,000 ppm significantly reduced expression. Incubation in continuous light caused lettuce to produce GUS protein more rapidly, but final levels did not exceed the GUS production in leaves incubated in continuous darkness after 72 h at 22 degrees C. The kinetics of GUS expression during incubation in continuous light and dark were represented well using a logistic model, with rate constants of 0.30 and 0.29/h, respectively. To semi-quantitatively measure the GUS expression in large numbers of leaf disks, a photometric enhancement of the standard histochemical staining method was developed. A linear relationship with an R2 value of 0.90 was determined between log10 (% leaf darkness) versus log10 (GUS activity). Although variability in expression level was observed, agroinfiltration appears to be a promising technology that could potentially be scaled up to produce high-value recombinant proteins in planta.

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

Positional cloning with microsatellite markers allowed further localization of the Darier disease gene to a 2-cM interval of chromosome 12, 12q23-24.1, between the polymorphic loci D12S234 and D12S129. A region this size is suitable for construction of a contig to identify the Darier disease gene. Use of a polymorphic intronic marker for nitric oxide synthetase 1 gene, which maps to the same chromosomal area as the Darier gene, allowed exclusion of that gene as the Darier disease gene.