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.

Morphometric and physiological studies were conducted to determine whether the wall ingrowths of transfer cells in the minor-vein phloem of Pisum sativum L. leaves increase the capacity of the cells for solute influx. Size and number of wall ingrowths are positively correlated to the photon flux density (PFD) at which the plants are grown. An analysis of plasmodesmatal frequencies indicated that numerous plasmodesmata are present at all interfaces except those between the sieveelement-transfer-cell complex (SE-TCC) and surrounding cells where plasmodesmata are present but few in number. Flux of exogenous sucrose into the SE-TCC was estimated from kinetic profiles of net sucrose influx into leaf discs, quantitative autoradiography, and measurements of sucrose translocation. Flux based both on the saturable (carrier-mediated) and the linear components of influx was 47% greater in leaves of plants grown at high PFD (1000 μmol·m(-2)·s(-1)) than those grown in low PFD (200 μmol·m(-2)·s(-1)) and was paralleled by a 47% increase in SE-TCC plasmalemma surface area. Flux of endogenous photosynthate across the SE-TCC plasmalemma was calculated from carbon balance and morphometric data. The increase in flux in high-light leaves over that in low-light leaves can be explained on the basis of an increase in plasmalemma surface area. In intact leaves, a 'standing osmotic gradient' may facilitate transport of solute into transfer cells with extensive wall elaborations.

The number of small proteins (SPs) encoded in the Escherichia coli genome is unknown, as current bioinformatics and biochemical techniques make short gene and small protein identification challenging. One method of small protein identification involves adding an epitope tag to the 3′ end of a short open reading frame (sORF) on the chromosome, with synthesis confirmed by immunoblot assays. In this study, this strategy was used to identify new E. coli small proteins, tagging 80 sORFs in the E. coli genome, and assayed for protein synthesis. The selected sORFs represent diverse sequence characteristics, including degrees of sORF conservation, predicted transmembrane domains, sORF direction with respect to flanking genes, ribosome binding site (RBS) prediction, and ribosome profiling results. Of 80 sORFs, 36 resulted in encoded synthesized proteins—a 45% success rate. Modeling of detected versus non‐detected small proteins analysis showed predictions based on RBS prediction, transcription data, and ribosome profiling had statistically‐significant correlation with protein synthesis; however, there was no correlation between current sORF annotation and protein synthesis. These results suggest substantial numbers of small proteins remain undiscovered in E. coli, and existing bioinformatics techniques must continue to improve to facilitate identification.

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

The nonchlorophyllous (albino) tissue of mature C. blumei leaves is a sink for photoassimilate. Transport from the green to the albino region of the same leaf was inhibited by cold and anoxia. When the green tissue of mature leaves was removed, the remaining albino portion imported labeled translocate from other mature leaves in the phloem. Photoassimilate unloading in the albino region of mature leaves was studied by quantitative autoradiography. The unloading was inhibited by cold but not by anoxia. No labeled photoassimilate could be detected in the free space of mature albino tissue by compartmental efflux analysis as phloem unloading proceeded in a N2 atmosphere, indicating that unloading, may occur by a symplastic pathway as it apparently does in sink leaves of other species. The minor veins of mature albino leaf tissue did not accumulate exogenous [(14)C]sucrose. Minor veins of green tissue in the same leaves accumulated [(14)C]sucrose but, in contrast to other species studied to date, this accumulation was insensitive to the inhibitor p-chloromercuribenzensulfonic acid (PCMBS).In its capacity to import and unload photoassimilate, and in the inability, of the minor veins to accumulate exogenous sucrose, the albino region of the mature C. blumei lamina differs from mature albino tobacco leaves and darkened mature leaves of other species. This, together with evidence indicating that phloem loading in C. blumei and other species may occur by different routes and with different sensitivity to PCMBS, indicates that the mechanism of transfer of photoassimilates between veins and surrounding tissues, and the mechanism of the sink-source transition, may not be the same in the leaves of all species. It is speculated that the unusual properties of the C. blumei leaf may be a consequence of the presence, in the minor veins, of "intermediary cells", large companion cells connected to the bundle sheath by abundant plasmodesmata.

Plant plasma membrane H + -ATPases (PM H + -ATPases) energize the secondary transport of Na + from the cytosol across the plasma membrane and so may play a role in the plant response to salt stress. A PM H + -ATPase gene in rice is closely linked to a locus responsible for increased salt resistance. PM H + -ATPases are encoded by large gene families, including 10-12 in tomato ( Lycopersicon esculentum ). Salt stress stimulates the accumulation of PM H + -ATPase transcripts in a variety of plants but the effect of salt stress on the expression of specific isoforms has not been investigated. We isolated a partial-length cDNA clone of a novel tomato PM H + -ATPase gene from salt-stressed expanded leaf tissue and characterized its expression in response to salt and osmotic stress. The gene, LHA8 , is a member of the subfamily including AHA1 , AHA2 , AHA3 , AHA4 , AHA9 , LHA4 , PHA2 and PMA4 , and is most closely related to the Nicotiana plumbaginifolia gene PMA6 . LHA8 transcript accumulation is induced by NaCl exposure. LHA8 is not expressed at detectable levels in roots or expanded leaves and is present at very low levels in unexpanded leaves. LHA8 expression is induced in expanded leaves, unexpanded leaves and roots. Induction appears to be specific to the ionic, rather than the osmotic, effects of NaCl because iso-osmotic levels of polyethylene glycol do not induce message accumulation.

l h e plant plasma membrane H+-ATPase energizes the secondary uptake of nutrients and may facilitate cell expansion by acidifying the cell wall. In yeast, Clc stimulates the accumulation of H+-ATPase mRNA, and the growth rate supported by various sugars is correlated with H+-ATPase protein abundance. Expression of three H+-ATPase genes, LHAI, LHA2, and LHA4, was previously detected in tomato (Lycopersicon esculentum). We have characterized the sequence of the LHA4 gene and examined the expression of these three tomato H+-ATPase genes in growing tissues and i n response to exogenous sugars. LHA4 is a member of the H+-ATPase subfamily, including the Arabidopsis fhaliana genes AHAl, AHA2, and AHA3. l h e 5' untranslated region of the deduced LHA4 cDNA contains a short, open reading frame very similar to that in the Nicofiana plumbaginifolia gene PMAl . LHA4 transcript abundance in seedlings is correlated with cell growth, being 2.5 times greater in hypocotyls of dark-versus light-grown plants. The accumulation of both LHA4 and LHA2 mRNAs is induced by the addition of exogenous sugars and this induction appears to be dependent on sugar uptake and metabolism, because mannitol and 3-Omethylglucose do not stimulate mRNA accumulation. These results suggest that the induction of expression of H+-ATPase genes by metabolizable sugars may be part of a generalized cellular response to increased cell growth and metabolism promoted by the availability of an abundant carbon source.H+-ATPases in the PM plays a critica1 role in the physiology of plants at both the cellular and organismal levels. They establish an electrical potential and pH gradient across the PM, which provides the force for the secondary transport of anions, cations, amino acids, and sugars (Serrano, 1989;Sussman and Harper, 1989). These secondary transport systems control physiological processes such as nutrient uptake by roots, phloem transport, and stomatal function. H+-ATPase in the PM may have other physiological roles, because the enzyme is subjected to control by physiological effectors such as hormones, light, and pathogens (Serrano, 1989;Sussman and Harper, 1989 control of the cell cycle by the regulation of cytosolic pH (Pichon and Desbiez, 1994) and by driving the auxin-induced cell expansion by cell-wall acidification (Rayle and Cleland, 1992). Total H+-ATPase levels in the PM may be directly correlated with growth. Hager et al. (1991) demonstrated by immunodetection that H+-ATPase levels in the PM increased 2-fold during auxin-induced maize coleoptile elongation. Rao et al. (1993) showed that the growth rate of liquid cultures of yeast supported by different sugars is closely correlated with PM H+-ATPase activity. Furthermore, the expression of the primary yeast PM H+-ATPase isoform PMAl was shown to be induced by the addition of growth-inducing exogenous sugars.Considerable progress has recently been made in the understanding of the molecular biology of plant PM H+-ATPases. Multiple genes encoding PM H+-ATPases have been cloned in tomato (Lycopers...

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