Na+ and Cl-are the principal solutes utilized for osmotic adjustment in cells of Nicotiana tabacum L. var Wisconsin 38 (tobacco) adapted to NaCi, accumulating to levels of 472 and 386 millimolar, respectively, in cells adapted to 428 millimolar NaCI. X-ray microanalysis of unetched frozen-hydrated cells adapted to salt indicated that Na+ and Cl-were compartmentalized in the vacuole, at concentrations of 780 and 624 millimolar, respectively, while cytoplasmic concentrations of the ions were maintained at 96 millimolar. The morphometric differences which existed between unadapted and salt adapted cells, (cytoplasmic volume of 22 and 45% of the cell, respectively), facilitated containment of the excited volume of the x-ray signal in the cytoplasm of the adapted cells. Confirmation of ion compartmentation in salt adapted cells was obtained based on kinetic analyses of 22NaI and 36CI-efflux from cells in steady state. These data provide evidence that ion compartmentation is a component of salt adaptation of glycophyte ceUs.A typical response of many plants to saline environments, particularly halophytes, is to accumulate high intracellular concentrations of Na+ and Cl- (9,11,23,26,28). Since the in vitro activities of enzymes isolated from glycophytes or halophytes are inhibited equally by NaCl (9, 11), it has been generally accepted that the accumulated ions are sequestered in the vacuole and 'compatible solutes,' such as sugars, proline, and glycinebetaine, function to balance the osmotic pressure of the cytoplasm (9,11,26). This ability to compartmentalize Na+ and Cl-has been considered to be a mechanism of tolerance of halophytes (26).Although salt tolerance of glycophytes, particularly agronomic species, is usually attributed to the ability to exclude Na+ and Cl-, especially from the shoot (8, 11), intracellular ion compartmentation also may occur in these species. Despite indications of negative correlations between ion accumulation and salt tolerance in glycophytes, such as tomato (22), rice (29), wheat (27), and maize (12), examination of the data reveals that, even in tolerant genotypes, the accumulated levels of Na+ and Clare substantial. In some instances whole cell Na+ and Cl-accumulation was similar for both tolerant and sensitive genotypes (22,27,29). Thus, the ability to compartmentalize Na+ and Clmay be an underlying determinant of the tolerance not only of halophytes but also of many crop species.Verification of ion compartmentation has been restricted by the difficulty in obtaining reliable measurements of subcellular ion concentrations (7,15,23,30). Despite this, cytoplasmic Na + concentration in leaf cells of Suaeda maritima was determined to be 165 mm by efflux analysis when the whole cell concentration was about 600 mM (28). Data obtained by x-ray microanalysis of frozen hydrated leaves of Atriplex spongiosa (23) revealed that cytoplasmic ion concentrations were considerably lower than those in the vacuole.Results from investigations of glycophytes exposed to salinity have been less conc...

It is widely reported that some humic substances behave as exogenous auxins influencing root growth by mechanisms that are not yet completely understood. This study explores the hypothesis that the humic acids' effects on root development involve a nitric oxide signaling. Maize seedlings were treated with HA 20 mg C L(-1), IAA 0.1 nM, and NO donors (SNP or GSNO), in combination with either the auxin-signaling inhibitor PCIB, the auxin efflux inhibitor TIBA, or the NO scavenger PTIO. H(+)-transport-competent plasma membrane vesicles were isolated from roots to investigate a possible link between NO-induced H(+)-pump and HA bioactivity. Plants treated with either HA or SNP stimulated similarly the lateral roots emergence even in the presence of the auxin inhibitors, whereas NO scavenger diminished this effect. These treatments induced H(+)-ATPase stimulation by threefold, which was abolished by PTIO and decreased by auxin inhibitors. HA-induced NO synthesis was also detected in the sites of lateral roots emergence. These data depict a new scenario where the root development stimulation and the H(+)-ATPase activation elicited by either HA or exogenous IAA depend essentially on mechanisms that use NO as a messenger induced site-specifically in the early stages of lateral root development.

Cell lines of tobacco (Nicotiana tabacum L. var Wisconsin 38) were obtained which are adapted to grow in media with varying concentrations of NaCI, up to 35 grams per liter (599 millimolar).

Cells of Nicotiana tabacum L. var Wisconsin 38 adapted to NaCi (up to 428 millimolar) which have undergone extensive osmotic adjustment accumulated Na' and a-as principal solutes for this adjustment. Although the intracellular concentrations of Na' and a-correlated well with the level of adaptation, these ions apparently did not contribute to the osmotic adjustment which occurred during a culture growth cycle, because the concentrations of Na' and a-did not increase during the period of most active osmotic adjustment. The average intracellular concentrations of soluble sugars and total free amino acids increased as a function of the level of adaptation; however, the levels of these solutes did not approach those observed for Na' and a-. The concentration of proline was positively correlated with cell osmotic potential, accumulating to an average concentration of 129 millimolar in cells adapted to 428 millimolar Naa and representing about 80% of the total free amino acid pool as compared to an average of 0.29 millimolar and about 4% of the pool in unadapted cells. These results indicate that although Na' and aare principal components of osmotic adjustment, organic solutes also may make significant contributions.Osmotic adjustment is a fundamental adaptive response of plant cells which are exposed to salinity (8,11,26) and is necessary for survival and growth under saline conditions. Osmotic adjustment in response to salinity is a result of solute accumulation which occurs through the uptake of solutes, the synthesis of organic compounds, or both. The identification of solutes which accumulate in response to salinity is an initial step toward elucidating the biochemical and physiological mechanisms which are responsible for and regulate osmotic adjustment. Halophytes typically utilize Na+ and Cl-as principal osmotica (8,11,26), although organic solutes apparently serve an important role in balancing the osmotic pressure ofthe cytoplasm with that of the vacuole, into which much of the Na+ and Cl-is thought to be compartmentalized (8,26,28). In response to moderate levels of salinity, many glycophytic plants appear to exclude Na+ and Cl-as a mechanism of tolerance (21), using instead the synthesis and accumulation of organic compounds for osmotic adjustment (1 1, 28). However, the ability of glycophytic plants to accumulate Na+ and Cl-and survive high levels of salinity has not been investigated thoroughly.The in vitro isolation ofcells ofglycophytes with enhanced salt tolerance has helped facilitate the study of cellular responses to salinity ( 1-3, 15, 24). These salt tolerant cells are especially useful

osmotica (3, 11, 14). Because cytosolic physiological and biochemical processes apparently are inhibited by high ion concentrations, mechanisms that permit compartmentation of these ions in the vacuole and regulate the concentrations of these ions in the cytosol are of substantial importance for adaptation and growth (11,14,15 for the uphill transport of ions and other solutes into the vacuole. The A/H. across the tonoplast is produced by electrogenic H+ pumps, the H+-ATPase (22), and the H+ translocating pyrophosphatase (17, 25). However, the significance of the pyrophosphatase H+ pump to the generation of the ,AH+ across the tonoplast under physiological conditions is not clear (17,25).H+-ATPases that have been categorized on the basis of similar biochemical properties as V-type enzymes act as H+ pumps in the membranes of a wide variety of acidic compartments in eukaryotes (22). The similarities of the protein sequences of these enzymes have recently been confirmed by analysis of cDNA and genomic clones encoding the major ATPase subunits (12,22). The V-type H+-ATPase is a multimeric enzyme of about 400 to 600 kD that consists of up to nine subunits (22,23). The putative catalytic 70-kD subunit forms a hydrophilic complex with a 60-kD subunit that is the principal component of ATPase activity. The 16-kD proteolipid subunits form a hydrophobic complex that functions in H+ conductance. Additional subunits are postulated to form a stalk connecting these two complexes or to be associated with the proton channel (22).It is reasonable to assume that the V-type H+-ATPase is an enzyme that has to be functional at all times during cell growth. Disruption of the genes encoding the 60-or 70-kD subunits of yeast vacuolar H+-ATPase caused conditional lethality. The mutants grew more slowly than the wild-type cells, failed to grow at neutral pH, and lacked the ability to acidify their vacuoles ( 18,28