As sessile organisms, plants are subject to numerous environmental insults. Of these, salinity is one of the most widespread and important in terms of limiting plant distribution and productivity. Molecular studies have established that plants challenged by high salinity respond by increasing expression of specific genes. A functional role for the products of such genes in stress tolerance has not been established, however, and little is known about the biochemical mechanisms that allow plants to tolerate osmotic stress. Mesembryanthemum crystallinum is a facultative halophyte capable of adjusting to and surviving in highly saline conditions. I have generated and screened a subtracted cDNA library to identify mRNAs that accumulate during this plant's adaptation to salt stress. Three mRNAs were identified that increased in abundance in leaf tissue of salt stressed plants. Patterns of induction for these mRNAs differed. The most dramatically-induced mRNA, Imt1, was characterized in depth. Imt1 expression was induced in leaves and, transiently, in roots. Nuclear run-on assays indicated that the gene is transcriptionally regulated. In several respects, the expression of Imt1 differed from that of other salinity-responsive genes involved in photosynthetic metabolism in M. crystallinum: The mRNA was induced by salinity and low temperature, but not by drought, and its induction by stress was not influenced by plant age. Imt1 encoded a predicted polypeptide of Mr 40,250 which exhibited sequence similarity to several hydroxymethyl transferases. The IMT1 protein was expressed in E. coli and identified by functional assay as a myo-inositol methyl transferase that catalyzes the first step in the biosynthesis of the cyclic sugar alcohol pinitol. The presence of high levels of sugar alcohols has been correlated with osmotolerance in a wide range of organisms, and the stress-initiated transcriptional induction of IMT1 expression in a facultative halophyte provides the strongest support to date for the importance of sugar alcohols in establishing tolerance to osmotic stress in higher plants. The ability of this methyl transferase to generate a putative osmoprotectant from a ubiquitous plant substrate makes it an attractive candidate enzyme for the creation of stress-resistant transgenic plants.

The halophyte Mesembryanthemum crystallinum L. accumulates high concentrations of NaCl (up to 1 M) in its leaf cells as a response to soil salinity. While there is evidence that vacuolar sodium transport is mediated by a tonoplast Na⁺/H⁺ antiporter (Barkla ^{et al.}, 1995), little is known about the transport of Cl^- transport into the vacuole. So far, it has been uncertain whether secondary active transport (e.g. H⁺/C1^- antiporter) is involved or whether a passive mechanism (Cl^- channel) is sufficient to mediate Cl^- accumulation in the vacuole of M.crystallinum. This thesis describes the use of tonoplast vesicles from leaf mesophyll cells of M. crystallinum to study vacuolar Cl^- transport. Cl^- uptake into the vesicles was measured using the Cl^--sensitive fluorescent dye lucigenin (N/N'-dimethyl-9,9'-bisacridinium dinitrate). This work was complemented by a patch-clamp study of ionic currents of leaf-mesophyll vacuoles from M.crystallinum. Cl^- transport into tonoplast vesicles showed saturation-type kinetics with an apparent Km between 10 and 36 mM and a maximum initial change of the intravesicular Cl^- concentration of 4.8 mM min^-, corresponding to an estimated Cl^- flux of 31 nmol m² s^-¹. Vacuolar chloride transport was not affected by sulphate, malate, or nitrate, indicating a high specificity of this transport process for chloride over other anions. By imposing insidepositive membrane potentials using a K⁺/valinomycin clamp revealed a sigmoidal voltagedependent relationship with the steepest increase in vacuolar Cl^- uptake around +30 mV. Only under severe salt treatment with 500 mM NaCl for 3 weeks did 9-week-old M. crystallinum plants show a significant increase (63%) of vacuolar C^- uptake, along with an increased V-type H⁺-ATPase hydrolytic activity (up to 65%). The apparent K_m of vacuolar Cl^- uptake was also increased from 27 mM to 44 mM under these conditions. An inside-acid pH gradient, generated by a K⁺/nigericin clamp, reduced the initial rate of chloride transport into tonoplast vesicles of M.crystallinum. External Cl^-, in contrast to external Na⁺ , did not dissipate an inside-acidic ΔpH generated by various techniques. This is strong evidence against a proton-driven antiport mechanism. The patch-clamp study of ionic currents of whole vacuoles and excised vacuolar membrane patches from M.crystallinum. leaf-mesophyll cells revealed a number of cation channels. At cytosolic free Ca ²⁺ concentrations of 1μM and above, ubiquitous slow-vacuolar type cation currents could be observed. In excised patches, eleven different single channel types, with conductances ranging from 2 up to 200 pS, could be described. However, no clear Cl^-conductance could be identified. The lack of observed vacuolar Cl^- channel activities is discussed in the light of possible lack of Cl^--channel activation due to the loss of a cytosolic factor. The results obtained from the biochemical work on tonoplast vesicles support the hypothesis that a passive transport mechanism (i.e. channel) is sufficient to mediate vacuolar chloride transport in M.crystallinum. The observed upregulation of vacuolar Cl^- transport under severe salt stress shows that it plays an important role in the salt adaptation of this halophytic plant.

No description available.

The genes that comprise the multi-gene family encoding the small subunit of the CO₂ fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in the facultative Crassulacean Acid Metabolism (CAM) plant Mesembryanthemum crystallinum were isolated and nucleotide sequences were determined. The gene family is comprised of six genes of which four are expressed (rbcS-1, rbcS-2, rbcS-3 and rbcS-4) based on the isolation of corresponding cDNAs and northern analysis. Sequence analysis of the genes reveals influences of gene duplication, gene conversion and selection pressure in the evolution of the gene family. Five of the genes are located in a tandem array within 20 kbp. Two are identical throughout the coding regions and intron sequences. The promoter regions of five of the genes lack regulatory sequence elements conserved in other dicot species. Gene specific probes were used to determine the expression properties of the rbcS-1, rbcS-2, rbcS-3 and rbcS-4 genes during environmental stress, development and the diurnal cycle. The four genes are differentially expressed and are co-regulated in response to stress and during development. Steady state rbcS mRNA amounts are down regulated by NaCl and cold stress. Comparison of changes in transcriptional activities relative to steady state mRNA amounts revealed that NaCl causes increased rbcS RNA turnover. RbcS expression is transcriptionally down regulated coincident with the developmental enablement of CAM induction by stress. The regulation of rbcS expression by changes in RNA turnover is itself developmentally regulated. The rbcS genes of M. crystallinum are transcriptionally regulated during development and are regulated at the level of RNA turnover by stress.