Convergent Induction of Osmotic Stress-Responses : Abscisic Acid, Cytokinin, and the Effects of NaCl

In Mesembryanthemum crystallinum, salt stress induces the accumulation of proline and a specific isoform of the enzyme phosphoenolpyruvate carboxylase (PEPCase) prior to the switch from C₃ to Crassulacean acid metabolism (CAM). To determine whether plant growth regulators initiate or imitate these responses, we have compared the effects elicited by NaCl, abscisic acid (ABA), and cytokinins using PEPCase and proline levels as diagnostic tools. Exogenously applied ABA is a poor substitute for NaCl in inducing proline and CAM-specific PEPCase accumulation. Even though ABA levels increase 8- to 10-fold in leaves during salt stress, inhibition of ABA accumulation does not affect these salt-induced responses. In contrast, the addition of cytokinins (6-benzylaminopurine, zeatin, 2-isopentyladenine) mimic salt by greatly increasing proline and PEPCase amounts. Endogenous zeatin levels remain unchanged during salt stress. We conclude: (a) The salt-induced accumulation of proline and PEPCase is coincident with, but is not attributable to, the rise in ABA or zeatin concentration. (b) For the first time, cytokinins and NaCl are implicated as independent initiators of a sensing pathway that signals leaves to alter PEPCase gene expression. (c) During stress, the sensing of osmotic imbalances leading to ABA, proline, and CAM-specific PEPCase accumulation may be mediated directly by NaCl.     

Salt Stress Increases the Level of Translatable mRNA for Phosphoenolpyruvate Carboxylase in Mesembryanthemum crystallinum

Mesembryanthemum crystallinum responds to salt stress by switching from C₃ photosynthesis to Crassulacean acid metabolism (CAM). During this transition the activity of phosphoenolpyruvate carboxylase (PEPCase) increases in soluble protein extracts from leaf tissue. We monitored CAM induction in plants irrigated with 0.5 molar NaCl for 5 days during the fourth, fifth, and sixth week after germination. Our results indicate that the age of the plant influenced the response to salt stress. There was no increase in PEPCase protein or PEPCase enzyme activity when plants were irrigated with 0.5 molar NaCl during the fourth and fifth week after germination. However, PEPCase activity increased within 2 to 3 days when plants were salt stressed during the sixth week after germination. Immunoblot analysis with anti-PEPCase antibodies showed that PEPCase synthesis was induced in both expanded leaves and in newly developing axillary shoot tissue. The increase in PEPCase protein was paralleled by an increase in PEPCase mRNA as assayed by immunoprecipitation of PEPCase from the in vitro translation products of RNA from salt-stressed plants. These results demonstrate that salinity increased the level of PEPCase in leaf and shoot tissue via a stress-induced increase in the steady-state level of translatable mRNA for this enzyme.     

A fruit-specific phosphoenolpyruvate carboxylase is related to rapid growth of tomato fruit

Malic and citric acids accumulate in cherry tomato (Lycopersicon esculentum Mill.) fruit during the period of rapid growth, from the end of cell division to the onset of ripening. The involvement of phospho enolpyruvate carboxylase (PEPCase, EC 4.1.1.31) in organic acid accumulation and tomato fruit development was investigated. Two PEPCases, named LYCes;Ppc1 and LYCes;Ppc2 and mapped to chromosomes 12 and 7, respectively, were shown to be differentially expressed during tomato fruit development. LYCes;Ppc1 mRNA was present in all fruit tissues and in all other plant organs examined. In contrast, LYCes;Ppc2 was strongly and specifically expressed in fruit from the end of cell division to ripening. No LYCes;Ppc2 expression was detected by northern blot in other plant tissues. In fruit, the increase in LYCes;Ppc2 mRNA was closely followed by an increase in fruit PEPCase protein and activity, and was coincident with the increased accumulation of malate and citrate during the initial period of rapid growth rate, from 8 to 20 days post anthesis. Localization of LYCes;Ppc2 mRNA in young tomato fruit by in situ hybridization revealed that LYCes;Ppc2 is preferentially expressed in large cells of the pericarp and in enlarging cells of the gel surrounding the seeds. Examination of the kinetic and regulatory properties of the PEPCases of growing and ripening fruit further showed that PEPCase in growing fruit is less sensitive to low pH and malate inhibition, indicating a high phosphorylation state and/or the presence of a PEPCase isoform with these characteristics. Taken together, these results indicate that in developing tomato fruit PEPCase is probably important in permitting the synthesis of organic acids to provide the turgor pressure necessary for cell expansion.     

Influence of NaCl on Growth, Proline, and Phosphoenolpyruvate Carboxylase Levels in Mesembryanthemum crystallinum Suspension Cultures

The facultative halophyte Mesembryanthemum crystallinum responds to salt stress by increasing the levels of phosphoenolpyruvate carboxylase (PEPCase) and other enzymes associated with Crassulacean acid metabolism. A more common response to salt stress in sensitive and tolerant species, including M. crystallinum, is the accumulation of proline. We have established M. crystallinum suspension cultures to investigate whether both these salt-induced responses occur at the cellular level. Leaf-and root-derived cultures maintain 5% of the total soluble amino acids as proline. Cell culture growth slows upon addition of 400 millimolar NaCl, and proline levels increase to 40% of the total soluble amino acids. These results suggest a functional salt-stress and response program in Mesembryanthemum cells. Suspension cultures grown with or without 400 millimolar NaCl have PEPCase levels that compare with those from roots and unstressed leaves. The predominant protein cross-reacting with an anti-PEPCase antibody corresponds to 105 kilodaltons (apparent molecular mass), whereas a second species of approximately 110 kilodaltons is present at low levels. In salt-stressed leaves, the 110 kilodalton protein is more prevalent. Levels of mRNA for both ppc1 (salt stress induced in leaves) and ppc2 (constitutive) genes in salt-treated suspensions cultures are equal to unstressed leaves, and only twice the levels found in untreated suspension cultures. Whereas cells accumulate proline in response to NaCl, PEPCase protein amounts remain similar in salt-treated and untreated cultures. The induction upon salt stress of the 110 kilodalton PEPCase protein and other Crassulacean acid metabolism enzymes in organized tissues is not observed in cell culture and may depend on tissue-dependent or photoautotrophy-dependent programs.