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.     

Increased Expression of a myo-Inositol Methyl Transferase in Mesembryanthemum crystallinum Is Part of a Stress Response Distinct from Crassulacean Acid Metabolism Induction

The facultative halophyte Mesembryanthemum crystallinum responds to osmotic stress by switching from C₃ photosynthesis to Crassulacean acid metabolism (CAM). This shift to CAM involves the stress-initiated up-regulation of mRNAs encoding CAM enzymes. The capability of the plants to induce a key CAM enzyme, phosphoenolpyruvate carboxylase, is influenced by plant age, and it has been suggested that adaptation to salinity in M. crystallinum may be modulated by a developmental program that controls molecular responses to stress. We have compared the effects of plant age on the expression of two salinity-induced genes: Gpdl, which encodes the photosynthesis-related enzyme glyceraldehyde 3-phosphate dehydrogenase, and Imtl, which encodes a methyl transferase involved in the biosynthesis of a putative osmoprotectant, pinitol. Imtl mRNA accumulation and the accompanying increase in pinitol in stressed Mesembryanthemum exhibit a pattern of induction distinct from that observed for CAM-related genes. We conclude that the molecular mechanisms that trigger Imtl and pinitol accumulation in response to salt stress in M. crystallinum differ in some respects from those that lead to CAM induction. There may be multiple signals or pathways that regulate inducible components of salinity tolerance in this facultative halophyte.     

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.     

Distinct responses to copper stress in the halophyte Mesembryanthemum crystallinum

Selective gene expression allows the halophyte Mesembryanthemum crystallinum to survive a salt stress. To broaden our understanding of the environmental cues initiating diverse stress responses in this higher plant, unstressed and 0.4 M NaCl‐stressed plants were compared to plants treated with several concentrations of copper (CuSO₄), an increasingly relevant environmental heavy metal pollutant. Comparisons of control and copper‐stressed plants included germination, chlorophyll content, accumulation of proline, heat shock protein (HSP) 60 and a Crassulacean acid metabolism (CAM)‐specific marker enzyme, phospho enol pyruvate carboxylase (PEPCase). In germination and whole plant tests, M. crystallinum was significantly more tolerant to copper than Arabidopsis thaliana. Mature M. crystallinum plants stressed with 50 ppm CuSO₄ for 48 h became dehydrated. These plants produced a 4‐fold increase in proline concentration and accumulated both the CAM‐specific PEPCase and HSP 60 compared to controls. Higher levels of copper stress resulted in a 10‐fold increase in leaf proline content, 10‐fold HSP 60 accumulation but no detectable PEPCase protein compared to unstressed controls. HSP 60 did not accumulate under NaCl stress. Concurrent with copper‐induced genetic responses to stress, copper was accumulated and concentrated in leaves (3 500 ppm). Together, these results suggest that this halophyte copes with copper metal exposure through distinct genetic mechanisms.     

水分胁迫对露花磷酸烯醇式丙酮酸羧化酶表达水平及特性的影响

水分胁迫能引起露花叶片PEP羧化酶的活力、酶蛋白和mRNA水平的提高。复水后,叶片PEP羧化酶表达量降低;茎中的PEP羧化酶在水分胁迫和恢复水分供应过程中变化情况与叶片相似,兼性CAM植物的碳代谢类型转变发生在植物的绿色组织中。露花叶片中除了250kD的PEP羧化酶同功酶外,还有300kD同功酶;主茎的叶片叶位越低,PEP羧化酶活力越高。     

The Irreversible C3 to CAM Shift in Well-watered and Salt-stressed Plants of Mesembryanthemum crystallinum is under Strict Ontogenetic Control

The shift from C₃ to CAM was investigated as a function of both leaf and plant age in well-watered and salt-stressed (300 mM NaCl solution) plants of Mesembryanthemum crystallinum. Initiation of a night-time accumulation of malic acid, the decisive criterion of CAM, was followed in plants that were continuously stressed at different points in their life cycle. The deinducibility of CAM was examined after the release from stress by extensively rinsing the potting soil with de-mineralized water. Our results show that in M. crystallinum CAM is under strict developmental control, since CAM appeared only when a certain stage of development of the whole plant was reached. CAM was not present in any plant before this threshold, which was the same in salt-stressed as well as in well-watered plants. The metabolic shift coincided with the change from the seedling to the juvenile growth phase, and not with that from vegetative to reproductive growth, represented by the start of branching. The latter is timed to the end of extension growth. In well-watered plants, after this decisive point in development, a weak nighttime accumulation of malic acid could be measured (? 0.05 mol kg_DW^?1) in the oldest, mature leaves but not in young, developing ones. This “CAM capacity” gradually increased up to 0.2 mol kg_DE^?1 with further plant ageing. Leaf senescence, characterized by wilting and yellowing, diminished the CAM activity. In mature leaves salt stress drastically enhanced the magnitude of diurnal fluctuation in malic acid content. Removal of salt stress did not deinduce CAM activity, but diminished the amplitude of malic acid oscillations to some extent in those plants which had been stressed from early in their life cycle. In these plants, salt stress delayed plant development and growth thus retarding the life cycle. Well-watered plants, for example, branched about three weeks earlier than those that had been stressed continuously from one week after germination. After removal of stress a quasi-preserved earlier developmental stage in relation to the control plants determined the weaker CAM expression.     

Effects of growth regulators on the induction of Crassulacean acid metabolism in the facultative halophyte Mesembryanthemum crystallinum L.

The classical induction of Crassulacean acid metabolism (CAM) in Mesembryanthemum crystallinum L. by water stress is observed within one week when fourto five-week-old plants (grown under a 16/8 h photoperiod at ca. 600 mol quanta · m^–2 · s^–1) are irrigated with 350 mM NaCl. The induction of CAM was evaluated by measuring phosphoenolpyruvate carboxylase (PEPCase, EC 4.1.1.31) and NADP-malic enzyme (NADP-ME, EC 4.1.1.82) activities and nocturnal increases in malate content and titratable acidity of leaf extracts, and the daily pattern of CO₂ exchange and stomatal conductance during the 7-d induction period. Three growth regulators, abscisic acid (ABA), farnesol (an antitranspirant and analog of ABA), and benzylaminopurine (BAP), were found to substitute for NaCl for induction of CAM when fed to plants in nutrient media. Daily irrigation with solutions containing micromolar levels (optimum ca. 10 micromolar) of these growth regulators led to the induction of CAM similar to that by high salt. Application of the growth regulators, like NaCl, caused large increases in the activity of NADP-ME and the activity and level of PEPCase, which are components of the biochemical machinery required for CAM. Western immunoblotting showed that the increased activity of PEPCase on addition of ABA, farnesol and BAP was mainly due to increased levels of the CAM-specific isoforms. Also, dehydration of cut leaves over 8.5 h under light resulted in a severalfold increase in PEPCase activity. An equivalent increase in PEPCase activity in excised leaves was also obtained by feeding 150 mM NaCl, or micromolar levels of ABA or BAP via the petiole, which supports results obtained by feeding the growth regulators to roots. However, the increase in PEPCase activity was inhibited by feeding high levels of BAP to cut leaves prior to dehydration, indicating a more complex response to the cytokinin. Abscisic acid may have a role in induction of CAM in M. crystallinum under natural conditions as there is previous evidence that induction by NaCl causes an increase in the content of ABA, but not cytokinins, in leaves of this species.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - CAM Crassulacean acid metabolism - Chl chlorophyll - 2,4D 2,4-dichlorophenoxyacetic acid - NADP-ME NADP-malic enzyme - PEPCase phosphoenolpyruvate carboxylase Methyl jasmonate was generously provided by Dr. Vincent Franceschi (Botany Department, Washington State University). The anti-maize leaf PEPCase was kindly supplied by Dr. Tatsuo Sugiyama (Department of Agricultural Chemistry, Nagoya University, Japan) and the anti-Flaveria trinervia leaf PEPCase was kindly supplied by Dr. Samuel Sun (Department of Plant Molecular Physiology, University of Hawaii, Honulu). This work was funded in part by U.S. Department of Agriculture Competitive Grant 90-37280-5706 and an equipment grant (DMB 8515521) from the National Science Foundation. Ziyu Dai was supported in part by Guangxi Agricultural College and Ministry of Agriculture of the People's Republic of China