Halophyte Mesembryanthemum Crystallinum Research Articles

Na+/H+ exchange in the halophyte Mesembryanthemum crystallinum is associated with cellular sites of Na+ storage.

The tonoplast Na+/H+ exchanger is involved in sequestering Na+ in plant vacuoles, providing solutes for osmotic adjustment while avoiding cytoplasmic Na+ toxicity. As such it is assumed to be one of the key mechanisms involved in salt-tolerance in plants. In this study, we measured tonoplast Na+/H+ exchange in roots and different leaf tissues of adult Mesembryanthemum crystallinum L. plants to determine if activity of the exchanger follows the gradient from roots to leaves previously observed for Na+ and pinitol accumulation. Na+/H+ exchange was absent from roots of control and NaCl-treated plants. In contrast, leaves showed constitutive Na+/H+ exchange that was enhanced by growth of the plants in NaCl. Highest activity was measured in the epidermal bladder cells in agreement with the highest concentrations of Na+ found in this tissue. Tonoplast H+-translocating ATPase activity was also greatest in this tissue, as were protein levels for myo-inositol-O-methyltransferase, a key enzyme in the pinitol biosynthesis pathway. The strong correlation between Na+/H+ exchange and Na+ accumulation confirms the role of this transporter in vacuolar sequestration of Na+ and plant salt tolerance.

Light-induced betacyanin and flavonol accumulation in bladder cells of Mesembryanthemum crystallinum.

Treatment of the halophyte Mesembryanthemum crystallinum L. (ice plant) (Aizoaceae) with high intensities of white light resulted in a rapid cell-specific accumulation of betacyanins and flavonoids with 6-methoxyisorhamnetin 3-O-¿[(2"'-E-feruloyl)-3"'-O-(beta-D- glucopyranosyl)](2"-O-beta-D-xylopyranosyl)]-beta-D-glucopyranoside (mesembryanthin) as the predominant component, within bladder cells of the leaf epidermis. Induced accumulation of these metabolites was first detected 18 h after the initiation of light treatment in bladder cells located at the tip of young leaves followed by the bladder cells located on the epidermis of fully expanded leaves. UV-A light apparently is sufficient to induce accumulation of betacyanins and flavonoids. Application of 2-aminoindan 2-phosphonic acid, a specific inhibitor of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5), not only inhibited the accumulation of flavonoids but also reduced betacyanin formation. Based on these observations we suggest these bladder cells as a model system to study regulation of betacyanin and flavonoid biosyntheses.

Tissue- and environmental response-specific expression of 10 PP2C transcripts in Mesembryanthemum crystallinum.

Ten transcripts (Mpc1-10) homologous to protein phosphatases of the 2C family have been isolated from the halophyte Mesembryanthemum crystallinum (common ice plant). Transcripts range in size from 1.6 to 2.6 kb, and encode proteins whose catalytic domains are between 24% and 62% identical to that of the Arabidopsis PP2C, ABI1. Transcript expression is tissue specific. Two isoforms are present only in roots (Mpc1 and Mpc5), three in young leaves (Mpc6, 8 and 9), two in old leaves (Mpc6 and Mpc8), and two in post-flowering leaves (Mpc8 and Mpc9). Mpc2 is strongly expressed in roots and also in seeds, meristematic tissues and mature flowers. Mpc3 is specific for leaf meristems, and Mpc4 is found in root and leaf meristems. Mpc7 is restricted to meristematic tissues. Mpc10 is only present in mature flowers. Mpc2 (in roots and leaves), Mpc5 (in roots) and Mpc8 (weakly in leaves) are induced by salinity stress and drought conditions with different kinetics in different tissues, but other Mpcs are downregulated by stress. Cold stress (4 degrees C) leads to a decline in Mpc5 and Mp6, but low temperature provoked a long-term (days) increase in Mpc2 levels in leaves and a transient increase (less than 24 h) in roots. Four full-length transcripts have been obtained. In each case, after over-expression in E. coli, the isolated proteins exhibited (Mg2+-dependent, okadeic acid-insensitive) protein phosphatase activity, although activity against 32P-phosphocasein varied among different PP2Cs. Determination of tissue developmental and stress response specificity of PP2C will facilitate functional studies of signal-transducing enzymes in this halophytic organism.

Salt stress in Mesembryanthemum crystallinum L. cell suspensions activates adaptive mechanisms similar to those observed in the whole plant.

A salt-tolerant stable cell-suspension culture from the halophyte Mesembryanthemum crystallinum L. has been established from calli generated from leaves of 6-week-old well-watered plants. Optimal cell growth was observed in the presence of 200 mM NaCl, and within 7 d cells were able to concentrate Na+ to levels exceeding those in the growth medium. Accumulation of Na+ was paralled by increases in the compatible solute pinitol and myo-inositol methyl transferase (IMT), a key enzyme in pinitol biosynthesis. Increasing concentrations of NaCl stimulated the activities of tonoplast and plasma-membrane H(+)-ATPases. Immunodetection of the ATPases showed that the increased activity was not due to changes in protein amount that could be attributed to treatment conditions. A specific role for these mechanisms in salt-adaptation is supported by the inability of mannitol-induced water stress to elicit the same responses, and the absence of enzyme activity and protein expression associated with Crassulacean acid metabolism in the cells. Results demonstrate that these M. crystallinum cell suspensions show a halophytic growth response, comparable to that of the whole plant, and thus provide a valuable tool for studying signaling and biochemical pathways involved in salt recognition and response.

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, phosphoenolpyruvate 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.

Salt‐induced changes in protein composition in light‐grown callus of Mesembryanthemum crystallinum

The halophyte Mesembryanthemum crystallinum (ice plant) has been suggested as a model for salt‐tolerance in higher plants. To investigate salt‐induced changes in polypeptide patterns at the cellular level, a light‐grown callus of M. crystallinum with substantial chlorophyll content, was established and the effect of NaCl on the composition of phenol‐extracted protein was examined by SDS‐ and 2D‐polyacrylamide gel electrophoresis (PAGE). SDS‐PAGE showed the accumulation of five polypeptides with estimated molecular masses of 40, 34, 32, 29 and 14 kDa was enhanced by the addition of 200 mM NaCl to the culture media. The addition of ABA (10 μM) or mannitol (400 mM) did not elicit the same degree of accumulation of these salt‐specific proteins. These polypeptides were classified into two groups according to their course of induction: early responsive (40, 34, 29 kDa) and late‐responsive (32, 14 kDa) proteins. In addition, two polypeptides (20, 18 kDa) were transiently accumulated during salt treatment. Further separation of soluble proteins by 2‐D gel electrophoresis, either isoelectric focusing (IEF) or non‐equilibrium pH‐gradient electrophoresis (NEPHGE) followed by SDS‐PAGE, showed more alterations in accumulation of polypeptides by NaCl than 1‐D gel electrophoresis. Overall, levels of more than 30% of basic polypeptides, detected by NEPHGE/SDS‐PAGE, were altered by 200 mM NaCl treatment, while only 10% of neutral and acidic polypeptides, detected by IEF/SDS‐PAGE, were changed. The enhanced expression of these proteins by salt in cultured cells is most likely related to the cellular responses to salinity, and not to the mechanism of CAM induction in this facultative halophyte.

The 32 kDa tonoplast polypeptide D i associated with the V0-type H +-ATPase of Mesembryanthemum crystallinum L. in the CAM state: A proteolytically processed subunit B?

In the facultative halophyte Mesembryanthemum crystallinum, the salt- or age-induced transition to crassulacean acid metabolism (CAM) leads to the occurrence of a tonoplast-bound 32 kDa polypeptide (D i). The alignment of its N-terminal protein sequence with protein sequences of recently cloned higher plant V-ATPase B-subunits indicates that D i may be derived from subunit B by proteolytic removal of a protein fragment of about 20 kDa from its N-terminus. Furthermore, an antiserum directed against D i cross-reacts with subunit B from Nicotiana tabacum. It inhibits both proton pumping and ATP hydrolysis of the holoenzyme in M. crystallinum. As D i remains firmly attached to the holoenzyme the proteolytic processing may have functional implications.

Coordinate transcriptional induction of myo-inositol metabolism during environmental stress.

The pathway from glucose 6-phosphate (G 6-P) to myoinositol 1-phosphate (Ins 1-P) and myo-inositol (Ins) is essential for the synthesis of various metabolites. In the halophyte Mesembryanthemum crystallinum (common ice plant), two enzymes, myo-inositol O-methyltransferase (IMT1) and ononitol epimerase (OEP1), extend this pathway and lead to the accumulation of methylated inositols, D-ononitol and D-pinitol, which serve as osmoprotectants. This paper describes transcripts for the enzyme, Inps1, encoding myo-inositol 1-phosphate synthase (INPS1), from the ice plant. Two Inps-like sequences are present in the genome. The deduced amino acid sequences of the cloned transcript are 49.5% and 87-90%, respectively, identical to those of yeast and other higher plant sequences. Inps1 RNA amounts are upregulated at least fivefold and amounts of free Ins accumulate approximately 10-fold during salinity stress. Inps1 induction is by transcription, similar to the induction of Imt1. In contrast, Arabidopsis thaliana does not show upregulation of Inps1 or increased amounts of Ins when salt-stressed. The lack of Inps1 induction in Arabidopsis exemplifies differences in glycophytic and halophytic regulation of gene expression at the point of entry into a pathway that leads to osmoprotection. The stress-induced coordinate upregulation of this pathway and its extension by novel enzymes in the ice plant also highlights biochemical differences.

A salinity-induced gene from the halophyte M. crystallinum encodes a glycolytic enzyme, cofactor-independent phosphoglyceromutase

In the facultative halophyte Mesembryanthemum crystallinum (ice plant), salinity stress triggers significant changes in gene expression, including increased expression of mRNAs encoding enzymes involved with osmotic adaptation to water stress and the crassulacean acid metabolism (CAM) photosynthetic pathway. To investigate adaptive stress responses in the ice plant at the molecular level, we generated a subtracted cDNA library from stressed plants and identified mRNAs that increase in expression upon salt stress. One full-length cDNA clone was found to encode cofactor-independent phosphoglyceromutase (PGM), an enzyme involved in glycolysis and gluconeogenesis. Pgm1 expression increased in leaves of plants exposed to either saline or drought conditions, whereas levels of the mRNA remained unchanged in roots of hydroponically grown plants. Pgm1 mRNA was also induced in response to treatment with either abscisic acid or cytokinin. Transcription run-on experiments confirmed that Pgm1 mRNA accumulation in leaves was due primarily to increased transcription rates. Immunoblot analysis indicated that Pgm1 mRNA accumulation was accompanied by a modest but reproductible increase in the level of PGM protein. The isolation of a salinity-induced gene encoding a basic enzyme of glycolysis and gluconeogenesis indicates that adaptation to salt stress in the ice plant involves adjustments in fundamental pathways of carbon metabolism and that these adjustments are controlled at the level of gene expression. We propose that the leaf-specific expression of Pgm1 contributes to the maintenance of efficient carbon flux through glycolysis/gluconeogenesis in conjunction with the stress-induced shift to CAM photosynthesis.

Characterization of IMT1, myo-Inositol O-methyltransferase, from Mesembryanthemum crystallinum

A full-length transcript, Imt1, encoding myo-inositol O-methyltransferase (EC 2.1.1.X) from the halophyte Mesembryanthemum crystallinum was expressed in Escherichia coli. The enzyme, IMT1, uses S-adenosyl-L-methionine to methylate myo-inositol to form D-ononitol. IMT1 with a monomeric mass of 41,000 was isolated by ammonium sulfate fractionation, gel filtration and ion exchange chromatography to apparent purity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified recombinant enzyme was identical to that encoded by the cDNA sequence. The apparent K m for S-adenosylmethionine was 0.18 mM with a V max of 1550 pkat/mg protein. The K m for myo-inositol was 1.32 mM. The reaction became substrate-inhibited by concentrations of S-adenosylmethionine greater than 0.5 mM. Inositol methyltransferase was competitively inhibited 50% with 0.01 mM S-adenosyl-homocysteine, while 1 mM homocysteine, homoserine, or adenosine did not inhibit. The enzyme exhibited a pH optimum of 7.8 and a temperature optimum of 37°C. Activity of the isolated inositol methyltransferase was stable when stored at 4°C.

Identification of enhancer and silencer regions involved in salt-responsive expression of Crassulacean acid metabolism (CAM) genes in the facultative halophyte Mesembryanthemum crystallinum

In response to salinity or drought stress, the facultative halophyte Mesembryanthemum crystallinum will switch from C3 photosynthesis to Crassulacean acid metabolism (CAM). During this switch, the transcription rates of many genes encoding glycolytic, gluconeoagenic, and malate metabolism enzymes are increased. In particular, transcription of the Ppc1 and Gap1 genes encoding a CAM-specific isozyme of phosphoenolpyruvate carboxylase and NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, respectively, is increased by salinity stress. To investigate the molecular basis of salt-induced gene regulation, we examined the Ppc1 and Gap1 promoters for cis-elements and trans-acting factors that may participate in their expression. Ppc1 or Gap1 promoter-beta-glucuronidase chimeric gene constructs containing various deletions were introduced into intact, detached M. crystallinum leaves by microprojectile bombardmen. The Ppc1 5'-flanking region contains several salt-responsive enhancer regions and one silencer region reflecting the complex regulation patterns exhibited by this promoter in vivo. A region localized between nucleotides -977 and -487 relative to the transcriptional start site appears to regulate the magnitude of salt-inducibility. In contrast, the Gap1 promoter contains a single region from -735 to -549 that confers salt-responsive gene expression. Alignment of these 5'-flanking regions reveals several common sequence motifs that resemble consensus binding sites for the Myb class of transcription factors. Electrophoretic gel mobility shift assays indicate that both the -877 to -679 region of Ppc1 and the -735 to -549 region of Gap1 form a DNA-protein complex unique to nuclear extracts from salt-stressed plants. The appearance of this DNA-protein complex upon salt stress suggests that it may participate in salt-induced transcriptional activation of Ppc1 and Gap1.

The Effects of High Salinity, Water-Deficit, and Abscisic Acid on Phosphoeno/pyruvate Carboxylase Activity and Proline Accumulation in Mesembryanthemum crystallinum Cell Cultures

It has been established from previous studies in the halophyte Mesembryanthemum crystallinum that saline conditions induce the synthesis of phospho enol pyruvate carboxylase (PEPC), the performance of Crassulacean acid metabolism (CAM), and the production of compatible solutes such as proline. The objective of the present study was to determine whether factors causing the induction of these processes in leaves were also effective in tissue cultures of M. crystallinum grown under different conditions. In heterotrophically (sucrose)-grown calli of M. crystallinum , low levels of NaCl (10 to 25 mM) were required for optimum growth, while increasing concentrations of salt up to 200 mM caused a progressive decline in growth. Addition of salt to the culture medium caused a progressive increase in proline accumulation (13 fold increase in calli grown in 200 mM NaCl compared with calli grown without salt) but had little affect on PEPC activity in calli grown in sucrose media in the dark or light. Addition of 10 -7 M abscisic acid in the presence of 200 mM NaCl doubled the proline content in light-grown calli, while abscisic acid alone had no affect. However, growth with 10 -7 to 10 -5 M abscisic acid had no affect on levels of PEPC in dark or light-grown calli, in contrast to the previously reported induction of this enzyme and CAM by provision of abscisic acid to whole plants. Water stress (imposed by addition of polyethylene glycol) and low levels of NaCl (10 mM) in combination caused a large increase in proline content in heterotrophically cultured cells, whereas neither were effective alone. In photomixotrophic cell cultures, obtained by replacement of sucrose by soluble starch as the external carbon supply, there was a much higher chlorophyll content, and a limited salt induced increase in PEPC activity (4 fold enhancement during a 12-d, 200-mM, salt-shock period).

Stress perception and response in a facultative halophyte: the regulation of salinity‐induced genes in Mesembryanthemum crystallinum

ABSTRACTThe facultative halophyte Mesembryanthemum crystallinum (ice plant) shifts from C3 to CAM photosynthesis in response to water stress. The switch to CAM can also be induced by high salinity, presumably because osmotic stress causes water loss from leaf and stem tissues. A number of mRNAs (Ppc1, Imt1, B5 and Gpd1) that encode proteins involved in different biochemical pathways accumulate in M. crystallinum leaf tissues when plants are salt‐stressed. We hypothesized that environmental challenges that result in water stress invoke a common mechanism that triggers the coordinated induction of mRNAs involved in different aspects of the ice plant's adaptive stress response. Nuclear run‐on experiments indicated that Ppc1, Imt1, B5 and Gpd1 are all transcriptional I y activated in salt‐stressed leaf tissue. However, a comparison of Ppc1, Imt1, and B5 transcript levels after exposure of plants to growth regulators (6‐benzylamino purine and abscisic acid) and to different environmental stress treatments that affect plant water status (drought, low temperature and salinity) indicated that, despite the coordinated transcriptional activation of these genes during salt stress, their repsonses to other stimuli that also upset water balance are not the same. Our results are consistent with a model involving multiple control mechanisms governing stress perception and molecular response in the ice plant.

Expression of a phosphoenolpyruvate carboxylase promoter from Mesembryanthemum crystallinum is not salt-inducible in mature transgenic tobacco

The 5' flanking region of a salt-stress-inducible, CAM-specific phosphoenolpyruvate carboxylase (PEPC) gene from the facultative halophyte Mesembryanthemum crystallinum, was fused to the beta-glucuronidase (GUS) reporter gene and introduced into Nicotiana tabacum SR1. The Ppc1 promoter displayed high levels of expression in transgenic tobacco quantitatively and qualitatively similar to a full-length 35S CaMV-GUS construct. Histochemical assays revealed that the full-length Ppc1-GUS fusions expressed GUS activity in all tissues except in root tips. While tobacco is capable of utilizing the Ppc1 cis-acting regulatory regions from M. crystallinum to yield high levels of constitutive expression, this glycophyte fails to direct a stress-inducible pattern of gene expression typical of this promoter in its native, facultative halophytic host.

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 C(3) 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.

A novel methyl transferase induced by osmotic stress in the facultative halophyte Mesembryanthemum crystallinum.

Molecular mechanisms of osmotic stress tolerance were studied in Mesembryanthemum crystallinum (ice plant), a facultative halophyte capable of adjusting to and surviving in highly saline conditions. We screened a subtracted cDNA library enriched for salt stress-induced mRNAs to identify transcripts involved in this plant's adaptation to salinity. One mRNA, Imt1, was found to be up-regulated in leaves and, transiently, in roots. Nuclear run-on assays indicated that this mRNA is transcriptionally regulated. Imt1 encoded a predicted polypeptide of M(r) 40,250 which exhibited sequence similarity to several hydroxymethyl transferases. Expression of the protein in Escherichia coli and subsequent activity assays identified the protein as a novel myoinositol O-methyl transferase which catalyzes the first step in the biosynthesis of the cyclic sugar alcohol pinitol. Pinitol accumulates in salt-stressed M.crystallinum and is abundant in a number of salt- and drought-tolerant plants. The presence of high levels of sugar alcohols correlates with osmotolerance in a diverse range of organisms, including bacteria, fungi and algae, as well as higher plants. The stress-initiated transcriptional induction of IMT1 expression in a facultative halophyte provides strong support for the importance of sugar alcohols in establishing tolerance to osmotic stress in higher plants.

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.

Expression during Salt Stress and Nucleotide Sequence of cDNA for Ferredoxin-NADP+ Reductase from Mesembryanthemum crystallinum

In the facultative halophyte Mesembryanthemum crystallinum (common ice plant) the enzyme ferredoxin-NADP(+)-reductase (FNR) is coded for by a small family of 2 to 3 genes. We have determined the expression characteristics as the plants adapt to high salt and the nucleotide sequence of a full-length cDNA coding for the precursor of this chloroplast-located enzyme. On a developmental scale amounts of FNR transcripts and protein are highest in young emerging leaves. The FNR cDNA is a member of a class of genes whose expression is only slightly affected by salt stress. Even less pronounced than mRNA fluctuations, the amount of FNR protein is unaffected by salt stress. The longest FNR cDNA found was 1,419 nucleotides in size. It consisted of 74 nucleotides 5'-leader sequence, 1,095 nucleotides of protein coding sequence encoding 365 amino acids, and 247 nucleotides 3-region excluding a short poly(A(+)) tail. As expected for a nucleus-coded chloroplast protein an amino terminal transit peptide (52 amino acids in length) was found. The mature FNR protein is predicted to contain 313 amino acids corresponding to a protein of M(r) 35,713. The deduced amino acid sequence of the mature FNR protein is 93.2 and 85.9% identical to those of spinach and pea. The transit peptide of pea and spinach have 55.8 and 69.2% identity with that from ice plant.

Time Course of mRNA Induction Elicited by Salt Stress in the Common Ice Plant (Mesembryanthemum crystallinum)

In the facultative halophyte Mesembryanthemum crystallinum (common ice plant), irrigation with solutions containing NaCl induces an alternate mode of carbon dioxide fixation, Crassulacean acid metabolism (CAM). The salt stress protocol which we have established facilitates the study of CAM induction and the correlation of changes in metabolism and gene expression. We have studied the time course of mRNA induction for phosphoenolpyruvate carboxylase (PEPCase) (gene: ppc) and several other enzymes of carbon metabolism during stress. While CAM is not fully established for at least 10 days after the start of stress, mRNA amounts for PEPCase and for other CAM enzymes, such as Pyruvate orthophosphate dikinase, increase between day 2 and 3 after stress induction. Increases continue for at least 5 days. Concomitant with the increase of CAM transcripts, fluctuations in the mRNA amounts for genes rbcS and cab were observed. Transcript levels for these proteins decreased several-fold during a 3 to 4 day period.

Sodium, potassium, chloride and proline concentrations of chloroplasts isolated from a halophyte, Mesembryanthemum crystallinum L.

Concentrations of four major solutes (Na(+), K(+), Cl(-), proline) were determined in isolated, intact chloroplasts from the halophyte Mesembryanthemum crystallinum L. following long-term exposure of plants to three levels of NaCl salinity in the rooting medium. Chloroplasts were obtained by gentle rupture of leaf protoplasts. There was either no or only small leakage of inorganic ions from the chloroplasts to the medium during three rapidly performed washing steps involving precipitation and re-suspension of chloroplast pellets. Increasing NaCl salinity of the rooting medium resulted in a rise of Na(+) und Cl(-) in the total leaf sap, up to approximately 500 and 400 mM, respectively, for plants grown at 400 mM NaCl. However, chloroplast levels of Na(+) und Cl(-) did not exceed 160-230 and 40-60 mM, respectively, based upon a chloroplast osmotic volume of 20-30 μl per mg chlorophyll. At 20 mM NaCl in the rooting medium, the Na(+)/K(+) ratio of the chloroplasts was about 1; at 400 mM NaCl the ratio was about 5. Growth at 400 mM NaCl led to markedly increased concentrations of proline in the leaf sap (8 mM) compared with the leaf sap of plants grown in culture solution without added NaCl (proline 0.25 mM). Although proline was fivefold more concentrated in the chloroplasts than in the total leaf sap of plants treated with 400 mM NaCl, the overall contribution of proline to the osmotic adjustment of chloroplasts was small. The capacity to limit chloroplast Cl(-) concentrations under conditions of high external salinity was in contrast to an apparent affinity of chloroplasts for Cl(-) under conditions of low Cl(-) availability.