Effect of hypermethylation of CCWGG sequences in DNA of Mesembryanthemum crystallinum plants on their adaptation to salt stress.

Under salt stress conditions, the level of CpNpG-methylation (N is any nucleoside) of the nuclear genome of the facultative halophyte Mesembryanthemum crystallinum in the CCWGG sequences (W = A or T) increases two-fold and is coupled with hypermethylation of satellite DNA on switching-over of C3-photosynthesis to the crassulacean acid metabolism (CAM) pathway of carbon dioxide assimilation. The methylation pattern of the CCWGG sequences is not changed in both the 5'-promoter region of the gene of phosphoenolpyruvate carboxylase, the key enzyme of C4-photosynthesis and CAM, and in the nuclear ribosomal DNA. Thus, a specific CpNpG-hypermethylation of satellite DNA has been found under conditions of expression of a new metabolic program. The functional role of the CpNpG-hypermethylation of satellite DNA is probably associated with formation of a specialized chromatin structure simultaneously regulating expression of a large number of genes in the cells of M. crystallinum plants on their adaptation to salt stress and switching-over to CAM metabolism.

Induction of Crassulacean acid metabolism by water limitation.

Crassulacean acid metabolism (CAM), a key adaptation of photosynthetic carbon fixation to limited water availability, is characterized by nocturnal CO2 fixation and daytime CO2 re-assimilation, which generally results in improved water-use efficiency. However, CAM plants display a remarkable degree of photosynthetic plasticity within a continuum of diel gas exchange patterns. Genotypic, ontogenetic and environmental factors combine to govern the extent to which CAM is expressed. The ecological diversity of CAM is mirrored by plasticity in a range of biochemical and physiological attributes. In C3/CAM-intermediate plants, limited water availability can induce or enhance the expression of CAM. CAM induction is controlled by a combination of transcriptional, post-transcriptional and post-translational regulatory events. Early events in CAM induction point to a requirement for calcium and calcium-dependent protein kinase activities. Gene discovery efforts, improved transformation technologies and genetic models for CAM plants, coupled with detailed physiological investigations, will lead to new insights into the molecular genetic basis of induction processes and the circadian oscillator that governs carbon flux during CAM. Future integration of genomic, biochemical and physiological approaches in selected CAM models promise to provide a detailed view of the complex regulatory dynamics involved in CAM induction and modulation by water deficit. Such information is expected to have broad significance as the ecological and agricultural importance of CAM species increases in the face of global warming trends and the associated expansion of desertification in semi-arid regions around the world.

Genes that are uniquely stress regulated in salt overly sensitive (sos) mutants.

Repetitive rounds of differential subtraction screening, followed by nucleotide sequence determination and northern-blot analysis, identified 84 salt-regulated (160 mM NaCl for 4 h) genes in Arabidopsis wild-type (Col-0 gl1) seedlings. Probes corresponding to these 84 genes and ACP1, RD22BP1, MYB2, STZ, and PAL were included in an analysis of salt responsive gene expression profiles in gl1 and the salt-hypersensitive mutant sos3. Six of 89 genes were expressed differentially in wild-type and sos3 seedlings; steady-state mRNA abundance of five genes (AD06C08/unknown, AD05E05/vegetative storage protein 2 [VSP2], AD05B11/S-adenosyl-L-Met:salicylic acid carboxyl methyltransferase [SAMT], AD03D05/cold regulated 6.6/inducible2 [COR6.6/KIN2], and salt tolerance zinc finger [STZ]) was induced and the abundance of one gene (AD05C10/circadian rhythm-RNA binding1 [CCR1]) was reduced in wild-type plants after salt treatment. The expression of CCR1, SAMT, COR6.6/KIN2, and STZ was higher in sos3 than in wild type, and VSP2 and AD06C08/unknown was lower in the mutant. Salt-induced expression of VSP2 in sos1 was similar to wild type, and AD06C08/unknown, CCR1, SAMT, COR6.6/KIN2, and STZ were similar to sos3. VSP2 is regulated presumably by SOS2/3 independent of SOS1, whereas the expression of the others is SOS1 dependent. AD06C08/unknown and VSP2 are postulated to be effectors of salt tolerance whereas CCR1, SAMT, COR6.6/KIN2, and STZ are determinants that must be negatively regulated during salt adaptation. The pivotal function of the SOS signal pathway to mediate ion homeostasis and salt tolerance implicates AD06C08/unknown, VSP2, SAMT, 6.6/KIN2, STZ, and CCR1 as determinates that are involved in salt adaptation.

A stress-induced calcium-dependent protein kinase from Mesembryanthemum crystallinum phosphorylates a two-component pseudo-response regulator.

McCDPK1 is a salinity- and drought-induced calcium-dependent protein kinase (CDPK) isolated from the common ice plant, Mesembryanthemum crystallinum. A yeast two-hybrid experiment was performed, using full-length McCDPK1 and truncated forms of McCDPK1 as baits, to identify interacting proteins. A catalytically impaired bait isolated a cDNA clone encoding a novel protein, CDPK substrate protein 1 (CSP1). CSP1 interacted with McCDPK1 in a substrate-like fashion in both yeast two-hybrid assays and wheat germ interaction assays. Furthermore, McCDPK1 was capable of phosphorylating CSP1 in vitro in a calcium-dependent manner. Our results demonstrate that the use of catalytically impaired and unregulated CDPKs with the yeast two-hybrid system can accelerate the discovery of CDPK substrates. The deduced CSP1 amino acid sequence indicated that it is a novel member of a class of pseudo-response regulator-like proteins that have a highly conserved helix-loop-helix DNA binding domain and a C-terminal activation domain. McCDPK1 and CSP1 co-localized to nuclei of NaCl-stressed ice plants. Csp1 transcript accumulation was not regulated by NaCl or dehydration stress. Our results strongly suggest that McCDPK1 may regulate the function of CSP1 by reversible phosphorylation.

A minimal serine/threonine protein kinase circadianly regulates phosphoenolpyruvate carboxylase activity in crassulacean acid metabolism-induced leaves of the common ice plant.

Plant phosphoenolpyruvate carboxylase (PEPc) activity and allosteric properties are regulated by PEPc kinase (PPcK) through reversible phosphorylation of a specific serine (Ser) residue near the N terminus. We report the molecular cloning of PPcK from the facultative Crassulacean acid metabolism (CAM) common ice plant (Mesembryanthemum crystallinum), using a protein-kinase-targeted differential display reverse transcriptase-polymerase chain reaction approach. M. crystallinum PPcK encodes a minimal, Ca(2+)-independent Ser/threonine protein kinase that is most closely related to calcium-dependent protein kinases, yet lacks both the calmodulin-like and auto-inhibitory domains typical of plant calcium-dependent protein kinase. In the common ice plant PPcK belongs to a small gene family containing two members. McPPcK transcript accumulation is controlled by a circadian oscillator in a light-dependent manner. McPPcK encodes a 31.8-kD polypeptide (279 amino acids), making it among the smallest protein kinases characterized to date. Initial biochemical analysis of the purified, recombinant McPPcK gene product documented that this protein kinase specifically phosphorylates PEPc from CAM and C(4) species at a single, N-terminal Ser (threonine) residue but fails to phosphorylate mutated forms of C(4) PEPc in which this specific site has been changed to tyrosine or aspartate. McPPcK activity was specific for PEPc, Ca(2+)-insensitive, and displayed an alkaline pH optimum. Furthermore, recombinant McPPcK was shown to reverse the sensitivity of PEPc activity to L-malate inhibition in CAM-leaf extracts prepared during the day, but not at night, documenting that PPcK contributes to the circadian regulation of photosynthetic carbon flux in CAM plants.

Genomic approaches to plant stress tolerance.

Past efforts to improve plant tolerance to drought, high salinity and low-temperature through breeding and genetic engineering have had limited success owing to the genetic complexity of stress responses. Progress is now anticipated through comparative genomics studies of an evolutionarily diverse set of model organisms, and through the use of techniques such as high-throughput analysis of expressed sequence tags, large-scale parallel analysis of gene expression, targeted or random mutagenesis, and gain-of-function or mutant complementation. The discovery of novel genes, determination of their expression patterns in response to abiotic stress, and an improved understanding of their roles in stress adaptation (obtained by the use of functional genomics) will provide the basis of effective engineering strategies leading to greater stress tolerance.

Efficient plant regeneration of Mesembryanthemum crystallinum via somatic embryogenesis.

An efficient plant regeneration procedure has been established from hypocotyl explants of the common ice plant, Mesembryanthemum crystallinum L, a halophytic leaf succulent that exhibits a stress-induced switch from C3 photosynthesis to crassulacean acid metabolism (CAM). Somatic embryos were initiated and developed up to globular and heart stages in Murashige and Skoog (MS) media supplemented with 3% sucrose, 0.6% bacto-agar, 80 mM NaCl, 5 µM 2,4-D and 1 µM kinetin. High frequency regeneration occurred when somatic embryos were germinated on media that lacked 2,4-D. High cytokinin treatment suppressed normal growth of embryos and favored abnormal embryo proliferation. Without growth regulators, regenerated plants rooted on MS medium with 100% efficiency. Mature, regenerated plants were fertile and morphologically identical to seed-derived plants.

cDNA clones encoding 1,3-beta-glucanase and a fimbrin-like cytoskeletal protein are induced by Al toxicity in wheat roots.

A cDNA library made from mRNA of Al-treated roots of an Al-sensitive wheat (Triticum aestivum cv Victory) cultivar was screened with a degenerate oligonucleotide probe derived from the partial amino acid sequence of the Al-induced protein TAI-18. Of seven clones that initially hybridized with the probe, one encoded a novel 1,3-beta-glucanase having a calculated molecular weight of 46.3 and an isoelectric point of 6.0. Like the A6 1,3-beta-glucanase gene products from Brassica napus and Arabidopsis thaliana, the predicted wheat protein had a C-terminal extension with three potential glycosylation sites. Northern analysis revealed that wheat 1,3-beta-glucanase mRNA was up-regulated in Al-intoxicated roots, with highest expression after 12 h. The antibody to A6 1,3-beta-glucanase from B. napus cross-reacted with a 56-kD protein that was induced after 24 h. A second partial cDNA clone showed similarity to genes encoding cytoskeletal fimbrin-like (actin-bundling) proteins. Although well studied in animals and fungi, fimbrins have not previously been described in plants. Fimbrin-like transcripts were up-regulated after 24 h of Al treatment in the Al-sensitive wheat cv Victory. In the Al-tolerant cv Atlas 66, fimbrin-like mRNA was up-regulated within 12 h by Al concentrations that did not inhibit root growth. Cellular stress associated with Al toxicity therefore causes up-regulation of a defense-related gene and a gene involved in the maintenance of cytoskeletal function.

Molecular Genetics of Crassulacean Acid Metabolism.

Most higher plants assimilate atmospheric CO2 through the C3 pathway of photosynthesis using ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). However, when CO2 availability is reduced by environmental stress conditions, the incomplete discrimination of CO2 over O2 by Rubisco leads to increased photorespiration, a process that reduces the efficiency of C3 photosynthesis. To overcome the wasteful process of photorespiration, approximately 10% of higher plant species have evolved two alternate strategies for photosynthetic CO2 assimilation, C4 photosynthesis and Crassulacean acid metabolism. Both of these biochemical pathways employ a "CO2 pump" to elevate intracellular CO2 concentrations in the vicinity of Rubisco, suppressing photorespiration and therefore improving the competitiveness of these plants under conditions of high light intensity, high temperature, or low water availability. This CO2 pump consists of a primary carboxylating enzyme, phosphoenolpyruvate carboxylase. In C4 plants, this CO2-concentrating mechanism is achieved by the coordination of two carboxylating reactions that are spatially separated into mesophyll and bundle-sheath cell types (for review, see R.T. Furbank, W.C. Taylor [1995] Plant Cell 7: 797-807;M.S.B. Ku, Y. Kano-Murakami, M. Matsuoka [1996] Plant Physiol 111: 949-957). In contrast, Crassulacean acid metabolism plants perform both carboxylation reactions within one cell type, but the two reactions are separated in time. Both pathways involve cell-specific changes in the expression of many genes that are not present in C3 plants.

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.

Posttranscriptional and posttranslational control of enolase expression in the facultative Crassulacean acid metabolism plant Mesembryanthemum Crystallinum L.

During the induction of Crassulacean acid and metabolism by environmental stresses in the common ice plant (Mesembryanthemum crystallinum L.), enzyme activities involved in glycolysis and gluconeogenesis, including enolase (2-phospho-D-glycerate hydrolase), increase significantly. In this study, we describe two nearly identical cDNA clones (Pgh1a and Pgh1b) encoding enolase from the common ice plant. This cytoplasmically localized enzyme is encoded by a gene family of at least two members. The polypeptides encoded by these cDNAs share a high degree of amino acid sequence identity (86.7-88.3%) with other higher plant enolases. Enolase activity increased more than 4-fold in leaves during salt stress. This increase was accompanied by a dramatic increase in Pgh1 transcription rate and the accumulation of enolase transcripts in leaves. Pgh1 transcript levels also increased in leaves in response to low temperature, drought, and anaerobic stress conditions and upon treatment of unstressed plants with the plant growth regulators abscisic acid and 6-benzylaminopurine. In roots, enolase transcripts increased in abundance in response to salt, low and high temperature, and anaerobic stresses. Surprisingly, we observed no increase in enolase protein levels, despite the increased levels of mRNA and enzyme activity during salt stress. The stress-induced increase in enolase activity is therefore due to posttranslational regulation of steady-state enzyme pools. Our results demonstrate that the stress-induced shift to Crassulacean acid metabolism in the ice plant involves complex regulatory control mechanisms that operate at the transcriptional, posttranscriptional, and postranslational levels.

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.

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.

Molecular cloning and expression of chloroplast NADP-malate dehydrogenase during Crassulacean acid metabolism induction by salt stress.

A full-length cDNA clone for NADP(+)-dependent malate dehydrogenase (NADP-MDH; EC 1.1.1.82) from the facultative CAM plant,Mesembryanthemum crystallinum has been isolated and characterized. NADP-MDH is responsible for the reduction of oxaloacetate to malate in the chloroplasts of higher plants. The cDNA clone is 1747 bp in size and contains a single open reading frame encoding a 441 amino acid long precursor polypeptide with a predicted molecular weight of 47 949. The predicted, mature NADP-MDH polypeptide sequence fromM. crystallinum shares 82.7% to 84% amino acid identity with other known higher plant sequences. Genomic Southern blot analysis ofM. crystallinum DNA indicates that MDH is encoded by a small gene family. Steady-state transcript levels for chloroplast NADP-MDH decrease transiently in the leaves after salt stress and then increase to levels greater than two-fold higher than in unstressed plants. Transcript levels in roots are extremely low and are unaffected by salt-stress treatment. In vitro transcription run-on experiments using isolated nuclei from leaf tissue confirm that the accumulation of NADP-MDH transcripts is, at least in part, the result of increased transcription of this gene during salt stress. The salt-stress-induced expression pattern of this enzyme suggests that it may participate in the CO2 fixation pathway during CAM.

Salt stress alters A/T-rich DNA-binding factor interactions within the phosphoenolpyruvate carboxylase promoter from Mesembryanthemum crystallinum.

The common ice plant, Mesembryanthemum crystallinum, shifts from C3 to crassulacean acid metabolism (CAM) photosynthesis in response to osmotic stress. The expression of a number of genes encoding enzymes involved in the CAM pathway increases as a result of increased transcription rates. To begin to investigate the mechanisms responsible for the transcriptional activation, we have characterized the 5' control region of a specific isoform of phosphoenolpyruvate carboxylase gene (Ppc1) that plays a key role in CAM. We have determined the nucleotide sequence of the 5' flanking region of this gene. Ppc1 contains a long 5'-leader sequence with the transcriptional start site located 332/333 nucleotides 5' of the translational initiation codon. Multiple DNA interactions with nuclear factors are detectable within the 5'-flanking region of Ppc1. We have used copper orthophenanthroline footprinting to demonstrate that one particularly abundant factor (designated PCAT-1) binds the Ppc1 promoter at two distinct A/T-rich sites located -128 to -158 and -187 to -205 bp upstream of the transcriptional start site. These binding sites share a loose consensus motif having the sequence AARTAAC(T/A)A(G/T)TTTY. Gel retardation competition experiments with oligonucleotides containing these A/T-rich binding sites suggest that both sites bind the same factor, but with different affinities. Fractionation of crude nuclear extracts by heparin-agarose chromatography indicates that PCAT-1 is more prevalent in extracts prepared from salt-stressed leaf tissue. Additional binding activities that interact with the PCAT-1 binding sites have been detected that either increase or decrease in abundance or binding affinity in response to salt stress.

Characterization and expression of a NADP-malic enzyme cDNA induced by salt stress from the facultative crassulacean acid metabolism plant, Mesembryanthemum crystallinum.

The facultative halophyte and crassulacean acid-metabolism plant, Mesembryanthemum crystallium shifts from C3 photosynthesis to crassulacean acid metabolism when exposed to high-salt or drought conditions. To study the molecular basis of this metabolic transition, the expression of NADP(+)-dependent malic enzyme (NADP-ME), which catalyzes the decarboxylation of malate to release pyruvate and CO2, has been investigated. The complete nucleotide sequence of a full-length cDNA clone was determined and found to contain a single open reading frame encoding a 585-amino-acid polypeptide of 64284 Da. The ice plant (M. crystallinum) NADP-ME shares amino acid identities in the range 72.5-79.0% when compared to other higher-plant enzymes and is more closely related to C3 rather than C4 forms of the enzyme. Genomic Southern-blot analysis of ice-plant DNA indicates that NADP-ME is encoded by a small gene family. Steady-state transcript levels increase 8-10-fold in response to salt stress in the leaves. Transcript levels in roots are extremely low and are unaffected by salt-stress treatment. Nuclear run-on experiments, using isolated nuclei from leaf tissue, confirm that the accumulation of NADP-ME transcripts is, in part, the result of increased transcription of this gene during salt stress.