Expression and stress-dependent induction of potassium channel transcripts in the common ice plant. (1/25)

We have characterized transcripts for three potassium channel homologs in the AKT/KAT subfamily (Shaker type) from the common ice plant (Mesembryanthemum crystallinum), with a focus on their expression during salt stress (up to 500 mM NaCl). Mkt1 and 2, Arabidopsis AKT homologs, and Kmt1, a KAT homolog, are members of small gene families with two to three isoforms each. Mkt1 is root specific; Mkt2 is found in leaves, flowers, and seed capsules; and Kmt1 is expressed in leaves and seed capsules. Mkt1 is present in all cells of the root, and in leaves a highly conserved isoform is detected present in all cells with highest abundance in the vasculature. MKT1 for which antibodies were made is localized to the plasma membrane. Following salt stress, MKT1 (transcripts and protein) is drastically down-regulated, Mkt2 transcripts do not change significantly, and Kmt1 is strongly and transiently (maximum at 6 h) up-regulated in leaves and stems. The detection and stress-dependent behavior of abundant transcripts representing subfamilies of potassium channels provides information about tissue specificity and the complex regulation of genes encoding potassium uptake systems in a halophytic plant.  (+info)

Early salt stress effects on the changes in chemical composition in leaves of ice plant and Arabidopsis. A Fourier transform infrared spectroscopy study. (2/25)

A technique based on Fourier transform infrared (FT-IR) spectrometry was developed to detect the corresponding changes in chemical composition associated with the rapid changes in sodium and water content in 200 mM NaCl-stressed halophyte ice plants (Mesembryanthemum crystallinum). The changes in glycophyte Arabidopsis stressed with 50 mM NaCl were also examined for comparison. The obtained IR spectra were further processed by deconvolution and curve fitting to examine the chemical nature of the responding sources in the leaves. Using three stages of ice plant leaves, absorption bands corresponding to carbohydrates, cell wall pectin, and proteins were identified, with distinct IR spectra representing each developmental stage. Within 48 h of mild salt stress, the absorption band intensities in the fingerprint region increased continuously in both plants, suggesting that the carbon assimilation was not affected at the early stage of stress. The intensities of ester and amide I absorption bands decreased slightly in Arabidopsis but increased in ice plant, suggesting that the cell expansion and protein synthesis ceased in Arabidopsis but continued in ice plant. In both plants, the shift in amide I absorption band was observed hourly after salt stress, indicating a rapid conformational change of cellular proteins. Analyses of the ratio between major and minor amide I absorption band revealed that ice plant was able to maintain a higher-ordered form of proteins under stress. Furthermore, the changes in protein conformation showed a positive correlation to the leaf sodium contents in ice plant, but not in Arabidopsis.  (+info)

A novel Mg(2+)-dependent O-methyltransferase in the phenylpropanoid metabolism of Mesembryanthemum crystallinum. (3/25)

Upon irradiation with elevated light intensities, the ice plant (Mesembryanthemum crystallinum) accumulates a complex pattern of methylated and glycosylated flavonol conjugates in the upper epidermal layer. Identification of a flavonol methylating activity, partial purification of the enzyme, and sequencing of the corresponding peptide fragments revealed a novel S-adenosyl-l-methionine-dependent O-methyltransferase that was specific for flavonoids and caffeoyl-CoA. Cloning and functional expression of the corresponding cDNA verified that the new methyltransferase is a multifunctional 26.6-kDa Mg(2+)-dependent enzyme, which shows a significant sequence similarity to the cluster of caffeoyl coenzyme A-methylating enzymes. Functional analysis of highly homologous members from chickweed (Stellaria longipes), Arabidopsis thaliana, and tobacco (Nicotiana tabacum) demonstrated that the enzymes from the ice plant, chickweed, and A. thaliana possess a broader substrate specificity toward o-hydroquinone-like structures than previously anticipated for Mg(2+)-dependent O-methyltransferases, and are distinctly different from the tobacco enzyme. Besides caffeoyl-CoA and flavonols, a high specificity was also observed for caffeoylglucose, a compound never before reported to be methylated by any plant O-methyltransferase. Based on phylogenetic analysis of the amino acid sequence and differences in acceptor specificities among both animal and plant O-methyltransferases, we propose that the enzymes from the Centrospermae, along with the predicted gene product from A. thaliana, form a novel subclass within the caffeoyl coenzyme A-dependent O-methyltransferases, with potential divergent functions not restricted to lignin monomer biosynthesis.  (+info)

The N-myristoylated Rab-GTPase m-Rabmc is involved in post-Golgi trafficking events to the lytic vacuole in plant cells. (4/25)

We report on the sub-cellular localisation and function of m-Rab(mc), a N-myristoylated plant-specific Rab-GTPase previously characterised at the molecular level and also by structural analysis in Mesembryanthemum crystallinum. By confocal laser scanning microscopy, we identified m-Rab(mc) predominantly on the prevacuolar compartment of the lytic vacuole but also on the Golgi apparatus in various plant cell types. Two complementary approaches were used immunocytochemistry and cyan fluorescent protein (CFP)/yellow fluorescent protein (YFP)-fusion proteins. Co-localisation studies of m-Rab(mc) with a salinity stress modulated integral calcium-ATPase suggest involvement of m-Rab(mc) in a plant-specific transport pathway to the prevacuolar compartment of the lytic vacuole. This hypothesis was strengthened by the inhibition of the transport of aleurain fused to green fluorescent protein (GFP), a marker of the lytic vacuole, in the presence of the dominant negative mutant m-Rab(mc)(N147I) in Arabidopsis thaliana protoplasts. The inhibitory effect of m-Rab(mc)(N147I) was specific for the transport pathway to the lytic vacuole, since the transport of chitinase-YFP, a marker for the neutral vacuole, was not hindered by the mutant.  (+info)

Regiospecificity and kinetic properties of a plant natural product O-methyltransferase are determined by its N-terminal domain. (5/25)

A recently discovered, S-adenosyl-L-methionine and bivalent cation-dependent O-methyltransferase from the ice plant, Mesembryanthemum crystallinum, is involved in the methylation of various flavonoid and phenylpropanoid conjugates. Differences in regiospecificity as well as altered kinetic properties of the recombinant as compared to the native plant O-methyltransferase can be attributed to differences in the N-terminal part of the protein. Upon cleavage of the first 11 amino acids, the recombinant protein displays essentially the same substrate specificity as observed earlier for the native plant enzyme. Product formation of the newly designed, truncated recombinant enzyme is consistent with light-induced accumulation of methylated flavonoid conjugates in the ice plant. Therefore, substrate affinity and regiospecificity of an O-methyltransferase in vivo and in vitro can be controlled by cleavage of an N-terminal domain.  (+info)

Novel regulation of aquaporins during osmotic stress. (6/25)

Aquaporin protein regulation and redistribution in response to osmotic stress was investigated. Ice plant (Mesembryanthemum crystallinum) McTIP1;2 (McMIPF) mediated water flux when expressed in Xenopus leavis oocytes. Mannitol-induced water imbalance resulted in increased protein amounts in tonoplast fractions and a shift in protein distribution to other membrane fractions, suggesting aquaporin relocalization. Indirect immunofluorescence labeling also supports a change in membrane distribution for McTIP1;2 and the appearance of a unique compartment where McTIP1;2 is expressed. Mannitol-induced redistribution of McTIP1;2 was arrested by pretreatment with brefeldin A, wortmannin, and cytochalasin D, inhibitors of vesicle trafficking-related processes. Evidence suggests a role for glycosylation and involvement of a cAMP-dependent signaling pathway in McTIP1;2 redistribution. McTIP1;2 redistribution to endosomal compartments may be part of a homeostatic process to restore and maintain cellular osmolarity under osmotic-stress conditions.  (+info)

Conservation and divergence of circadian clock operation in a stress-inducible Crassulacean acid metabolism species reveals clock compensation against stress. (7/25)

One of the best-characterized physiological rhythms in plants is the circadian rhythm of CO(2) metabolism in Crassulacean acid metabolism (CAM) plants, which is the focus here. The central components of the plant circadian clock have been studied in detail only in Arabidopsis (Arabidopsis thaliana). Full-length cDNAs have been obtained encoding orthologs of CIRCADIAN CLOCK-ASSOCIATED1 (CCA1)/LATE ELONGATED HYPOCOTYL (LHY), TIMING OF CAB EXPRESSION1 (TOC1), EARLY FLOWERING4 (ELF4), ZEITLUPE (ZTL), FLAVIN-BINDING KELCH REPEAT F-BOX1 (FKF1), EARLY FLOWERING3 (ELF3), and a partial cDNA encoding GIGANTEA in the model stress-inducible CAM plant, Mesembryanthemum crystallinum (Common Ice Plant). TOC1 and LHY/CCA1 are under reciprocal circadian control in a manner similar to their regulation in Arabidopsis. ELF4, FKF1, ZTL, GIGANTEA, and ELF3 are under circadian control in C(3) and CAM leaves. ELF4 transcripts peak in the evening and are unaffected by CAM induction. FKF1 shows an abrupt transcript peak 3 h before subjective dusk. ELF3 transcripts appear in the evening, consistent with their role in gating light input to the circadian clock. Intriguingly, ZTL transcripts do not oscillate in Arabidopsis, but do in M. crystallinum. The transcript abundance of the clock-associated genes in M. crystallinum is largely unaffected by development and salt stress, revealing compensation of the central circadian clock against development and abiotic stress in addition to the well-known temperature compensation. Importantly, the clock in M. crystallinum is very similar to that in Arabidopsis, indicating that such a clock could control CAM without requiring additional components of the central oscillator or a novel CAM oscillator.  (+info)

Are the metabolic components of crassulacean acid metabolism up-regulated in response to an increase in oxidative burden? (8/25)

In the halophytic species Mesembryanthemum crystallinum, crassulacean acid metabolism (CAM) may be induced by a range of abiotic factors including drought, salinity, high light intensity, low temperature, and anoxia. A key biotic consequence of all these environmental changes is the generation of reactive oxygen species in planta that can elicit potentially damaging oxidative reactions and/or act as signals for engaging mechanisms that alleviate oxidative stress. However, induction of CAM per se also has the potential for increasing the oxidative burden via the enhanced internal O2 concentrations that develop behind closed stomata during daytime decarboxylation. The aim of this paper was to test two hypotheses. The first one, that reactive oxygen species are key signals for up-regulating the major genes and proteins required for the operation of CAM as part of an integrated strategy for alleviating oxidative burden, was tested using gaseous ozone to increase the oxidative burden at a cellular level. The second hypothesis, that CAM potentially increases oxidative load, was tested using a CAM-deficient mutant of M. crystallinum. The data indicate that ozone, like salinity, elicits an increase in the transcript and protein abundance of myo-inositol o-methyl transferase (a key enzyme of cyclitol synthesis), together with phosphoenolpyruvate carboxylase and other 'CAM-related' enzymes. However, ozone, unlike salinity, does not induce functional CAM, implying that the various metabolic components required for CAM respond to different signals. Comparing the activities of different subcellular isoforms of superoxide dismutase in wild-type and CAM-deficient mutants of M. crystallinum suggests that the induction of CAM potentially curtails the oxidative load in planta.  (+info)

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