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Mesembryanthemum crystallinum (common ice plant), a facultative CAM plant, shifts from C 3 to CAM photosynthesis under salt stress, enhancing water use efficiency. Here we used transcriptomics, proteomics, and targeted metabolomics to profile molecular changes during the diel cycle of C 3 to CAM transition. The results confirmed expected changes associated with CAM photosynthesis, starch biosynthesis and degradation, and glycolysis/gluconeogenesis. Importantly, they yielded new discoveries: 1) Transcripts displayed greater circadian regulation than proteins. 2) Oxidative phosphorylation and inositol methylation may play important roles in initiating the transition. 3) V-type H + -ATPases showed consistent transcriptional regulation, aiding in vacuolar malate uptake. 4) A protein phosphatase 2C, a major component in the ABA signaling pathway, may trigger the C 3 to CAM transition. Our work highlights the potential molecular switches in the C 3 to CAM transition, including the potential role of ABA signaling. SIGNIFICANCE: The common ice plant is a model facultative CAM plant, and under stress conditions it can shift from C 3 to CAM photosynthesis within a three-day period. However, knowledge about the molecular changes during the transition and the molecular switches enabling the transition is lacking. Multi-omic analyses not only revealed the molecular changes during the transition, but also highlighted the importance of ABA signaling, inositol methylation, V-type H + -ATPase in initiating the shift. The findings may explain physiological changes and nocturnal stomatal opening, and inform future synthetic biology effort in improving crop water use efficiency and stress resilience., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The mechanism of ethylene (ET)-regulated salinity stress response remains largely unexplained, especially for semi-halophytes and halophytes. Here, we present the results of the multifaceted analysis of the model semi-halophyte Mesembryanthemum crystallinum L. (common ice plant) ET biosynthesis pathway key components' response to prolonged (14 days) salinity stress. Transcriptomic analysis revealed that the expression of 3280 ice plant genes was altered during 14-day long salinity (0.4 M NaCl) stress. A thorough analysis of differentially expressed genes (DEGs) showed that the expression of genes involved in ET biosynthesis and perception (ET receptors), the abscisic acid (ABA) catabolic process, and photosynthetic apparatus was significantly modified with prolonged stressor presence. To some point this result was supported with the expression analysis of the transcript amount (qPCR) of key ET biosynthesis pathway genes, namely ACS6 (1-aminocyclopropane-1-carboxylate synthase) and ACO1 (1-aminocyclopropane-1-carboxylate oxidase) orthologs. However, the pronounced circadian rhythm observed in the expression of both genes in unaffected (control) plants was distorted and an evident downregulation of both orthologs' was induced with prolonged salinity stress. The UPLC-MS analysis of the ET biosynthesis pathway rate-limiting semi-product, namely of 1-aminocyclopropane-1-carboxylic acid (ACC) content, confirmed the results assessed with molecular tools. The circadian rhythm of the ACC production of NaCl-treated semi-halophytes remained largely unaffected by the prolonged salinity stress episode. We speculate that the obtained results represent an image of the steady state established over the past 14 days, while during the first hours of the salinity stress response, the view could be completely different.

Five decades ago, the first report of a shift from C 3 to CAM (crassulacean acid metabolism) photosynthesis following the imposition of stress was published in this journal. The annual, Mesembryanthemum crystallinum (Aizoaceae), was shown to be a C 3 plant when grown under non-saline conditions, and a CAM plant when exposed to high soil salinity. This observation of environmentally triggered CAM eventually led to the introduction of the term facultative CAM, which categorises CAM that is induced or upregulated in response to water-deficit stress and is lost or downregulated when the stress is removed. Reversibility of C 3 -to-CAM shifts distinguishes stress-driven facultative-CAM responses from purely ontogenetic increases of CAM activity. We briefly review how the understanding of facultative CAM has developed, evaluate the current state of knowledge, and highlight questions of continuing interest. We demonstrate that the long-lived leaves of a perennial facultative-CAM arborescent species, Clusia pratensis, can repeatedly switch between C 3 and CAM in response to multiple wet-dry-wet cycles. Undoubtedly, this is a dedicated response to environment, independent of ontogeny. We highlight the potential for engineering facultative CAM into C 3 crops to provide a flexible capacity for drought tolerance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Mesembryanthemum crystallinum L. (ice plant) develops salt tolerance during the transition from the juvenile to the adult stage through progressive morphological, physiological, biochemical, and molecular changes. Myo -inositol is the precursor for the synthesis of compatible solute D-pinitol and promotes Na+ transport in ice plants. We previously showed that supplying myo -inositol to 9-day-old seedlings alleviates salt damage by coordinating the expression of genes involved in inositol synthesis and transport, affecting osmotic adjustment and the Na/K balance. In this study, we examined the effects of myo -inositol on physiological parameters and inositol-related gene expression in early- and late-stage juvenile plants. The addition of myo -inositol to salt-treated, hydroponically grown late juvenile plants had no significant effects on growth or photosynthesis. In contrast, supplying exogenous myo -inositol to salt-treated early juvenile plants increased leaf biomass, relative water content, and chlorophyll content and improved PSII activity and CO2 assimilation. The treatment combining high salt and myo -inositol synergistically induced the expression of myo -inositol phosphate synthase (INPS ), myo -inositol O -methyltransferase (IMT ), and inositol transporters (INTs ), which modulated root-to-shoot Na/K ratio and increased leaf D-pinitol content. The results indicate that sufficient myo -inositol is a prerequisite for high salt tolerance in ice plant.

Plant-growth-promoting bacteria (PGPB) help plants thrive in polluted environments and increase crops yield using fewer inputs. Therefore, the design of tailored biofertilizers is of the utmost importance. The purpose of this work was to test two different bacterial synthetic communities (SynComs) from the microbiome of Mesembryanthemum crystallinum , a moderate halophyte with cosmetic, pharmaceutical, and nutraceutical applications. The SynComs were composed of specific metal-resistant plant-growth-promoting rhizobacteria and endophytes. In addition, the possibility of modulating the accumulation of nutraceutical substances by the synergetic effect of metal stress and inoculation with selected bacteria was tested. One of the SynComs was isolated on standard tryptone soy agar (TSA), whereas the other was isolated following a culturomics approach. For that, a culture medium based on M. crystallinum biomass, called Mesem Agar (MA), was elaborated. Bacteria of three compartments (rhizosphere soil, root endophytes, and shoot endophytes) were isolated on standard TSA and MA media, stablishing two independent collections. All bacteria were tested for PGP properties, secreted enzymatic activities, and resistance towards As, Cd, Cu, and Zn. The three best bacteria from each collection were selected in order to produce two different consortiums (denominated TSA- and MA-SynComs, respectively), whose effect on plant growth and physiology, metal accumulation, and metabolomics was evaluated. Both SynComs, particularly MA, improved plant growth and physiological parameters under stress by a mixture of As, Cd, Cu, and Zn. Regarding metal accumulation, the concentrations of all metals/metalloids in plant tissues were below the threshold for plant metal toxicity, indicating that this plant is able to thrive in polluted soils when assisted by metal/metalloid-resistant SynComs and could be safely used for pharmaceutical purposes. Initial metabolomics analyses depict changes in plant metabolome upon exposure to metal stress and inoculation, suggesting the possibility of modulating the concentration of high-value metabolites. In addition, the usefulness of both SynComs was tested in a crop plant, namely Medicago sativa (alfalfa). The results demonstrate the effectiveness of these biofertilizers in alfalfa, improving plant growth, physiology, and metal accumulation.

Mesembryanthemum crystallinum (common ice plant) is one of the facultative halophyte plants, and it serves as a model for investigating the molecular mechanisms underlying its salt stress response and tolerance. Here we cloned one of the homeobox transcription factor (TF) genes, McHB7 , from the ice plant, which has 60% similarity with the Arabidopsis AtHB7 . Overexpression of the McHB7 in Arabidopsis (OE) showed that the plants had significantly elevated relative water content (RWC), chlorophyll content, superoxide dismutase (SOD), and peroxidase (POD) activities after salt stress treatment. Our proteomic analysis identified 145 proteins to be significantly changed in abundance, and 66 were exclusively increased in the OE plants compared to the wild type (WT). After salt treatment, 979 and 959 metabolites were significantly increased and decreased, respectively, in the OE plants compared to the WT. The results demonstrate that the McHB7 can improve photosynthesis, increase the leaf chlorophyll content, and affect the TCA cycle by regulating metabolites (e.g., pyruvate) and proteins (e.g., citrate synthase). Moreover, Mc HB7 modulates the expression of stress-related proteins (e.g., superoxide dismutase, dehydroascorbate reductase, and pyrroline-5-carboxylate synthase B) to scavenge reactive oxygen species and enhance plant salt tolerance.

Background: Ice plant is a halophyte, known for its antioxidant activity and for being a highly functional food. It is capable of increasing its contents of health-promoting compounds when subjected to certain stresses such as salinity. The objective of this work was to determine the plant's best growing conditions to achieve both an optimal production of bioactive metabolites and high crop yield. Mesembryanthemum crystallinum were grown under semi-controlled conditions and four saline treatments were applied at: 0, 100, 200 and 300 mmol L -1 sodium chloride (NaCl), respectively., Results: The 100 mmol L -1 NaCl treatment induced a slight increase in shoot dry weight (DW) and enhanced the leaf area. At higher salinity levels, however, the shoot biomass decreased. The concentration of starch and total proteins declined as the concentration of salt increased, while the total soluble sugars (TSS) content was lower in 100 and 300 mmol L -1 NaCl treatments. Proline increased in conditions over 100 mmol L -1 NaCl. Furthermore, plants grown with 300 mmol L -1 of NaCl presented the highest values of glutathione, ascorbic acid and vitamin C. Antioxidant enzymes activity and total phenolics increased with the severity of the salinity., Conclusion: Ice plant accumulates high levels of health-promoting compounds when grown with 300 mmol L -1 NaCl. A high concentration of beneficial compounds, however, is detrimental to the plant's growth. Moreover, 100 mmol L -1 NaCl treatment not only improved the concentration of bioactive and antioxidant compounds but also preserved the crop yield. It could thus be interesting to promote the cultivation of this high nutritional value plant in environments of moderate salinity. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Among all post-translational modifications of proteins, phosphorylation is one of the most common and most studied. Since plants are sessile organisms, many physiological processes on which their survival depends are regulated by phosphorylation and dephosphorylation. Understanding the extent to which a plant proteome is phosphorylated at specific developmental stages and/or under certain environmental conditions is essential for identifying molecular switches that regulate physiological processes and responses. While most phosphoproteomic workflows proposed in the literature provide tools to exclusively analyze phosphorylated proteins, it is imperative to examine both the proteome and the phosphoproteome to reveal the true complexity of a biological process. Here we describe a mass spectrometry-based phosphoproteomics workflow to analyze both total and phosphorylated proteins. Our method includes phenol-based protein extraction, as well as techniques to measure the quantity and quality of protein extracts. In addition, we compare in detail the efficiency and suitability of in-gel and in-solution trypsin digestion methods. A metal oxide affinity chromatography technique for rapid and efficient enrichment of phosphorylated peptides and an LC-MS/MS method for analysis of the phosphorylated peptides are described. Finally, we present and discuss the results generated by applying this workflow to our study of the C 3 to CAM transition in the common ice plant (Mesembryanthemum crystallinum). Overall, our workflow provides robust methods for the identification of phosphoproteins and total proteins. It can be broadly applied to many other organisms and sample types, and the results provide a more accurate picture of the molecular switches that regulate different biological processes., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The shoot epidermal cell wall in land-plants is associated with a polyester, cutin, which controls water loss and possibly organ expansion. Covalent bonds between cutin and its neighbouring cell-wall polysaccharides have long been proposed. However, the lack of biochemical evidence makes cutin-polysaccharide linkages largely conjectural. Here we optimised a portfolio of radiochemical assays to look for cutin-polysaccharide ester bonds in the epidermis of pea epicotyls, ice-plant leaves and tomato fruits, based on the hypothesis that a transacylase remodels cutin in a similar fashion to cutin synthase and cutin:cutin transacylase activities. Through in-situ enzyme assays and chemical degradations coupled with chromatographic analysis of the 3 H-labelled products, we observed that among several wall-related oligosaccharides tested, only a xyloglucan oligosaccharide ([ 3 H]XXXGol) could acquire ester-bonds from endogenous cutin, suggesting a cutin:xyloglucan transacylase (CXT). CXT activity was heat-labile, time-dependent, and maximal at near-neutral pH values. In-situ CXT activity peaked in nearly fully expanded tomato fruits and ice-plant leaves. CXT activity positively correlated with organ growth rate, suggesting that it contributes to epidermal integrity during rapid expansion. This study uncovers hitherto unappreciated re-structuring processes in the plant epidermis and provides a step towards the identification of CXT and its engineering for biotechnological applications., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

The halophyte ice plant (Mesembryanthemum crystallinum) converts its mode of photosynthesis from C 3 to crassulacean acid metabolism (CAM) during severe water stress. During the transition to CAM, the plant induces CAM-related genes and changes its diurnal stomatal behavior to take up CO 2 efficiently at night. However, limited information concerning this signaling exists. Here, we investigated the changes in the diurnal stomatal behavior of M. crystallinum during its shift in photosynthesis using a detached epidermis. M. crystallinum plants grown under C 3 conditions opened their stomata during the day and closed them at night. However, CAM-induced plants closed their stomata during the day and opened them at night. Quantitative analysis of endogenous phytohormones revealed that trans-zeatin levels were high in CAM-induced plants. In contrast, the levels of jasmonic acid (JA) and JA-isoleucine were severely reduced in CAM-induced plants, specifically at night. CAM induction did not alter the levels of abscisic acid; however, inhibitors of abscisic acid synthesis suppressed CAM-induced stomatal closure. These results indicate that M. crystallinum regulates the diurnal balance of cytokinin and JA during CAM transition to alter stomatal behavior., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Mesembryanthemum crystallinum (common ice plant) is a halophyte species that has adapted to extreme conditions. In this study, we cloned a McHB7 transcription factor gene from the ice plant. The expression of McHB7 was significantly induced by 500 mM NaCl and it reached the peak under salt treatment for 7 days. The McHB7 protein was targeted to the nucleus. McHB7 -overexpressing in ice plant leaves through Agrobacterium -mediated transformation led to 25 times more McHB7 transcripts than the non-transformed wild type (WT). After 500 mM NaCl treatment for 7 days, the activities of superoxide dismutase (SOD) and peroxidase (POD) and water content of the transgenic plants were higher than the WT, while malondialdehyde (MDA) was decreased in the transgenic plants. A total of 1082 and 1072 proteins were profiled by proteomics under control and salt treatment, respectively, with 22 and 11 proteins uniquely identified under control and salt stress, respectively. Among the 11 proteins, 7 were increased and 4 were decreased after salt treatment. Most of the proteins whose expression increased in the McHB7 overexpression (OE) ice plants under high salinity were involved in transport regulation, catalytic activities, biosynthesis of secondary metabolites, and response to stimulus. The results demonstrate that the McHB7 transcription factor plays a positive role in improving plant salt tolerance.

Physiology and nutritional quality of a facultative CAM plant Mesembryanthemum crystallinum under drought stress alone are poorly understood. To induce drought, M. crystallinum was cultured aeroponically with different nutrient spraying intervals such as 5, 30, 60 and 240 min. The long spraying interval such as 240 min resulted in lower mass of root and shoot, shorter total root length with less tips and smaller surface area, compared to short interval of 5 min. Grown under the longest spraying interval of 240 min, M. crystallinumalso had significantly higher leaf dry matter content but lower leaf succulence. However, CAM acidity was undetectable for any plants. Although M. crystallinum grown under extended spraying intervals had higher photosynthetic pigments, they utilized lesser light energy and did not dissipate heat as effectively as those grown under 5 min. Compare to other shorter spraying intervals, photosynthetic gas exchange rates were significantly reduced under 240 min spraying interval, indicating signs of water deficit stress. Shoot nitrate, total reduced nitrogen, total soluble protein and Rubisco concentrations were similar for all plants. For phytochemicals and dietary minerals, plants grown under 240 min spraying interval had significantly higher values than the other plants. Therefore, drought does not result in the induction of CAM but regulates photosynthetic performance and enhances nutritional quality of M. crystallinum., Competing Interests: The authors have declared that no competing interests exist.

Roasting improved the determined protein and carbohydrate content of the flour compared to raw flour (p < 0.05). Baking enhanced the determined moisture and ash content of the flour compared to all treatments (p < 0.05). Similar amino acid content was found in both raw and treated flours with glutamic acid, glycine, arginine, and aspartic acid being predominant. Cooking reduced the total aromatic and non-essential amino acid content whereas roasting reduced the total essential amino acid content of samh flour. All treatments significantly (p < 0.05) decreased the antinutritional factors compared to untreated raw flour. Baking decreased the trypsin inhibitor activity by almost 98.7% whereas cooking reduced phytate and tannin content by 38.5% and 10.8, respectively. Roasting and baking significantly (p < 0.05) improved the in vitro protein digestibility of the flour. In vivo, the true faecal nitrogen digestibility of rats was significantly (p < 0.05) enhanced by all treatments. Baking and cooking increased (p < 0.05) the net protein utilization and biological value of the flour. Overall, the treatments improved the nutritional quality of samh flour.

Many areas exhibiting increased concentrations of soluble salts are simultaneously polluted with heavy metals (HM), and halophytes with extended tolerance to heavy metal toxicity seem to represent a promising tool for their phytoremediation. In this study, the response of the soil-grown C 3 -CAM (Crassulacean acid metabolism) intermediate halophyte Mesembryanthemum crystallinum (common ice plant) to increased concentrations of Cd (0.01-1 mM) was investigated. None of the tested Cd treatments affected growth parameters or tissue water content of either C 3 or CAM-performing plants. Chlorophyll a fluorescence confirmed high tolerance of the photosynthetic apparatus of both metabolic states towards Cd. Plants performing both photosynthesis types accumulated significant Cd amounts only under the highest (1 mM) treatment, and the metal was primarily deposited in the roots, which are features typical of an excluding strategy. Upon the application of 1 mM Cd solution CAM-performing plants, due to the NaCl pre-treatment applied for CAM induction, were exposed to significantly higher amounts of bioavailable Cd in comparison with those of C 3 -performing plants. As a result, roots of CAM plants accumulated over 4-fold higher Cd amounts when compared with C 3 plants. In our opinion, enhanced Cd-accumulating potential observed in CAM-performing plants was the effect of osmotic stress episode and resulting modifications e.g. in the detoxifying capacity of the antioxidative system. Increased antioxidative potential of NaCl pre-treated plants was pronounced with significantly higher activity of CuZnSOD (copper-zinc superoxide dismutase), not achievable in C 3 plants subjected to high Cd concentrations. Moreover, the applied Cd doses induced SOD activity in a compartment-dependent manner only in C 3 plants. We confirmed that none of the applied Cd concentrations initiated the metabolic shift from C 3 to CAM., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Key Message: Transgenic callus and roots of ice plant with altered SnRK1 function were established using Agrobacterium-mediated transformation. The role of McSnRK1 in controlling Na + influx and Na/K ratio was demonstrated. SnRK1 kinases (SNF1-related protein kinase1) control metabolic adaptation during energy deprivation and regulate protective mechanisms against environmental stress. Yeast SNF1 activates a P-type ATPase, the Na + exclusion pump, under glucose starvation. The involvement of plant SnRK1 in salt stress response is largely unknown. We previously identified a salt-induced McSnRK1 in the halophyte ice plant (Mesembryanthemum crystallinum). In the current study, the function of McSnRK1 in salt tolerance was analyzed in transgenic cultured cells and roots of ice plant. Ice plant callus constitutively expressed a high level of McSnRK1 and introducing the full-length McSnRK1 did not alter the Na/K ratio at 24 h after 200 mM NaCl treatment. However, interfering with McSnRK1 activity by introducing a truncate McSnRK1 to produce a dominant-negative form of McSnRK1 increased cellular Na + accumulation and Na/K ratio. As a result, the growth of cultured cells diminished under salt treatment. Hydroponically grown ice plants with roots expressing full-length McSnRK1 had better growth and lowered Na/K ratio compared to the wild-type or vector-only plants. Roots expressing a truncate McSnRK1 had reduced growth and high Na/K ratio under 400 mM NaCl treatment. The changes in Na/K ratio in transgenic cells and whole plants demonstrated the function of SnRK1 in controlling Na + flux and maintaining Na/K homeostasis under salinity. The Agrobacterium-mediated transformation system could be a versatile tool for functional analysis of genes involved in salt tolerance in the ice plant.

Microbially enhanced availability and phytoextraction is a promising technique for phytoremediation of lead (Pb). In this study, Pb resistant strains were isolated and investigated for potential effects on plant growth and Pb phytoextraction. Incubation experiments were carried for inoculated and un-inoculated soil containing different levels of Pb. Results revealed that 20% of the isolated bacteria could tolerate Pb up to 800 mg L -1 . Five Pb resistant strains with plant growth promoting (PGP) abilities were evaluated for possible influence on water soluble Pb through soil incubation experiments and significant increase i.e. 1.85- and 1.49-folds in water soluble Pb was observed for NCCP-1848 and NCCP-1862 strains, respectively. Pot experiments indicated significantly higher uptake by Pelargonium hortorum than that by Mesembryanthemum criniflorrum at all levels of soil Pb concentrations with the highest increase (1.9-folds) in plants inoculated with NCCP-1848 followed by NCCP-1862 (1.8-folds increase) compared to the control (Pb and without bacterial strain). The strains NCCP-1848 and NCCP-1862 were identified by 16S rRNA gene sequencing as Microbacterium sp. and Klebsiella sp. The results of present study suggest that Pb resistant plant growth promoting bacteria can serve as an effective bio-inoculant through wide action spectrum for maximizing efficiency of phytoremediation system., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Ice plant (Mesembryanthemum crystallinum) extract (IPE) is a rich source of d-pinitol, which is widely known to have potential anti-diabetic effects. In the present study, response surface methodology (RSM) was used to optimize d-pinitol extraction conditions with the Box-Behnken design. We then evaluated the anti-diabetic effects properties of IPE that was extracted under optimized conditions (53 °C, 119 min extraction time, and 1 : 11 dilution) in type 2 diabetic Goto-Kakizaki (GK) rats. IPE (400 mg kg-1 day-1) effectively controlled the increased fasting blood glucose level (decreased by 45% vs. GK-control rats) and impaired glucose tolerance (decreased area under curve (AUC) of glucose values by 24%, p < 0.05 vs. GK-control rats) after eight weeks of treatment. Furthermore, IPE significantly improved pancreatic islet morphology, β-cell survival, and insulin secretion in diabetic rats, thus contributing to the antihyperglycemic effect. Finally, prebiotic effects of IPE on gut microbiota were observed and included increased abundance of the beneficial bacteria Bacteroidales&#95;S24-7 and Ruminococcaceae&#95;UCG-014 and decreased abundance of Treponema&#95;2 and Lactobacillus. Overall, IPE has a substantial effect on attenuating hyperglycemia and modulating gut microbiota composition in diabetic GK rats. Therefore, IPE might be a promising functional food for the prevention of diabetes.

Carbon isotope (13C) fractionations occurring during and after photosynthetic CO2 fixation shape the carbon isotope composition (δ13C) of plant material and respired CO2. However, responses of 13C fractionations to diel variation in starch metabolism in the leaf are not fully understood. Here we measured δ13C of organic matter (δ13COM), concentrations and δ13C of potential respiratory substrates, δ13C of dark-respired CO2 (δ13CR), and gas exchange in leaves of starch-deficient plastidial phosphoglucomutase (pgm) mutants and wild-type plants of four species (Arabidopsis thaliana, Mesembryanthemum crystallinum, Nicotiana sylvestris, and Pisum sativum). The strongest δ13C response to the pgm-induced starch deficiency was observed in N. sylvestris, with more negative δ13COM, δ13CR, and δ13C values for assimilates (i.e. sugars and starch) and organic acids (i.e. malate and citrate) in pgm mutants than in wild-type plants during a diel cycle. The genotype differences in δ13C values could be largely explained by differences in leaf gas exchange. In contrast, the PGM-knockout effect on post-photosynthetic 13C fractionations via the plastidic fructose-1,6-bisphosphate aldolase reaction or during respiration was small. Taken together, our results show that the δ13C variations in starch-deficient mutants are primarily explained by photosynthetic 13C fractionations and that the combination of knockout mutants and isotope analyses allows additional insights into plant metabolism., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

This paper presents a simultaneous isolation of pure, intact chloroplasts and mitochondria from mature leaves of Ice plant (Mesembryanthemum crystallinum) and mitochondrial protein preparation for two-dimensional electrophoresis (2DE) analysis under well watered and water -deficit stressed treatments. The washed chloroplasts and mitochondria were purified with Percoll gradients prepared using a Master flex R pump. The chloroplast and mitochondrial proteins were extracted in lysis buffer containing a protease inhibitor mix supplemented with 1 μM Leupeptin and 1 μM E64, followed by precipitation with ice-cold acetone. The protein contents were determined by an EZQ protein quantitation kit. The results show that chloroplast and mitochondria isolated from Ice plant leaves via this protocol have pure and intact. The shape of chloroplast and mitochondria observed by microscopy were clear and sharp. This procedure was employed for assessing the significant differences in mitochondrial protein expression patterns from the well watered and water-deficit stressed treatment leaves collected at dawn (6 a.m.) and dusk (6 p.m.). The results showed 71 and 20 differentially abundant spots between control and CAM for 6 a.m. and 6 p.m., respectively. In addition, 32 protein spots were differentially abundant for 6 a.m. control compared with 6 p.m. control, and 45 protein spots were differentially abundant for 6 a.m. CAM compared with 6 p.m. CAM. Spots that displayed differential abundance for control compared with CAM likely included proteins involved in mitochondrial processes necessary for CAM function. Through further analysis, these proteins will be identified and characterized in the near future using mass-spectrometry-based techniques., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Salt stress causes dramatic changes in the organization and dynamic properties of membranes, however, little is known about the underlying mechanisms involved. Modified trichomes, known as epidermal bladder cells (EBC), on the leaves and stems of the halophyte Mesembryanthemum crystallinum can be successfully exploited as a single-cell-type system to investigate salt-induced changes to cellular lipid composition. In this study, alterations in key molecular species from different lipid classes highlighted an increase in phospholipid species, particularly those from phosphatidylcholine and phosphatidic acid (PA), where the latter is central to the synthesis of membrane lipids. Triacylglycerol (TG) species decreased during salinity, while there was little change in plastidic galactolipids. EBC transcriptomic and proteomic data mining revealed changes in genes and proteins involved in lipid metabolism and the upregulation of transcripts for PIPKIB, PI5PII, PIPKIII, and phospholipase D delta suggested the induction of signalling processes mediated by phosphoinositides and PA. TEM and flow cytometry showed the dynamic nature of lipid droplets in these cells under salt stress. Altogether, this work indicates that the metabolism of TG might play an important role in EBC response to salinity as either an energy reserve for sodium accumulation and/or driving membrane biosynthesis for EBC expansion., (© 2018 John Wiley & Sons Ltd.)

Mesembryanthemum crystallinum, which switches the mode of photosynthesis from C3 to crassulacean acid metabolism (CAM) upon high salt stress, was shown here to exhibit diurnal changes in not only the CO2 fixation pathway but also Chl fluorescence parameters under CAM-induced conditions. We conducted comprehensive time course measurements of M. crystallinum leaf Chl fluorescence using the same leaf throughout the CAM induction period. By doing so, we were able to distinguish the effect of CAM induction from that of photoinhibition and avoid the possible effects of differences in foliar age. We found that the diurnal change in the status of electron transfer could be ascribed to the formation of a proton gradient across thylakoid membranes presumably resulting from diurnal changes in the ATP/ADP ratio reported earlier. The electron transport by actinic illumination thus became limited at the step of plastoquinol oxidation by the Cyt b6/f complex in the 'night' period upon CAM induction, resulting in high levels of non-photochemical quenching. The actinically induced non-photochemical quenching in the 'night' period correlated well with the degree of CAM induction. Chl fluorescence parameters, such as NPQ or qN, could be used as a simple indexing system for the CAM induction.

Abiotic drivers of environmental stress have been found to induce CAM expression (nocturnal carboxylation) in facultative CAM species such as Mesembryanthemum crystallinum. The role played by biotic factors such as competition with non-CAM species in affecting CAM expression, however, remains largely understudied. This research investigated the effects of salt and water conditions on the competition between M. crystallinum and the C 3 grass Bromus mollis with which it is found to coexist in California's coastal grasslands. We also investigated the extent to which CAM expression in M. crystallinum was affected by the intensity of the competition with B. mollis. We found that M. crystallinum had a competitive advantage over B. mollis in drought and saline conditions, while B. mollis exerted strong competitive effects on M. crystallinum in access to light and soil nutrients in high water conditions. This strong competitive effect even outweighed the favorable effects of salt or water additions in increasing the biomass and productivity of M. crystallinum in mixture. Regardless of salt conditions, M. crystallinum did not switch to CAM photosynthesis in response to this strong competitive effect from B. mollis. Disturbance (i.e., grass cutting) reduced the competitive pressure by B. mollis and allowed for CAM expression in M. crystallinum when it was grown mixed with B. mollis. We suggest that moderate competition with other functional groups can enhance CAM expression in M. crystallinum, thereby affecting its plasticity and ability to cope with biological stress.

Soil salinity remains a major threat to global food security, and the progress in crop breeding for salinity stress tolerance may be achieved only by pyramiding key traits mediating plant adaptive responses to high amounts of dissolved salts in the rhizosphere. This task may be facilitated by studying natural variation in salinity tolerance among plant species and, specifically, exploring mechanisms of salinity tolerance in halophytes. The aim of this work was to establish the causal link between mesophyll ion transport activity and plant salt tolerance in a range of evolutionary contrasting halophyte and glycophyte species. Plants were grown under saline conditions in a glasshouse, followed by assessing their growth and photosynthetic performance. In a parallel set of experiments, net K + and H + transport across leaf mesophyll and their modulation by light were studied in control and salt-treated mesophyll segments using vibrating non-invasive ion selective microelectrode (the MIFE) technique. The reported results show that mesophyll cells in glycophyte species loses 2-6 fold more K + compared with their halophyte counterparts. This decline was reflected in a reduced maximum photochemical efficiency of photosystem II, chlorophyll content and growth observed in the glasshouse experiments. In addition to reduced K + efflux, the more tolerant species also exhibited reduced H + efflux, which is interpreted as an energy-saving strategy allowing more resources to be redirected towards plant growth. It is concluded that the ability of mesophyll to retain K + without a need to activate plasma membrane H + -ATPase is an essential component of salinity tolerance in halophytes and halophytic crop plants., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Background: Epidermal bladder cells (EBC) are large single-celled, specialized, and modified trichomes found on the aerial parts of the halophyte Mesembryanthemum crystallinum. Recent development of a simple but high throughput technique to extract the contents from these cells has provided an opportunity to conduct detailed single-cell-type analyses of their molecular characteristics at high resolution to gain insight into the role of these cells in the salt tolerance of the plant., Results: In this study, we carry out large-scale complementary quantitative proteomic studies using both a label (DIGE) and label-free (GeLC-MS) approach to identify salt-responsive proteins in the EBC extract. Additionally we perform an ionomics analysis (ICP-MS) to follow changes in the amounts of 27 different elements. Using these methods, we were able to identify 54 proteins and nine elements that showed statistically significant changes in the EBC from salt-treated plants. GO enrichment analysis identified a large number of transport proteins but also proteins involved in photosynthesis, primary metabolism and Crassulacean acid metabolism (CAM). Validation of results by western blot, confocal microscopy and enzyme analysis helped to strengthen findings and further our understanding into the role of these specialized cells. As expected EBC accumulated large quantities of sodium, however, the most abundant element was chloride suggesting the sequestration of this ion into the EBC vacuole is just as important for salt tolerance., Conclusions: This single-cell type omics approach shows that epidermal bladder cells of M. crystallinum are metabolically active modified trichomes, with primary metabolism supporting cell growth, ion accumulation, compatible solute synthesis and CAM. Data are available via ProteomeXchange with identifier PXD004045.

Citrate, malate and histidine have been involved in many processes including metal tolerance and accumulation in plants. These molecules have been frequently reported to be the potential nickel chelators, which most likely facilitate metal transport through xylem. In this context, we assess here, the relationship between organics acids and histidine content and nickel accumulation in Mesembryanthemum crystallinum and Brassica juncea grown in hydroponic media added with 25, 50 and 100 µM NiCl2. Results showed that M. crystallinum is relatively more tolerant to Ni toxicity than B. juncea. For both species, xylem transport rate of Ni increased with increasing Ni supply. A positive correlation was established between nickel and citrate concentrations in the xylem sap. In the shoot of B. juncea, citric and malic acids concentrations were significantly higher than in the shoot of M. crystallinum. Also, the shoots and roots of B. juncea accumulated much more histidine. In contrast, a higher root citrate concentration was observed in M. crystallinum. These findings suggest a specific involvement of malic and citric acid in Ni translocation and accumulation in M. crystallinum and B. juncea. The high citrate and histidine accumulation especially at 100µM NiCl2, in the roots of M. crystallinum might be among the important factors associated with the tolerance of this halophyte to toxic Ni levels., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Although plant responses to drought stress have been studied in detail in several plant species, including CAM plants, the occurrence of stress memory and possible mechanisms for its regulation are still very poorly understood. In an attempt to better understand the occurrence and possible mechanisms of regulation of stress memory in plants, we measured the concentrations of phytohormones in Aptenia cordifolia exposed to reiterated drought, together with various stress indicators, including leaf water contents, photosynthesis and mechanisms of photo- and antioxidant protection. Results showed that plants exposed to drought stress responded differently if previously challenged with a first drought. Gibberellin levels decreased upon exposure to the first drought and remained lower in double-stressed plants compared with those exposed to stress for the first time. In contrast, abscisic acid levels were higher in double- than single-stressed plants. This occurred in parallel with alterations in hydroperoxide levels, but not with malondialdehyde levels, thus suggesting an increased oxidation state that did not result in oxidative damage in double-stressed plants. It is concluded that (i) drought stress memory occurs in double-stressed A. cordifolia plants, (ii) both gibberellins and abscisic acid may play a role in plant response to repeated periods of drought, and (iii) changes in abscisic acid levels in double-stressed plants may have a positive effect by modulating changes in the cellular redox state with a role in signalling, rather than cause oxidative damage to the cell.

According to microscopic observations, germinating hyphae of Botrytis cinerea, though easily penetrating Mesembryanthemum crystallinum mesophyll tissue, are limited in growth in mid-ribs and only occasionally reach vascular bundles. In mid-ribs of C3 and CAM leaves, we found significantly lower rbcL (large RubisCO subunit) abundance. Moreover, in CAM leaves, minute transcript contents for pepc1 (phosphoenolpyruvate carboxylase) and nadpme1 (malic enzyme) genes found in the mid-ribs suggest that they perform β-carboxylation at a low rate. The gene of the main H2O2-scavenging enzyme, catL (catalase), showed lower expression in C3 mid-rib parts in comparison to mesophyll. This allows maintenance of higher H2O2 quantities in mid-rib parts. In C3 leaves, pathogen infection does not impact photosynthesis. However, in CAM plants, the expression profiles of rbcL and nadpme1 were similar under biotic stress, with transcript down-regulation in mid-ribs and up-regulation in mesophyll (however, in case of rbcL not significant). After B. cinerea infection in C3 plants, transcripts for both antioxidative proteins strongly increased in mid-ribs, but not in mesophyll. In infected CAM plants, a significant transcript increase in the mesophyll was parallel to its decrease in the mid-rib region (however, in the case of catL this was not significant). Pathogen infection modified the expression of carbon and ROS metabolism genes in mid-ribs and mesophyll, resulting in the establishment of successful leaf defense., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

The effect of abscisic acid (ABA) and fluridone on the content of endogenous phytohormones and free polyamines and the intensity of oxidative stress was studied in plants of Mesembryanthemum crystallinum L. under salinity. It was shown that the pretreatment of plant roots with 1 μM ABA, followed by the action of 300 mM NaCl, caused a protective effect and improved the physiological state of the plants, which was manifested in increased biomass and content of available cytokinins and reduced values of the indicators of oxidative stress. It was noted that the inhibitor fluridone reduced the effect of ABA and acted as a pro-oxidant.

Saline soils often constitute sites of accumulation of industrial and urban wastes contaminated by heavy metals. Halophytes, i.e. native salt-tolerant species, could be more suitable for heavy metal phytoextraction from saline areas than glycophytes, most frequently used so far. In the framework of this approach, we assess here the Ni phytoextraction potential in the halophyte Mesembryanthemum crystallinum compared with the model species Brassica juncea. Plants were hydroponically maintained for 21 days at 0, 25, 50, and 100μM NiCl2. Nickel addition significantly restricted the growth activity of both species, and to a higher extent in M. crystallinum, which did not, however, show Ni-related toxicity symptoms on leaves. Interestingly, photosynthesis activity, chlorophyll content and photosystem II integrity assessed by chlorophyll fluorescence were less impacted in Ni-treated M. crystallinum as compared to B. juncea. The plant mineral nutrition was differently affected by NiCl2 exposure depending on the element, the species investigated and even the organ. In both species, roots were the preferential sites of Ni(2+) accumulation, but the fraction translocated to shoots was higher in B. juncea than in M. crystallinum. The relatively good tolerance of M. crystallinum to Ni suggests that this halophyte species could be used in the phytoextraction of moderately polluted saline soils., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

RING-type copines are a small family of plant-specific RING-type ubiquitin ligases. They contain an N-terminal myristoylation site for membrane anchoring, a central copine domain for substrate recognition, and a C-terminal RING domain for E2 docking. RING-type copine McCPN1 (copine1) from halophyte ice plant (Mesembryanthemum crystallinum L.) was previously identified from a salt-induced cDNA library. In this work, we characterize the activity, expression, and localization of McCPN1 in ice plant. An in vitro ubiquitination assay of McCPN1 was performed using two ice plant UBCs, McUBC1 and McUBC2, characterized from the same salt-induced cDNA library. The results showed that McUBC2, a member of the UBC8 family, stimulated the autoubiquitination activity of McCPN1, while McUBC1, a homolog of the UBC35 family, did not. The results indicate that McCPN1 has selective E2-dependent E3 ligase activity. We found that McCPN1 localizes primarily on the plasma membrane and in the nucleus of plant cells. Under salt stress, the accumulation of McCPN1 in the roots increases. A yeast two-hybrid screen was used to search for potential McCPN1-interacting partners using a library constructed from salt-stressed ice plants. Screening with full-length McCPN1 identified several independent clones containing partial Argonaute 4 (AGO4) sequence. Subsequent agro-infiltration, protoplast two-hybrid analysis, and bimolecular fluorescence complementation assay confirmed that McCPN1 and AGO4 interacted in vivo in the nucleus of plant cells. The possible involvement of a catalyzed degradation of AGO4 by McCPN1 in response to salt stress is discussed., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

SKD1 (suppressor of K+ transport growth defect 1) is an AAA-type ATPase that functions as a molecular motor. It was previously shown that SKD1 accumulates in epidermal bladder cells of the halophyte Mesembryanthemum crystallinum. SKD1 knock-down Arabidopsis mutants showed an imbalanced Na+/K+ ratio under salt stress. Two enzymes involved in protein post-translational modifications that physically interacted with McSKD1 were identified. McCPN1 (copine 1), a RING-type ubiquitin ligase, has an N-terminal myristoylation site that links to the plasma membrane, a central copine domain that interacts with McSKD1, and a C-terminal RING domain that catalyses protein ubiquitination. In vitro ubiquitination assay demonstrated that McCPN1 was capable of mediating ubiquitination of McSKD1. McSnRK1 (sucrose non-fermenting 1-related protein kinase) is a Ser/Thr protein kinase that contains an N-terminal STKc catalytic domain to phosphorylate McSKD1, and C-terminal UBA and KA1 domains to interact with McSKD1. The transcript and protein levels of McSnRK1 increased as NaCl concentrations increased. The formation of an SKD1-SnRK1-CPN1 ternary complex was demonstrated by yeast three-hybrid and bimolecular fluorescence complementation. It was found that McSKD1 preferentially interacts with McSnRK1 in the cytosol, and salt induced the re-distribution of McSKD1 and McSnRK1 towards the plasma membrane via the microtubule cytoskeleton and subsequently interacted with RING-type E3 McCPN1. The potential effects of ubiquitination and phosphorylation on McSKD1, such as changes in the ATPase activity and cellular localization, and how they relate to the functions of SKD1 in the maintenance of Na+/K+ homeostasis under salt stress, are discussed.

The halophyte Mesembryanthemum crystallinum adapts to salt stress by salt uptake and switching from C3 photosynthesis to CAM (crassulacean acid metabolism). An important role in this process is played by transport proteins in the tonoplast of the central vacuole. In the present study we examine dynamic changes in the protein composition during salt-stress adaptation in microsomes from M. crystallinum leaves. Plants challenged with 400 mM NaCl accumulate salt by day 4 of treatment and malic acid only at day 12; a switching to CAM hence follows any initial steps of salt adaptation with a delay. Using a label-free and semiquantitative approach, we identified the most dramatic changes between the proteome of control plants and plants harvested after 12 days of the treatment; the abundance of 14 proteins was significantly affected. The proteomic data revealed that the majority of the subunits of V-ATPase (vacuolar H(+)-ATPase) holoenzyme. The salt treatment somewhat decreased the abundance of all subunits in the short term (4 days). Long-term adaptation, including the switching to CAM, goes together with a strong increase in the representation of all detectable subunits. Because this increase is subunit-specific, with the highest rise occurring for subunits E and c, the data suggest that long-term adaptation to salt stress correlates with a change in V-ATPase subunit stoichiometry and highlight the structural plasticity of this holoenzyme.

The response of Mesembryanthemum crystallinum plants performing C3 photosynthesis and crassulacean acid metabolism (CAM) to the non-host necrotrophic pathogen Botrytis cinerea was analyzed at the local and systemic levels. The induction of programmed cell death, lignin and callose deposition, changes in salicylic acid, glutathione and cysteinylglycine pools as well as the content of thiolated proteins were studied. The infected C3 and CAM plants exhibited hypersensitive-like defence response, however fluorescence staining with acridine orange and ethidium bromide revealed programmed cell death events in C3 plants only. The local immune response was not related to callose and lignin deposition. In the infected plants, salicylic acid, glutathione and cysteinylglycine, the first product of glutathione catabolism, as well as protein S-thiolation, predominantly S-glutathionylation, contributed to local defence at sites of inoculation. They (except protein thiolation) were also active in the establishment of systemic acclimation response monitored in the non-treated upper leaves. The extent to which they were involved in the local and systemic responses induced by B. cinerea differed in C3 and CAM plants. The accumulation of free salicylic acid, both in treated and upper leaves of the infected plants, was much more pronounced in CAM plants. The results have been discussed with respect to redox regulations in defence against necrotrophic pathogens and to stress acclimation., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

In the halophytic plant Mesembryanthemum crystallinum salinity or drought can change the mode of photosynthesis from C(3) to crassulacean acid metabolism (CAM). These two stress factors are linked to oxidative stress, however, the induction of CAM by oxidative stress per se is not straightforward. Treatment with high light (HL) did not lead to the induction of CAM, as documented by a low night/day difference in malate level and a low expression of the CAM-related form of phosphoenolcarboxylase (Ppc1), despite causing some oxidative damage (elevated MDA level, malondialdehyde). In contrast to the action of high salinity (0.4M NaCl), HL treatment did not activate neither the cytosolic NADP-malic enzyme nor the chloroplastic form of NADP-dependent malate dehydrogenase (NADP-MDH). In plastids of HL-treated plants a huge amount of starch was accumulated. This was associated with a weak stimulation of hydrolytic and phosphorolytic starch-degrading enzymes, in contrast to their strong up-regulation under high salinity. It is concluded that HL alone is not able to activate starch degradation necessary for CAM performance. Moreover, in the absence of salinity in C(3)M. crystallinum plants an age-dependent increase in energy dissipation from PSII was documented under high irradiance, as illustrated by non-photochemical quenching (NPQ). Obtained data suggest that in this halophytic species several photoprotective strategies are strictly salinity-dependent., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

In plant Mesembryanthemum crystallinum, which has the inducible crassulacean acid metabolism (CAM), isoforms of plastidic phosphate translocators (pPTs) are categorized into three subfamilies: the triose phosphate/phosphate translocator (McTPT1), the phosphoenolpyruvate/phosphate translocator (McPPT1), and the glucose 6-phosphate/phosphate translocator (McGPT1 and McGPT2). In order to elucidate the physiological roles of these pPTs in M. crystallinum, we determined the substrate specificity of each pPT isoform. The substrate specificities of McTPT1, McPPT1, and McGPT1 showed overall similarities to those of orthologs that have been characterized. In contrast, for glucose 6-phosphate, McGPT2 showed higher selectivity than McGPT1 and other GPT orthologs. Because the expression of McGTP2 is specific to CAM while that of McGTP1 is constitutively expressed in both the C3- and the CAM-state in M. crystallinum, we propose that McGPT2 functions as a CAM system-specific GPT in this plant.

Plant epidermal trichomes are as varied in morphology as they are in function. In the halophyte Mesembryanthemum crystallinum, specialized trichomes called epidermal bladder cells (EBC) line the surface of leaves and stems, and increase dramatically in size and volume upon plant salt-treatment. These cells have been proposed to have roles in plant defense and UV protection, but primarily in sodium sequestration and as water reservoirs. To gain further understanding into the roles of EBC, a cell-type-specific proteomics approach was taken in which precision single-cell sampling of cell sap from individual EBC was combined with shotgun peptide sequencing (LC-MS/MS). Identified proteins showed diverse biological functions and cellular locations, with a high representation of proteins involved in H(+)-transport, carbohydrate metabolism, and photosynthesis. The proteome of EBC provides insight into the roles of these cells in ion and water homeostasis and raises the possibility that they are photosynthetically active and functioning in Crassulacean acid metabolism., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The temporal co-ordination of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco) and phosphoenolpyruvate carboxylase (PEPc) activities by Mesembryanthemum crystallinum L. in C(3) and crassulacean acid metabolism (CAM) modes was investigated under conventional light-dark (LD) and continuous light (LL) conditions. When C(3) , net CO(2) assimilation rate increased during each subjective night under LL with maximum carboxylation unrelated to Rubisco activation state. The CAM circadian rhythm of CO(2) uptake was more pronounced, with CO(2) assimilation rate maximal towards the end of each subjective night. In vivo and in vitro techniques were integrated to map carboxylase enzyme regulation to the framework provided by CAM LL gas exchange activity. Rubisco was activated in vitro throughout each subjective dark period and consistently deactivated at each subjective dawn, similar to that observed at true dawn in constitutive CAM species. Instantaneous carbon isotope discrimination showed in vivo carboxylase co-dominance during the CAM subjective night, initially by Rubisco and latterly C(4) (PEPc), despite both enzymes seemingly activated in vitro. The circadian rhythm in titratable acidity accumulation was progressively damped over successive subjective nights, but maintenance of PEPc carboxylation capacity ensures that CAM plants do not become progressively more 'C(3) -like' with time under LL., (© 2012 Blackwell Publishing Ltd.)

Organic wastes were successfully used as soil amendment to improve agrosystems productivity. Yet, the effectiveness of this practice to enhance plant antioxidant capacities has received little attention. Here, we assess the effect of municipal solid waste (MSW) compost (at 40 t ha(-1)) on growth, polyphenol contents and antioxidant activities of Mesembryanthemum edule. MSW compost application significantly increased the soil contents of carbon, nitrogen, calcium, phosphorus and potassium. This was associated with higher nutrient (N, P, and K) uptake, which likely led to the significant improvement of the plant biomass and relative growth rate (RGR) (+93% on average) as compared to the control. In the same way, the fertilizing effect of the added organic matter significantly enhanced the antioxidant potential M. edule, assessed by radical scavenging activity, iron reducing power and β-carotene bleaching capacity. This was associated with significantly higher antioxidant contents, mainly total phenols and flavonoids. Heavy metal (Pb, Cd, Cu, and Zn) concentrations were slightly increased upon compost application, but remained lower than phytotoxic values. Overall, our results point out that short-term MSW compost application at 40 t ha(-1) is efficient in enhancing the productivity together with the antioxidant potentiality of M. edule without any adverse environmental impact., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Mesembryathemum crystallinum plants performing C(3) or CAM (crassulacean acid metabolism) appear to be highly resistant to Botrytis cinerea as well as to Pseudomonas syringae. Fungal hyphae growth was restricted to 48h post-inoculation (hpi) in both metabolic types and morphology of hyphae differed between those growing in C(3) and CAM plants. Growth of bacteria was inhibited significantly 24 hpi in both C(3) and CAM plants. B. cinerea and P. syringae infection led to an increase in the concentration of H(2)O(2) in C(3) plants 3 hpi, while a decrease in H(2)O(2) content was observed in CAM performing plants. The concentration of H(2)O(2) returned to the control level 24 and 48 hpi. Changes in H(2)O(2) content corresponded with the activity of guaiacol peroxidase (POD), mostly 3 hpi. We noted that its activity decreased significantly in C(3) plants and increased in CAM plants in response to inoculation with both pathogens. On the contrary, changes in the activity of CAT did not correlate with H(2)O(2) level. It increased significantly after interaction of C(3) plants with B. cinerea or P. syringae, but in CAM performing plants, the activity of this enzyme was unchanged. Inoculation with B. cinerea or P. syringae led to an increase in the total SOD activity in C(3) plants while CAM plants did not exhibit changes in the total SOD activity after interaction with both pathogens. In conclusion, the pathogen-induced changes in H(2)O(2) content and in SOD, POD and CAT activities in M. crystallinum leaves, were related to the photosynthetic metabolism type of the stressed plants rather than to the lifestyle of the invading pathogen., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Stable transformation of Mesembryanthemum crystallinum L. (common ice plant) with a green fluorescent protein (GFP) construct targeted to the endoplasmic reticulum was obtained. Seven and fourteen days after germination seedlings were infected with Agrobacterium rhizogenes strain ARqua1 either by direct coating of the cut radicles with bacteria growing on solid medium or by immersion of the cut surface in bacterial suspension at different optical densities. Both methods of infection resulted in production of GFP-positive roots with a frequency ranging from 6 to 20% according to the age of the explants and the application procedure. The green fluorescing roots displayed the typical hairy root phenotype and were easily maintained in liquid medium without growth regulators for over 2 years. Stable expression of the transgene in the roots was confirmed by polymerase chain reaction (PCR), immunoblotting and the capacity of roots to grow and produce callus on kanamycin-enriched medium. Nineteen endogenous cytokinins were determined in transgenic and non-transformed roots. The results revealed significantly lower levels of the free bases of isopentenyladenine, dihydrozeatin, cis- and trans-zeatin, as well as a conspicuous decline in concentrations of the corresponding nucleosides and most nucleotides in transgenic roots compared to the wild type. Comparison of the cytokinin profiles in transgenic and non-transformed roots suggested that transformation by A. rhizogenes disturbed cytokinin metabolism during the early steps of biosynthesis. Calli obtained from transformed roots were GFP-positive and remained non-regenerative or displayed high rhizogenic potential depending on the auxin/cytokinin ratio in the medium. Calli and callus-derived roots showed a strong GFP signal for over 2 years., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Crassulacean acid metabolism (CAM) is an intriguing metabolic strategy to maintain photosynthesis under conditions of closed stomata. A shift from C(3) photosynthesis to CAM in Mesembryanthemum crystallinum plants was induced by high salinity (0.4 M NaCl). In CAM-performing plants, the quantum efficiencies of photosystem II and I were observed to undergo distinct diurnal fluctuations that were characterized by a strong decline at the onset of the day, midday recovery, and an evening drop. The temporal recovery of both photosystems' efficiency at midday was associated with a more rapid induction of the electron transport rate at PSII. This recovery of the photosynthetic apparatus at midday was observed to be accompanied by extreme swelling of thylakoids. Despite these fluctuations, a persistent effect of CAM was the acceptor side limitation of PSI during the day, which was accompanied by a strongly decreased level of Rubisco protein. Diurnal changes in the efficiency of photosystems were parallel to corresponding changes in the levels of mRNAs for proteins of PSII and PSI reaction centers and for rbcL, reaching a maximum in CAM plants at midday. This might reflect a high demand for new protein synthesis at this time of the day. Hybridization of run-on transcripts with specific probes for plastid genes of M. crystallinum revealed that the changes in plastidic mRNA levels were regulated at the level of transcription.

Inositols are indispensable components of cellular signaling molecules, and impaired cytoplasmic inositol concentrations affect cellular development. Although most cells can synthesize inositol de novo, plasma membrane-localized inositol uptake systems are indispensable for normal development. Here, we present in-depth functional analyses of plasma membrane-localized H(+)-inositol symporters from human and from the higher plant Arabidopsis thaliana. Sequence comparisons, structural and phylogenetic analyses revealed that these transporters possess conserved extracellular loop domains that represent homologs of plexins/semaphorin/integrin (PSI) domains from animal type I receptors. In these receptors, PSI domains modulate intracellular signaling via extracellular protein-protein interactions. Comparisons of H(+)-inositol symporters with wild type, mutated and truncated PSI domains in different expression systems showed that removal of the entire loop domain increased the V(max) of inositol uptake. Finally, we show that the PSI domains are targets for Ni(++) ions that cause a complete loss of transport activity. A possible role of Ni(++)-binding to PSI domains in Ni(++)-induced carcinogenicity is discussed.

The increased expression of McPIP2;1 (MipC), a root-specific aquaporin (AQP) from Mesembryanthemum crystallinum, under salt stress has suggested a role for this AQP in the salt tolerance of the plant. However, whether McPIP2;1 transports water or another solute and how its activity is regulated are so far unknown. Therefore, wild type (wt) or mutated McPIP2;1 protein was expressed in Xenopus laevis oocytes. Then, the osmotic water permeability (P(f)) of the oocytes membrane was assessed by hypotonic challenges. Selectivity of McPIP2;1 to water was determined by radiolabeled glycerol or urea uptake assays. Moreover, swelling and in vitro phosphorylation assays revealed that both water permeation and phosphorylation status of McPIP2;1 were significantly increased by the phosphorylation agonists okadaic acid (OA), phorbol myristate acetate (PMA), and 8-Br-cAMP, and markedly decreased by the inhibitory peptides PKI 14-22 and PKC 20-28, inhibitors of protein kinases A (PKA) and C (PKC), respectively. Substitution of Ser(123) or both, Ser(123) and Ser(282), abolished the water channel activity of McPIP2;1 while substitution of Ser(282) only partially inhibited it (51.9% inhibition). Despite lacking Ser(123) and/or Ser(282), the McPIP2;1 mutant forms were still phosphorylated in vitro, which suggests that phosphorylation may have a dual role on this AQP. Our results indicate that McPIP2;1 water permeability depends completely on Ser(123) and is positively regulated by PKA- and PKC-mediated phosphorylation. Regulation of the phosphorylation status of McPIP2;1 may contribute to control water transport through root cells when the plant is subjected to high salinity conditions.

Halophyte ability to withstand salt-triggered oxidative stress is governed by multiple biochemical mechanisms that facilitate retention and/or acquisition of water, protect chloroplast functioning, and maintain ion homeostasis. Most essential traits include the synthesis of osmolytes, specific proteins, and antioxidant molecules. This might explain the utilization of some halophytes as traditional medicinal and dietary plants. The present study aimed at assessing the phenolic content and antioxidant activities of some Tunisian halophytes (Cakile maritima, Limoniastrum monopetalum, Mesembryanthemum crystallinum, M. edule, Salsola kali, and Tamarix gallica), depending on biological (species, organ and developmental stage), environmental, and technical (extraction solvent) factors. The total polyphenol contents and antioxidant activities (DPPH and superoxide radicals scavenging activities, and iron chelating and reducing powers) were strongly affected by the above-cited factors. Such variability might be of great importance in terms of valorising these halophytes as a source of naturally secondary metabolites, and the methods for phenolic and antioxidant production.

Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that improves water use efficiency by shifting part or all of net atmospheric CO2 uptake to the night. Genetic dissection of regulatory and metabolic attributes of CAM has been limited by the difficulty of identifying a reliable phenotype for mutant screening. We developed a novel and simple colorimetric assay to measure leaf pH to screen fast neutron-mutagenized populations of common ice plant (Mesembryanthemum crystallinum), a facultative CAM species, to detect CAM-deficient mutants with limited nocturnal acidification. The isolated CAM-deficient mutants showed negligible net dark CO2 uptake compared with wild-type plants following the imposition of salinity stress. The mutants and wild-type plants accumulated nearly comparable levels of sodium in leaves, but the mutants grew more slowly than the wild-type plants. The mutants also had substantially reduced seed set and seed weight relative to wild type under salinity stress. Carbon-isotope ratios of seed collected from 4-month-old plants indicated that C3 photosynthesis made a greater contribution to seed production in mutants compared to wild type. The CAM-deficient mutants were deficient in leaf starch and lacked plastidic phosphoglucomutase, an enzyme critical for gluconeogenesis and starch formation, resulting in substrate limitation of nocturnal C4 acid formation. The restoration of nocturnal acidification by feeding detached leaves of salt-stressed mutants with glucose or sucrose supported this defect and served to illustrate the flexibility of CAM. The CAM-deficient mutants described here constitute important models for exploring regulatory features and metabolic consequences of CAM.

Mechanical wounding of Mesembryanthemum crystallinum leaves in planta induced a fast decrease in stomatal conductance, which was related to accumulation of hydrogen peroxide (H(2)O(2)). Higher levels of H(2)O(2) were accompanied by an increase in total activity of superoxide dismutase (SOD) and a decrease in catalase (CAT) activity. Among SOD forms, manganese SOD (MnSOD) and copper/zinc SOD (Cu/ZnSOD) seem to be especially important sources of H(2)O(2) at early stages of wounding response. Moreover, NADP-malic enzyme (NADP-ME), one of the key enzymes of primary carbon metabolism, which is also involved in stress responses, showed a strong increase in activity in wounded leaves. All these symptoms: high accumulation of H(2)O(2), high activities of Cu/ZnSOD and NADP-ME, together with the decrease of CAT activity, were also observed in the major veins of unwounded leaves. The potential role of veinal tissues as an important source of H(2)O(2) during wounding response is discussed.

Proline (Pro) is one of the most accumulated osmolytes in salinity and water deficit conditions in plants. In the present study, we measured the Pro content, the activity and the expression level of delta 1-pyrroline-5-carboxylate synthetase (P5CS: gamma-glutamyl kinase, EC 2.7.2.11 and glutamate-5-semialdehyde dehydrogenase, EC 1.2.1.41), a key regulatory enzyme involved in the biosynthesis of Pro, in cactus pear (Opuntia streptacantha) subjected to 6, 9 and 11 days of salt stress. Treatment with NaCl of O. streptacantha young plants resulted in a decrease in the cladode thickness and root length, and in a significant and gradual accumulation of Pro in young cladodes, in a time- and concentration-dependent manner. P5CS activity, studied as gamma-glutamyl kinase, was reduced at all times as a consequence of salt treatment, except at the sixth day at 75 and 150mM of NaCl, where a slight increase was observed. We isolated an open reading frame (ORF) fragment of p5cs gene. The deduced amino acid sequence of the P5CS protein exhibited 90.4% of identity with the P5CS protein from Mesembryanthemum crystallinum. RT-PCR analysis revealed that the Osp5cs gene of O. streptacantha was induced by salt stress at 9 and 11 days of treatment. Furthermore, ABA-induced Osp5cs gene expression was observed in cladodes of cactus pear young plants. We observed an evident correlation between the transcript up-regulation and the Pro accumulation under salt stress; however, these results do not parallel with the changes in P5CS enzymatic activity. This Pro accumulation might function as an osmolyte for the intracellular osmotic adjustment and might be playing a critical role in protecting photosynthetic activity in O. streptacantha plants under salt stress.

The common ice plant (Mesembryanthemum crystallinum L.) has emerged as a useful model for molecular genetic studies of Crassulacean acid metabolism (CAM) because CAM can be induced in this species by water deficit or salinity stress. Non-redundant sequence information from expressed sequence tag data was used to fabricate a custom oligonucleotide microarray to compare large-scale mRNA expression patterns in M. crystallinum plants conducting C(3) photosynthesis versus CAM. Samples were collected every 4 h over a 24 h time period at the start of the subjective second day from plants grown under constant light and temperature conditions in order to capture variation in mRNA expression due to salinity stress and circadian clock control. Of 8455 genes, a total of 2343 genes (approximately 28%) showed a significant change as judged by analysis of variance (ANOVA) in steady-state mRNA abundance at one or more time points over the 24 h period. Of these, 858 (10%) and 599 (7%) exhibited a greater than two-fold ratio (TFR) increase or decrease in mRNA abundance, respectively. Functional categorization of these TFR genes revealed that many genes encoding products that function in CAM-related C(4) acid carboxylation/decarboxylation, glycolysis/gluconeogenesis, polysaccharide, polyol, and starch biosynthesis/degradation, protein degradation, transcriptional activation, signalling, stress response, and transport facilitation, and novel, unclassified proteins exhibited stress-induced increases in mRNA abundance. In contrast, salt stress resulted in a significant decrease in transcript abundance for genes encoding photosynthetic functions, protein synthesis, and cellular biogenesis functions. Many genes with CAM-related functions exhibited phase shifts in their putative circadian expression patterns following CAM induction. This report establishes an extensive catalogue of gene expression patterns for future investigations aimed at understanding the complex, transcriptional hierarchies that govern CAM-specific expression patterns. A novel graph-theoretic approach called 'Max Clique Builder' is introduced that identifies and organizes sets of coordinately regulated genes, such as those encoding subunits of the vacuolar H(+)-ATPase complex, into tighter functionally related clusters with more similar expression patterns compared with standard hierarchical clustering methods.

Mesembryanthemum crystallinum L. (Aizoaceae) is a facultative annual halophyte and a C(3)-photosynthesis/crassulacean acid metabolism intermediate species currently used as a model plant in stress physiology. Both salinity and high light irradiance stress are known to induce CAM in this species. The present study was performed to provide a diagnosis of alterations at the photosystem II level during salinity and irradiance stress. Plants were subjected for up to 13 days to either 0.4M NaCl salinity or high irradiance of 1000 micromol m(-2)s(-1), as well as to both stress factors combined (LLSA=low light plus salt; HLCO=high light of 1000 micromol m(-2)s(-1), no salt; HLSA=high light plus salt). A control of LLCO=low light of 200 micromol m(-2)s(-1), no salt was used. Parameters of chlorophyll a fluorescence of photosystem II (PSII) were measured with a pulse amplitude modulated fluorometer. HLCO and LLSA conditions induced a weak degree of CAM with day/night changes of malate levels (Deltamalate) of approximately 12mM in the course of the experiment, while HLSA induced stronger CAM of Deltamalate approximately 20 mM. Effective quantum yield of PSII, DeltaF/F'(m), was only slightly affected by LLSA, somewhat reduced during the course of the experiment by HLCO and clearly reduced by HLSA. Potential quantum efficiency of PSII, F(v)/F(m), at predawn times was not affected by any of the conditions, always remaining at 0.8, showing that there was no acute photoinhibition. During the course of the days HL alone (HLCO) also did not elicit photoinhibition; salt alone (LLSA) caused acute photoinhibition which was amplified by the combination of the two stresses (HLSA). Non-photochemical, NPQ, quenching remained low (<0.5) under LLCO, LLSA and HLCO and increased during the course of the experiment under HLSA to 1-2. Maximum apparent photosynthetic electron transport rates, ETR(max), declined during the daily courses and were reduced by LLSA and to a similar extent by HLSA. It is concluded that M. crystallinum expresses effective stress tolerance mechanisms but photosynthetic capacity is reduced by the synergistic effects of salinity and light irradiance stress combined.