Membrane Profiling by Free Flow Electrophoresis and SWATH-MS to Characterize Subcellular Compartment Proteomes in Mesembryanthemum crystallinum.

The study of subcellular membrane structure and function facilitates investigations into how biological processes are divided within the cell. However, work in this area has been hampered by the limited techniques available to fractionate the different membranes. Free Flow Electrophoresis (FFE) allows for the fractionation of membranes based on their different surface charges, a property made up primarily of their varied lipid and protein compositions. In this study, high-resolution plant membrane fractionation by FFE, combined with mass spectrometry-based proteomics, allowed the simultaneous profiling of multiple cellular membranes from the leaf tissue of the plant Mesembryanthemum crystallinum. Comparisons of the fractionated membranes' protein profile to that of known markers for specific cellular compartments sheds light on the functions of proteins, as well as provides new evidence for multiple subcellular localization of several proteins, including those involved in lipid metabolism.

Molecular changes in Mesembryanthemum crystallinum guard cells underlying the C3 to CAM transition.

ABSTARCT: KEY MESSAGE: The timing and transcriptomic changes during the C3 to CAM transition of common ice plant support the notion that guard cells themselves can shift from C3 to CAM. Crassulacean acid metabolism (CAM) is a specialized type of photosynthesis: stomata close during the day, enhancing water conservation, and open at night, allowing CO2 uptake. Mesembryanthemum crystallinum (common ice plant) is a facultative CAM species that can shift from C3 photosynthesis to CAM under salt or drought stresses. However, the molecular mechanisms underlying the stress induced transition from C3 to CAM remain unknown. Here we determined the transition time from C3 to CAM in M. crystallinum under salt stress. In parallel, single-cell-type transcriptomic profiling by 3'-mRNA sequencing was conducted in isolated stomatal guard cells to determine the molecular changes in this key cell type during the transition. In total, 495 transcripts showed differential expression between control and salt-treated samples during the transition, including 285 known guard cell genes, seven CAM-related genes, 18 transcription factors, and 185 other genes previously not found to be expressed in guard cells. PEPC1 and PPCK1, which encode key enzymes of CAM photosynthesis, were up-regulated in guard cells after seven days of salt treatment, indicating that guard cells themselves can shift from C3 to CAM. This study provides important information towards introducing CAM stomatal behavior into C3 crops to enhance water use efficiency.

Making Epidermal Bladder Cells Bigger: Developmental- and Salinity-Induced Endopolyploidy in a Model Halophyte.

Endopolyploidy occurs when DNA replication takes place without subsequent mitotic nuclear division, resulting in cell-specific ploidy levels within tissues. In plants, endopolyploidy plays an important role in sustaining growth and development, but only a few studies have demonstrated a role in abiotic stress response. In this study, we investigated the function of ploidy level and nuclear and cell size in leaf expansion throughout development and tracked cell type-specific ploidy in the halophyte Mesembryanthemum crystallinum In addition to developmental endopolyploidy, we examined the effects of salinity stress on ploidy level. We focused specifically on epidermal bladder cells (EBC), which are modified balloon-like trichomes, due to their large size and role in salt accumulation. Our results demonstrate that ploidy increases as the leaves expand in a similar manner for each leaf type, and ploidy levels up to 512C were recorded for nuclei in EBC of leaves of adult plants. Salt treatment led to a significant increase in ploidy levels in the EBC, and these cells showed spatially related differences in their ploidy and nuclear and cell size depending on the positions on the leaf and stem surface. Transcriptome analysis highlighted salinity-induced changes in genes involved in DNA replication, cell cycle, endoreduplication, and trichome development in EBC. The increase in cell size and ploidy observed in M. crystallinum under salinity stress may contribute to salt tolerance by increasing the storage capacity for sodium sequestration brought about by higher metabolic activity driving rapid cell enlargement in the leaf tissue and EBC.

Photosynthesis-related characteristics of the midrib and the interveinal lamina in leaves of the C3-CAM intermediate plant Mesembryanthemum crystallinum.

BACKGROUND AND AIMS: Leaf veins are usually encircled by specialized bundle sheath cells. In C4 plants, they play an important role in CO2 assimilation, and the photosynthetic activity is compartmentalized between the mesophyll and the bundle sheath. In C3 and CAM (Crassulacean acid metabolism) plants, the photosynthetic activity is generally attributed to the leaf mesophyll cells, and the vascular parenchymal cells are rarely considered for their role in photosynthesis. Recent studies demonstrate that enzymes required for C4 photosynthesis are also active in the veins of C3 plants, and their vascular system contains photosynthetically competent parenchyma cells. However, our understanding of photosynthesis in veins of C3 and CAM plants still remains insufficient. Here spatial analysis of photosynthesis-related properties were applied to the midrib and the interveinal lamina cells in leaves of Mesembryanthemum crystallinum, a C3-CAM intermediate plant. METHODS: The midrib anatomy as well as chloroplast structure and chlorophyll fluorescence, diurnal gas exchange profiles, the immunoblot patterns of PEPC (phosphoenolpyruvate carboxylase) and RubisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), H2O2 localization and antioxidant enzyme activities were compared in the midrib and in the interveinal mesophyll cells in leaves of C3 and CAM plants. KEY RESULTS: Leaf midribs were structurally competent to perform photosynthesis in C3 and CAM plants. The midrib chloroplasts resembled those in the bundle sheath cells of C4 plants and were characterized by limited photosynthetic activity. CONCLUSIONS: The metabolic roles of midrib chloroplasts differ in C3 and CAM plants. It is suggested that in leaves of C3 plants the midrib chloroplasts could be involved in the supply of CO2 for carboxylation, and in CAM plants they could provide malate to different metabolic processes and mediate H2O2 signalling.

Suppressor of K+ transport growth defect 1 (SKD1) interacts with RING-type ubiquitin ligase and sucrose non-fermenting 1-related protein kinase (SnRK1) in the halophyte ice plant.

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.

Day/night regulation of aquaporins during the CAM cycle in Mesembryanthemum crystallinum.

Mesembryanthemum crystallinum exhibits induction of Crassulacean acid metabolism (CAM) after a threshold stage of development, by exposure to long days with high light intensities or by water and salt stress. During the CAM cycle, fluctuations in carbon partitioning within the cell lead to transient drops in osmotic potential, which are likely stabilized/balanced by passive movement of water via aquaporins (AQPs). Protoplast swelling assays were used to detect changes in water permeability during the day/night cycle of CAM. To assess the role of AQPs during the same period, we followed transcript accumulation and protein abundance of four plasma membrane intrinsic proteins (PIPs) and one tonoplast intrinsic protein (TIP). CAM plants showed a persistent rhythm of specific AQP protein abundance changes throughout the day/night cycle, including changes in amount of McPIP2;1, McTIP1;2, McPIP1;4 and McPIP1;5, while the abundance of McPIP1;2 was unchanged. These protein changes did not appear to be coordinated with transcript levels for any of the AQPs analysed; however, they did occur in parrallel to alterations in water permeability, as well as variations in cell osmolarity, pinitol, glucose, fructose and phosphoenolpyruvate carboxylase (PEPc) levels measured throughout the day/night CAM cycle. Results suggest a role for AQPs in maintaining water balance during CAM and highlight the complexity of protein expression during the CAM cycle.

A CAM- and starch-deficient mutant of the facultative CAM species Mesembryanthemum crystallinum reconciles sink demands by repartitioning carbon during acclimation to salinity.

In the halophytic species Mesembryanthemum crystallinum, the induction of crassulacean acid metabolism (CAM) by salinity requires a substantial investment of resources in storage carbohydrates to provide substrate for nocturnal CO(2) uptake. Acclimation to salinity also requires the synthesis and accumulation of cyclitols as compatible solutes, maintenance of root respiration, and nitrate assimilation. This study assessed the hierarchy and coordination of sinks for carbohydrate in leaves and roots during acclimation to salinity in M. crystallinum. By comparing wild type and a CAM-/starch-deficient mutant of this species, it was sought to determine if other metabolic sinks could compensate for a curtailment in CAM and enable acclimation to salinity. Under salinity, CAM deficiency reduced 24 h photosynthetic carbon gain by >50%. Cyclitols were accumulated to comparable levels in leaves and roots of both the wild type and mutant, but represented only 5% of 24 h carbon balance. Dark respiration of leaves and roots was a stronger sink for carbohydrate in the mutant compared with the wild type and implied higher maintenance costs for the metabolic processes underpinning acclimation to salinity when CAM was curtailed. CAM required the nocturnal mobilization of >70% of primary carbohydrate in the wild type and >85% of carbohydrate in the mutant. The substantial allocation of carbohydrate to CAM limited the export of sugars to roots, and the root:shoot ratio declined under salinity. The data suggest a key role for the vacuole in regulating the supply and demand for carbohydrate over the day/night cycle in the starch-/CAM-deficient mutant.

Root system architecture analysis in Mesembryanthemum crystallinum (ice plant) seedlings reveals characteristic root halotropic response.

One of the major environmental stress factors that affect root growth is salinity. Arabidopsis thaliana, a glycophyte, shows halotropism, whereby it alters the direction of root growth in a non-gravitropic pattern to evade high soil salinity. Asymmetric auxin distribution regulated by the relocation of auxin-efflux carrier proteins is a key cellular event in the halotropic response. However, there are no reports of halotropism in halophytes. Here, we investigated root growth traits in Mesembryanthemum crystallinum (ice plant), under high salinity conditions. We hypothesized that ice plant roots would show halotropic responses different from those of Arabidopsis Notably, similar to halotropism observed in Arabidopsis, ice plant roots showed continuous root bending under salinity stress. However, the root elongation rate did not change in ice plants. Expression analyses of several genes revealed that auxin transport might be partially involved in ice plant halotropism. This study enhances our understanding of halophyte root adaptation to high salinity stress.

Possible roles for phytohormones in controlling the stomatal behavior of Mesembryanthemum crystallinum during the salt-induced transition from C3 to crassulacean acid metabolism.

The halophyte ice plant (Mesembryanthemum crystallinum) converts its mode of photosynthesis from C3 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 CO2 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 C3 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.

Leaf carbohydrates influence transcriptional and post-transcriptional regulation of nocturnal carboxylation and starch degradation in the facultative CAM plant, Mesembryanthemum crystallinum.

Nocturnal degradation of transitory starch is a limiting factor for the optimal function of crassulacean acid metabolism and must be coordinated with phosphoenolypyruvate carboxylase (PEPC)-mediated CO2 uptake to optimise carbon gain over the diel cycle. The aim of this study was to test the hypothesis that nocturnal carboxylation is coordinated with starch degradation in CAM via a mechanism whereby the products of these pathways regulate diel transcript abundance and enzyme activities for both processes. To test this hypothesis, a starch and CAM-deficient mutant of Mesembryanthemum crystallinum was compared with wild type plants under well-watered and saline (CAM-inducing) conditions. Exposure to salinity increased the transcript abundance of genes required for nocturnal carboxylation, starch and sucrose degradation in both wild type and mutant, but the transcript abundance of several of these genes was not sustained over the dark period in the low-carbohydrate, CAM-deficient mutant. The diel pattern of transcript abundance for PEPC mirrored that of PEPC protein, as did the transcripts, protein, and activity of chloroplastic starch phosphorylase in both wild type and mutant, suggesting robust diel coordination of these metabolic processes. Activities of several amylase isoforms were low or lacking in the mutant, whilst the activity of a cytosolic isoform of starch phosphorylase was significantly elevated, indicating contrasting modes of metabolic regulation for the hydrolytic and phosphorylytic routes of starch degradation. Externally supplied sucrose resulted in an increase in nocturnal transcript abundance of genes required for nocturnal carboxylation and starch degradation. These results demonstrate that carbohydrates impact on transcriptional and post-transcriptional regulation of nocturnal carboxylation and starch degradation in CAM.

Honeycomb Actuators Inspired by the Unfolding of Ice Plant Seed Capsules.

Plant hydro-actuated systems provide a rich source of inspiration for designing autonomously morphing devices. One such example, the pentagonal ice plant seed capsule, achieves complex mechanical actuation which is critically dependent on its hierarchical organization. The functional core of this actuation system involves the controlled expansion of a highly swellable cellulosic layer, which is surrounded by a non-swellable honeycomb framework. In this work, we extract the design principles behind the unfolding of the ice plant seed capsules, and use two different approaches to develop autonomously deforming honeycomb devices as a proof of concept. By combining swelling experiments with analytical and finite element modelling, we elucidate the role of each design parameter on the actuation of the prototypes. Through these approaches, we demonstrate potential pathways to design/develop/construct autonomously morphing systems by tailoring and amplifying the initial material's response to external stimuli through simple geometric design of the system at two different length scales.

Effects of exogenously applied hydrogen peroxide on antioxidant and osmoprotectant profiles and the C3-CAM shift in the halophyte Mesembryanthemum crystallinum L.

Exogenously applied H2O2 (50, 100 and 200mM) to Mesembryanthemum crystallinum root medium induced a transition from C3 to Crassulacean Acid Metabolism (CAM), as evaluated by diurnal malate (Δmal) fluctuations. A very high concentration of H2O2 (400mM) reduced Δmal below the value measured in control plants. An increase of malate content during the night in 400mM H2O2-treated plants might suggest that malate decarboxylation is crucial for CAM functioning. We conclude that malate plays a dual role: i) a protective and signaling function before CAM expression, and ii) a storage form of CO2 in plants performing CAM. A slight stimulation of photosystem II (PSII) photochemistry and net photosynthesis observed during the C3-CAM shift indicated that neither photoinhibition nor reduction of the photosynthetic rate were prerequisites for CAM. Moreover, CAM induction corresponded to a decrease of catalase activity. In CAM-performing plants, α-tocopherol, polyamines (putrescine and spermidine) and proline showed daily alterations and the content of α-tocopherol and polyamines was lower at the end of the day. In contrast, the proline concentration correlated with the applied H2O2 concentration and was higher at the end of the day in treated plants. The dynamic changes of antioxidant and osmolyte levels suggest their active role in preventing oxidative damage, stress acclimation mechanisms and involvement in metabolic regulation and/or signal transduction cascades.

RNA-seq analysis of the response of the halophyte, Mesembryanthemum crystallinum (ice plant) to high salinity.

Understanding the molecular mechanisms that convey salt tolerance in plants is a crucial issue for increasing crop yield. The ice plant (Mesembryanthemum crystallinum) is a halophyte that is capable of growing under high salt conditions. For example, the roots of ice plant seedlings continue to grow in 140 mM NaCl, a salt concentration that completely inhibits Arabidopsis thaliana root growth. Identifying the molecular mechanisms responsible for this high level of salt tolerance in a halophyte has the potential of revealing tolerance mechanisms that have been evolutionarily successful. In the present study, deep sequencing (RNAseq) was used to examine gene expression in ice plant roots treated with various concentrations of NaCl. Sequencing resulted in the identification of 53,516 contigs, 10,818 of which were orthologs of Arabidopsis genes. In addition to the expression analysis, a web-based ice plant database was constructed that allows broad public access to the data. The results obtained from an analysis of the RNAseq data were confirmed by RT-qPCR. Novel patterns of gene expression in response to high salinity within 24 hours were identified in the ice plant when the RNAseq data from the ice plant was compared to gene expression data obtained from Arabidopsis plants exposed to high salt. Although ABA responsive genes and a sodium transporter protein (HKT1), are up-regulated and down-regulated respectively in both Arabidopsis and the ice plant; peroxidase genes exhibit opposite responses. The results of this study provide an important first step towards analyzing environmental tolerance mechanisms in a non-model organism and provide a useful dataset for predicting novel gene functions.

Effect of salt treatment on phenolic compounds and antioxidant activity of two Mesembryanthemum edule provenances.

Mesembryanthemum edule L. is an edible and medicinal halophyte widespread in Tunisia seashore. In this study, parameters of oxidative stress, phenolic compounds and antioxidant activities were comparatively investigated in two M. edule provenances (Jerba and Bizerte, respectively sampled from arid and humid bioclimatic stages). Plants were subjected to 0, 300 and 600mM NaCl treatment under glasshouse conditions. Results showed that M. edule response to salinity depends on provenance (P), salt treatment (T) and their interaction (P×T). (T) affected more significantly the oxidative stress parameters and antioxidant activities than (P) and (P×T). Conversely, (P) was much affluent for tannin polymerization degree and interaction between the two factors (P×T) was more determinants for analyzed antioxidant parameters. The higher salt tolerance of Jerba plants was associated with low levels of malondialdehyde and of electrolyte leakage mainly at 600mM NaCl. Besides, antioxidant activities of Jerba provenance, were more efficient than Bizerte. In addition, avicularin was the major phenolic in both provenances. This compound concentration increased with salinity in Jerba shoots, while it was reduced in Bizerte especially at 600mM NaCl. Overall, the higher salt tolerance of plants from Jerba provenance, and to a lower extent of those from Bizerte, may be partly related to their better capacity to limit oxidative damage when salt-challenged, and this is likely the result of redistribution in phenolic composition. Besides, abiotic factors such as salinity could be determinant in antioxidant potentiality of this medicinal plant.

Religión y salud: la intervención pública de agentes religiosos católicos formados en bioética en el debate parlamentario sobre la muerte digna en la Argentina / Religion and health: the public intervention of Catholic religious agents trained in bioethics in the parliamentary debate on death with dignity in Argentina

Desde una perspectiva sociológica, este trabajo aborda una de las aristas de la intervención pública de ciertos sectores del catolicismo en la elaboración y sanción de leyes de salud. En particular se hace foco en el debate en comisiones parlamentarias sobre la llamada ley de "muerte digna" (Ley 26742) en el cual se convocó a un grupo de expertos en bioética para asesorar a los senadores sobre los alcances y límites de la ley. La mayoría de los expertos invitados pregonan la perspectiva de la bioética personalista, un desarrollo teológico de la bioética del catolicismo contemporáneo. En el debate no participaron representantes de otros credos consolidando la ampliamente estudiada imbricación entre el catolicismo y lo político en Argentina.

This paper discusses from a sociological perspective one of Catholicism's fronts of public intervention in the development and enactment of health legislation. In particular we analyze the debate in parliamentary committees on the so-called "death with dignity" law (No. 26742), for which a group of bioethics experts was convened to counsel senators regarding the scope and limits of the law. The majority of the invited experts advocated a personalist bioethics perspective, which is a theological bioethics development of contemporary Catholicism. In the debate no representatives of other faiths were present, reinforcing the widely studied overlap between Catholicism and politics in Argentina.

Two functionally different vacuoles for static and dynamic purposes in one plant mesophyll leaf cell.

It is a common belief that plant mesophyll cells are occupied up to 95% by a single multipurpose vacuole. The common ice plant, Mesembryanthemum crystallinum L., however, requires two contrasting functions of the vacuole under salt stress. Large amounts of NaCl have to be sequestered permanently for osmotic purpose and for protecting the cytoplasm from NaCl toxicity. A dynamic exchange with the cytoplasm is required because photosynthesis proceeds under these conditions via the metabolic cycle of crassulacean acid metabolism (CAM). Nocturnally acquired CO2 must be kept as malate in the vacuole and re-mobilized in the daytime. Here, we show that two large independent types of vacuoles with different transport properties meet the requirements for the contrasting functions within the same cell.

Novel regulation of aquaporins during osmotic stress.

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.

Expression and stress-dependent induction of potassium channel transcripts in the common ice plant.

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.