How the Ethylene Biosynthesis Pathway of Semi-Halophytes Is Modified with Prolonged Salinity Stress Occurrence?

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.

Exogenous myo-inositol increases salt tolerance and accelerates CAM induction in the early juvenile stage of the facultative halophyte Mesembryanthemum crystallinum but not in the late juvenile stage.

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.

Ice plant root plasma membrane aquaporins are regulated by clathrin-coated vesicles in response to salt stress.

The regulation of root Plasma membrane (PM) Intrinsic Protein (PIP)-type aquaporins (AQPs) is potentially important for salinity tolerance. However, the molecular and cellular details underlying this process in halophytes remain unclear. Using free-flow electrophoresis and label-free proteomics, we report that the increased abundance of PIPs at the PM of the halophyte ice plant (Mesembryanthemum crystallinum L.) roots under salinity conditions is regulated by clathrin-coated vesicles (CCV). To understand this regulation, we analyzed several components of the M. crystallinum CCV complexes: clathrin light chain (McCLC) and subunits µ1 and µ2 of the adaptor protein (AP) complex (McAP1µ and McAP2µ). Co-localization analyses revealed the association between McPIP1;4 and McAP2µ and between McPIP2;1 and McAP1µ, observations corroborated by mbSUS assays, suggesting that AQP abundance at the PM is under the control of CCV. The ability of McPIP1;4 and McPIP2;1 to form homo- and hetero-oligomers was tested and confirmed, as well as their activity as water channels. Also, we found increased phosphorylation of McPIP2;1 only at the PM in response to salt stress. Our results indicate root PIPs from halophytes might be regulated through CCV trafficking and phosphorylation, impacting their localization, transport activity, and abundance under salinity conditions.

Overexpression of McHB7 Transcription Factor from Mesembryanthemum crystallinum Improves Plant Salt Tolerance.

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, McHB7 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.

High-quality ice plant reference genome analysis provides insights into genome evolution and allows exploration of genes involved in the transition from C3 to CAM pathways.

Ice plant (Mesembryanthemum crystallinum), a member of the Aizoaceae family, is a typical halophyte crop and a model plant for studying the mechanism of transition from C3 photosynthesis to crassulacean acid metabolism (CAM). Here, we report a high-quality chromosome-level ice plant genome sequence. This 98.05% genome sequence is anchored to nine chromosomes, with a total length of 377.97 Mb and an N50 scaffold of 40.45 Mb. Almost half of the genome (48.04%) is composed of repetitive sequences, and 24 234 genes have been annotated. Subsequent to the ancient whole-genome triplication (WGT) that occurred in eudicots, there has been no recent whole-genome duplication (WGD) or WGT in ice plants. However, we detected a novel WGT event that occurred in the same order in Simmondsia chinensis, which was previously overlooked. Our findings revealed that ice plants have undergone chromosome rearrangements and gene removal during evolution. Combined with transcriptome and comparative genomic data and expression verification, we identified several key genes involved in the CAM pathway and constructed a comprehensive network. As the first genome of the Aizoaceae family to be released, this report will provide a rich data resource for comparative and functional genomic studies of Aizoaceae, especially for studies on salt tolerance and C3-to-CAM transitions to improve crop yield and resistance.

At the Edges of Photosynthetic Metabolic Plasticity-On the Rapidity and Extent of Changes Accompanying Salinity Stress-Induced CAM Photosynthesis Withdrawal.

The common ice plant (Mesembryanthemum crystallinum L.) is a facultative crassulacean acid metabolism (CAM) plant, and its ability to recover from stress-induced CAM has been confirmed. We analysed the photosynthetic metabolism of this plant during the 72-h response period following salinity stress removal from three perspectives. In plants under salinity stress (CAM) we found a decline of the quantum efficiencies of PSII (Y(II)) and PSI (Y(I)) by 17% and 15%, respectively, and an increase in nonphotochemical quenching (NPQ) by almost 25% in comparison to untreated control. However, 48 h after salinity stress removal, the PSII and PSI efficiencies, specifically Y(II) and Y(I), elevated nonphotochemical quenching (NPQ) and donor side limitation of PSI (YND), were restored to the level observed in control (C3 plants). Swelling of the thylakoid membranes, as well as changes in starch grain quantity and size, have been found to be components of the salinity stress response in CAM plants. Salinity stress induced an over 3-fold increase in average starch area and over 50% decline of average seed number in comparison to untreated control. However, in plants withdrawn from salinity stress, during the first 24 h of recovery, we observed chloroplast ultrastructures closely resembling those found in intact (control) ice plants. Rapid changes in photosystem functionality and chloroplast ultrastructure were accompanied by the induction of the expression (within 24 h) of structural genes related to the PSI and PSII reaction centres, including PSAA, PSAB, PSBA (D1), PSBD (D2) and cp43. Our findings describe one of the most flexible photosynthetic metabolic pathways among facultative CAM plants and reveal the extent of the plasticity of the photosynthetic metabolism and related structures in the common ice plant.

Proteomics of Homeobox7 Enhanced Salt Tolerance in Mesembryanthemum crystallinum.

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.

Evaluation of potential impacts on biodiversity of the salt-tolerant transgenic Eucalyptus camaldulensis harboring an RNA chaperonic RNA-Binding-Protein gene derived from common ice plant.

We recently reported that a genetic transformation of the RNA-Binding-Protein (McRBP), an RNA chaperone gene derived from common ice plant (Mesembryanthemum crystallinum), alleviated injury and loss of biomass production by salt stress in Eucalyptus camaldulensis in a semi-confined screen house trial. In this study, we assessed the potential environmental impact of the transgenic Eucalyptus in a manner complying with Japanese biosafety regulatory framework required for getting permission for experimental confined field trials. Two kinds of bioassays for the effects of allelopathic activity on the growth of other plants, i.e., the sandwich assay and the succeeding crop assay, were performed for three transgenic lines and three non-transgenic lines. No significant differences were observed between transgenic and non-transgenic plants. No significant difference in the numbers of cultivable microorganisms analyzed by the spread plate method were observed among the six transgenic and non-transgenic lines. These results suggested that there is no significant difference in the potential impact on biodiversity between the transgenic McRBP-E. camaldulensis lines and their non-transgenic comparators.

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.

Functional analysis of McSnRK1 (SNF1-related protein kinase 1) in regulating Na/K homeostasis in transgenic cultured cells and roots of halophyte Mesembryanthemum crystallinum.

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.

Development and evaluation of novel salt-tolerant Eucalyptus trees by molecular breeding using an RNA-Binding-Protein gene derived from common ice plant (Mesembryanthemum crystallinum L.).

The breeding of plantation forestry trees for the possible afforestation of marginal land would be one approach to addressing global warming issues. Here, we developed novel transgenic Eucalyptus trees (Eucalyptus camaldulensis Dehnh.) harbouring an RNA-Binding-Protein (McRBP) gene derived from a halophyte plant, common ice plant (Mesembryanthemum crystallinum L.). We conducted screened-house trials of the transgenic Eucalyptus using two different stringency salinity stress conditions to evaluate the plants' acute and chronic salt stress tolerances. Treatment with 400 mM NaCl, as the high-stringency salinity stress, resulted in soil electrical conductivity (EC) levels >20 mS/cm within 4 weeks. With the 400 mM NaCl treatment, >70% of the transgenic plants were intact, whereas >40% of the non-transgenic plants were withered. Treatment with 70 mM NaCl, as the moderate-stringency salinity stress, resulted in soil EC levels of approx. 9 mS/cm after 2 months, and these salinity levels were maintained for the next 4 months. All plants regardless of transgenic or non-transgenic status survived the 70 mM NaCl treatment, but after 6-month treatment the transgenic plants showed significantly higher growth and quantum yield of photosynthesis levels compared to the non-transgenic plants. In addition, the salt accumulation in the leaves of the transgenic plants was 30% lower than that of non-transgenic plants after 15-week moderate salt stress treatment. There results suggest that McRBP expression in the transgenic Eucalyptus enhances their salt tolerance both acutely and chronically.

Crassulacean Acid Metabolism Induction in Mesembryanthemum crystallinum Can Be Estimated by Non-Photochemical Quenching upon Actinic Illumination During the Dark Period.

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.

Ectopic expression of Mesembryanthemum crystallinum sodium transporter McHKT2 provides salt stress tolerance in Arabidopsis thaliana.

Most plants do not tolerate highly saline environments; the development of salt stress tolerance is crucial for improving crop yield. An efficient way of finding genes involved in salt tolerance is to study and use data from halophytes. In this study, we used the Mesembryanthemum crystallinum (ice plant) expression data-set and selected for further study the gene McHKT2, which encodes for the Arabidopsis sodium transporter ortholog AtHKT1. In comparison with the HKT1 amino acid sequences from other plants, McHKT2 has several unique features. It seems to be localized to the plasma membrane, and its overexpression confers strong salt tolerance in Arabidopsis thaliana. Our results indicate that McHKT2 is a suitable candidate protein that can induce salt tolerance in non-halophytes. Like McHKT2, using transcriptome data-sets from halophytes such as ice plant give us an efficiency way to obtain new gene resources that might involve in plant salt tolerance.

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.

Single-cell-type quantitative proteomic and ionomic analysis of epidermal bladder cells from the halophyte model plant Mesembryanthemum crystallinum to identify salt-responsive proteins.

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.

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.

Veinal-mesophyll interaction under biotic stress.

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 ß-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.

Cell type-specific responses to salinity - the epidermal bladder cell transcriptome of Mesembryanthemum crystallinum.

Mesembryanthemum crystallinum (ice plant) exhibits extreme tolerance to salt. Epidermal bladder cells (EBCs), developing on the surface of aerial tissues and specialized in sodium sequestration and other protective functions, are critical for the plant's stress adaptation. We present the first transcriptome analysis of EBCs isolated from intact plants, to investigate cell type-specific responses during plant salt adaptation. We developed a de novo assembled, nonredundant EBC reference transcriptome. Using RNAseq, we compared the expression patterns of the EBC-specific transcriptome between control and salt-treated plants. The EBC reference transcriptome consists of 37 341 transcript-contigs, of which 7% showed significantly different expression between salt-treated and control samples. We identified significant changes in ion transport, metabolism related to energy generation and osmolyte accumulation, stress signalling, and organelle functions, as well as a number of lineage-specific genes of unknown function, in response to salt treatment. The salinity-induced EBC transcriptome includes active transcript clusters, refuting the view of EBCs as passive storage compartments in the whole-plant stress response. EBC transcriptomes, differing from those of whole plants or leaf tissue, exemplify the importance of cell type-specific resolution in understanding stress adaptive mechanisms.

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.

Isolation and characterization of a polyubiquitin gene and its promoter region from Mesembryanthemum crystallinum.

Transcript levels of the polyubiquitin gene McUBI1 had been reported to be constant during Crassulacean acid metabolism (CAM) induction in the facultative CAM plant, Mesembryanthemum crystallinum. Here, we report the sequences of the full-length cDNA of McUBI1 and its promoter, and validation of the McUBI1 promoter as an internal control driving constitutive expression in transient assays using the dual-luciferase system to investigate the regulation of CAM-related gene expression. The McUBI1 promoter drove strong, constitutive expression during CAM induction. We compared the activities of this promoter with those of the cauliflower mosaic virus (CaMV) 35S promoter in detached C3- and CAM-performing M. crystallinum and tobacco leaves. We confirmed stable expression of the genes controlled by the McUBI1 promoter with far less variability than under the CaMV 35S promoter in M. crystallinum, whereas both promoters worked well in tobacco. We found the McUBI1 promoter more suitable than the CaMV 35S promoter as an internal control for transient expression assays in M. crystallinum.