Current Understanding of Role of Vesicular Transport in Salt Secretion by Salt Glands in Recretohalophytes.

Soil salinization is a serious and growing problem around the world. Some plants, recognized as the recretohalophytes, can normally grow on saline-alkali soil without adverse effects by secreting excessive salt out of the body. The elucidation of the salt secretion process is of great significance for understanding the salt tolerance mechanism adopted by the recretohalophytes. Between the 1950s and the 1970s, three hypotheses, including the osmotic potential hypothesis, the transfer system similar to liquid flow in animals, and vesicle-mediated exocytosis, were proposed to explain the salt secretion process of plant salt glands. More recently, increasing evidence has indicated that vesicular transport plays vital roles in salt secretion of recretohalophytes. Here, we summarize recent findings, especially regarding the molecular evidence on the functional roles of vesicular trafficking in the salt secretion process of plant salt glands. A model of salt secretion in salt gland is also proposed.

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.

Single cell-type analysis of cellular lipid remodelling in response to salinity in the epidermal bladder cells of the model halophyte Mesembryanthemum crystallinum.

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.

Cellular mechanisms to survive salt in the halophyte Cakile maritima.

We recently identified two behaviours in cultured cells of the salt accumulating halophyte Cakile maritima: one related to a sustained depolarization due to Na+ influx through the non-selective cation channels leading to programmed cell death of these cells, a second one related to a transient depolarization allowing cells to survive (Ben Hamed-Laouti, 2016). In this study, we considered at the cellular level mechanisms that could participate to the exclusion of Na+ out of the cell and thus participate in the regulation of the internal contents of Na+ and cell survival. Upon addition of NaCl in the culture medium of suspension cells of C. maritima, we observed a rapid influx of Na+ followed by an efflux dependent of the activity of plasma membrane H+-ATPases, in accordance with the functioning of a Na+/H+ antiporter and the ability of some cells to repolarize. The Na+ efflux was shown to be dependent on Na+-dependent on Ca2+ influx like the SOS1 Na+/H+ antiporter. We further could observe in response to salt addition, an early production of singlet oxygen (1O2) probably due to peroxidase activities. This early 1O2 production seemed to be a prerequisite to the Na+ efflux. Our findings suggest that in addition to the pathway leading to PCD (Ben Hamed-Laouti, 2016), a second pathway comprising an SOS-like system could participate to the survival of a part of the C. maritima cultured cells challenged by salt stress.

Detection of lipid-protein microdomains (rafts) and investigation of their functional role in the chloroplast membranes of halophytes.

To study the characteristics of the structure and the functional role of chloroplast membranes of halophytes with different salt tolerance strategy, raft structures were isolated. Lipid analysis data provided evidence for the presence of rafts in the chloroplast membranes. The discovered significant differences in the content of raft-forming lipids in different halophytes suggest a relationship between the functions of rafts and the mechanisms of salt tolerance in plants.

Pleiotropic phenotypes of the salt-tolerant and cytosine hypomethylated leafless inflorescence, evergreen dwarf and irregular leaf lamina mutants of Catharanthus roseus possessing Mendelian inheritance.

In Catharanthus roseus, three morphological cum salt-tolerant chemically induced mutants of Mendelian inheritance and their wild-type parent cv Nirmal were characterized for overall cytosine methylation at DNA repeats, expression of 119 protein coding and seven miRNA-coding genes and 50 quantitative traits. The mutants, named after their principal morphological feature(s), were leafless inflorescence (lli), evergreen dwarf (egd) and irregular leaf lamina (ill). The Southern-blot analysis of MspI digested DNAs of mutants probed with centromeric and 5S and 18S rDNA probes indicated that, in comparison to wild type, the mutants were extensively demethylated at cytosine sites. Among the 126 genes investigated for transcriptional expression, 85 were upregulated and 41 were downregulated in mutants. All of the five genes known to be stress responsive had increased expression in mutants. Several miRNA genes showed either increased or decreased expression in mutants. The C. roseus counterparts of CMT3, DRM2 and RDR2 were downregulated in mutants. Among the cell, organ and plant size, photosynthesis and metabolism related traits studied, 28 traits were similarly affected in mutants as compared to wild type. Each of the mutants also expressed some traits distinctively. The egd mutant possessed superior photosynthesis and water retention abilities. Biomass was hyperaccumulated in roots, stems, leaves and seeds of the lli mutant. The ill mutant was richest in the pharmaceutical alkaloids catharanthine, vindoline, vincristine and vinblastine. The nature of mutations, origins of mutant phenotypes and evolutionary importance of these mutants are discussed.

Protein profiling of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum.

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.

The rolB gene suppresses reactive oxygen species in transformed plant cells through the sustained activation of antioxidant defense.

The rolB (for rooting locus of Agrobacterium rhizogenes) oncogene has previously been identified as a key player in the formation of hairy roots during the plant-A. rhizogenes interaction. In this study, using single-cell assays based on confocal microscopy, we demonstrated reduced levels of reactive oxygen species (ROS) in rolB-expressing Rubia cordifolia, Panax ginseng, and Arabidopsis (Arabidopsis thaliana) cells. The expression of rolB was sufficient to inhibit excessive elevations of ROS induced by paraquat, menadione, and light stress and prevent cell death induced by chronic oxidative stress. In rolB-expressing cells, we detected the enhanced expression of antioxidant genes encoding cytosolic ascorbate peroxidase, catalase, and superoxide dismutase. We conclude that, similar to pathogenic determinants in other pathogenic bacteria, rolB suppresses ROS and plays a role not only in cell differentiation but also in ROS metabolism.

Mechanisms of plant salt response: insights from proteomics.

Soil salinity is a major abiotic stress that limits plant growth and agriculture productivity. To cope with salt stress, plants have evolved complex salt-responsive signaling and metabolic processes at the cellular, organ, and whole-plant levels. Investigation of the physiological and molecular mechanisms underlying plant salinity tolerance will provide valuable information for effective engineering strategies. Current proteomics provides a high-throughput approach to study sophisticated molecular networks in plants. In this review, we describe a salt-responsive protein database by an integrated analysis of proteomics-based studies. The database contains 2171 salt-responsive protein identities representing 561 unique proteins. These proteins have been identified from leaves, roots, shoots, seedlings, unicells, grains, hypocotyls, radicles, and panicles from 34 plant species. The identified proteins provide invaluable information toward understanding the complex and fine-tuned plant salt-tolerance mechanisms in photosynthesis, reactive oxygen species (ROS) scavenging, ion homeostasis, osmotic modulation, signaling transduction, transcription, protein synthesis/turnover, cytoskeleton dynamics, and cross-tolerance to different stress conditions.

Rising from the sea: correlations between sulfated polysaccharides and salinity in plants.

High salinity soils inhibit crop production worldwide and represent a serious agricultural problem. To meet our ever-increasing demand for food, it is essential to understand and engineer salt-resistant crops. In this study, we evaluated the occurrence and function of sulfated polysaccharides in plants. Although ubiquitously present in marine algae, the presence of sulfated polysaccharides among the species tested was restricted to halophytes, suggesting a possible correlation with salt stress or resistance. To test this hypothesis, sulfated polysaccharides from plants artificially and naturally exposed to different salinities were analyzed. Our results revealed that the sulfated polysaccharide concentration, as well as the degree to which these compounds were sulfated in halophytic species, were positively correlated with salinity. We found that sulfated polysaccharides produced by Ruppia maritima Loisel disappeared when the plant was cultivated in the absence of salt. However, subjecting the glycophyte Oryza sativa Linnaeus to salt stress did not induce the biosynthesis of sulfated polysaccharides but increased the concentration of the carboxylated polysaccharides; this finding suggests that negatively charged cell wall polysaccharides might play a role in coping with salt stress. These data suggest that the presence of sulfated polysaccharides in plants is an adaptation to high salt environments, which may have been conserved during plant evolution from marine green algae. Our results address a practical biological concept; additionally, we suggest future strategies that may be beneficial when engineering salt-resistant crops.

The extreme halophyte Salicornia veneta is depleted of the extrinsic PsbQ and PsbP proteins of the oxygen-evolving complex without loss of functional activity.

BACKGROUND AND AIMS: Photosystem II of oxygenic organisms is a multi-subunit protein complex made up of at least 20 subunits and requires Ca(2+) and Cl(-) as essential co-factors. While most subunits form the catalytic core responsible for water oxidation, PsbO, PsbP and PsbQ form an extrinsic domain exposed to the luminal side of the membrane. In vitro studies have shown that these subunits have a role in modulating the function of Cl(-) and Ca(2+), but their role(s) in vivo remains to be elucidated, as the relationships between ion concentrations and extrinsic polypeptides are not clear. With the aim of understanding these relationships, the photosynthetic apparatus of the extreme halophyte Salicornia veneta has been compared with that of spinach. Compared to glycophytes, halophytes have a different ionic composition, which could be expected to modulate the role of extrinsic polypeptides. METHODS: Structure and function of in vivo and in vitro PSII in S. veneta were investigated and compared to spinach. Light and electron microscopy, oxygen evolution, gel electrophoresis, immunoblotting, DNA sequencing, RT-PCR and time-resolved chlorophyll fluorescence were used. KEY RESULTS: Thylakoids of S. veneta did not contain PsbQ protein and its mRNA was absent. When compared to spinach, PsbP was partly depleted (30 %), as was its mRNA. All other thylakoid subunits were present in similar amounts in both species. PSII electron transfer was not affected. Fluorescence was strongly quenched upon irradiation of plants with high light, and relaxed only after prolonged dark incubation. Quenching of fluorescence was not linked to degradation of D1 protein. CONCLUSIONS: In S. veneta the PsbQ protein is not necessary for photosynthesis in vivo. As the amount of PsbP is sub-stoichiometric with other PSII subunits, this protein too is largely dispensable from a catalytic standpoint. One possibility is that PsbP acts as an assembly factor for PSII.

Tolerance of combined submergence and salinity in the halophytic stem-succulent Tecticornia pergranulata.

BACKGROUND AND AIMS: Habitats occupied by many halophytes are not only saline, but are also prone to flooding. Few studies have evaluated submergence tolerance in halophytes. METHODS: Responses to submergence, at a range of salinity levels, were studied for the halophytic stem-succulent Tecticornia pergranulata subsp. pergranulata (syn. Halosarcia pergranulata subsp. pergranulata). Growth and total sugars in succulent stems were assessed as a function of time after submergence. Underwater net photosynthesis, dark respiration, total sugars, glycinebetaine, Na(+), Cl(-) and K(+), in succulent stems, were assessed in a NaCl dose-response experiment. KEY RESULTS: Submerged plants ceased to grow, and tissue sugars declined. Photosynthesis by succulent stems was reduced markedly when underwater, as compared with in air. Capacity for underwater net photosynthesis (P(N)) was not affected by 10-400 mM NaCl, but it was reduced by 30 % at 800 mM. Dark respiration, underwater, increased in succulent stems at 200-800 mM NaCl, as compared with those at 10 mM NaCl. On an ethanol-insoluble dry mass basis, K(+) concentration in succulent stems of submerged plants was equal to that in drained controls, across all NaCl treatments. Na(+) and Cl(-) concentrations, however, were elevated in stems of submerged plants, but so was glycinebetaine. Submerged stems increased in succulence, so solutes would have been 'diluted' on a tissue-water basis. CONCLUSIONS: Tecticornia pergranulata tolerates complete submergence, even in waters of high salinity. A 'quiescence response', i.e. no shoot growth, would conserve carbohydrates, but tissue sugars still declined with time. A low K(+) : Na(+) ratio, typical for tissues of succulent halophytes, was tolerated even during prolonged submergence, as evidenced by maintenance of underwater P(N) at up to 400 mM NaCl. Underwater P(N) provides O(2) and sugars, and thus should enhance survival of submerged plants.

Isolation and characterization of plasma membrane Na(+)/H(+) antiporter genes from salt-sensitive and salt-tolerant reed plants.

We isolated cDNAs for Na(+)/H(+) antiporter genes (PhaNHA1s) from salt-sensitive and salt-tolerant reed plants. A phylogenetic analysis and localization analysis using yeast strains expressing PhaNHA1-GFP protein showed that PhaNHA1s were plasma membrane Na(+)/H(+) antiporters. Yeast strains expressing PhaNHA1 from salt-tolerant reed plants (PhaNHA1-n) grew well than yeast strains expressing PhaNHA1 from salt-sensitive reed plants (PhaNHA1-u) in the presence of 100mM NaCl. Furthermore, Na(+) contents of yeast cells expressing PhaNHA1-n were less than half of those of yeast cells expressing PhaNHA1-u. These results suggest that PhaNHA1-n is more efficient at excluding Na(+) from the cells than PhaNHA1-u.

Contrasting responses of photosynthesis to salt stress in the glycophyte Arabidopsis and the halophyte thellungiella: role of the plastid terminal oxidase as an alternative electron sink.

The effects of short-term salt stress on gas exchange and the regulation of photosynthetic electron transport were examined in Arabidopsis (Arabidopsis thaliana) and its salt-tolerant close relative Thellungiella (Thellungiella halophila). Plants cultivated on soil were challenged for 2 weeks with NaCl. Arabidopsis showed a much higher sensitivity to salt than Thellungiella; while Arabidopsis plants were unable to survive exposure to greater than 150 mM salt, Thellugiella could tolerate concentrations as high as 500 mM with only minimal effects on gas exchange. Exposure of Arabidopsis to sublethal salt concentrations resulted in stomatal closure and inhibition of CO2 fixation. This lead to an inhibition of electron transport though photosystem II (PSII), an increase in cyclic electron flow involving only PSI, and increased nonphotochemical quenching of chlorophyll fluorescence. In contrast, in Thellungiella, although gas exchange was marginally inhibited by high salt and PSI was unaffected, there was a large increase in electron flow involving PSII. This additional electron transport activity is oxygen dependent and sensitive to the alternative oxidase inhibitor n-propyl gallate. PSII electron transport in Thellungiella showed a reduced sensitivity to 2'-iodo-6-isopropyl-3-methyl-2',4,4'-trinitrodiphenylether, an inhibitor of the cytochrome b6f complex. At the same time, we observed a substantial up-regulation of a protein reacting with antibodies raised against the plastid terminal oxidase. No such up-regulation was seen in Arabidopsis. We conclude that in salt-stressed Thellungiella, plastid terminal oxidase acts as an alternative electron sink, accounting for up to 30% of total PSII electron flow.