Guard Cell Transcriptome Reveals Membrane Transport, Stomatal Development and Cell Wall Modifications as Key Traits Involved in Salinity Tolerance in Halophytic Chenopodium quinoa.

A comparative investigation was conducted to evaluate transcriptional changes in guard cells (GCs) of closely related halophytic (Chenopodium quinoa) and glycophytic (Spinacia oleracea) species. Plants were exposed to 3 weeks of 250 mM sodium chloride treatment, and GC-enriched epidermal fragments were mechanically prepared. In both species, salt-responsive genes were mainly related to categories of protein metabolism, secondary metabolites, signal transduction and transport systems. Genes related to abscisic acid (ABA) signaling and ABA biosynthesis were strongly induced in quinoa but not in spinach GCs. Also, expression of the genes encoding transporters of amino acids, proline, sugars, sucrose and potassium increased in quinoa GCs under salinity stress. Analysis of cell-wall-related genes suggests that genes involved in lignin synthesis (e.g. lignin biosynthesis LACCASE 4) were highly upregulated by salt in spinach GCs. In contrast, transcripts related to cell wall plasticity Pectin methylesterase3 (PME3) were highly induced in quinoa. Faster stomatal response to light and dark measured by observing kinetics of changes in stomatal conductance in quinoa might be associated with higher plasticity of the cell wall regulated by PME3 Furthermore, genes involved in the inhibition of stomatal development and differentiation were highly expressed by salt in quinoa, but not in spinach. These changes correlated with reduced stomatal density and index in quinoa, thus improving its water use efficiency. The fine modulation of transporters, cell wall modification and controlling stomatal development in GCs of quinoa may have resulted in high K+/Na+ ratio, lower stomatal conductance and higher stomatal speed for better adaptation to salinity stress in quinoa.

Isoprenoid biosynthesis regulation in poplars by methylerythritol phosphate and mevalonic acid pathways.

It is critical to develop plant isoprenoid production when dealing with human-demanded industries such as flavoring, aroma, pigment, pharmaceuticals, and biomass used for biofuels. The methylerythritol phosphate (MEP) and mevalonic acid (MVA) plant pathways contribute to the dynamic production of isoprenoid compounds. Still, the cross-talk between MVA and MEP in isoprenoid biosynthesis is not quite recognized. Regarding the rate-limiting steps in the MEP pathway through catalyzing 1-deoxy-D-xylulose5-phosphate synthase and 1-deoxy-D-xylulose5-phosphate reductoisomerase (DXR) and also the rate-limiting step in the MVA pathway through catalyzing 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the characterization and function of HMGR from Populus trichocarpa (PtHMGR) were analyzed. The results indicated that PtHMGR overexpressors (OEs) displayed various MEP and MVA-related gene expressions compared to NT poplars. The overexpression of PtDXR upregulated MEP-related genes and downregulated MVA-related genes. The overexpression of PtDXR and PtHMGR affected the isoprenoid production involved in both MVA and MEP pathways. Here, results illustrated that the PtHMGR and PtDXR play significant roles in regulating MEP and MVA-related genes and derived isoprenoids. This study clarifies cross-talk between MVA and MEP pathways. It demonstrates the key functions of HMGR and DXR in this cross-talk, which significantly contribute to regulate isoprenoid biosynthesis in poplars.

Early responses to salt stress in quinoa genotypes with opposite behavior.

Soil salinity is among the major abiotic stresses that plants must cope with, mainly in arid and semiarid regions. The tolerance to high salinity is an important agronomic trait to sustain food production. Quinoa is a halophytic annual pseudo-cereal species with high nutritional value that can secrete salt out of young leaves in external non-glandular cells called epidermal bladder cells (EBC). Previous work showed high salt tolerance, but low EBC density was associated with an improved response in the early phases of salinity stress, mediated by tissue-tolerance traits mainly in roots. We compared the transcript profiling of two quinoa genotypes with contrasting salt tolerance patterning to identify the candidate genes involved in the differentially early response among genotypes. The transcriptome profiling, supported by in vitro physiological analyses, provided insights into the early-stage molecular mechanisms, both at the shoot and root level, based on the sensitive/tolerance traits. Results showed the presence of numerous differentially expressed genes among genotypes, tissues, and treatments, with genes involved in hormonal and stress response upregulated mainly in the sensitive genotype, suggesting that tolerance may be correlated to restricted changes in gene expression, at least after a short salt stress. These data, showing constitutive differences between the two genotypes, represent a solid basis for further studies to characterize the salt tolerance traits. Additionally, new information provided by this work might be useful for the development of plant breeding or genome engineering programs in quinoa.

Salinity Effects on Guard Cell Proteome in Chenopodium quinoa.

Epidermal fragments enriched in guard cells (GCs) were isolated from the halophyte quinoa (Chenopodium quinoa Wild.) species, and the response at the proteome level was studied after salinity treatment of 300 mM NaCl for 3 weeks. In total, 2147 proteins were identified, of which 36% were differentially expressed in response to salinity stress in GCs. Up and downregulated proteins included signaling molecules, enzyme modulators, transcription factors and oxidoreductases. The most abundant proteins induced by salt treatment were desiccation-responsive protein 29B (50-fold), osmotin-like protein OSML13 (13-fold), polycystin-1, lipoxygenase, alpha-toxin, and triacylglycerol lipase (PLAT) domain-containing protein 3-like (eight-fold), and dehydrin early responsive to dehydration (ERD14) (eight-fold). Ten proteins related to the gene ontology term "response to ABA" were upregulated in quinoa GC; this included aspartic protease, phospholipase D and plastid-lipid-associated protein. Additionally, seven proteins in the sucrose-starch pathway were upregulated in the GC in response to salinity stress, and accumulation of tryptophan synthase and L-methionine synthase (enzymes involved in the amino acid biosynthesis) was observed. Exogenous application of sucrose and tryptophan, L-methionine resulted in reduction in stomatal aperture and conductance, which could be advantageous for plants under salt stress. Eight aspartic proteinase proteins were highly upregulated in GCs of quinoa, and exogenous application of pepstatin A (an inhibitor of aspartic proteinase) was accompanied by higher oxidative stress and extremely low stomatal aperture and conductance, suggesting a possible role of aspartic proteinase in mitigating oxidative stress induced by saline conditions.

Developing and validating protocols for mechanical isolation of guard-cell enriched epidermal peels for omics studies.

Stomata, which are microscopic valves on the leaf surface formed by two guard cells (GC), play a critical role in the regulation of leaf water and gas exchange and, hence, determine plant adaptive potential. However, little data is available on GC biochemistry, protein abundance and gene expression, mainly due to technical difficulties and challenges in isolating sufficient amounts of high-quality pure GC. In the present study we applied some modifications to the mechanical isolation of guard-cell to generalise this method for diverse growth conditions as well as plant species. Epidermal peel fragments enriched in guard cells were mechanically isolated from quinoa, spinach and sugar beet leaves grown at two conditions (normal and salt stress). Multiple analysis was performed to confirm the suitability and superiority of the modified technique to the original method. At the first step, the viability and purity of GC-enriched epidermal fragments were assessed under the microscope. Then, the RNA integrity, gene expression, and 1D SDS-PAGE tests were performed to validate the suitability of this technique for omics studies. The data revealed a wide range of proteins as well as a high integrity of RNA extracted from guard cell samples. The expression level of several GC-specific genes and mesophyll-dominant genes were investigated using a comparative analysis of transcriptome datasets of GC and whole-leaf samples. We found that Rubisco and photosynthesis-related proteins such as chlorophyll a/b binding protein were substantially higher in the whole leaf compared with the GCs. More importantly, GC-specific genes such as OST1, SLAC1, MYB60, FAMA and HT1 were highly expressed in the GCs, confirming that our guard cell preparation was highly enriched in GC gene transcripts. Real-time quantitative reverse transcription PCR further confirmed the efficacy of the GC isolation technique for exploring responses of GC to diverse types of stress at the molecular level.

Understanding the role of root-related traits in salinity tolerance of quinoa accessions with contrasting epidermal bladder cell patterning.

MAIN CONCLUSION: To compensate for the lack of capacity for external salt storage in the epidermal bladder cells, quinoa plants employ tissue-tolerance traits, to confer salinity stress tolerance. Our previous studies indicated that sequestration of toxic Na+ and Cl- ions into epidermal bladder cells (EBCs) is an efficient mechanism conferring salinity tolerance in quinoa. However, some halophytes do not develop EBCs but still possess superior salinity tolerance. To elucidate the possible compensation mechanism(s) underlying superior salinity tolerance in the absence of the external salt storage capacity, we have selected four quinoa accessions with contrasting patterns of EBC development. Whole-plant physiological and electrophysiological characteristics were assessed after 2 days and 3 weeks of 400 mM NaCl stress. Both accessions with low EBC volume utilised Na+ exclusion at the root level and could maintain low Na+ concentration in leaves to compensate for the inability to sequester Na+ load in EBC. These conclusions were further confirmed by electrophysiological experiments showing higher Na+ efflux from roots of these varieties (measured by a non-invasive microelectrode MIFE technique) as compared to accessions with high EBC volume. Furthermore, accessions with low EBC volume had significantly higher K+ concentration in their leaves upon long-term salinity exposures compared to plants with high EBC sequestration ability, suggesting that the ability to maintain high K+ content in the leaf mesophyll was as another important compensation mechanism.

Sugar Beet (Beta vulgaris) Guard Cells Responses to Salinity Stress: A Proteomic Analysis.

Soil salinity is a major environmental constraint affecting crop growth and threatening global food security. Plants adapt to salinity by optimizing the performance of stomata. Stomata are formed by two guard cells (GCs) that are morphologically and functionally distinct from the other leaf cells. These microscopic sphincters inserted into the wax-covered epidermis of the shoot balance CO2 intake for photosynthetic carbon gain and concomitant water loss. In order to better understand the molecular mechanisms underlying stomatal function under saline conditions, we used proteomics approach to study isolated GCs from the salt-tolerant sugar beet species. Of the 2088 proteins identified in sugar beet GCs, 82 were differentially regulated by salt treatment. According to bioinformatics analysis (GO enrichment analysis and protein classification), these proteins were involved in lipid metabolism, cell wall modification, ATP biosynthesis, and signaling. Among the significant differentially abundant proteins, several proteins classified as "stress proteins" were upregulated, including non-specific lipid transfer protein, chaperone proteins, heat shock proteins, inorganic pyrophosphatase 2, responsible for energized vacuole membrane for ion transportation. Moreover, several antioxidant enzymes (peroxide, superoxidase dismutase) were highly upregulated. Furthermore, cell wall proteins detected in GCs provided some evidence that GC walls were more flexible in response to salt stress. Proteins such as L-ascorbate oxidase that were constitutively high under both control and high salinity conditions may contribute to the ability of sugar beet GCs to adapt to salinity by mitigating salinity-induced oxidative stress.

Stomatal traits as a determinant of superior salinity tolerance in wild barley.

Wild barley Hordeum spontaneum (WB) is the progenitor of a cultivated barley Hordeum vulgare (CB). Understanding efficient mechanisms evolved by WB to cope with abiotic stresses may open prospects of transferring these promising traits to the high yielding CB genotypes. This study aimed to investigate the strategies that WB plants utilise in regard to the control of stomatal operation and ionic homeostasis to deal with salinity stress, one of the major threats to the global food security. Twenty-six genotypes of WB and CB were grown under glasshouse conditions and exposed to 300 mM NaCl salinity treatment for 5 weeks followed by their comprehensive physiological assessment. WB had higher relative biomass than CB when exposed to salinity stress. Under saline conditions, WB plants were able to keep constant stomatal density (SD) while SD significantly decreased in CB. The higher SD in WB also resulted in a higher stomatal conductance (gs) under saline conditions, with gs reduction being 51% and 72% in WB and CB, respectively. Furthermore, WB showed faster stomatal response to light, indicating their better ability to adapt to changing environmental conditions. Experiments with isolated epidermal strips indicated that CB genotypes have the higher stomatal aperture when incubated in 80 mM KCl solution, and its aperture declined when KCl was substituted by NaCl. On the contrary, WB genotype had the highest stomatal aperture being exposed to 80 mM NaCl suggesting that WB plants may use Na+ instead of K+ for stomata movements. Overall, our data suggest that CB employ a stress-escaping strategy by reducing stomata density, to conserve water, when grown under salinity conditions. WB, on a contrary, is capable of maintaining relatively constant stomata density, faster stomatal movement and higher gs under saline conditions.

Epidermal bladder cells confer salinity stress tolerance in the halophyte quinoa and Atriplex species.

Epidermal bladder cells (EBCs) have been postulated to assist halophytes in coping with saline environments. However, little direct supporting evidence is available. Here, Chenopodium quinoa plants were grown under saline conditions for 5 weeks. One day prior to salinity treatment, EBCs from all leaves and petioles were gently removed by using a soft cosmetic brush and physiological, ionic and metabolic changes in brushed and non-brushed leaves were compared. Gentle removal of EBC neither initiated wound metabolism nor affected the physiology and biochemistry of control-grown plants but did have a pronounced effect on salt-grown plants, resulting in a salt-sensitive phenotype. Of 91 detected metabolites, more than half were significantly affected by salinity. Removal of EBC dramatically modified these metabolic changes, with the biggest differences reported for gamma-aminobutyric acid (GABA), proline, sucrose and inositol, affecting ion transport across cellular membranes (as shown in electrophysiological experiments). This work provides the first direct evidence for a role of EBC in salt tolerance in halophytes and attributes this to (1) a key role of EBC as a salt dump for external sequestration of sodium; (2) improved K+ retention in leaf mesophyll and (3) EBC as a storage space for several metabolites known to modulate plant ionic relations.

Plant ionic relation and whole-plant physiological responses to waterlogging, salinity and their combination in barley.

Waterlogging and salinity stresses significantly affect crop growth and global food production, and these stresses are often interrelated because waterlogging can lead to land salinisation by transporting salts to the surface. Although the physiological and molecular mechanisms of plant responses to each of these environmental constraints have been studied in detail, fewer studies have dealt with potential mechanisms underlying plant tolerance to the combined stress. This gap in knowledge is jeopardising the success of breeding programs. In the present work we studied the physiological and agronomical responses of 12 barley varieties contrasting in salinity stress tolerance to waterlogging (WL), salinity (NaCl) and combined (WL/NaCl) stresses. Stress damage symptoms were much greater in plants under combined WL/NaCl stress than those under separate stresses. The shoot biomass, chlorophyll content, maximum photochemical efficiency of PSII and shoot K+ concentration were significantly reduced under WL/NaCl conditions, whereas shoot Na+ concentration increased. Plants exposed to salinity stress showed lower damage indexes compared with WL. Chlorophyll fluorescence Fv/Fm value showed the highest correlation with the stress damage index under WL/NaCl conditions (r=-0.751) compared with other measured physiological traits, so was nominated as a good parameter to rank the tolerance of varieties. Average FW was reduced to 73±2, 52±1 and 23±2 percent of the control under NaCl, WL and combined WL/NaCl treatments respectively. Generally, the adverse effect of WL/NaCl stress was much greater in salt-sensitive varieties than in more tolerant varieties. Na+ concentrations of the shoot under control conditions were 97±10µmolg-1 DW, and increased to 1519±123, 179±11 and 2733±248µmolg-1 under NaCl, WL and combined WL/NaCl stresses respectively. K+ concentrations were 1378±66, 1260±74, 1270±79 and 411±92µmolg-1 DW under control, NaCl, WL and combined WL/NaCl stresses respectively. No significant correlation was found between the overall salinity stress tolerance and amount of Na+ accumulated in plant shoots after 15 days of exposure to 250mM NaCl stress. However, plants exposed to combined salinity and WL stress showed a negative correlation between shoot Na+ accumulation and extent of salinity damage. Overall, the reported results indicate that K+ reduction in the plants under combined WL/NaCl stress, but not stress-induced Na+ accumulation in the shoot, was the most critical feature in determining the overall plant performance under combined stress conditions.

Hydrogeochemistry and water quality assessment of the Kor-Sivand Basin, Fars province, Iran.

In order to assess the quality and suitability of waters in the Kor-Sivand river basin, 60 water samples from the Kor river and 90 water samples from wells in the basin were studied. Assessments were based on Piper's and Gibbs' diagrams for water quality, Food and Agricultural Organization's (FAO) guidelines, and US Salinity Laboratory diagram for water suitability. The results showed that the river water is of Ca-HCO(3) type, while well water is of Ca-Cl and Na-Cl type. Based on Gibbs' diagram, the source of soluble ions in the river water samples is the weathering of stones over which water flows, while evaporation was found to be the dominant process in the ion concentration of the well samples. According to the FAO Guidelines, the salinity of surface water for irrigation did not cause great restrictions; however, many of these waters could create potential permeability problems. In the groundwater samples, a high salt concentration is more important than the infiltration problem. Mg hazard values at some sites limit its use for agricultural purposes. One third of the river water samples and two thirds of well waters had more than 50% magnesium. Saturation indices showed that 94% of the analyzed water samples are supersaturated with calcite, aragonite, and dolomite. Based on the US Salinity Laboratory diagram, river water samples were classified as C(2)S(1) and C(3)S(1), while C(4)S(3), C(4)S(4), C(2)S(1), and C(3)S(1) were the most dominant classes in well samples. Some management practices necessary for sustainable development of water resources in the study area were discussed briefly, including appropriate selection of crops, adequate drainage, leaching, blending and cyclic use of saline water, proper irrigation method, and addition of soil amendment.