Extreme Halophyte Research Articles

Mitigation of salt stress in Sorghum bicolor L. by the halotolerant endophyte Pseudomonas stutzeri ISE12.

Increasing soil salinity, exacerbated by climate change, threatens seed germination and crop growth, causing significant agricultural losses. Using bioinoculants based on halotolerant plant growth-promoting endophytes (PGPEs) in modern agriculture is the most promising and sustainable method for supporting plant growth under salt-stress conditions. Our study evaluated the efficacy of Pseudomonas stutzeri ISE12, an endophyte derived from the extreme halophyte Salicornia europaea, in enhancing the salinity tolerance of sorghum (Sorghum bicolor L.). We hypothesized that P. stutzeri ISE12 would improve sorghum salt tolerance to salinity, with the extent of the increase in tolerance depending on the genotype's sensitivity to salt stress. Experiments were conducted for two sorghum genotypes differing in salinity tolerance (Pegah - salt tolerant, and Payam - salt sensitive), which were inoculated with a selected bacterium at different salinity concentrations (0, 100, 150, and 200 mM NaCl). For germination, we measured germination percentage and index, mean germination time, vigor, shoot and root length of seedlings, and fresh and dry weight. In pot experiments, we assessed the number of leaves, leaf area, specific leaf area, leaf weight ratio, relative root weight, plantlet shoot and root length, fresh and dry weight, proline and hydrogen peroxide concentrations, and peroxidase enzyme activity. Our study demonstrated that inoculation significantly enhanced germination and growth for both sorghum genotypes. The salinity-sensitive genotype (Payam) responded better to bacterial inoculation during germination and early seedling growth stages, showing approximately 1.4 to 1.8 times greater improvement than the salinity-tolerant genotype (Pegah). Payam also displayed better performance at the plantlet growth stage, between 1.1 and 2.6 times higher than Pegah. Furthermore, inoculation significantly reduced hydrogen peroxide, peroxidase activity, and proline levels in both sorghum genotypes. These reductions were notably more pronounced in Payam, with up to 1.5, 1.3, and 1.5 times greater reductions than in Pegah. These results highlight the efficacy of P. stutzeri ISE12 in alleviating oxidative stress and reducing energy expenditure on defense mechanisms in sorghum, particularly benefiting salt-sensitive genotypes. Our findings highlight the potential of the bacterial endophyte P. stutzeri ISE12 as a valuable bioinoculant to promote sorghum growth under saline conditions.

Proteomic profiling of an extreme halophyte Schrenkiella parvula with accelerated root elongation under mild salt stress.

Increased salinity in soil is one of the impacts of climate change and a major problem for crop cultivation. Halophytes have the ability to survive in hypersaline environments, and investigating their adaptation mechanisms is effective in imparting salt tolerance to plants. Recently, we discovered a strategy by the extreme halophyte Schrenkiella parvula to promote primary root elongation, a morpho-physiological response that may be given to have access to groundwater sources, while reducing meristem DNA replication, root hair development, and biomass at moderate salinities around 100 mM NaCl. However, when NaCl concentration exceeds 200 mM, seedling root elongation is inhibited, and seedlings change to respond to severe stress induced by salinity. To understand the interesting physiological and molecular mechanisms underlying primary root elongation at moderate salinity, we performed a proteomic analysis using two-dimensional gel electrophoresis and MALDI-TOF MS. Ultimately, a total of 300 different proteins were identified, of which 20 showed significant increases and 25 showed significant decreases at 100 mM NaCl. Among the increased proteins, proteins responding to abiotic stress such as glutathione transferases were found, and among the decreased proteins, proteins involved in glycolysis, purine nucleotide synthesis, and protein synthesis were found. Accumulation levels of proline, an osmotic regulator that inhibits root growth, were lower in S. parvula than in A. thaliana. On the other hand, interestingly, the expression levels of fructose-bisphosphate aldolase, sucrose phosphatase, and α-subunit of acetyl-CoA carboxylase increased. In addition, increases in P5CDH, an enzyme in the proline catabolism process, and decreases in GLN and GDH in glutamate synthesis in S. parvula suggest that these may lead to a fine-tuning of proline content. For annexins, a family of calcium-binding and membrane-bound proteins that regulate plant tolerance, moderate salt treatment showed a significant decrease in SpANN7, a non-significant downtrend for SpANN2, but no change for SpANN1. These findings suggest that the 100 mM NaCl does not create a serious stress for S. parvula. We also performed gene expression analysis of these altered proteins between S. parvula and A. thaliana. Taken together, in S. parvula roots, 100 mM NaCl partially induced the redox homeostasis system, stress response, and proline-mediated osmoregulation, moderately suppressing carbon metabolism, nucleotide, and protein synthesis to accelerate primary root elongation.

Genome-wide identification of the mitogen-activated kinase gene family from Limonium bicolor and functional characterization of LbMAPK2 under salt stress

BackgroundMitogen-activated protein kinases (MAPKs) are ubiquitous signal transduction components in eukaryotes. In plants, MAPKs play an essential role in growth and development, phytohormone regulation, and abiotic stress responses. The typical recretohalophyte Limonium bicolor (Bunge) Kuntze has multicellular salt glands on its stems and leaves; these glands secrete excess salt ions from its cells to mitigate salt damage. The number, type, and biological function of L. bicolor MAPK genes are unknown.ResultsWe identified 20 candidate L. bicolor MAPK genes, which can be divided into four groups. Of these 20 genes, 17 were anchored to 7 chromosomes, while LbMAPK18, LbMAPK19, and LbMAPK20 mapped to distinct scaffolds. Structure analysis showed that the predicted protein LbMAPK19 contains the special structural motif TNY in its activation loop, whereas the other LbMAPK members harbor the conserved TEY or TDY motif. The promoters of most LbMAPK genes carry cis-acting elements related to growth and development, phytohormones, and abiotic stress. LbMAPK1, LbMAPK2, LbMAPK16, and LbMAPK20 are highly expressed in the early stages of salt gland development, whereas LbMAPK4, LbMAPK5, LbMAPK6, LbMAPK7, LbMAPK11, LbMAPK14, and LbMAPK15 are highly expressed during the late stages. These 20 LbMAPK genes all responded to salt, drought and ABA stress. We explored the function of LbMAPK2 via virus-induced gene silencing: knocking down LbMAPK2 transcript levels in L. bicolor resulted in fewer salt glands, lower salt secretion ability from leaves, and decreased salt tolerance. The expression of several genes [LbTTG1 (TRANSPARENT TESTA OF GL1), LbCPC (CAPRICE), and LbGL2 (GLABRA2)] related to salt gland development was significantly upregulated in LbMAPK2 knockdown lines, while the expression of LbEGL3 (ENHANCER OF GL3) was significantly downregulated.ConclusionThese findings increase our understanding of the LbMAPK gene family and will be useful for in-depth studies of the molecular mechanisms behind salt gland development and salt secretion in L. bicolor. In addition, our analysis lays the foundation for exploring the biological functions of MAPKs in an extreme halophyte.

Morfo-anatomía e histoquímica de órganos vegetativos de Lycium humile (Solanaceae): rasgos adaptativos a humedales salinos de altura

Lycium humile Phil., is an extreme halophyte plant, endemic to Argentina, Bolivia, and Chile, which grows between 2000 and 4000 m a.s.l. in saline wetlands environments. The aim of this work is to identify morpho-anatomical and histochemical adaptive characteristics of the vegetative organs of L. humile linked to its tolerance to extreme environments of high altitude saline wetlands, in order to evaluate its potential as source of metabolites with commercial value or as source of resistance genes related to abiotic stress tolerance. To carry out the analysis, during the period of February 2018, samples were obtained from plants belonging to 4 populations of the species (5 individuals per population) located at the departments of Antofagasta de la Sierra, Santa María and Ambato, province of Catamarca, Argentina. Leaf and stems were studied using histochemical tests, conventional histological preparations and optical microscopy. Different foliar tissues were analyzed by scanning electron microscopy coupled to elemental X-ray diffraction spectroscopy (EDS). The leaves and stems of the analyzed populations showed uniform anatomical and histochemical features. It was stated for the first time that the succulence of the leaf is reached by the folding of the tissues evidenced in a tridimensional venation pattern. The presence of isolateral-radial mesophyll and aquifer parenchyma is highlighted as a unique feature characteristic for the species. The glandular trichomes and palisade parenchyma revealed complex contents of phenols, flavonoids, proteins, lipids, terpenes, and alkaloids; while the outer layer of the cuticles presented phenolic, terpenic and lipidic deposits. The EDS analysis showed crystalliferous idioblasts with high contents of Ca2+ and Na+. The stems exhibited hypodermis and air chambers formed in the cortical and medullary layers. These anatomical and histochemical characteristics of L. humile represent adaptive traits to saline high-altitude wetland environments. Its functionality in relation to the environment and its potential as source of resistance genes and metabolites interesting for the cosmetic and pharmaceutical industries are discussed.

The genome of the recretohalophyte Limonium bicolor provides insights into salt gland development and salinity adaptation during terrestrial evolution

Halophytes have evolved specialized strategies to cope with high salinity. The extreme halophyte sea lavender (Limonium bicolor) lacks trichomes but possesses salt glands on its epidermis that can excrete harmful ions, such as sodium, to avoid salt damage. Here, we report a high-quality, 2.92-Gb, chromosome-scale L. bicolor genome assembly based on a combination of Illumina short reads, single-molecule, real-time long reads, chromosome conformation capture (Hi-C) data, and Bionano genome maps, greatly enriching the genomic information on recretohalophytes with multicellular salt glands. Although the L. bicolor genome contains genes that show similarity to trichome fate genes from Arabidopsis thaliana, it lacks homologs of the decision fate genes GLABRA3, ENHANCER OF GLABRA3, GLABRA2, TRANSPARENT TESTA GLABRA2, and SIAMESE, providing a molecular explanation for the absence of trichomes in this species. We identified key genes (LbHLH and LbTTG1) controlling salt gland development among classical trichome homologous genes and confirmed their roles by showing that their mutations markedly disrupted salt gland initiation, salt secretion, and salt tolerance, thus offering genetic support for the long-standing hypothesis that salt glands and trichomes may share a common origin. In addition, a whole-genome duplication event occurred in the L. bicolor genome after its divergence from Tartary buckwheat and may have contributed to its adaptation to high salinity. The L. bicolor genome resource and genetic evidence reported in this study provide profound insights into plant salt tolerance mechanisms that may facilitate the engineering of salt-tolerant crops.

Introgression of SbERD4 Gene Encodes an Early-Responsive Dehydration-Stress Protein That Confers Tolerance against Different Types of Abiotic Stresses in Transgenic Tobacco.

Salicornia brachiata is an extreme halophyte that commonly grows on marsh conditions and is also considered a promising resource for drought and salt-responsive genes. To unveil a glimpse of stress endurance by plants, it is of the utmost importance to develop an understanding of stress tolerance mechanisms. ‘Early Responsive to Dehydration’ (ERD) genes are defined as a group of genes involved in stress tolerance and the development of plants. To increase this understanding, parallel to this expedited thought, a novel SbERD4 gene was cloned from S. brachiata, characterized, and functionally validated in the model plant tobacco. The study showed that SbERD4 is a plasma-membrane bound protein, and its overexpression in tobacco plants improved salinity and osmotic stress tolerance. Transgenic plants showed high relative water, chlorophylls, sugars, starch, polyphenols, proline, free amino acids, and low electrolyte leakage and H2O2 content compared to control plants (wild type and vector control) under different abiotic stress conditions. Furthermore, the transcript expression of antioxidant enzyme encoding genes NtCAT, NtSOD, NtGR, and NtAPX showed higher expression in transgenic compared to wild-type and vector controls under varying stress conditions. Overall, the overexpression of a novel early responsive to dehydration stress protein 4-encoding gene (SbERD4) enhanced the tolerance of the plant against multiple abiotic stresses. In conclusion, the overexpression of the SbERD4 gene mitigates plant physiology by enduring stress tolerance and might be considered as a promising key gene for engineering salinity and drought stress tolerance in crops.

A hyperaccumulation pathway to hierarchically porous carbon nanosheets from halophyte biomass for wastewater remediation

Sustainable and eco-friendly production is vital to meet the ever-increasing demand for porous carbon materials. We propose a single step process to obtain porous carbon nanosheets (CNS) by exploiting the hyperaccumulating pathway in extreme halophyte, Salicornia brachiata. The process was designed so as to achieve seamless hybridization with already existing technologies of salt production while reducing the overall carbon and chemical footprint. Further, CNS were tested as an efficient adsorbent for wastewater remediation. The raw material, dried Salicornia brachiata biomass consisted of roughly ~30 w/w% hyperaccumulated inorganic salts while the organic constituents (cellulose, lignin etc.) made up for the rest. Compartmentalized salt in the plant structure acted as a templating agent while water washing after pyrolysis resulted in exfoliation and pore formation due to its dissolution. Morphology and texture analysis of the carbon material obtained from water washed biomass (salts removed) exemplified the structure and pore directing effect of intrinsic salt. The unwashed pyrolyzed sample (S7--UW) exhibited nominal BET area of ~90 m2/g while the value increased to ~700 m2/g for exfoliated CNS obtained after water washing (S7--W). Intrinsic salts not only act as a template, but also catalyses the carbonization process, increases carbon retention during pyrolysis and inhibits restacking of exfoliated graphitized carbon sheets after pyrolysis. S7--W displayed high removal capacity for Pb2+ (55 mg/g), Hg2+(~100 mg/g), Cd2+(~250–300 mg/g), maintaining its efficiency under seawater and simulated wastewater conditions.

Genetic diversity in westernmost European populations of Halimione verrucifera (M. Bieb.) Aellen

Halimione verrucifera – an extreme halophyte plant species spread mainly in Asia with a few locations in South Eastern Europe – have the westernmost marginal populations in Romania. In order to asses population fitness and conservation status, genetic diversity among and within population was estimated using ISSR markers.A set of 6 primers were utilized after selection from a primer set which showed clear and reproducible banding patterns, generating a total number of 86 loci. A presence/absence matrix was generated by evaluating the gel electrophoresis banding patterns of PCR products. From this, genetic distances between individuals and populations were calculated, which were used in all subsequent generation of genetic diversity indices, assuming the Hardy-Weinberg equilibrium. Result revealed a low expected heterozygosity for both populations. Further testing by constructing an UPGMA dendrogram and a PCoA analysis confirmed that populations are clearly separated and with levels of genetic diversity that could ensure population survival in time and space.

Introgression of a novel cold and drought regulatory-protein encoding CORA-like gene, SbCDR, induced osmotic tolerance in transgenic tobacco.

A potent cold and drought regulatory-protein encoding gene, SbCDR was cloned from an extreme halophyte Salicornia brachiata. In vitro localisation study, performed with SbCDR::RFP gene-construct revealed that SbCDR is a membrane protein. Overexpression of the SbCDR gene in tobacco plants confirmed tolerance against major environmental constraints such as salinity, drought and cold, as evidenced by improved chlorophyll contents, plant morphology, plant biomass, root length, shoot length and seed germination efficiency. Transgenic lines also exhibited high accumulation of proline, total sugar, reducing sugar, free amino acid and polyphenol, besides the low level of malondialdehyde (MDA) contents. SbCDR transgenic lines showed better relative water contents, membrane stability index and osmotic water potential. Furthermore, higher expression of ROS scavenging genes was observed in transgenic lines under stress. Moreover, microarray analysis revealed that several host genes were upregulated and downregulated under drought and salt stress conditions in SbCDR transgenic line compared with control (WT) plants. The results demonstrated that the overexpression of the halophytic SbCDR gene has intense effects on the abiotic stress tolerance of transgenic tobacco plants. However, the exact mode of action of SbCDR in multiple abiotic stress tolerance of plants is yet to be unveiled. It is believed that the precise role of SbCDR gene will provide additional information to comprehend the abiotic stress tolerance mechanism. Furthermore, it will appear as a promising candidate gene for improving stress tolerance in different crop plants for sustainable agriculture and crop productivity.

Exposure to High Salinity During Seed Development Markedly Enhances Seedling Emergence and Fitness of the Progeny of the Extreme Halophyte Suaeda salsa.

Irrigation with 200 mM NaCl significantly increases vegetative and reproductive growth of the extreme halophyte Suaeda salsa. However, little is known about how the progeny of S. salsa plants grown under a continuous NaCl supply behave in terms of growth and seed set parameters. We investigated various plant growth and reproductive parameters of the progeny that germinated from seeds harvested from mother plants grown under 0 or 200 mM NaCl over three generations. Seedling emergence, plant height, stem diameter, total branch length, flowering branch length, flowering branch ratio, and seed production were all significantly enhanced in the progeny produced by mother plants grown with 200 mM NaCl compared to progeny of mother plants grown on low salinity conditions. Therefore, irrigation with 200 mM of NaCl is beneficial to seed development in the halophyte S. salsa and possibly contributes to population establishment in high salinity environments. Likewise, the prolonged absence of NaCl in the growth environment inhibits seed development, results in lower seed quality, and thus limits seedling growth of the progeny, thereby restricting S. salsa to a high salinity ecological niche.

Effect of prior salt experience on desalination capacity of the halophyte Arthrocnemum macrostachyum

We studied the capacity of extreme halophyte, Arthrocnemum macrostachyum, to desalinize an experimentally-salinized soil under non-leaching conditions. Furthermore, we tested the effect of salt pre-exposure of this species on its phytodesalination capacity. The presence of the halophyte, independently salt pre-exposure, decreased salinity of the sanilized soil by 31% after 30 days; reducing the negative effects on emergence of both test cultures, barley and wheat. Plants of A. macrostachyum pre-treated without salt showed 4-fold higher relative desalination rate than those grown with optimum salinity (with higher initial biomass). Thus, previous salt exposure was as determinant as plant biomass for desalinizing capacity. In conclusion, pre-exposure to salt of halophytes must be taken into account for desalination programs.

Polyamines in Halophytes.

Polyamines (PAs) are related to many aspects of the plant’s life cycle, including responses to biotic and abiotic stress. On the other hand, halophytic plants are useful models for studying salt tolerance mechanisms related to the adaptive strategies that these plants present in adverse environments. Furthermore, some halophytes have high economic value, being recommended instead of glycophytes as alternative agricultural crops in salt-affected coastal zones or saline farmlands. In recent years, the understanding of the role of PAs in salt-tolerant plants has greatly advanced. This mini review reports on the advances in the knowledge of PAs and their participation in achieving better salt tolerance in 10 halophytes. PAs are associated with responses to heavy metals in phytoremediation processes using certain salt-tolerant species (Atriplex atacamensis, A. halimus, Inula chrithmoides, and Kosteletzkya pentacarpos). In crops with exceptional nutritional properties such as Chenopodium quinoa, PAs may be useful markers of salt-tolerant genotypes. The signaling and protection mechanisms of PAs have been investigated in depth in the extreme halophyte Mesembryanthemum crystallinum and Thellungiella spp., enabling genetic manipulation of PA biosynthesis. In Prosopis strombulifera, different biochemical and physiological responses have been reported, depending on the type of salt (NaCl, Na2SO4). Increases in spermidine and spermine have been positively associated with stress tolerance as these compounds provide protection in Cymodocea nodosa, and Solanum chilense, respectively. In addition, abscisic acid and salicylic acid can improve the beneficial effect of PAs in these plants. Therefore, these results indicate the great potential of PAs and their contribution to stress tolerance.

Engineering of a novel gene from a halophyte: Potential for agriculture in degraded coastal saline soil

AbstractTo feed the ever increasing world population, more crops production per hectare has become a necessity. Soil salinity is one of the important factors causing land degradation leading to low soil aeration and water conductance. It adversely impacts plant growth and development. Adaption through modern molecular tools is an important strategy for alternative use for degraded land. To achieve this aim, we have cloned a novel salt responsive gene Salicornia brachiata RNA Poly III Complex 5 Like subunit (SbRPC5L) from an extreme halophyte and transformed it into tobacco for abiotic stress tolerance. The gene is intronless, 1,202 bp long, membrane‐localized, encoding a protein of 196 amino acids and shows upregulation under salt and osmotic stress. Tobacco plants harboring SbRPC5L perform better under stress and have more water content, membrane stability, and stress tolerance indices as compared with control plants. The transgenics exhibited lower electrolyte leakage, malondialdehyde, reactive oxygen species (ROS), H2O2, and Na+, more negative value of osmotic potential, higher K+ content, and K+/Na+ ratio. Some of the important antioxidant genes, namely, NtAPX, NtPOX, and NtSOD, get upregulated in transgenic lines. The results suggest that this novel gene has a potential to be used as a promising alternative to impart abiotic stress tolerance for climate resilient agriculture in degraded costal saline areas.

Cloning and functional characterization of the Na+/H+ antiporter (NHX1) gene promoter from an extreme halophyte Salicornia brachiata

Salinity is one of the major abiotic stresses which affect plant growth and productivity by imposing dual stress, ionic and osmotic stress, on plants. Halophytes which are adapted to complete their life cycle in saline soil keep the transcript expression of stress-responsive genes constitutively higher in the optimum growth environments, which can be further increased by several folds under stress conditions. The transcript expression of SbNHX1 gene, cloned from a leafless succulent halophyte Salicornia brachiata, was up-regulated under salinity stress, but its transcriptional regulation has not been studied so far. In the present study, a 1727 bp putative promoter (upstream to translation start site) of the SbNHX1 gene was cloned using a genome walking method. The bioinformatics analysis identified important stress-responsive cis-regulatory motifs, GT1, MBS, LTR and ARE, in addition to two leaf-specific enhancer motifs. The GUS expression analysis of stable transgenic tobacco plants, transformed with a transcriptional fusion of GUS with the full SbNHX1 promoter (NP1) or any of its five deletion fragments (NP2 to NP6), showed that the deletion of two enhancer motifs resulted in the sudden decrease in GUS expression in leaves but not in the stem or root tissues. In contrast, under salinity stress, the higher induction of GUS expression observed in NP1 and NP2 was correlated by the presence of salt-inducible GT1- and MBS-motifs which is distributed only in NP1 and NP2 deletion promoter fragments. Finally, we concluded that the SbNHX1 promoter has a 624 bp (−1727 to −1103 bp) regulatory region which contains the two leaf-specific enhancer motifs and salinity stress-inducible GT-1 and MBS motifs. We suggest the SbNHX1 gene promoter and fragments as a candidate alternative promoter/s for crop engineering for better stress tolerance, which can be amended according to the desired level of expression needed.

Isolation and characterization of a photosystem II preparation from thylakoid membranes of the extreme halophyte Salicornia veneta Pignatti et Lausi

Salicornia veneta (Pignatti et Lausi) is an extreme halophyte living in salt marsh where NaCl concentration may be as high as 1 M. Here we report on the isolation and characterization of a PSII preparation obtained by Triton X-100 solubilisation of the thylakoid membrane. By a combination of gel electrophoresis, immunoblotting and mass spectrometry, the depletion of a number of PSII proteins such as PsbQ, PsbM and PsbT was highlighted. Moreover, the requirement of Cl− and Ca2+ for optimal oxygen evolution was determined, showing that in absence of PsbQ a higher level of these ions are required. At high Cl− concentrations, oxygen evolution was inhibited in the same way in Salicornia veneta and spinach. Reconstitution of Salicornia veneta PSII preparation with partially purified spinach PsbP and PsbQ restored oxygen evolution activity at low Cl− and Ca2+ concentrations. Adaptation to high salt makes several PSII proteins dispensable.

Draft genome sequence of first monocot-halophytic species Oryza coarctata reveals stress-specific genes

Oryza coarctata (KKLL; 2n = 4x = 48, 665 Mb) also known as Porteresia coarctata is an extreme halophyte species of genus Oryza. Using Illumina and Nanopore reads, we achieved the assembled genome size of 569.9 Mb, accounting 85.69% of the estimated genome size with N50 of 1.85 Mb and 19.89% repetitive region. We also found 230,968 simple sequence repeats (SSRs) and 5,512 non-coding RNAs (ncRNAs). The functional annotation of predicted 33,627 protein-coding genes and 4,916 transcription factors revealed that high salinity adaptation of this species is due to the exclusive or excessive presence of stress-specific genes as compared to rice. We have identified 8 homologs to salt-tolerant SOS1 genes, one of the three main components of salt overly sensitive (SOS) signal pathway. On the other hand, the phylogenetic analysis of the assembled chloroplast (134.75 kb) and mitochondrial genome (491.06 kb) favours the conservative nature of these organelle genomes within Oryza taxon.

Photosynthetic pigments, betalains, proteins, sugars, and minerals during Salicornia brachiata senescence

Senescence is the last developmental stage in plants during which recycling of nutrients takes place from senescing organs to newly formed organs such as young leaves and developing seeds. In the present work, senescence induced alterations in mineral ions, chlorophylls, carotenoids, betacyanin, betaxanthin, proteins, amino acids, sugars, starch, and polyphenols were monitored in shoots of an extreme halophyte Salicornia brachiata. A sharp decline in the content of chlorophylls, carotenoids, and proteins in the shoot was noticed at middle and late stages of senescence in comparison with early stage. However, the content of betacyanin, betaxanthin, total soluble sugars, reducing sugars, and starch increased significantly in senescing shoots. The total free amino acid content decreased gradually with the progress of senescence. The content of major minerals did not change significantly with the progress of senescence, whereas marked changes in content of minor minerals were observed. From this study, it was concluded that the sugars and starch accumulating in senescing shoots might be transported into developing seeds to serve as storage nutrients. The accumulation of betacyanin and betaxanthin in senescing shoots suggests that these pigments may act as scavengers of reactive oxygen species during senescence. This study provides comprehensive information on the variations in the utilization of mineral nutrients and organic metabolites with progressing senescence in the halophyte S. brachiata.

Transcriptomic Profiling and Physiological Responses of Halophyte Kochia sieversiana Provide Insights into Salt Tolerance.

Halophytes are remarkable plants that can tolerate extremely high-salinity conditions, and have different salinity tolerance mechanisms from those of glycophytic plants. In this work, we investigated the mechanisms of salinity tolerance of an extreme halophyte, Kochia sieversiana (Pall.) C. A. M, using RNA sequencing and physiological tests. The results showed that moderate salinity stimulated the growth and water uptake of K. sieversiana and, even under 480-mM salinity condition, K. sieversiana maintained an extremely high water content. This high water content may be a specific adaptive strategy of K. sieversiana to high salinity. The physiological analysis indicated that increasing succulence and great accumulations of sodium, alanine, sucrose, and maltose may be favorable to the water uptake and osmotic regulation of K. sieversiana under high-salinity stress. Transcriptome data indicated that some aquaporin genes and potassium (K+) transporter genes may be important for water uptake and ion balance, respectively, while different members of those gene families were employed under low- and high-salinity stresses. In addition, several aquaporin genes were up-regulated in low- but not high-salinity stressed roots. The highly expressed aquaporin genes may allow low-salinity stressed K. sieversiana plants to uptake more water than control plants. The leaf K+/root K+ ratio was enhanced under low- but not high-salinity stress, which suggested that low salinity might promote K+ transport from the roots to the shoots. Hence, we speculated that low salinity might allow K. sieversiana to uptake more water and transport more K+ from roots to shoots, increasing the growth rate of K. sieversiana.

Inorganic solutes contribute more than organic solutes to the osmotic adjustment in Salicornia brachiata (Roxb.) under natural saline conditions

Salicornia brachiata (Roxb.) is an extreme halophyte that grows in the inundated intertidal zone. The osmotic traits were studied in S. brachiata to understand the ecophysiological responses under natural saline condition. The improved growth and biomass yield in S. brachiata under saline conditions proved the true halophytic nature. The relaxometry and diffusion map analysis showed deposition of salt in the outer cortical regions of the stem of S. brachiata with increasing salinity in the greenhouse. The field growing plant did not show any salt deposition. At 100% of seawater salinity level accumulation of total solutes, inorganic- and organic solutes increased 168.47, 177.88 and 28.57% respectively in S. brachiata under greenhouse conditions. While the accumulation of total solutes, inorganic- and organic solutes increased 176.58, 181.73 and 100% respectively in S. brachiata under field condition. The accumulation of solutes helped the plant to maintain the osmotic homeostasis thus salt adaptation. The increase in the contribution of proline (CNTpro) among organic and Na+ (CNTNa) among inorganic solutes to the osmotic adjustment was estimated the highest under saline conditions. The present results supported significantly higher contribution of inorganic solutes than organic solutes to the osmotic adjustment. As the synthesis of organic solutes costs more energy as compared to the uptake of the different inorganic solutes from surrounding and accumulation theirof, thus use of the accumulated inorganic solutes in osmotic homeostasis for salt adaptation in S. brachiata is an energy-efficient process.

In planta Transformed Cumin (Cuminum cyminum L.) Plants, Overexpressing the SbNHX1 Gene Showed Enhanced Salt Endurance.

Cumin is an annual, herbaceous, medicinal, aromatic, spice glycophyte that contains diverse applications as a food and flavoring additive, and therapeutic agents. An efficient, less time consuming, Agrobacterium-mediated, a tissue culture-independent in planta genetic transformation method was established for the first time using cumin seeds. The SbNHX1 gene, cloned from an extreme halophyte Salicornia brachiata was transformed in cumin using optimized in planta transformation method. The SbNHX1 gene encodes a vacuolar Na+/H+ antiporter and is involved in the compartmentalization of excess Na+ ions into the vacuole and maintenance of ion homeostasis Transgenic cumin plants were confirmed by PCR using gene (SbNHX1, uidA and hptII) specific primers. The single gene integration event and overexpression of the gene were confirmed by Southern hybridization and competitive RT-PCR, respectively. Transgenic lines L3 and L13 showed high expression of the SbNHX1 gene compared to L6 whereas moderate expression was detected in L5 and L10 transgenic lines. Transgenic lines (L3, L5, L10 and L13), overexpressing the SbNHX1 gene, showed higher photosynthetic pigments (chlorophyll a, b and carotenoid), and lower electrolytic leakage, lipid peroxidation (MDA content) and proline content as compared to wild type plants under salinity stress. Though transgenic lines were also affected by salinity stress but performed better compared to WT plants. The ectopic expression of the SbNHX1 gene confirmed enhanced salinity stress tolerance in cumin as compared to wild type plants under stress condition. The present study is the first report of engineering salt tolerance in cumin, so far and the plant may be utilized for the cultivation in saline areas.