Structural and transcriptional characterization of Thaumatin-Like Proteins in Cenostigma pyramidale under salt stress.

BackgroundAlfalfa (Medicago sativa L.) production decreases under salt stress. Identification of genes associated with salt tolerance in alfalfa is essential for the development of molecular markers used for breeding and genetic improvement.ResultAn RNA-Seq technique was applied to identify the differentially expressed genes (DEGs) associated with salt stress in two alfalfa cultivars: salt tolerant ‘Halo’ and salt intolerant ‘Vernal’. Leaf and root tissues were sampled for RNA extraction at 0 h, 3 h, and 27 h under 12 dS m− 1 salt stress maintained by NaCl. The sequencing generated a total of 381 million clean sequence reads and 84.8% were mapped on to the alfalfa reference genome. A total of 237 DEGs were identified in leaves and 295 DEGs in roots of the two alfalfa cultivars. In leaf tissue, the two cultivars had a similar number of DEGs at 3 h and 27 h of salt stress, with 31 and 49 DEGs for ‘Halo’, 34 and 50 for ‘Vernal’, respectively. In root tissue, ‘Halo’ maintained 55 and 56 DEGs at 3 h and 27 h, respectively, while the number of DEGs decreased from 42 to 10 for ‘Vernal’. This differential expression pattern highlights different genetic responses of the two cultivars to salt stress at different time points. Interestingly, 28 (leaf) and 31 (root) salt responsive candidate genes were highly expressed in ‘Halo’ compared to ‘Vernal’ under salt stress, of which 13 candidate genes were common for leaf and root tissues. About 60% of DEGs were assigned to known gene ontology (GO) categories. The genes were involved in transmembrane protein function, photosynthesis, carbohydrate metabolism, defense against oxidative damage, cell wall modification and protection against lipid peroxidation. Ion binding was found to be a key molecular activity for salt tolerance in alfalfa under salt stress.ConclusionThe identified DEGs are significant for understanding the genetic basis of salt tolerance in alfalfa. The generated genomic information is useful for molecular marker development for alfalfa genetic improvement for salt tolerance.

Salt-stress-induced ABA accumulation in maize root tissues was compared with that in leaf tissues. While salt stress with NaCl resulted in a significant ABA accumulation in root tissues (up to 10-fold), the same stress only led to a small ABA accumulation in leaf tissues (about 1-fold). Pretreatment with ethylene glycol (EG), a permeable and inert monomer of PEG, could prevent the shrinkage of cell volume and completely block the ABA accumulation in leaf tissues under salt stress, but substantial salt-induced ABA accumulation was still observed in root tissues following such pretreatment. Hypotonic salt solutions, i.e. below 100 mM NaCl, still induced a significant ABA accumulation (more than 3-fold) in roots, but showed no effect on that in leaf tissues. Results suggest that the salt-stress-induced ABA accumulation in roots may also be triggered by an osmosensing mechanism, which is in addition to the perception of the changes in reduced cellular volume or plasmalemma tension that leads to ABA accumulation in leaves. When leaf and root tissues were immersed into salt solutions, salt entered into the cells as a function of time and salt concentrations. Such entrance apparently led to a loss of sensitivity of leaf tissues to accumulate ABA under the salt stress, and also prevented the leaf tissues from responding to further air-drying in terms of ABA accumulation. Roots showed no such responses. Results suggest that the entrance of salt into leaf cells brought about some toxic effect that might have reduced the capability of leaf cells to produce ABA under dehydration.

Salt affected soil inhibits plant growth, development and productivity, especially in case of rice crop. Ion homeostasis is a candidate defense mechanism in the salt tolerant plants or halophyte species, where the salt toxic ions are stored in the vacuoles. The aim of this investigation was to determine the OsNHX1 (a vacuolar Na+/H+ exchanger) and OsHKT2;1 (Na+/K+ transporter) regulation by salt stress (200mM NaCl) in two rice cultivars, i.e. Pokkali (salt tolerant) and IR29 (salt susceptible), the accumulation of Na+ in the root and leaf tissues using CoroNa Green® staining dye and the associated physiological changes in test plants. Na+ content was largely increased in the root tissues of rice seedlings cv. Pokkali (15min after salt stress) due to the higher expression of OsHKT2;1 gene (by 2.5 folds) in the root tissues. The expression of OsNHX1 gene in the leaf tissues was evidently increased in salt stressed seedlings of Pokkali, whereas it was unchanged in salt stressed seedlings of IR29. Na+ in the root tissues of both Pokkali and IR29 was enriched, when subjected to 200mM NaCl for 12h and easily detected in the leaf tissues of salt stressed plants exposed for 24h, especially in cv. Pokkali. Moreover, the overexpression of OsNHX1 gene regulated the translocation of Na+ from root to leaf tissues, and compartmentation of Na+ into vacuoles, thereby maintaining the photosynthetic abilities in cv. Pokkali. Overall growth performance, maximum quantum yield (Fv/Fm), photon yield of PSII (ΦPSII) and net photosynthetic rate (Pn) was improved in salt stressed leaves of Pokkali than those in salt stressed IR29.

BackgroundQuantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) is a sensitive technique for quantifying gene expression levels. One or more appropriate reference genes must be selected to accurately compare mRNA transcripts across different samples and tissues. Thus far, only actin-2 has been used as a reference gene for qRT-PCR in chicory, and a full comparison of several candidate reference genes in chicory has not yet been reported.ResultsSeven candidate reference genes, including nicotinamide adenine dinucleotide dehydrogenase (NADHD), actin (ACT), β-tubulin (TUB), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), histone H3 (H3), elongation factor 1-alpha (EF) and 18S rRNA (rRNA) were selected to study the expression stability for normalisation of gene expression in chicory. Primer specificity and amplification efficiency were verified for each gene. The expression stability of these genes was analysed across chicory root and leaf tissues using geNorm, NormFinder and BestKeeper software. ACT, EF, and rRNA were the most stable genes as identified by the three different analysis methods. In addition, the use of ACT, EF and GAPDH as reference genes was illustrated by analysing 1-FEHII (FEHII) expression in chicory root and leaf tissues. These analyses revealed the biological variation in FEHII transcript expression among the tissues studied, and between individual plants.ConclusionsgeNorm, NormFinder, and BestKeeper analyses indicated that ACT, EF and rRNA had the highest expression stability across leaf and root tissues, while GAPDH and NADHD showed relatively low expression stability. The results of this study emphasise the importance of validating reference genes for qRT-PCR analysis in chicory. The use of the most stable reference genes such as ACT and EF allows accurate normalisation of gene expression in chicory leaf and root tissues.

The changes in the activity of antioxidant enzymes, like superoxide dismutase, ascorbate peroxidase, catalase and glutathione reductase, and growth parameters such as length, fresh and dry weight, proline and H2O2 contents, chlorophyll fluorescence (Fv/Fm), quantum yield of PSII and the rate of lipid peroxidation in terms of malondialdehyde in leaf and root tissues of a chickpea cultivar (Cicer arietinum L. cv. Gokce) under salt treatment were investigated. Plants were subjected to 0.1, 0.2 and 0.5 M NaCl treatments for 2 and 4 days. Compared to controls, salinity resulted in the reduction of length and of the fresh and dry weights of shoot and root tissues. Salinity caused significant (P < 0.05) changes in proline and MDA levels in leaf tissue. In general, a dose-dependent decrease was observed in H2O2 content, Fv/Fm and quantum yield of photosynthesis under salt stress. Leaf tissue extracts exhibited three activity bands, of which the higher band was identified as MnSOD and the others as FeSOD and Cu/ZnSOD. A significant enhancement was detected in the activities of Cu/ZnSOD and MnSOD isozymes in both tissues. APX and GR activities exhibited significant increases (P < 0.05) in leaf tissue under all stress treatments, whereas no significant change was observed in root tissue. The activity of CAT was significantly increased under 0.5 M NaCl stress in root tissue, while its activity was decreased in leaf tissue under 0.5 M NaCl stress for 4 days. These results suggest that CAT and SOD activities play an essential protective role against salt stress in chickpea seedlings.

Expression patterns of four candidate AREB/ABF genes and four DREB/CBF genes were evaluated in leaf and root tissues of five grape varieties (‘Qalati’, ‘Kaj Angoor’, ‘Sabz Angoor’, ‘Siahe Zarghan’, ‘Bidane Safid’) with differential response to drought stress. Among the AREB/ABF genes, AREB1 and ABF2 showed up-regulation in response to drought stress in leaf and root tissues of all varieties while AREB2 and ABF1 showed down-regulation in both leaf and root tissues of the sensitive variety ‘Bidane Sefid’ in response to drought and salt stress. Among the DREB/CBF genes, CBF4 was the most responsive to drought stress in both leaf and root tissues. CBF2 and CBF3 showed up-regulation in all varieties in response to drought stress in leaf except in ‘Bidane Sefid’. Under salinity stress, AREB2 and ABF2 showed up-regulation in response to the increasing level of salinity in the leaf tissues but in the root tissues ABF2 was up-regulated in response to increasing NaCl concentration while AREB2 was down-regulated. Therefore, it seems AREB2 has tissue-specific response to salinity stress. All CBF genes were up-regulated in response to salinity stress in the leaf and root tissues. Expression data suggested that CBF2 is more responsive to NaCl stress. Among all four promising and stress tolerant varieties ‘Siah Zarghan’ and ‘Kaj Angoor’ were more tolerant than ‘Qalati’ and ‘Sabz Angoor’ to drought and salinity.

High salinity is a major environmental stressor for crops. To understand the regulatory mechanisms underlying salt tolerance, we conducted a comparative transcriptome analysis between salt-tolerant and salt-sensitive jute (Corchorus spp.) genotypes in leaf and root tissues under salt stress and control conditions. In total, 68,961 unigenes were identified. Additionally, 11,100 unigenes (including 385 transcription factors (TFs)) exhibited significant differential expression in salt-tolerant or salt-sensitive genotypes. Numerous common and unique differentially expressed unigenes (DEGs) between the two genotypes were discovered. Fewer DEGs were observed in salt-tolerant jute genotypes whether in root or leaf tissues. These DEGs were involved in various pathways, such as ABA signaling, amino acid metabolism, etc. Among the enriched pathways, plant hormone signal transduction (ko04075) and cysteine/methionine metabolism (ko00270) were the most notable. Eight common DEGs across both tissues and genotypes with similar expression profiles were part of the PYL-ABA-PP2C (pyrabactin resistant-like/regulatory components of ABA receptors-abscisic acid-protein phosphatase 2C). The methionine metabolism pathway was only enriched in salt-tolerant jute root tissue. Twenty-three DEGs were involved in methionine metabolism. Overall, numerous common and unique salt-stress response DEGs and pathways between salt-tolerant and salt-sensitive jute have been discovered, which will provide valuable information regarding salt-stress response mechanisms and help improve salt-resistance molecular breeding in jute.

Plant microRNAs (miRs) have emerged as important regulators of gene expression under normal as well as stressful environments. Rice is an important cereal crop whose productivity is compromised due to various abiotic stress factors such as salt, heat and drought. In the present study, we have investigated the role of rice-specific Osa-miR820, in indica rice cultivars showing contrasting response to salt stress. The dissection of expression patterns indicated that the miR is present in all the tissues but is enriched in the anther tissues. In salinity, the miR levels are up-regulated in the leaf tissues but down-regulated in the root tissues. To map the deregulation under salt stress comprehensive time kinetics of expression was performed in the leaf and root tissues. The reproductive stages were also analyzed under salt stress. It emerged that a common regulatory scheme for Osa-miR820 expression is present in the salt-susceptible Pusa Basmati 1 and salt-tolerant Pokkali varieties, although there is a variation in the levels of the miR and its target transcript, OsDRM2. The regulation of Osa-miR820 and its target were also studied under other abiotic stresses. This study thus captures the window for the miR-target correlation and the putative role of this regulation is discussed. This will help in gaining useful insights on the role of species specific miRs in plant development and abiotic stress response.

iTRAQ-based comparative proteomic analysis reveals tissue-specific and novel early-stage molecular mechanisms of salt stress response in Carex rigescens

The development of salt-tolerant rice has become urgent due to climate change and rising global rice consumption. A large-scale analysis using different but related platforms has become imperative to filter out candidate genes responsible for salinity tolerance and salinity stress-responsive pathways. Such genes can be used to find prospective candidate salt resistance genes in donor rice genotypes and transfer them to high-yielding rice varieties. We performed a meta-analysis to screen out candidate genes using stress-related three microarray and one RNASeq datasets from NCBI. As different genotypes of rice and different salinity stress conditions were considered in our analysis, the sensitivity of the results is expected to be multi-fold higher. Our analysis revealed the differentially expressed genes (DEGs) OsbZIP52 and OsLTP2.5 to be common between leaf and root tissues. These genes were further compared with those of the wild halophytic rice Oryza coarctata expression data in stress conditions to understand the significance of these genes. The OsbZIP52 gene homolog of Oryza coarctata was the only one found to be differentially expressed. The expression level of OsbZIP52 was quantified using RT-qPCR and observed downregulated expression in salt stress in root and leaf tissues of four rice cultivars (2 salt-tolerant and 2 salt-sensitive). Promoter and motif analysis revealed a high number of variations in promoter and motif regions of the gene in IR29 salt-sensitive rice. Expression correlation analysis and Gene Ontology study suggested that OsbZIP52 interacts with genes that are engaged in stress response and participate in stress-responsive pathways. Collectively this study increases our understanding of the differential gene expression in various stress conditions in root and leaf tissues. It also helped identify a critical regulatory transcription factor in assisting the plant in combating salinity stress.

Salinity, recognized as a major threat in agriculture, causes 4.0–6.3% yield loss annually across the world. The problem is aggravated due to increasing irrigation with suboptimal quality of irrigation water and more salinization of coastal area due to the rise in sea level because of climate change. In saline soil, excessive concentrations of Na+ and Cl− impair absorption of other beneficial ions such as K+ and Ca2+ that in turn inhibit plant growth and productivity. Maintenance of cellular K+ level and K+/Na+ ratio is still considered the most important factor for salt tolerance. Under high-Na+ environment, excess Na+ competes with K+ thereby hindering its uptake. Tolerant plants by employing a number of strategies restrict Na+ movement to young meristematic tissues and allow greater movement and/or tissue retention of K+ to physiologically more active tissues. Under salt stress different K+- and Na+-specific transporters, viz. SOS, NHX, and HKT family transporters (regulate cellular Na+ movement) and HAK, AKT, KT, and KUP (regulate K+ movement), either by upregulation or downregulation, control the cellular ion homeostasis and salt tolerance in plants. SOS1, a plasma membrane-bound Na+/H+ antiporter, mostly active in root tissue, removes the excess salt from the plant body by pumping them back to the rhizosphere in an energy-dependent process. Tonoplast-bound vacuolar Na+/H+ antiporters (NHX family transporters) play crucial role in Na+ compartmentalization inside the vacuole in mature cell in both root and leaf tissues. Storing excess salts in vacuole imparts tolerance in multifaceted manner, viz. imparting tissue and osmo-tolerance. Biosynthesis of organic osmolytes, a more energy-expensive process, is sometimes substituted by the accumulation of excess Na+ in non-active tissues under salt stress. Improved Ca2+ status inside the plant tissue is another important factor associated with salt tolerance and acts as a key signalling molecule to initiate Na+ exclusion. Several QTLs and miRNAs were reported to impart salt tolerance in several crops. Managing salinity beyond crop improvement strategies was also deliberated, e.g. lowering salt effect through K+ supplementation and phytohormones, etc. In this compilation, emphasis has been given on how nutrient/ionic imbalance causes deleterious effects on plants under saline conditions and what are the possible adaptive strategies plants employ to maintain the ionic homeostasis in saline environment.

The effect of salt stress (100 mM and 200 mM NaCl) on antioxidant responses in shoots and roots of 14-day-old lentil (Lens culinaris M.) seedlings was investigated. Salt stress caused a significant decrease in length, wet-dry weight and an increase in proline content of both shoot and root tissues. In leaf tissues, high salinity treatment resulted in a 4.4 fold increase in H2O2 content which was accompanied by a significant level of lipid peroxidation and an increase in electrolyte leakage. Root tissues were less affected with respect to these parameters. Leaf tissue extracts exhibited four activity bands, of which two were identified as Cu/Zn-SOD and others as Fe-SOD and Mn-SOD. Fe-SOD activity was missing in root extracts. In both tissues Cu/Zn-SOD activity comprised 70–75% of total SOD activity. Salt stress did not cause a significant increase in total SOD activity of leaf tissues but a significant enhancement (88%) was observed in roots mainly due to an enhancement in Cu/ZnSOD isoforms. Compared to leaf tissues a significantly higher constitutive ascorbate peroxidase (APX) and glutathion reductase (GR) activity was observed in root tissues. Upon salt stress no significant change in the activity of APX, catalase (CAT) and GR was observed in root tissues but a higher APX activity was present when compared to leaf tissues. On the other hand, in leaf tissues, with the exception of CAT, salt stress caused significant enhancement in the activity of other antioxidant enzymes. These results suggested that, root tissues of lentil are protected better from NaCl stress induced oxidative damage due to enhanced total SOD activity together with a higher level of APX activity under salinity stress. To our knowledge this is the first report describing antioxidant enzyme activities in lentil.

Quinoa is gaining importance on global scale due to its excellent nutritious profile and environmental stress‐enduring potential. Its production decreases under high salt stress but can be improved with paclobutrazol application. This study showed involvement of some potential protective mechanisms in root and leaf tissues of quinoa plants treated with paclobutrazol (PBZ) against high salinity. The treatment levels were based on preliminary experiments, and it was found that salt stress (400 mm NaCl) markedly reduced growth and photosynthetic pigments while PBZ (20 mg/L) application significantly improved these attributes. Stomata density and aperture declined on adaxial and abaxial surfaces of leaves due to salinity. Paclobutrazol application significantly improved the stomatal density on both surfaces of leaves. Concentration of proline and soluble sugars increased in root and leaf tissues under salinity, which was more obvious in PBZ‐treated plants. Salinity stress induced the oxidative damage by increasing lipid peroxidation (MDA) level in roots and more specifically in leaf tissues. However, PBZ treatments ameliorated the drastic effects of salinity and markedly reduced oxidative damage in salt‐stressed quinoa plants. Enhanced activity of enzymatic antioxidants such as superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) was triggered by PBZ application, more pronounced in leaf than root tissues. Based on these findings, we conclude that PBZ application improves the salt tolerance in quinoa by activation of the above‐mentioned physiological and biochemical mechanisms specifically in leaves.

The cotton diploid species, Gossypium arboreum, shows important properties of stress tolerance and good genetic stability. In this study, through mRNA-seq, we de novo assembled the unigenes of multiple samples with 3h H2O, NaCl, or PEG treatments in leaf, stem and root tissues and successfully obtained 123,579 transcripts of G. arboreum, 89,128 of which were with hits through BLAST against known cotton ESTs and draft genome of G. raimondii. About 36,961 transcripts (including 1,958 possible transcription factor members) were identified with differential expression under water stresses. Principal component analysis of differential expression levels in multiple samples suggested tissue selective signalling responding to water stresses. Venn diagram analysis showed the specificity and intersection of transcripts’ response to NaCl and PEG treatments in different tissues. Self-organized mapping and hierarchical cluster analysis of the data also revealed strong tissue selectivity of transcripts under salt and osmotic stresses. In addition, the enriched gene ontology (GO) terms for the selected tissue groups were differed, including some unique enriched GO terms such as photosynthesis and tetrapyrrole binding only in leaf tissues, while the stem-specific genes showed unique GO terms related to plant-type cell wall biogenesis, and root-specific genes showed unique GO terms such as monooxygenase activity. Furthermore, there were multiple hormone cross-talks in response to osmotic and salt stress. In summary, our multidimensional mRNA sequencing revealed tissue selective signalling and hormone crosstalk in response to salt and osmotic stresses in G. arboreum. To our knowledge, this is the first such report of spatial resolution of transcriptome analysis in G. arboreum. Our study will potentially advance understanding of possible transcriptional networks associated with water stress in cotton and other crop species.

Plant protein phosphatase 2C (PP2C) plays vital roles in responding to various stresses, stimulating growth factors, phytohormones, and metabolic activities in many important plant species. However, the PP2C gene family has not been investigated in the economically valuable plant species sunflower (Helianthus annuus L.). This study used comprehensive bioinformatics tools to identify and characterize the PP2C gene family members in the sunflower genome (H. annuus r1.2). Additionally, we analyzed the expression profiles of these genes using RNA-seq data under four different stress conditions in both leaf and root tissues. A total of 121 PP2C genes were identified in the sunflower genome distributed unevenly across the 17 chromosomes, all containing the Type-2C phosphatase domain. HanPP2C genes are divided into 15 subgroups (A-L) based on phylogenetic tree analysis. Analyses of conserved domains, gene structures, and motifs revealed higher structural and functional similarities within various subgroups. Gene duplication and collinearity analysis showed that among the 53 HanPP2C gene pairs, 48 demonstrated segmental duplications under strong purifying selection pressure, with only five gene pairs showing tandem duplications. The abundant segmental duplication was observed compared to tandem duplication, which was the major factor underlying the dispersion of the PP2C gene family in sunflowers. Most HanPP2C proteins were localized in the nucleus, cytoplasm, and chloroplast. Among the 121 HanPP2C genes, we identified 71 miRNAs targeting 86 HanPP2C genes involved in plant developmental processes and response to abiotic stresses. By analyzing cis-elements, we identified 63 cis-regulatory elements in the promoter regions of HanPP2C genes associated with light responsiveness, tissue-specificity, phytohormone, and stress responses. Based on RNA-seq data from two sunflower tissues (leaf and root), 47 HanPP2C genes exhibited varying expression levels in leaf tissue, while 49 HanPP2C genes showed differential expression patterns in root tissue across all stress conditions. Transcriptome profiling revealed that nine HanPP2C genes (HanPP2C12, HanPP2C36, HanPP2C38, HanPP2C47, HanPP2C48, HanPP2C53, HanPP2C54, HanPP2C59, and HanPP2C73) exhibited higher expression in leaf tissue, and five HanPP2C genes (HanPP2C13, HanPP2C47, HanPP2C48, HanPP2C54, and HanPP2C95) showed enhanced expression in root tissue in response to the four stress treatments, compared to the control conditions. These results suggest that these HanPP2C genes may be potential candidates for conferring tolerance to multiple stresses and further detailed characterization to elucidate their functions. From these candidates, 3D structures were predicted for six HanPP2C proteins (HanPP2C47, HanPP2C48, HanPP2C53, HanPP2C54, HanPP2C59, and HanPP2C73), which provided satisfactory models. Our findings provide valuable insights into the PP2C gene family in the sunflower genome, which could play a crucial role in responding to various stresses. This information can be exploited in sunflower breeding programs to develop improved cultivars with increased abiotic stress tolerance.