Characterization of Differentially Expressed Genes under Salt Stress in Olive

Climate change, currently taking place worldwide and also in the Mediterranean area, is leading to a reduction in water availability and to groundwater salinization. Olive represents one of the most efficient tree crops to face these scenarios, thanks to its natural ability to tolerate moderate salinity and drought. In the present work, four olive cultivars (Koroneiki, Picual, Royal de Cazorla and Fadak86) were exposed to high salt stress conditions (200 mM of NaCl) in greenhouse, in order to evaluate their tolerance level and to identify key genes involved in salt stress response. Molecular and physiological parameters, as well as plant growth and leaves’ ions Na+ and K+ content were measured. Results of the physiological measurements showed Royal de Cazorla as the most tolerant cultivar, and Fadak86 and Picual as the most susceptible ones. Ten candidate genes were analyzed and their complete genomic, CDS and protein sequences were identified. The expression analysis of their transcripts through reverse transcriptase quantitative PCR (RT-qPCR) demonstrated that only OeNHX7, OeP5CS, OeRD19A and OePetD were upregulated in tolerant cultivars, thus suggesting their key role in the activation of a salt tolerance mechanism.

Characterization of the Glehnia littoralis Non-specific Phospholipase C Gene GlNPC3 and Its Involvement in the Salt Stress Response

Glehnia littoralis is a medicinal halophyte that inhabits sandy beaches and has high ecological and commercial value. However, the molecular mechanism of salt adaptation in G. littoralis remains largely unknown. Here, we cloned and identified a non-specific phospholipase C gene (GlNPC3) from G. littoralis, which conferred lipid-mediated signaling during the salt stress response. The expression of GlNPC3 was induced continuously by salt treatment. Overexpression of GlNPC3 in Arabidopsis thaliana increased salt tolerance compared to wild-type (WT) plants. GlNPC3-overexpressing plants had longer roots and higher fresh and dry masses under the salt treatment. The GlNPC3 expression pattern revealed that the gene was expressed in most G. littoralis tissues, particularly in roots. The subcellular localization of GlNPC3 was mainly at the plasma membrane, and partially at the tonoplast. GlNPC3 hydrolyzed common membrane phospholipids, such as phosphotidylserine (PS), phosphoethanolamine (PE), and phosphocholine (PC). In vitro enzymatic assay showed salt-induced total non-specific phospholipase C (NPC) activation in A. thaliana GlNPC3-overexpressing plants. Plant lipid profiling showed a significant change in the membrane-lipid composition of A. thaliana GlNPC3-overexpressing plants compared to WT after the salt treatment. Furthermore, downregulation of GlNPC3 expression by virus-induced gene silencing in G. littoralis reduced the expression levels of some stress-related genes, such as SnRK2, P5SC5, TPC1, and SOS1. Together, these results indicated that GlNPC3 and GlNPC3-mediated membrane lipid change played a positive role in the response of G. littoralis to a saline environment.

Integrative analysis of the salt stress response in cyanobacteria

Microorganisms evolved specific acclimation strategies to thrive in environments of high or fluctuating salinities. Here, salt acclimation in the model cyanobacterium Synechocystis sp. PCC 6803 was analyzed by integrating transcriptomic, proteomic and metabolomic data. A dynamic reorganization of the transcriptome occurred during the first hours after salt shock, e.g. involving the upregulation of genes to activate compatible solute biochemistry balancing osmotic pressure. The massive accumulation of glucosylglycerol then had a measurable impact on the overall carbon and nitrogen metabolism. In addition, we observed the coordinated induction of putative regulatory RNAs and of several proteins known for their involvement in other stress responses. Overall, salt-induced changes in the proteome and transcriptome showed good correlations, especially among the stably up-regulated proteins and their transcripts. We define an extended salt stimulon comprising proteins directly or indirectly related to compatible solute metabolism, ion and water movements, and a distinct set of regulatory RNAs involved in post-transcriptional regulation. Our comprehensive data set provides the basis for engineering cyanobacterial salt tolerance and to further understand its regulation.

BpTCP3 Transcription Factor Improves Salt Tolerance of Betula platyphylla by Reducing Reactive Oxygen Species Damage

The plant-specific transcription factors TEOSINTE BRANCHED1/CYCLO IDEA/PROLIFERATING CELL FACTOR1 (TCP) act as developmental regulators that have many roles in the growth and development processes throughout the entire life span of plants. TCP transcription factors are responsive to endogenous and environmental signals, such as salt stress. However, studies on the role of the TCP genes in salt stress response have rarely focused on woody plants, especially forest trees. In this study, the BpTCP3 gene, a CYC/TB1 subfamily member, isolated from Betula platyphylla Sukaczev, was significantly influenced by salt stress. The β-glucuronidase (GUS) staining analysis of transgenic B. platyphylla harboring the BpTCP3 promoter fused to the reporter gene GUS (pBpTCP3::GUS) further confirmed that the BpTCP3 gene acts a positive regulatory position in salt stress. Under salt stress, we found that the BpTCP3 overexpressed lines had increased relative/absolute high growth but decreased salt damage index, hydrogen peroxide (H2O2), and malondialdehyde (MDA) levels versus wild-type (WT) plants. Conversely, the BpTCP3 suppressed lines exhibited sensitivity to salt stress. These results indicate that the BpTCP3 transcription factor improves the salt tolerance of B. platyphylla by reducing reactive oxygen species damage, which provides useful clues for the functions of the CYC/TB1 subfamily gene in the salt stress response of B. platyphylla.

Identification of Raf-Like Kinases B Subfamily Genes in Gossypium Species Revealed GhRAF42 Enhanced Salt Tolerance in Cotton

Salinity is a critical abiotic factor that significantly reduces agricultural production. Cotton is an important fiber crop and a pioneer on saline soil, hence genetic architecture that underpins salt tolerance should be thoroughly investigated. The Raf-like kinase B-subfamily (RAF) genes were discovered to regulate the salt stress response in cotton plants. However, understanding the RAFs in cotton, such as Enhanced Disease Resistance 1 and Constitutive Triple Response 1 kinase, remains a mystery. This study obtained 29, 28, 56, and 54 RAF genes from G. arboreum, G. raimondii, G. hirsutum, and G. barbadense, respectively. The RAF gene family described allopolyploidy and hybridization events in allotetraploid cotton evolutionary connections. Ka/Ks analysis advocates that cotton evolution was subjected to an intense purifying selection of the RAF gene family. Interestingly, integrated analysis of synteny and gene collinearity suggested dispersed and segmental duplication events involved in the extension of RAFs in cotton. Transcriptome studies, functional validation, and virus-induced gene silencing on salt treatments revealed that GhRAF42 is engaged in salt tolerance in upland cotton. This research might lead to a better understanding of the role of RAFs in plants and the identification of suitable candidate salt-tolerant genes for cotton breeding.

Transcriptome Profiling of the Salt Stress Response in the Leaves and Roots of Halophytic Eutrema salsugineum

Eutrema salsugineum can grow in natural harsh environments; however, the underlying mechanisms for salt tolerance of Eutrema need to be further understood. Herein, the transcriptome profiling of Eutrema leaves and roots exposed to 300 mM NaCl is investigated, and the result emphasized the role of genes involved in lignin biosynthesis, autophagy, peroxisome, and sugar metabolism upon salt stress. Furthermore, the expression of the lignin biosynthesis and autophagy-related genes, as well as 16 random selected genes, was validated by qRT-PCR. Notably, the transcript abundance of a large number of lignin biosynthesis genes such as CCoAOMT, C4H, CCR, CAD, POD, and C3′H in leaves was markedly elevated by salt shock. And the examined lignin content in leaves and roots demonstrated salt stress led to lignin accumulation, which indicated the enhanced lignin level could be an important mechanism for Eutrema responding to salt stress. Additionally, the differentially expressed genes (DEGs) assigned in the autophagy pathway including Vac8, Atg8, and Atg4, as well as DEGs enriched in the peroxisome pathway such as EsPEX7, EsCAT, and EsSOD2, were markedly induced in leaves and/or roots. In sugar metabolism pathways, the transcript levels of most DEGs associated with the synthesis of sucrose, trehalose, raffinose, and xylose were significantly enhanced. Furthermore, the expression of various stress-related transcription factor genes including WRKY, AP2/ERF-ERF, NAC, bZIP, MYB, C2H2, and HSF was strikingly improved. Collectively, the increased expression of biosynthesis genes of lignin and soluble sugars, as well as the genes in the autophagy and peroxisome pathways, suggested that Eutrema encountering salt shock possibly possess a higher capacity to adjust osmotically and facilitate water transport and scavenge reactive oxidative species and oxidative proteins to cope with the salt environment. Thus, this study provides a new insight for exploring the salt tolerance mechanism of halophytic Eutrema and discovering new gene targets for the genetic improvement of crops.

Salt responsive alternative splicing of a RING finger E3 ligase modulates the salt stress tolerance by fine-tuning the balance of COP9 signalosome subunit 5A

Increasing evidence points to the tight relationship between alternative splicing (AS) and the salt stress response in plants. However, the mechanisms linking these two phenomena remain unclear. In this study, we have found that Salt-Responsive Alternatively Spliced gene 1 (SRAS1), encoding a RING-Type E3 ligase, generates two splicing variants: SRAS1.1 and SRAS1.2, which exhibit opposing responses to salt stress. The salt stress-responsive AS event resulted in greater accumulation of SRAS1.1 and a lower level of SRAS1.2. Comprehensive phenotype analysis showed that overexpression of SRAS1.1 made the plants more tolerant to salt stress, whereas overexpression of SRAS1.2 made them more sensitive. In addition, we successfully identified the COP9 signalosome 5A (CSN5A) as the target of SRAS1. CSN5A is an essential player in the regulation of plant development and stress. The full-length SRAS1.1 promoted degradation of CSN5A by the 26S proteasome. By contrast, SRAS1.2 protected CSN5A by competing with SRAS1.1 on the same binding site. Thus, the salt stress-triggered AS controls the ratio of SRAS1.1/SRAS1.2 and switches on and off the degradation of CSN5A to balance the plant development and salt tolerance. Together, these results provide insights that salt-responsive AS acts as post-transcriptional regulation in mediating the function of E3 ligase.

Genome-wide identification and functional prediction of salt- stress related long non-coding RNAs (lncRNAs) in chickpea (Cicer arietinum L.)

LncRNAs (long noncoding RNAs) are 200 bp length crucial RNA molecules, lacking coding potential and having important roles in regulating gene expression, particularly in response to abiotic stresses. In this study, we identified salt stress-induced lncRNAs in chickpea roots and predicted their intricate regulatory roles. A total of 3452 novel lncRNAs were identified to be distributed across all 08 chickpea chromosomes. On comparing salt-tolerant (ICCV 10, JG 11) and salt-sensitive cultivars (DCP 92–3, Pusa 256), 4446 differentially expressed lncRNAs were detected under various salt treatments. We predicted 3373 lncRNAs to be regulating their target genes in cis regulating manner and 80 unique lncRNAs were observed as interacting with 136 different miRNAs, as eTMs (endogenous target mimic) targets of miRNAs and implicated them in the regulatory network of salt stress response. Functional analysis of these lncRNA revealed their association in targeting salt stress response-related genes like potassium transporter, transporter family genes, serine/threonine-protein kinase, aquaporins like TIP1-2, PIP2-5 and transcription factors like, AP2, NAC, bZIP, ERF, MYB and WRKY. Furthermore, about 614 lncRNA-SSRs (simple sequence repeats) were identified as a new generation of molecular markers with higher efficiency and specificity in chickpea. Overall, these findings will pave the understanding of comprehensive functional role of potential lncRNAs, which can help in providing insight into the molecular mechanism of salt tolerance in chickpea.

Transcriptome analysis and functional identification of GmMYB46 in soybean seedlings under salt stress

Salinity is one of the major abiotic stress that limits crop growth and productivity. We investigated the transcriptomes of salt-treated soybean seedlings versus a control using RNA-seq to better understand the molecular mechanisms of the soybean (Glycine max L.) response to salt stress. Transcriptome analysis revealed 1,235 differentially expressed genes (DEGs) under salt stress. Several important pathways and key candidate genes were identified by KEGG enrichment. A total of 116 differentially expressed transcription factors (TFs) were identified, and 17 TFs were found to belong to MYB families. Phylogenetic analysis revealed that these TFs may be involved in salt stress adaptation. Further analysis revealed that GmMYB46 was up-regulated by salt and mannitol and was localized in the nucleus. The salt tolerance of transgenic Arabidopsis overexpressing GmMYB46 was significantly enhanced compared to wild-type (WT). GmMYB46 activates the expression of salt stress response genes (P5CS1, SOD, POD, NCED3) in Arabidopsis under salt stress, indicating that the GmMYB46 protein mediates the salt stress response through complex regulatory mechanisms. This study provides information with which to better understand the molecular mechanism of salt tolerance in soybeans and to genetically improve the crop.