Transcriptome-Metabolome Integration Uncovers Salt Stress Effects on Flavonoid Biosynthesis in Two Self-Selected Breeding Alfalfa Varieties.

The flavonoids play important roles in plant salt tolerance. Blueberries (Vaccinium spp.) are extremely sensitive to soil salt increases. Therefore, improving the salt resistance of blueberries by increasing the flavonoid content is crucial for the development of the blueberry industry. To explore the underlying molecular mechanism, we performed an integrated analysis of the metabolome and transcriptome of blueberry leaves under salt stress. We identified 525 differentially accumulated metabolites (DAMs) under salt stress vs. control treatment, primarily including members of the flavonoid class. We also identified 20,920 differentially expressed genes (DEGs) based on transcriptome data; of these, 568 differentially expressed transcription factors (TFs) were annotated, and bHLH123, OsHSP20, and HSP20 TFs might be responsible for blueberry leaf salt tolerance. DEGs involved in the flavonoid biosynthesis pathway were significantly enriched at almost all stages of salt stress. Salt treatment upregulated the expression of most flavonoid biosynthetic pathway genes and promoted the accumulation of flavonols, flavonol glycosides, flavans, proanthocyanidins, and anthocyanins. Correlation analysis suggested that 4-coumarate CoA ligases (4CL5 and 4CL1) play important roles in the accumulation of flavonols (quercetin and pinoquercetin) and flavan-3-ol (epicatechin and prodelphinidin C2) under salt stress, respectively. The flavonoid 3′5′-hydroxylases (F3′5′H) regulate anthocyanin (cyanidin 3-O-beta-D-sambubioside and delphinidin-3-O-glucoside chloride) biosynthesis, and leucoanthocyanidin reductases (LAR) are crucial for the biosynthesis of epicatechin and prodelphinidin C2 during salt stress. Taken together, it is one of the future breeding goals to cultivate salt-resistant blueberry varieties by increasing the expression of flavonoid biosynthetic genes, especially 4CL, F3′5′H, and LAR genes, to promote flavonoid content in blueberry leaves.

Comparative analyses of plant responses to drought and salt stress in related taxa: A useful approach to study stress tolerance mechanisms.

Abiotic stresses affect crop productivity worldwide. Plants have developed defense mechanisms against environmental stresses by altering the gene expression pattern which leads to regulation of certain metabolic and defensive pathways. Sorghum [Sorghum bicolor (L.) Moench] is an important crop in those regions irrigated by salty water. Sweet sorghum is a variant of common grain sorghum and is relatively more adapted to marginal growing conditions. Here, we compared the different response to salt stress of sweet and grain sorghum. We investigated six traits related with seed germination under salt-stress and normal conditions, conducted a genome-wide research on the salt effect on the gene expression of a landrace sweet and two grain sorghum by RNA-sequencing at seedling stage. The results showed that salt stress had significant inhibition to sorghum seed germination capability, and the inhibition to grain sorghum was greater. By comparing sweet and grain sorghum and the KEGG pathway analysis based on the DEGs, six genes involved in flavonoid biosynthesis pathway to tannins and anthocyanins from phenylalanine were identified in the landrace sweet sorghum, which expression was significant different with that in grain sorghum. Quantitative real-time PCR (qRT-PCR) data were closely in accordance with the transcript patterns estimated from the RNA-seq data. Tannins accumulation changes were associated with the genes expression under salt stress and control. These suggested that flavonoid biosynthesis pathway was involved in the sorghum resistance to salt stress. The present results suggested that flavonoid biosynthesis plays an important role in the sweet sorghum capacity for salt tolerance.

A meeting on the molecular basis of ionic homeostasis and salt tolerance in plants took place in Madrid, Spain, October 22–24, 2001. This meeting was organized by Eduardo Blumwald (Davis, CA) and Alonso Rodriguez‐Navarro (Madrid, Spain) at the Centre for International Meetings on Biology (‘Instituto Juan March de Estudios e Investigaciones’). ![][1] Ionic homeostasis is a fundamental cellular phenomenon. All living cells maintain an intracellular ionic composition compatible with their constituent molecules, and this requires the regulation of multiple membrane transporters and signal transduction pathways. Other biophysical parameters such as turgor and electrical potential are also part of this essential regulation. How ionic homeostasis is achieved, however, is not completely understood. Although most transporters have already been identified, their physiological function is only starting to be demonstrated and the receptors and most components of the regulatory pathways that effect ionic homeostasis remain unknown. In the case of plants, this problem is related to mineral nutrition and salinity tolerance, both of which have great relevance for agriculture. In fact, as demonstrated by this meeting, salinity stress has been one of the keys to opening the black box of ionic homeostasis in general. Another has been the novel molecular genetics of the plant Arabidopsis thaliana . Of course, other approaches have also contributed to our present understanding of ion homeostasis in plants and were represented at the meeting. For further details, see Blumwald (2000), Hasegawa et al . (2000), Bohnert et al . (2001), Serrano and Rodriguez‐Navarro (2001) and Zhu (2001). ### Some physiology of salt tolerance Salt stress is an important threat to the future of agriculture in many productive areas of the planet. In countries such as Australia and Pakistan, salinity is already a national concern, as it was in the past in ancient Mesopotamia. Areas of California and the Mediterranean region are also threatened. … [1]: /embed/graphic-1.gif

Soybean (Glycine max) production is severely affected in unfavorable environments. Identification of the regulatory factors conferring stress tolerance would facilitate soybean breeding. In this study, through coexpression network analysis of salt-tolerant wild soybeans, together with molecular and genetic approaches, we revealed a previously unidentified function of a class B heat shock factor, HSFB2b, in soybean salt stress response. We showed that HSFB2b improves salt tolerance through the promotion of flavonoid accumulation by activating one subset of flavonoid biosynthesis-related genes and by inhibiting the repressor gene GmNAC2 to release another subset of genes in the flavonoid biosynthesis pathway. Moreover, four promoter haplotypes of HSFB2b were identified from wild and cultivated soybeans. Promoter haplotype II from salt-tolerant wild soybean Y20, with high promoter activity under salt stress, is probably selected for during domestication. Another promoter haplotype, III, from salt-tolerant wild soybean Y55, had the highest promoter activity under salt stress, had a low distribution frequency and may be subjected to the next wave of selection. Together, our results revealed the mechanism of HSFB2b in soybean salt stress tolerance. Its promoter variations were identified, and the haplotype with high activity may be adopted for breeding better soybean cultivars that are adapted to stress conditions.

Soybean (Glycine max) is a globally important crop for oil and protein production, but its growth and yield are severely affected by abiotic stresses, such as drought and salinity. We investigated subcellular localization of the DRB0118 gene from Deinococcus radiodurans, and phenotypic, physiological and biochemical indicators of DRB0118 overexpressing soybean plants under salt and drought stresses. Combined with transcriptome data, the results showed that overexpression of DRB0118 improved salt and drought tolerance of soybean. Subcellular localization revealed that the DRB0118 protein is localized in the nucleus and cell membrane. Overexpressed DRB0118 soybean lines had significantly improved survival under drought and salt stress, accompanied by enhanced superoxide dismutase (SOD) and peroxidase (POD) activity, reduced malondialdehyde (MDA) content, and lower reactive oxygen species (ROS) accumulation, as well as tighter closure of the stomatal aperture and a stronger root system. Transcriptome profiling further revealed that DRB0118 upregulated photosynthesis-related pathways under drought stress, and flavonoid biosynthesis under salt stress, both critical for mitigating oxidative damage. These findings highlight DRB0118 as a promising candidate gene for engineering crops with enhanced resilience to abiotic stresses. We discuss the potential mechanism of overexpressing DRB0118-enhanced salt and drought tolerance in soybean, including changes in antioxidants, stomata and roots, and enrichment of photosynthetic and flavonoid synthesis pathways.

Long non-coding RNAs (lncRNAs) are pivotal regulators of the abiotic stress responses in plants, yet their specific involvement in salt/alkali stress during alfalfa germination remains incompletely understood. Here, we subjected Zhongmu No.1 alfalfa (Medicago sativa L.) seeds to salt stress (20 mM NaCl and 20 mM Na2SO4 solutions) or alkali stress (5 mM NaHCO3 and 5 mM Na2CO3 solutions) treatments for 3 days, followed by total RNA extraction and RNA-seq analysis to delineate stress-responsive alfalfa lncRNAs. We identified 17,473 novel alfalfa lncRNAs, among which 101 and 123 were differentially expressed lncRNAs (DElncRNAs) under salt and alkali stress, respectively, compared to the control. Furthermore, we predicted 16 and 237 differentially expressed target genes regulated by DElncRNAs through cis/trans-regulatory mechanisms under salt or alkali stress, respectively. A functional enrichment analysis of DElncRNA target genes indicated that lncRNAs were implicated in the fatty acid metabolism pathway under salt stress, while they played a significant role in the phenylpropanoid and flavonoid biosynthesis pathway under alkali stress. Notably, lncRNAs were found to participate in the plant hormone signal transduction pathway, a common regulatory mechanism in both salt and alkali stress responses. These findings contribute to a deeper understanding of the mechanisms underlying alfalfa’s response to salt and alkali stresses.

(1) Background: Alfalfa is an important legume forage throughout the world. Although alfalfa is considered moderately tolerant to salinity, its production and nitrogen-fixing activity are greatly limited by salt stress. (2) Methods: We examined the physiological changes and proteomic profiles of alfalfa with active nodules (NA) and without nodules (NN) under NaCl treatment. (3) Results: Our data suggested that NA roots showed upregulation of the pathways of abiotic and biotic stress responses (e.g., heat shock proteins and pathogenesis-related proteins), antioxidant enzyme synthesis, protein synthesis and degradation, cell wall degradation and modification, acid phosphatases, and porin transport when compared with NN plants under salt stress conditions. NA roots also upregulated the processes or proteins of lipid metabolism, heat shock proteins, protein degradation and folding, and cell cytoskeleton, downregulated the DNA and protein synthesis process, and vacuolar H+-ATPase proteins under salt stress. Besides, NA roots displayed a net H+ influx and low level of K+ efflux under salt stress, which may enhance the salt tolerance of NA plants. (4) Conclusions: The rhizobium symbiosis conferred the host plant salt tolerance by regulating a series of physiological processes to enhance stress response, improve antioxidant ability and energy use efficiency, and maintain ion homeostasis.

BackgroundSalt and drought are the primary environmental stress factors that severely threaten plant growth, development, and yield. bZIP transcription factors reportedly play crucial roles in plant responses to both biotic and abiotic stressors. However, the biological function of bZIP transcription factors in oil crops, particularly walnuts, under salt and drought stress remains unclear.ResultsIn this study, members of the walnut bZIP gene family were identified based on the walnut genome Chandler 2.0. Transcriptome data and RT-qPCR results were used to analyze the expression patterns of various JrbZIP genes under biotic and abiotic stress, revealing that JrbZIP40 was strongly induced by both drought and salt stress. Subcellular localization and transcriptional activation assays demonstrated that JrbZIP40 localized to the nucleus and exhibited transcriptional activation activity. Overexpression of JrbZIP40 in transgenic Arabidopsis seedlings significantly enhanced resistance to salt and drought stress. DAP-seq and Dual-luciferase results indicated that JrbZIP40 may bind to the JrHB7 and JrATG8G promoters and activate their expression, contributing to stress resistance.ConclusionsOverall, this study elucidates the regulatory network and biological functions of JrbZIP40 in drought and salt tolerance, providing a theoretical foundation and candidate gene resources for the future use of genetic engineering to improve walnut stress resistance.

The content of flavonoids could increase in A. canescens under saline conditions. Overexpression of AcCHI in transgenic A. thaliana promotes flavonoid biosynthesis, thereby functioning in the tolerance of transgenic plants to salt and osmotic stress by maintaining ROS homeostasis. Atriplex canescens is a halophytic forage shrub with excellent adaptation to saline environment. Our previous study showed that a large number of genes related to the biosynthesis of flavonoids in A. canescens were significantly up-regulated by NaCl treatments. However, it remains unclear whether flavonoids are involved in A. canescens response to salinity. In this study, we found that the accumulation of flavonoids significantly increased in either the leaves or roots of A. canescens seedling under 100 and 300mM NaCl treatments. Correspondingly, AcCHS, AcCHI and AcF3H, which encode three key enzymes (chalcone synthases (CHS), chalcone isomerase (CHI), and flavanone 3-hydroxylase (F3H), respectively) of flavonoids biosynthesis, were significantly induced in the roots or leaves of A. canescens by 100 or 300mM NaCl. Then, we generated the transgenic Arabidopsis thaliana overexpressing AcCHI and found that transgenic plants accumulated more flavonoids through enhancing the pathway of flavonoids biosynthesis. Furthermore, overexpression of AcCHI conferred salt and osmotic stress tolerance in transgenic A. thaliana. Contrasted with wild-type A. thaliana, transgenic lines grew better with greater biomass, less H2O2 content as well as lower relative plasma permeability in either salt or osmotic stress conditions. In conclusion, our results indicate that flavonoids play an important role in A. canescens response to salt stress through reactive oxygen species (ROS) scavenging and the key enzyme gene AcCHI in flavonoids biosynthesis pathway of A. canescens has the potential to improve the stress tolerance of forages and crops.

The sugar beet monosomic addition line M14 is a unique germplasm that contains genetic materials from Beta vulgaris L. and Beta corolliflora Zoss, and shows tolerance to salt stress. Our study focuses on exploring the molecular mechanism of the salt tolerance of the sugar beet M14. In order to identify differentially expressed genes in M14 under salt stress, a subtractive cDNA library was generated by suppression subtractive hybridization (SSH). A total of 36 unique sequences were identified in the library and their putative functions were analyzed. One of the genes, S-adenosylmethionine synthetase (SAMS), is the key enzyme involved in the biosynthesis of S-adenosylmethionine (SAM), a precursor of polyamines. To determine the potential role of SAMS in salt tolerance, we isolated BvM14-SAMS2 from the salt-tolerant sugar beet M14. The expression of BvM14-SAMS2 in leaves and roots was greatly induced by salt stress. Overexpression of BvM14-SAMS2 in Arabidopsis resulted in enhanced salt and H2O2 tolerance. Furthermore, we obtained a knock-down T-DNA insertion mutant of AtSAMS3, which shares the highest homology with BvM14-SAMS2. Interestingly, the mutant atsam3 showed sensitivity to salt and H2O2 stress. We also found that the antioxidant system and polyamine metabolism play an important role in salt and H2O2 tolerance in the BvM14-SAMS2-overexpressed plants. To our knowledge, the function of the sugar beet SAMS has not been reported before. Our results have provided new insights into SAMS functions in sugar beet.

Plant growth and development are significantly affected by salt stress. Chrysanthemum lavandulifolium is a halophyte species and one of the ancestors of chrysanthemum ( C. ×morifolium ). Understanding how this species tolerates salt stress could provide vital insight for clarifying the salt response systems of higher plants, and chrysanthemum-breeding programs could be improved. In this study, salt tolerance was compared among C. lavandulifolium and three chrysanthemum cultivars by physiological experiments, among which C. lavandulifolium and Jinba displayed better tolerance to salt stress than the other two cultivars, whereas Xueshan was a salt-sensitive cultivar. Using the transcriptome database of C. lavandulifolium as a reference, we used digital gene expression technology to analyze the global gene expression changes in C. lavandulifolium seedlings treated with 200 m m NaCl for 12 hours compared with seedlings cultured in normal conditions. In total, 2254 differentially expressed genes (DEGs), including 1418 up-regulated and 836 down-regulated genes, were identified. These DEGs were significantly enriched in 35 gene ontology terms and 29 Kyoto Encyclopedia of Genes and Genomes pathways. Genes related to signal transduction, ion transport, proline biosynthesis, reactive oxygen species scavenging systems, and flavonoid biosynthesis pathways were relevant to the salt tolerance of C. lavandulifolium . Furthermore, comparative gene expression analysis was conducted using reverse transcription polymerase chain reaction to compare the transcriptional levels of significantly up-regulated DEGs in C. lavandulifolium and the salt-sensitive cultivar Xueshan, and species-specific differences were observed. The analysis of one of the DEGs, ClAKT , an important K + transport gene, was found to enable transgenic Arabidopsis thaliana to absorb K + and efflux Na + under salt stress and to absorb K + under drought stress. The present study investigated potential genes and pathways involved in salt tolerance in C. lavandulifolium and provided a hereditary resource for the confinement of genes and pathways responsible for salt tolerance in this species. This study provided a valuable source of reference genes for chrysanthemum cultivar transgenesis breeding.

Overexpression of high affinity K+ transporter from Nitraria sibirica enhanced salt tolerance of transgenic plants

As a multifunctional tree species, Cyclocarya paliurus leaves are rich in bioactive substances with precious healthy values. To meet the huge requirement of C. paliurus leaf production, sites with some environmental stresses would be potential land for developing its plantations due to the limitation of land resources in China. Nitric oxide (NO) and hydrogen sulfide (H2S) are common gas messengers used to alleviate abiotic stress damage, whereas the mechanism of these messengers in regulating salt resistance of C. paliurus still remains unclear. We performed a comprehensive study to reveal the physiological response and molecular regulatory mechanism of C. paliurus seedlings to the application of exogenous NO and H2S under salt stress. The results showed that the application of sodium hydrosulfide (NaHS) and sodium nitroprusside (SNP) not only maintained the photosynthetic capacity and reduced the loss of leaf biomass, but also promoted endogenous NO synthesis and reduced oxidative damage by activating antioxidant enzyme activity and increasing the content of soluble protein and flavonoids. Moreover, transcriptome and metabolome analysis indicated the expression of genes encoding phenylalanine ammonia lyase (PAL), cytochromeP450 (CYP), chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR) and flavonol synthase (FLS) in flavonoid biosynthesis pathway was all up-regulated by the application of NO and H2S. Meanwhile, 15 transcriptional factors (TFs) such as WRKY, ERF, bHLH and HY5 induced by NO were found to regulated the activities of several key enzymes in flavonoid biosynthesis pathway under salt stress, via the constructed co-expression network. Our findings revealed the underlying mechanism of NO and H2S to alleviate salt stress and regulate flavonoid biosynthesis, which provides a theoretical basis for establishing C. paliurus plantations in the salt stress areas.

Plant acclimation to salt and alkali stress is closely linked to the ability of the antioxidant system to mediate the scavenging of reactive oxygen species (ROS). In this study, we investigated the effects of salt stress and alkali stress on ROS, antioxidant enzymes, transcriptome, and metabolome. The results showed that the levels of superoxide anions, hydrogen peroxide, malondialdehyde, and electrolyte leakage increased under salt and alkali stress, with higher concentrations observed under alkali stress than salt stress. The activities of superoxide dismutase (EC 1.15.1.1), peroxidase (EC 1.11.1.7), catalase (EC 1.11.1.6), ascorbate peroxidase (EC 1.11.1.11), glutathione reductase (EC 1.6.4.2), dehydroascorbate reductase (EC 1.8.5.1), and monodehydroascorbate reductase (EC 1.6.5.4) varied under salt and alkali stress. The transcriptome analysis revealed the induction of signal transduction and metabolic processes and differential expression of genes encoding antioxidant enzymes in response to salt and alkali stress. The metabolome analysis demonstrated increased ascorbic acid and glutathione under salt stress, while most phenolic acids, flavonoids, and alkaloids increased under salt and alkali stress. Integrative analysis of the metabolome and transcriptome data revealed that the flavonoid biosynthesis pathway played a key role in the grapevine's response to salt stress. The total flavonoid content increased under salt and alkali stress, but the accumulation of flavonoids was higher under salt stress than alkali stress. In conclusion, our findings indicate significant differences in the antioxidant defense of grapevines under these two stresses, providing insight into distinct acclimation mechanisms in grapevine under salt and alkali stress. This article is protected by copyright. All rights reserved.