Genome-Wide Identification of the WRKY Gene Family in Four Cotton Varieties and the Positive Role of GhWRKY31 in Response to Salt and Drought Stress.

The WRKY gene family is ubiquitously distributed in plants, serving crucial functions in stress responses. Nevertheless, the structural organization and evolutionary dynamics of WRKY genes in cotton have not been fully elucidated. In this study, a total of 112, 119, 217, and 222 WRKY genes were identified in Gossypium arboreum, Gossypium raimondii, Gossypium hirsutum, and Gossypium barbadense, respectively. These 670 WRKY genes were categorized into seven distinct subgroups and unequally distributed across chromosomes. Examination of conserved motifs, domains, cis-acting elements, and gene architecture collectively highlighted the evolutionary conservation and divergence within the WRKY gene family in cotton. Analysis of synteny and collinearity further confirmed instances of expansion, duplication, and loss events among WRKY genes during cotton evolution. Furthermore, GhWRKY31 transgenic Arabidopsis exhibited heightened germination rates and longer root lengths under drought and salt stress. Silencing GhWRKY31 in cotton led to reduced levels of ABA, proline, POD, and SOD, along with downregulated expression of stress-responsive genes. Yeast one-hybrid and molecular docking assays confirmed the binding capacity of GhWRKY31 to the W box of GhABF1, GhDREB2, and GhRD29. The findings collectively offer a systematic and comprehensive insight into the evolutionary patterns of cotton WRKYs, proposing a suitable regulatory framework for developing cotton cultivars with enhanced resilience to drought and salinity stress.

Genome-Wide Analysis of Cotton MYB Transcription Factors and the Functional Validation of GhMYB in Response to Drought Stress.

MYB transcription factors play important roles during abiotic stress responses in plants. However, little is known about the accurate systematic analysis of MYB genes in the four cotton species, Gossypium hirsutum, G. barbadense, G. arboreum and G. raimondii. Herein, we performed phylogenetic analysis and showed that cotton MYBs and Arabidopsis MYBs were clustered in the same subfamilies for each species. The identified cotton MYBs were distributed unevenly on chromosomes in various densities for each species, wherein genome-wide tandem and segment duplications were the main driving force of MYB family expansion. Synteny analysis suggested that the abundant collinearity pairs of MYBs were identified between G. hirsutum and the other three species, and that they might have undergone strong purification selection. Characteristics of conserved motifs, along with their consensus sequence, promoter cis elements and gene structure, revealed that MYB proteins might be highly conserved in the same subgroups for each species. Subsequent analysis of differentially expressed genes and expression patterns indicated that most GhMYBs might be involved in response to drought (especially) and salt stress, which was supported by the expression levels of nine GhMYBs using real-time quantitative PCR. Finally, we performed a workflow that combined virus-induced gene silencing and the heterologous transformation of Arabidopsis, which confirmed the positive roles of GhMYBs under drought conditions, as validated by determining the drought-tolerant phenotypes, damage index and/or water loss rate. Collectively, our findings not only expand our understanding of the relationships between evolution and function of MYB genes, but they also provide candidate genes for cotton breeding.

Genome-wide identification and expression analysis of DREB family genes in cotton.

BACKGROUND: Dehydration responsive element-binding (DREB) transcription factors are widely present in plants, and involve in signalling transduction, plant growth and development, and stress response. DREB genes have been characterized in multiple species. However, only a few DREB genes have been studied in cotton, one of the most important fibre crops. Herein, the genome­wide identification, phylogeny, and expression analysis of DREB family genes are performed in diploid and tetraploid cotton species. RESULTS: In total, 193, 183, 80, and 79 putative genes containing the AP2 domain were identified using bioinformatics approaches in G. barbadense, G. hirsutum, G. arboretum, and G. raimondii, respectively. Phylogenetic analysis showed that based on the categorization of Arabidopsis DREB genes, 535 DREB genes were divided into six subgroups (A1-A6) by using MEGA 7.0. The identified DREB genes were distributed unevenly across 13/26 chromosomes of A and/or D genomes. Synteny and collinearity analysis confirmed that during the evolution, the whole genome duplications, segmental duplications, and/or tandem duplications occurred in cotton DREB genes, and then DREB gene family was further expanded. Further, the evolutionary trees with conserved motifs, cis-acting elements, and gene structure of cotton DREB gene family were predicted, and these results suggested that DREB genes might be involved in the hormone and abiotic stresses responses. The subcellular localization showed that in four cotton species, DREB proteins were predominantly located in the nucleus. Further, the analysis of DREB gene expression was carried out by real-time quantitative PCR, confirming that the identified DREB genes of cotton were involved in response to early salinity and osmotic stress. CONCLUSIONS: Collectively, our results presented a comprehensive and systematic understanding in the evolution of cotton DREB genes, and demonstrated the potential roles of DREB family genes in stress and hormone response.

Effects of 5-azaC on Iridoid Glycoside Accumulation and DNA Methylation in Rehmannia glutinosa.

Iridoid glycoside is the important secondary metabolite and the main active component in Rehmannia glutinosa. However, the mechanisms that underlie the regulation of iridoid glycoside biosynthesis remain poorly understood in R. glutinosa. Herein, the analysis of RNA-seq data revealed that 3,394 unigenes related to the biosynthesis of secondary metabolites were identified in R. glutinosa. A total of 357 unigenes were involved in iridoid glycoside synthesis, in which the highly conservative genes, such as DXS, DXR, GPPS, G10H, and 10HGO, in organisms were overexpressed. The analysis of the above genes confirmed that the co-occurrence ratio of DXS, DXR, and GPPS was high in plants. Further, our results showed that under normal and 5-azacytidine (5-azaC) treatment, the expression levels of DXS, DXR, GPPS, G10H, and 10HGO were consistent with the iridoid glycoside accumulation in R. glutinosa, in which the application of the different concentrations of 5-azaC, especially 50 µM 5-azaC, could significantly upregulate the expression of five genes above and iridoid glycoside content. In addition, the changes in the spatiotemporal specificity of degree and levels of DNA methylation were observed in R. glutinosa, in which the hemi-methylation was the main reason for the change in DNA methylation levels. Similar to the changes in 5-methyl cytosine (5mC) content, the DNA demethylation could be induced by 5-azaC and responded in a dose-dependent manner to 15, 50, and 100 µM 5-azaC. Taken together, the expression of iridoid glycoside synthesis gene was upregulated by the demethylation in R. glutinosa, followed by triggering the iridoid glycoside accumulation. These findings not only identify the key genes of iridoid glycoside synthesis from R. glutinosa, but also expand our current knowledge of the function of methylation in iridoid glycoside accumulation.

Methane control of cadmium tolerance in alfalfa roots requires hydrogen sulfide.

Hydrogen sulfide (H2S) is well known as a gaseous signal in response to heavy metal stress, while methane (CH4), the most prevalent greenhouse gas, confers cadmium (Cd) tolerance. In this report, the causal link between CH4 and H2S controlling Cd tolerance in alfalfa (Medicago sativa) plants was assessed. Our results observed that the administration of CH4 not only intensifies H2S metabolism, but also attenuates Cd-triggered growth inhibition in alfalfa seedlings, which were parallel to the alleviated roles in the redox imbalance and cell death in root tissues. Above results were not observed in roots after the removal of endogenous H2S, either in the presence of either hypotaurine (HT; a H2S scavenger) or DL-propargylglycine (PAG; a H2S biosynthesis inhibitor). Using in situ noninvasive microtest technology (NMT) and inductively coupled plasma mass spectroscopy (ICP-MS), subsequent results confirmed the participation of H2S in CH4-inhibited Cd influx and accumulation in roots, which could be explained by reestablishing glutathione (GSH) pool (reduced/oxidized GSH and homoglutathione) homeostasis and promoting antioxidant defence. Overall, our results clearly revealed that H2S operates downstream of CH4 enhancing tolerance against Cd stress, which are significant for both fundamental and applied plant biology.

Molecular hydrogen-induced salinity tolerance requires melatonin signalling in Arabidopsis thaliana.

Melatonin (MT) plays positive roles in salinity stress tolerance. However, the upstream signalling components that regulate MT are poorly understood. Here, we report that endogenous MT acts downstream of molecular hydrogen (H2 ) in the salinity response in Arabidopsis. The addition of hydrogen-rich water and expression of the hydrogenase1 gene (CrHYD1) from Chlamydomonas reinhardtii increased endogenous H2 and MT levels and enhanced salinity tolerance. These results were not observed in the absence of serotonin N-acetyltransferase gene (SNAT). H2 increased the levels of SNAT transcripts in the wild-type and CrHYD1 lines, which had lower Na+ /K+ ratios and higher levels of ion transport-related gene transcripts. These changes were not observed in atsnat/CrHYD1-4 hybrids. The increased MT-dependent Na+ extrusion observed in the CrHYD1 plants resulted, at least in part, from enhanced Na+ /H+ antiport across the plasma membrane. The endogenous H2 -induced MT-dependent regulation of ion and redox homeostasis was impaired in the atsnat/CrHYD1-4 hybrids. Taken together, these results demonstrate that MT-induced salinity tolerance is induced by a H2 signalling cascade that regulates ion and redox homeostasis in response to salinity.

Genetic elucidation of hydrogen signaling in plant osmotic tolerance and stomatal closure via hydrogen sulfide.

Although ample evidence showed that exogenous hydrogen gas (H2) controls a diverse range of physiological functions in both animals and plants, the selective antioxidant mechanism, in some cases, is questioned. Importantly, most of the experiments on the function of H2 in plants were based on pharmacological approaches due to the synthesis pathway(s) in plants are still unclear. Here, we observed that the seedling growth inhibition of Arabidopsis caused by low doses of mannitol could progressively recover by recuperation, accompanied with the increased hydrogenase activity and H2 synthesis. To investigate the functions of endogenous H2, a hydrogenase gene (CrHYD1) for H2 biosynthesis from Chlamydomonas reinhardtii was expressed in Arabidopsis. Transgenic plants could intensify higher H2 synthesis compared with wild type and Arabidopsis transformed with the empty vector, and exhibited enhanced osmotic tolerance in both germination and post-germination stages. In response to mannitol, transgenic plants enhanced L-Cys desulfhydrase (DES)-dependent hydrogen sulfide (H2S) synthesis in guard cells and thereafter stomatal closure. The application of des mutant further highlights H2S acting as a downstream molecule of endogenous H2 control of stomatal closure. These results thus open a new window for increasing plant tolerance to osmotic stress.

Nitric Oxide Enhances Rice Resistance to Rice Black-Streaked Dwarf Virus Infection.

BACKGROUND: Rice black-streaked dwarf virus (RBSDV) causes one of the most important rice virus diseases of plants in East Asia. However, molecular mechanism(s)controlling rice resistance to infection is largely unknown. RESULTS: In this paper, we showed that RBSDV infection in rice significantly induced nitric oxide (NO) production. This finding was further validated through a genetic approach using a RBSDV susceptible (Nipponbare) and a RBSDV resistant (15HPO187) cultivar. The production of endogenous NO was muchhigher in the 15HPO187 plants, leading to a much lower RBSDV disease incidence. Pharmacological studies showed that the applications of NO-releasingcompounds (i.e., sodium nitroprusside [SNP] and nitrosoglutathione [GSNO]) to rice plants reduced RBSDV disease incidence. After RBSDV infection, the levels of OsICS1, OsPR1b and OsWRKY 45 transcripts were significantly up-regulated by NO in Nipponbare. The increased salicylic acid contents were also observed. After the SNP treatment, protein S-nitrosylation in rice plants was also increased, suggesting that the NO-triggered resistance to RBSDV infection was partially mediated at the post-translational level. Although Osnia2 mutant rice produced less endogenous NO after RBSDV inoculation and showed a higher RBSDV disease incidence, its RBSDV susceptibility could be reduced by SNP treatment. CONCLUSIONS: Collectively, our genetic and molecular evidence revealed that endogenous NO was a vital signal responsible for rice resistance to RBSDV infection.

Methyl-coenzyme M reductase-dependent endogenous methane enhances plant tolerance against abiotic stress and alters ABA sensitivity in Arabidopsis thaliana.

KEY MESSAGE: Our study firstly elaborated the underlying mechanism of endogenous CH4-induced abiotic tolerance, along with an alteration of ABA sensitivity by mimicking the endogenous CH4 production in MtMCR transgenic Arabidopsis. Endogenous methane (CH4) production and/or emission have been ubiquitously observed in stressed plants. However, their physiological roles remain unclear. Here, the methyl-coenzyme M reductase gene from Methanobacterium thermoautotrophicum (MtMCR), encoding the enzyme of methanogenesis, was expressed in Arabidopsis thaliana, to mimic the production of endogenous CH4. In response to salinity and osmotic stress, MtMCR expression was up-regulated in transgenic plants, resulting in significant increase of endogenous CH4 levels. Similar results were observed in abscisic acid (ABA) treatment. The functions of endogenous CH4 were characterized by the changes in plant phenotypes related to stress and ABA sensitivity during the germination and post-germination periods. When challenged with osmotic stress, a reduction in water loss and stomatal closure, were observed. Redox homeostasis was reestablished during osmotic and salinity stress, and ion imbalance was also restored in salinity conditions. The expression of several stress/ABA-responsive genes was up-regulated, and ABA sensitivity, in particularly, was significantly altered in the MtMCR transgenic plants. Together, our genetic study for the first time elaborated the possible mechanism of endogenous CH4-enhanced salinity and osmotic tolerance, along with an alteration of ABA sensitivity. These findings thus provided novel cues for understanding the possible roles of endogenous CH4 in plants.

Nitrate reductase-dependent nitric oxide is crucial for multi-walled carbon nanotube-induced plant tolerance against salinity.

Although there have been some studies on the plant-carbonaceous nanomaterials (CNMs) interactions, related conclusions were controversial. Here, we report that multi-walled carbon nanotubes (MWCNTs) can enter into rapeseed (Brassica napus L.) seedling root, and transport to stem. Further results showed that salinity-inhibited rapeseed seedling growth was obviously alleviated by MWCNTs. Meanwhile, NaCl-induced nitrate reductase (NR)-dependent NO production was significantly intensified by MWCNTs. The redox and ion imbalance was reestablished as well, confirmed by the reduction in reactive oxygen species (ROS) overproduction, the decrease in thiobarbituric acid reactive substance production, and the lower Na+/K+ ratio. These beneficial effects could be explained by the changes in related antioxidant defense genes, sodium hydrogen exchanger 1 (NHX1), salt overly sensitive 1 (SOS1), and K+transporter 1 (KT1) transcripts. The above responses were separately abolished after the removal of endogenous NO with its scavengers or the addition of the NR inhibitor. Genetic evidence revealed that the NaCl-triggered NO level in wild-type seedling roots was partly abolished in either the nitric reductase mutant (nia1/2) or noa1 mutant (exhibiting indirectly a reduced endogenous NO level). Treatment with MWCNTs could totally rescue the impaired NO production in the noa1 mutant rather than the nia1/2 mutant, suggesting that NR-dependent NO acts as a downstream signaling molecule in MWCNT signaling. This point was verified by phenotypic analyses, histochemical staining, and ion analysis. qPCR analysis further demonstrated that MWCNTs stimulated antioxidant genes and ion balance-related genes through NR-mediated NO. The above molecular and genetic evidence indicated that NR-dependent NO acts downstream of MWCNTs in salinity tolerance, which requires the reestablishment of redox and ion homeostasis.

Nitric oxide contributes to methane-induced osmotic stress tolerance in mung bean.

BACKGROUND: Osmotic stress is a major abiotic stress limiting crop production by affecting plant growth and development. Although previous reports discovered that methane (CH4) has a beneficial effect on osmotic stress, the corresponding downstream signal(s) is still elusive. RESULTS: Polyethylene glycol (PEG) treatment progressively stimulated the production of CH4 in germinating mung bean seeds. Exogenous CH4 and sodium nitroprusside (SNP) not only triggered nitric oxide (NO) production in PEG-stressed plants, but also alleviated the inhibition of seed germination. Meanwhile, amylase activity was activated, thus accelerating the formation of reducing sugar and total soluble sugar. Above responses could be impaired by NO scavenger(s), suggesting that CH4-induced stress tolerance was dependent on NO. Subsequent tests showed that CH4 could reestablish redox balance in a NO-dependent fashion. The addition of inhibitors of the nitrate reductase (NR) and NO synthase in mammalian (NOS), suggested that NR and NOS-like protein might be partially involved in CH4-alleviated seed germination inhibition. In vitro and scavenger tests showed that NO-mediated S-nitrosylation might be associated with above CH4 responses. CONCLUSIONS: Together, these results indicated an important role of endogenous NO in CH4-enhanced plant tolerance against osmotic stress, and NO-regulated redox homeostasis and S-nitrosylation might be involved in above CH4 action.