Analysis of PAT1 subfamily members in the GRAS family of upland cotton and functional characterization of GhSCL13-2A in Verticillium dahliae resistance.

KEY MESSAGE: GhSCL13-2A, a member of the PAT1 subfamily in the GRAS family, positively regulates cotton resistance to Verticillium dahliae by mediating the jasmonic acid and salicylic acid signaling pathways and accumulation of reactive oxygen species. Verticillium wilt (VW) is a devastating disease of upland cotton (Gossypium hirsutum) that is primarily caused by the soil-borne fungus Verticillium dahliae. Scarecrow-like (SCL) proteins are known to be involved in plant abiotic and biotic stress responses, but their roles in cotton defense responses are still unclear. In this study, a total of 25 GhPAT1 subfamily members in the GRAS family were identified in upland cotton. Gene organization and protein domain analysis showed that GhPAT1 members were highly conserved. GhPAT1 genes were widely expressed in various tissues and at multiple developmental stages, and they were responsive to jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) signals. Furthermore, GhSCL13-2A was induced by V. dahliae infection. V. dahliae resistance was enhanced in Arabidopsis thaliana by ectopic overexpression of GhSCL13-2A, whereas cotton GhSCL13-2A knockdowns showed increased susceptibility. Levels of reactive oxygen species (ROS) and JA were also increased and SA content was decreased in GhSCL13-2A knockdowns. At the gene expression level, PR genes and SA signaling marker genes were down-regulated and JA signaling marker genes were upregulated in GhSCL13-2A knockdowns. GhSCL13-2A was shown to be localized to the cell membrane and the nucleus. Yeast two-hybrid and luciferase complementation assays indicated that GhSCL13-2A interacted with GhERF5. In Arabidopsis, V. dahliae resistance was enhanced by GhERF5 overexpression; in cotton, resistance was reduced in GhERF5 knockdowns. This study revealed a positive role of GhSCL13-2A in V. dahliae resistance, establishing it as a strong candidate gene for future breeding of V. dahliae-resistant cotton cultivars.

Combined transcriptome and metabolome analysis revealed pathways involved in improved salt tolerance of Gossypium hirsutum L. seedlings in response to exogenous melatonin application.

BACKGROUND: Salinization is major abiotic stress limiting cotton production. Melatonin (MT) has been implicated in salt stress tolerance in multiple crops including upland cotton. Here, we explored the transcriptomic and metabolomic response of a salt-tolerant self-bred high-yielding cotton line SDS-01, which was exogenously sprayed with four MT concentrations (50, 100, 200, and 500 µM). RESULTS: Here we found that MT improves plant biomass and growth under salt stress. The combined transcriptome sequencing and metabolome profiling approach revealed that photosynthetic efficiency is improved by increasing the expressions of chlorophyll metabolism and antenna proteins in MT-treated seedlings. Additionally, linoleic acid and flavonoid biosynthesis were improved after MT treatment. The Na+/K+ homeostasis-related genes were increasingly expressed in salt-stressed seedlings treated with MT as compared to the ones experiencing only salt stress. Melatonin treatment activated a cascade of plant-hormone signal transduction and reactive oxygen scavenging genes to alleviate the detrimental effects of salt stress. The global metabolome profile revealed an increased accumulation of flavonoids, organic acids, amino acids and derivatives, saccharides, and phenolic acids in MT-treated seedlings. Interestingly, N, N'-Diferuloylputrescine a known antioxidative compound was highly accumulated after MT treatment. CONCLUSION: Collectively, our study concludes that MT is a salt stress regulator in upland cotton and alleviates salt-stress effects by modulating the expressions of photosynthesis (and related pathways), flavonoid, ROS scavenging, hormone signaling, linoleic acid metabolism, and ion homeostasis-related genes.

[Cloning and functional analysis of the cotton Trihelix transcription factor GhGT29].

Trihelix transcription factors are important proteins involved in response to abiotic stresses in plants. Understanding the molecular mechanisms of Trihelix in cottons will lay the foundation to improve stress tolerance by gene engineering. In this study, a gene encoding Trihelix transcription factor was isolated in upland cottons using reverse transcription PCR according to bioinformatic analysis. The gene was named as GhGT29 (GenBank accession No. JQ013097), which was 1 092 bp, contained a 1 089 bp open reading frame and encoded a protein of 363 amino acids with a predicted molecular weight of 40.9 kDa and a isoelectric point of 5.45. SMART analysis showed GhGT29 contained one typical SANT motif. Phylogenetic analysis showed that GhGT29 belonged to the SH4 subfamily of the Trihelix family and was most closely related to AtSH4-like1 and AtSH4-like2. Quantitative real-time PCR (qRT-PCR) analysis revealed that GhGT29 was induced by high salt, drought, cold and abscisic acid. The expression profile also revealed that GhGT29 was constitutively expressed in all tested tissues, such as roots, stems, leaves, flowers, ovules (0 DPA) and fibers (12 DPA). The expression level of GhGT29 was the highest in flowers and the lowest in stems. Using the Arabidopsis protoplasts assay system, we found that the GhGT29 protein was located in cell nuclei and had trans-activation activity. These results revealed that GhGT29 might be involved in the regulation of stress resistance-related genes in stress signaling pathways in upland cottons.

Soybean Trihelix transcription factors GmGT-2A and GmGT-2B improve plant tolerance to abiotic stresses in transgenic Arabidopsis.

BACKGROUND: Trihelix transcription factors play important roles in light-regulated responses and other developmental processes. However, their functions in abiotic stress response are largely unclear. In this study, we identified two trihelix transcription factor genes GmGT-2A and GmGT-2B from soybean and further characterized their roles in abiotic stress tolerance. FINDINGS: Both genes can be induced by various abiotic stresses, and the encoded proteins were localized in nuclear region. In yeast assay, GmGT-2B but not GmGT-2A exhibits ability of transcriptional activation and dimerization. The N-terminal peptide of 153 residues in GmGT-2B was the minimal activation domain and the middle region between the two trihelices mediated the dimerization of the GmGT-2B. Transactivation activity of the GmGT-2B was also confirmed in plant cells. DNA binding analysis using yeast one-hybrid assay revealed that GmGT-2A could bind to GT-1bx, GT-2bx, mGT-2bx-2 and D1 whereas GmGT-2B could bind to the latter three elements. Overexpression of the GmGT-2A and GmGT-2B improved plant tolerance to salt, freezing and drought stress in transgenic Arabidopsis plants. Moreover, GmGT-2B-transgenic plants had more green seedlings compared to Col-0 under ABA treatment. Many stress-responsive genes were altered in GmGT-2A- and GmGT-2B-transgenic plants. CONCLUSION: These results indicate that GmGT-2A and GmGT-2B confer stress tolerance through regulation of a common set of genes and specific sets of genes. GmGT-2B also affects ABA sensitivity.

Soybean WRKY-type transcription factor genes, GmWRKY13, GmWRKY21, and GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants.

WRKY-type transcription factors have multiple roles in the plant defence response and developmental processes. Their roles in the abiotic stress response remain obscure. In this study, 64 GmWRKY genes from soybean were identified, and were found to be differentially expressed under abiotic stresses. Nine GmWRKY proteins were tested for their transcription activation in the yeast assay system, and five showed such ability. In a DNA-binding assay, three proteins (GmWRKY13, GmWRKY27 and GmWRKY54) with a conserved WRKYGQK sequence in their DNA-binding domain could bind to the W-box (TTGAC). However, GmWRKY6 and GmWRKY21, with an altered sequence WRKYGKK, lost the ability to bind to the W-box. The function of three stress-induced genes, GmWRKY13, GmWRKY21 and GmWRKY54, was further investigated using a transgenic approach. GmWRKY21-transgenic Arabidopsis plants were tolerant to cold stress, whereas GmWRKY54 conferred salt and drought tolerance, possibly through the regulation of DREB2A and STZ/Zat10. Transgenic plants over-expressing GmWRKY13 showed increased sensitivity to salt and mannitol stress, but decreased sensitivity to abscisic acid, when compared with wild-type plants. In addition, GmWRKY13-transgenic plants showed an increase in lateral roots. These results indicate that the three GmWRKY genes play differential roles in abiotic stress tolerance, and that GmWRKY13 may function in both lateral root development and the abiotic stress response.

Characterization of a novel cell cycle-related gene from Arabidopsis.

Cell division is a fundamental biological process sharing conserved features and controls in all eukaryotes. The cell cycle is usually divided into four phases: G1, S, G2, and M. Regulated gene expression is an important mechanism for controlling cell cycle progression and genes involved in cell division-related processes often show transcriptional regulation dependent on cell cycle position. In the present report, a novel cell cycle-related gene (AtCPR) from Arabidopsis thaliana was isolated and characterized. Sequence analysis revealed that the deduced amino acid sequence of AtCPR showed 53.2% identity with p38-2G4, a mouse G1-to-S cell cycle specifically modulated and proliferation-associated nuclear protein. Assay of expression of AtCPR in partially synchronized cells suggested that AtCPR mRNA was expressed in the G1-to-S phase. In the AtCPR transgenic plants, no apparent phenotypic change was observed. By fusing a GFP tag to the AtCPR protein, it was found that AtCPR was mainly located in the nucleus. However, AtCPR does not have any transcriptional activation ability. cDNA microarray analysis showed that a total of 17 and 30 genes were identified as up-regulated and down-regulated, respectively.