GmBSK1-GmGSK1-GmBES1.5 regulatory module controls heat tolerance in soybean.

INTRODUCTION: Heat stress poses a severe threat to the growth and production of soybean (Glycine max). Brassinosteroids (BRs) actively participate in plant responses to abiotic stresses, however, the role of BR signaling pathway genes in response to heat stress in soybean remains poorly understood. OBJECTIVES: In this study, we investigate the regulatory mechanisms of GmBSK1 and GmBES1.5 in response to heat stress and the physiological characteristics and yield performance under heat stress conditions. METHODS: Transgenic technology and CRISPR/Cas9 technology were used to generated GmBSK1-OE, GmBES1.5-OE and gmbsk1 transgenic soybean plants, and transcriptome analysis, LUC activity assay and EMSA assay were carried out to elucidate the potential molecular mechanism underlying GmBSK1-GmBES1.5-mediated heat stress tolerance in soybean. RESULTS: CRISPR/Cas9-generated gmbsk1 knockout mutants exhibited increased sensitivity to heat stress due to a reduction in their ability to scavenge reactive oxygen species (ROS). The expression of GmBES1.5 was up-regulated in GmBSK1-OE plants under heat stress conditions, and it directly binds to the E-box motif present in the promoters of abiotic stress-related genes, thereby enhancing heat stress tolerance in soybean plants. Furthermore, we identified an interaction between GmGSK1 and GmBES1.5, while GmGSK1 inhibits the transcriptional activity of GmBES1.5. Interestingly, the interaction between GmBSK1 and GmGSK1 promotes the localization of GmGSK1 to the plasma membrane and releases the transcriptional activity of GmBES1.5. CONCLUSION: Our findings suggest that both GmBSK1 and GmBES1.5 play crucial roles in conferring heat stress tolerance, highlighting a potential strategy for breeding heat-tolerant soybean crops involving the regulatory module consisting of GmBSK1-GmGSK1-GmBES1.5.

TaMYB-CC5 gene specifically expressed in root improve tolerance of phosphorus deficiency and drought stress in wheat.

Phosphate deficiency and drought are significant environmental constraints that impact both the productivity and quality of wheat. The interaction between phosphorus and water facilitates their mutual absorption processes in plants. Under conditions of both phosphorus deficiency and drought stress, we observed a significant upregulation in the expression of wheat MYB-CC transcription factors through the transcriptome analysis. 52 TaMYB-CC genes in wheat were identified and analyzed their evolutionary relationships, structures, and expression patterns. The TaMYB-CC5 gene exhibited specific expression in roots and demonstrated significant upregulation under phosphorus deficiency and drought stress compared to other TaMYB-CC genes. The overexpression of TaMYB-CC5A in Arabidopsis resulted in a significant increase of root length under stress conditions, thereby enhancing tolerance to phosphate starvation and drought stress. The wheat lines with silenced TaMYB-CC5 genes exhibited reduced root length under stress conditions and increased sensitivity to phosphate deficiency and drought stress. In addition, silencing the TaMYB-CC5 genes resulted in altered phosphorus content in leaves but did not lead to a reduction in phosphorus content in roots. Enrichment analysis the co-expression genes of TaMYB-CC5 transcription factors, we found the zinc-induced facilitator-like (ZIFL) genes were prominent associated with TaMYB-CC5 gene. The TaZIFL1, TaZIFL2, and TaZIFL5 genes were verified specifically expressed in roots and regulated by TaMYB-CC5 transcript factor. Our study reveals the pivotal role of the TaMYB-CC5 gene in regulating TaZIFL genes, which is crucial for maintaining normal root growth under phosphorus deficiency and drought stress, thereby enhanced resistance to these abiotic stresses in wheat.

Soybean steroids improve crop abiotic stress tolerance and increase yield.

Sterols have long been associated with diverse fields, such as cancer treatment, drug development, and plant growth; however, their underlying mechanisms and functions remain enigmatic. Here, we unveil a critical role played by a GmNF-YC9-mediated CCAAT-box transcription complex in modulating the steroid metabolism pathway within soybeans. Specifically, this complex directly activates squalene monooxygenase (GmSQE1), which is a rate-limiting enzyme in steroid synthesis. Our findings demonstrate that overexpression of either GmNF-YC9 or GmSQE1 significantly enhances soybean stress tolerance, while the inhibition of SQE weakens this tolerance. Field experiments conducted over two seasons further reveal increased yields per plant in both GmNF-YC9 and GmSQE1 overexpressing plants under drought stress conditions. This enhanced stress tolerance is attributed to the reduction of abiotic stress-induced cell oxidative damage. Transcriptome and metabolome analyses shed light on the upregulation of multiple sterol compounds, including fucosterol and soyasaponin II, in GmNF-YC9 and GmSQE1 overexpressing soybean plants under stress conditions. Intriguingly, the application of soybean steroids, including fucosterol and soyasaponin II, significantly improves drought tolerance in soybean, wheat, foxtail millet, and maize. These findings underscore the pivotal role of soybean steroids in countering oxidative stress in plants and offer a new research strategy for enhancing crop stress tolerance and quality from gene regulation to chemical intervention.

Heat shock protein TaHSP17.4, a TaHOP interactor in wheat, improves plant stress tolerance.

Adaptation to drought and salt stresses is a fundamental part of plant cell physiology and is of great significance for crop production under environmental stress. Heat shock proteins (HSPs) are molecular chaperones that play a crucial role in folding, assembling, translocating, and degrading proteins. However, their underlying mechanisms and functions in stress tolerance remain elusive. Here, we identified the HSP TaHSP17.4 in wheat by analyzing the heat stress-induced transcriptome. Further analysis showed that TaHSP17.4 was significantly induced under drought, salt, and heat stress treatments. Intriguingly, yeast-two-hybrid analysis showed that TaHSP17.4 interacts with the HSP70/HSP90 organizing protein (HOP) TaHOP, which plays a significant role in linking HSP70 and HSP90. We found that TaHSP17.4- and TaHOP-overexpressing plants have a higher proline content and a lower malondialdehyde content than wild-type plants under stress conditions and display strong tolerance to drought, salt, and heat stress. Additionally, qRT-PCR analysis showed that stress-responsive genes relevant to reactive oxygen species scavenging and abscisic acid signaling pathways were significantly induced in TaHSP17.4- and TaHOP-overexpressing plants under stress conditions. Together, our findings provide insight into HSP functions in wheat and two novel candidate genes for improvement of wheat varieties.

Transformation and Detection of Soybean Hairy Roots.

Agrobacterium rhizogenes is a soil bacteria with extensive infectivity, which can infect almost all dicotyledonous plants and a few monocotyledonous plants to induce root nodules. This is caused by the root-inducing plasmid, which contains genes responsible for the autonomous growth of root nodules and crown gall base synthesis. Structurally, it is similar to the tumor-inducing plasmid in that it mainly contains the Vir region, the T-DNA region, and the functional region of crown gall base synthesis. Its T-DNA is integrated into the nuclear genome of the plant with the assistance of Vir genes, causing hairy root disease in the host plant and the formation of hairy roots. The roots produced by Agrobacterium rhizogenes-infested plants are characterized by a fast growth rate, high degree of differentiation, physiological, biochemical, and genetic stability, and ease of manipulation and control. In particular, the hairy root system is an efficient and rapid research tool for plants that have no affinity for transformation by Agrobacterium rhizogenes and low transformation efficiency. The establishment of germinating root culture system for the production of secondary metabolites in the original plants through the genetic transformation of natural plants mediated by root-inducing plasmid in Agrobacterium rhizogenes has become a new technology combining plant genetic engineering and cell engineering. It has been widely used in a variety of plants for different molecular purposes, such as pathological analysis, gene function verification, and secondary metabolite research. Chimeric plants obtained by induction of Agrobacterium rhizogenes that can be expressed instantaneously and contemporarily are more rapidly obtained, compared to tissue culture and stably inheritable transgenic strains. In general, transgenic plants can be obtained in approximately one month.

GmDof41 regulated by the DREB1-type protein improves drought and salt tolerance by regulating the DREB2-type protein in soybean.

Despite their essential and multiple roles in biological processes, the molecular mechanism of Dof transcription factors (TFs) for responding to abiotic stresses is rarely reported in plants. We identified a soybean Dof gene GmDof41 which was involved in the responses to drought, salt, and exogenous ABA stresses. Overexpression of GmDof41 in soybean transgenic hairy roots attenuated H2O2 accumulation and regulated proline homeostasis, resulting in the drought and salt tolerance. Yeast one-hybrid and electrophoretic mobility shift assay (EMSA) illustrated that GmDof41 was regulated by the DREB1-type protein GmDREB1B;1 that could improve drought and salt tolerance in plants. Further studies illustrated GmDof41 can directly bind to the promoter of GmDREB2A which encodes a DREB2-type protein and affects abiotic stress tolerance in plants. Collectively, our results suggested that GmDof41 positively regulated drought and salt tolerance by correlating with GmDREB1B;1 and GmDREB2A. This study provides an important basis for further exploring the abiotic stress-tolerance mechanism of Dof TFs in soybean.

Foxtail millet MYB-like transcription factor SiMYB16 confers salt tolerance in transgenic rice by regulating phenylpropane pathway.

R2R3-MYB transcription factors play an important role in the synthesis of phenylpropanoid-derived compounds, which in turn provide salt tolerance in plant. In this study, we found that the expression of foxtail millet R2R3-MYB factor SiMYB16 can be induced by salt and drought. SiMYB16 is localized in the nucleus and acts as a transcriptional activator. Phylogenetic analysis indicates that SiMYB16 belongs to the R2R3-MYB transcription factor family subgroup 24. Transgenic rice expressing SiMYB16 (OX16) had a higher survival rate, lower malondialdehyde content, and heavier fresh weight compared with type (WT) under salt stress conditions. The transgenic plants also had a higher germination rate in salt treatment conditions and higher yield in the field compared with wild-type plants. Transcriptome analysis revealed that the up-regulated differential expression genes in the transgenic rice were mainly involved in phenylpropanoid biosynthesis, fatty acid elongation, phenylalanine metabolism, and flavonoid biosynthesis pathways. Quantitative real-time PCR analysis also showed that the genes encoding the major enzymes in the lignin and suberin biosynthesis pathways had higher expression level in SiMYB16 transgenic plants. Correspondingly, the content of flavonoid and lignin, and the activity of fatty acid synthase increased in SiMYB16 transgenic rice compared with wild-type plants under salt stress treatment. These results indicate that SiMYB16 gene can enhance plant salt tolerance by regulating the biosynthesis of lignin and suberin.

Mitogen-activated protein kinase TaMPK3 suppresses ABA response by destabilising TaPYL4 receptor in wheat.

Abscisic acid (ABA) receptors are considered as the targeted manipulation of ABA sensitivity and water productivity in plants. Regulation of their stability or activity will directly affect ABA signalling. Mitogen-activated protein kinase (MAPK) cascades link multiple environmental and plant developmental cues. However, the molecular mechanism of ABA signalling and MAPK cascade interaction remains largely elusive. TaMPK3 overexpression decreases drought tolerance and wheat sensitivity to ABA, significantly weakening ABA's inhibitory effects on growth. Under drought stress, overexpression lines show lower survival rates, shoot fresh weight and proline content, but higher malondialdehyde levels at seedling stage, as well as decreased grain width and 1000 grain weight in both glasshouse and field conditions at the adult stage. TaMPK3-RNAi increases drought tolerance. TaMPK3 interaction with TaPYL4 leads to decreased TaPYL4 levels by promoting its ubiquitin-mediated degradation, whereas ABA treatment diminishes TaMPK3-TaPYL interactions. In addition, the expression of ABA signalling proteins is impaired in TaMPK3-overexpressing wheat plants under ABA treatment. The MPK3-PYL interaction module was found to be conserved across monocots and dicots. Our results suggest that the MPK3-PYL module could serve as a negative regulatory mechanism for balancing appropriate drought stress response with normal plant growth signalling in wheat.

Comprehensive Profiling of Tubby-Like Proteins in Soybean and Roles of the GmTLP8 Gene in Abiotic Stress Responses.

Tubby-like proteins (TLPs) are transcription factors that are widely present in eukaryotes and generally participate in growth and developmental processes. Using genome databases, a total of 22 putative TLP genes were identified in the soybean genome, and unevenly distributed across 13 chromosomes. Phylogenetic analysis demonstrated that the predicted GmTLP proteins were divided into five groups (I-V). Gene structure, protein motifs, and conserved domains were analyzed to identify differences and common features among the GmTLPs. A three-dimensional protein model was built to show the typical structure of TLPs. Analysis of publicly available gene expression data showed that GmTLP genes were differentially expressed in response to abiotic stresses. Based on those data, GmTLP8 was selected to further explore the role of TLPs in soybean drought and salt stress responses. GmTLP8 overexpressors had improved tolerance to drought and salt stresses, whereas the opposite was true of GmTLP8-RNAi lines. 3,3-diaminobenzidine and nitro blue tetrazolium staining and physiological indexes also showed that overexpression of GmTLP8 enhanced the tolerance of soybean to drought and salt stresses; in addition, downstream stress-responsive genes were upregulated in response to drought and salt stresses. This study provides new insights into the function of GmTLPs in response to abiotic stresses.

Genome-Wide Analysis of the Soybean TIFY Family and Identification of GmTIFY10e and GmTIFY10g Response to Salt Stress.

TIFY proteins play crucial roles in plant abiotic and biotic stress responses. Our transcriptome data revealed several TIFY family genes with significantly upregulated expression under drought, salt, and ABA treatments. However, the functions of the GmTIFY family genes are still unknown in abiotic stresses. We identified 38 GmTIFY genes and found that TIFY10 homologous genes have the most duplication events, higher selection pressure, and more obvious response to abiotic stresses compared with other homologous genes. Expression pattern analysis showed that GmTIFY10e and GmTIFY10g genes were significantly induced by salt stress. Under salt stress, GmTIFY10e and GmTIFY10g transgenic Arabidopsis plants showed higher root lengths and fresh weights and had significantly better growth than the wild type (WT). In addition, overexpression of GmTIFY10e and GmTIFY10g genes in soybean improved salt tolerance by increasing the PRO, POD, and CAT contents and decreasing the MDA content; on the contrary, RNA interference plants showed sensitivity to salt stress. Overexpression of GmTIFY10e and GmTIFY10g in Arabidopsis and soybean could improve the salt tolerance of plants, while the RNAi of GmTIFY10e and GmTIFY10g significantly increased sensitivity to salt stress in soybean. Further analysis demonstrated that GmTIFY10e and GmTIFY10g genes changed the expression levels of genes related to the ABA signal pathway, including GmSnRK2, GmPP2C, GmMYC2, GmCAT1, and GmPOD. This study provides a basis for comprehensive analysis of the role of soybean TIFY genes in stress response in the future.