MicroRNAs (miRNAs) are versatile regulators of gene expression at both the transcription and post-transcription levels. The microRNA miR396 plays vital roles in growth, development, and resistance to abiotic stresses in many plant species. However, the roles and functions of miR396 in soybeans are not well understood. Here, we report that Gm-miR396a influences soybean development and salinity tolerance. We found that soybean miR396a was responsive to salt stress. Gm-miR396a gene-edited lines (miR396a-GEs), created using CRISPR/Cas9, exhibited more branches, higher grain yields, and greater salinity tolerance than control plants. The transcripts in lines with altered abundance of miR396a-GE were significantly enriched for biological processes related to hormone regulation. Overexpression of the Gm-miR396a precursor (pre-miR396a-OE) resulted in developmental deficiencies including dwarfness, abnormal inflorescences and flowers, smaller and fewer seeds, and small leaves with larger and more numerous stomata. Transcriptome analysis indicated photosynthesis-related genes were downregulated in pre-miR396a-OE plants. These results contribute valuable insights into the function of Gm-miR396a in soybeans and hold promise for enhancing soybean yield and salinity tolerance through germplasm innovation.

DREBs are transcription factors that regulate abiotic stress tolerance in plants. Previously, we reported that wheat transgenic lines overexpressing GmDREB1 showed increased tolerance to drought and salt stress. However, the molecular basis of increased tolerance is still poorly understood, and whether the overexpression of DREB will cause unexpected effects is also of concern. We performed seed metabolic profiling of the genetically modified (GM) wheat T349 and three non-GM cultivars with LC-MS to identify the metabolic basis of stress tolerance and to assess the unexpected effects of exogenous gene insertion. Although we did not note the appearance of novel metabolites or the disappearance of existing metabolites, overexpression of the transcription factor GmDREB1 in T349 wheat influenced metabolite levels in seeds. Increased levels of stress tolerance-associated metabolites were found in the stress-sensitive non-transgenic acceptor counterpart J19, while metabolites associated with cell membrane structure and stability accumulated in T349. Among these metabolites in T349, most showed levels similar to those in the non-GM wheats. Overexpression of GmDREB1 in T349 may cause a shift in its metabolic profile leading to down-regulation of several energy-consuming processes to favor increased yield under stress conditions, which is a reasonable expectation of breeders while creating the GM wheat and GmDREB1 overexpression did not cause unexpected effects in T349 seeds. These results may be helpful for GM crop research and risk assessment.

Small auxin-upregulated RNAs (SAURs) are genes regulated by auxin and environmental factors. In this study, we identified a SAUR gene in wheat, TaSAUR75. Under salt stress, TaSAUR75 is downregulated in wheat roots. Subcellular localization revealed that TaSAUR75 was localized in both the cytoplasm and nucleus. Overexpression of TaSAUR75 increased drought and salt tolerance in Arabidopsis. Transgenic lines showed higher root length and survival rate and higher expression of some stress-responsive genes than control plants under salt and drought stress. Less H₂O₂ accumulated in transgenic lines than in control plants under drought stress. Our findings reveal a positive regulatory role of the auxin-responsive gene TaSAUR75 in plant responses to drought and salt stress and provide a candidate gene for improvement of abiotic stress tolerance in crop breeding.

The engineering of plants with enhanced tolerance to abiotic stresses typically involves complex multigene networks and may therefore have a greater potential to introduce unintended effects than the genetic modification for simple monogenic traits. For this reason, it is essential to study the unintended effects in transgenic plants engineered for stress tolerance. We selected drought- and salt-tolerant transgenic wheat overexpressing the transcription factor, GmDREB1, to investigate unintended pleiotropic effects using RNA-seq analysis. We compared the transcriptome alteration of transgenic plants with that of wild-type plants subjected to salt stress as a control. We found that GmDREB1 overexpression had a minimal impact on gene expression under normal conditions. GmDREB1 overexpression resulted in transcriptional reprogramming of the salt response, but many of the genes with differential expression are known to mitigate salt stress and contribute incrementally to the enhanced stress tolerance of transgenic wheat. GmDREB1 overexpression did not activate unintended gene networks with respect to gene expression in the roots of transgenic wheat. This work is important for establishing a method of detecting unintended effects of genetic engineering and the safety of such traits with the development of marketable transgenic crops in the near future.

The transcription factor dehydration-responsive element binding protein (DREB) is able to improve tolerance to abiotic stress in plants by regulating the expression of downstream genes involved in environmental stress resistance. The objectives of this study were to evaluate the salt tolerance of GmDREB1 transgenic wheat (Triticum aestivum L.) and to evaluate its physiological and protein responses to salt stress. Compared with the wild type, the transgenic lines overexpressing GmDREB1 showed longer coleoptiles and radicles and a greater radicle number at the germination stage, as well as greater root length, fresh weight, and tiller number per plant at the seedling stage. The yield-related traits of transgenic lines were also improved compared with the wild type, indicating enhanced salt tolerance in transgenic lines overexpressing GmDREB1. Proteomics analysis revealed that osmotic- and oxidative-stress-related proteins were up-regulated in transgenic wheat leaves under salt stress conditions. Transgenic wheat had higher levels of proline and betaine and lower levels of malondialdehyde and relative electrolyte leakage than the wild type. These results suggest that GmDREB1 regulates the expression of osmotic- and oxidative-stress-related proteins that reduce the occurrence of cell injury caused by high salinity, thus improving the salt tolerance of transgenic wheat.