Agropyron mongolicum Keng, a perennial diploid grass with high drought tolerance, belongs to the genus Agropyron, tribe Triticeae. It has made tremendous contributions toward reseeding natural pasture and seeding artificial grassland in China, especially in the arid and semi-arid area of northern China. As a wild relative of wheat, A. mongolicum is also a valuable resource for the genetic improvement of wheat crops. MicroRNAs are small non-coding RNA molecules ubiquitous in plants, which have been involved in responses to a wide variety of stresses including drought, salinity, chilling temperature. To date, little research has been done on drought-responsive miRNAs in A. mongolicum. In this study, two miRNA libraries of A. mongolicum under drought and normal conditions were constructed, and drought-responsive miRNAs were screened via Solexa high throughput sequencing and bioinformatic analysis. A total of 114 new miRNAs were identified in A. mongolicum including 53 conservative and 61 unconservative miRNAs, and 1393 target genes of 98 miRNAs were predicted. Seventeen miRNAs were found to be differentially expressed under drought stress, seven (amo-miR21, amo-miR62, amo-miR82, amo-miR5, amo-miR77, amo-miR44 and amo-miR17) of which were predicted to target on genes involved in drought tolerance. QRT-PCR analysis confirmed the expression changes of the seven drought related miRNAs in A. mongolicum. We then transformed the seven miRNAs into Arabidopsis thaliana plants, and three of them (amo-miR21, amo-miR5 and amo-miR62) were genetically stable. The three miRNAs demonstrated the same expression pattern in A. thaliana as that in A. mongolicum under drought stress. Findings from this study will better our understanding of the molecular mechanism of miRNAs in drought tolerance and promote molecular breeding of forage grass with improved adaption to drought.

Agropyron mongolicum Keng is a diploid perennial grass of triticeae in gramineae. It has strong drought resistance and developed roots that can effectively fix the soil and prevent soil erosion. GDSL lipase or esterases/lipase has a variety of functions, mainly focusing on plant abiotic stress response. In this study, a GDSL gene from A. mongolicum, designated as AmGDSL1, was successfully cloned and isolated. The subcellular localization of the AmGDSL1 gene (pCAMBIA1302-AmGDSL1-EGFP) results showed that the AmGDSL1 protein of A. mongolicum was only localized in the cytoplasm. When transferred into tobacco (Nicotiana benthamiana), the heterologous expression of AmGDSL1 led to enhanced drought tolerance. Under drought stress, AmGDSL1 overexpressing plants showed fewer wilting leaves, longer roots, and larger root surface area. These overexpression lines possessed higher superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and proline (PRO) activities. At the same time, the malondialdehyde (MDA) content was lower than that in wild-type (WT) tobacco. These findings shed light on the molecular mechanisms involved in the GDSL gene’s role in drought resistance, contributing to the discovery and utilization of drought-resistant genes in A. mongolicum for enhancing crop drought resistance.

While bHLH transcription factors have been linked to the regulation of various abiotic stressors, research on drought-related bHLH proteins and their molecular processes in Agropyron mongolicum has remained limited. In this study, a bHLH gene from A. mongolicum, designated as AmbHLH148, was successfully cloned and isolated. AmbHLH148 was exclusively localized within the nucleus. Additionally, qRT-PCR analysis demonstrated a significant upregulation of AmbHLH148 in response to drought stress. When transferred into tobacco (Nicotiana benthamiana), the heterologous expression of AmbHLH148 led to enhanced drought tolerance. Under drought stress conditions, AmbHLH148-OE transgenic tobacco plants exhibited increased activities of antioxidant defense enzymes, such as SOD (superoxide dismutase), POD (peroxidase), and CAT (catalase). These enzymes efficiently mitigated the accumulation of reactive oxygen species (ROS) compared to wild-type plants. Furthermore, AmbHLH148-OE transgenic tobacco showed elevated levels of PRO (proline) and reduced MDA (malondialdehyde) content, contributing to enhanced stability in the plant’s cell membrane system during drought stress. In summary, this study underscores that the overexpression of AmbHLH148 in transgenic tobacco acts as a positive regulator under drought stress by enhancing the plant’s antioxidant capacity. These findings shed light on the molecular mechanisms involved in bHLH transcription factors’ role in drought resistance, contributing to the discovery and utilization of drought-resistant genes in A. mongolicum for enhancing crop drought resistance.