Agropyron mongolicum (A. mongolicum) is an excellent gramineous forage with extreme drought tolerance, which lives in arid and semiarid desert areas. However, the mechanism that underlies the response of microRNAs (miRNAs) and their targets in A. mongolicum to drought stress is not well understood. In this study, we analyzed the transcriptome, small RNAome (specifically the miRNAome) and degradome to generate a comprehensive resource that focused on identifying key regulatory miRNA-target circuits under drought stress. The most extended transcript in each collection is known as the UniGene, and a total of 41,792 UniGenes and 1,104 miRNAs were identified, and 99 differentially expressed miRNAs negatively regulated 1,474 differentially expressed target genes. Among them, eight miRNAs were unique to A. mongolicum, and there were 36 target genes. A weighted gene co-expression network analysis identified five hub genes. The miRNAs of five hub genes were screened with an integration analysis of the degradome and sRNAs, such as osa-miR444a-3p.2-MADS47, bdi-miR408-5p_1ss19TA-CCX1, tae-miR9774_L-2R-1_1ss11GT-carC, ata-miR169a-3p-PAO2, and bdi-miR528-p3_2ss15TG20CA-HOX24. The functional annotations revealed that they were involved in mediating the brassinosteroid signal pathway, transporting and exchanging sodium and potassium ions and regulating the oxidation–reduction process, hydrolase activity, plant response to water deprivation, abscisic acid (ABA) and the ABA-activated signaling pathway to regulate drought stress. Five hub genes were discovered, which could play central roles in the regulation of drought-responsive genes. These results show that the combined analysis of miRNA, the transcriptome and degradation group provides a useful platform to investigate the molecular mechanism of drought resistance in A. mongolicum and could provide new insights into the genetic engineering of Poaceae crops in the future.

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.

Drought stress is a major environmental constraint affecting crop yields. Plants in agricultural and natural environments have developed various mechanisms to cope with drought stress. Identifying genes associated with drought stress tolerance in potato and elucidating their regulatory mechanisms is crucial for the breeding of new potato germplasms. The bHLH transcription factors involved play crucial roles not only in plant development and growth but also in responsesresponse to abiotic stress. In this study, the StbHLH47 gene, which is highly expressed in potato leaves, was cloned and isolated. Subcellular localization assays revealed that the gene StbHLH47 performs transcriptional functions in the nucleus, as evidenced by increased malondialdehyde (MDA) content and relative conductivity under drought stress. These findings indicate that overexpressing plants are more sensitive to drought stress. Differential gene expression analysis of wild-type plants (WT) and plants overexpressing StbHLH47 (OE-StbHLH47) under drought stress revealed that the significantly differentially expressed genes were enriched in metabolic pathways, biosynthesis of various plant secondary metabolites, biosynthesis of metabolites, plant hormone signal transduction, mitogen-activated protein kinase (MAPK) signalling pathway-plant, phenylpropanoid biosynthesis, and plant‒pathogen interactions. Among these pathways, the phenylalanine and abscisic acid (ABA) signal transduction pathways were enriched in a greater number of differentially expressed genes, and the expression trends of these differentially expressed genes (DEGs) were significantly different between WT and OE-StbHLH47. Therefore, it is speculated that StbHLH47 may regulate drought resistance mainly through these two pathways. Additionally, RT‒qPCR was used for fluorescence quantification of the expression of StNCED1 and StERD11, which are known for their drought resistance, and the results revealed that the expression levels were much lower in OE-StbHLH47 than in WT plants. RNA-seq, RT‒qPCR, and physiological index analyses under drought conditions revealed that overexpression of the StbHLH47 gene increased the sensitivity of potato plants to drought stress, indicating that StbHLH47 negatively regulates drought tolerance in potato plants. In summary, our results indicate that StbHLH47 is a negative regulator of drought tolerance and provide a theoretical basis for further studies on the molecular mechanism involved.

MicroRNAs (miRNAs) are types of endogenous non‐coding small RNAs found in eukaryotes that are 18–25 nucleotides long. miRNAs are considered to be key regulatory factors of the expression of target mRNA. The roles of miRNAs involved in the regulation of anthocyanin accumulation in pigmented potatoes have not been systematically reported. In this study, the differentially expressed miRNAs and their target genes involved in the accumulation of anthocyanin during different developmental stages in purple potato (Solanum tuberosum L.) were identified using small RNA (sRNA) and degradome sequencing. A total of 275 differentially expressed miRNAs were identified in the sRNA libraries. A total of 69,387,200 raw reads were obtained from three degradome libraries. The anthocyanin responsive miRNA–mRNA modules were analyzed, and 37 miRNAs and 23 target genes were obtained. Different miRNAs regulate the key enzymes of anthocyanin synthesis in purple potato. The structural genes included phenylalanine ammonia lyase, chalcone isomerase, flavanone 3‐hydroxylase, and anthocyanidin 3‐O‐glucosyltransferase. The regulatory genes included WD40, MYB, and SPL9. stu‐miR172e‐5p_L‐1R‐1, stu‐miR828a, stu‐miR29b‐4‐5p, stu‐miR8019‐5p_L‐4R‐3, stu‐miR396b‐5p, stu‐miR5303f_L‐7R + 2, stu‐miR7997a_L‐3, stu‐miR7997b_L‐3, stu‐miR7997c_L + 3R‐5_2ss2TA3AG, stu‐miR156f‐5p_L + 1, stu‐miR156a, stu‐miR156a_R‐1, stu‐miR156e, stu‐miR858, stu‐miR5021, stu‐miR828 and their target genes were validated by qRT‐PCR. They play important roles in the coloration and accumulation of purple potatoes. These results provide new insights into the biosynthesis of anthocyanins in pigmented potatoes.

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.