MicroRNAs (miRNAs) are small, non-coding RNAs playing essential roles in plant growth, development, and stress responses. Sequencing of small RNAs is a starting point for understanding their number, diversity, expression and possible roles in plants. In this study, we conducted a genome-wide survey of wheat miRNAs from 11 tissues, characterizing a total of 323 novel miRNAs belonging to 276 families in wheat. A miRNA conservation analysis identified 191 wheat-specific miRNAs, 2 monocot-specific miRNAs, and 30 wheat-specific variants from 9 highly conserved miRNA families. To understand possible roles of wheat miRNAs, we determined 524 potential targets for 124 miRNA families through degradome sequencing, and cleavage of a subset of them was validated via 5′ RACE. Based on the genome-wide identification and characterization of miRNAs and their associated target genes, we further identified 64 miRNAs preferentially expressing in developing or germinating grains, which could play important roles in grain development. We discovered 323 wheat novel miRNAs and 524 target genes for 124 miRNA families in a genome-wide level, and our data will serve as a foundation for future research into the functional roles of miRNAs in wheat.

Background: MicroRNAs are a class of small, non-coding RNAs that regulate gene expression by binding target mRNA, which leads to cleavage or translational inhibition. The NAC proteins, which include NAM, ATAF, and CUC, are a plant-specific transcription factor family with diverse roles in development and stress regulation. It has been reported that miR164 negatively regulates NAC1 expression, which in turn affects lateral root development in Arabidopsis; however, little is known about the involvement of the maize NAC family and miR164 in lateral root development. Results: We collected 175 maize transcripts with NAC domains. Of these, 7 ZmNACs were putative targets for regulation by miR164. We isolated one gene, called TC258020 (designated ZmNAC1) from 2 maize inbred lines, 87-1 and Zong3. ZmNAC1 had a high expression level in roots and showed higher abundance (1.8 fold) in Zong3 relative to 87-1, which had less lateral roots than Zong3. There was a significant correlation between the expression level of ZmNAC1 and the lateral root density in the recombinant inbred line (RIL) population. Transgenic Arabidopsis that overexpressed ZmNAC1 had increased lateral roots in comparison to the wild type. These findings suggest that ZmNAC1 played a significant role in lateral root development. An allelic expression assay showed that trans-regulatory elements were the dominant mediators of ZmNAC1 differential expression in 87-1 and Zong3, and further analysis revealed that miR164 was a trans-element that guided the cleavage of endogenous ZmNAC1 mRNA. Both mature miR164 and miR164 precursors had higher expression in 87-1 than Zong3, which was the opposite of the expression pattern of ZmNAC1. Additionally, the allelic assay showed that the cis-regulatory element most likely affected Zm-miR164b's expression pattern. A β-glucuronidase (GUS) assay showed that the Zm-miR164b promoter had higher GUS activity in 87-1 than in Zong3. In addition, we detected miR164b expression in the RIL population, and the results indicated that miR164b had a higher expression level in the RILs containing 87-1 promoter than those containing Zong3 promoter. Conclusion: Our results indicate one possible pathway in maize by which differences in miR164b promoter activity resulted in a different expression pattern for mature miR164 which negatively regulates ZmNAC1 expression in 87-1 and Zong3, thereby contributing to a significantly different lateral root phenotype.

Root architectural traits at the seedling stage have been demonstrated to be crucial for the efficient uptake of nutrients and drought tolerance in wheat. To dissect the genetic basis of these traits from the D genome, 182 recombinant inbred lines (RILs) derived from the common wheat TAA10 crossed with resynthesized allohexaploid wheat XX329 possessed similar AABB genomes were used for QTL mapping of five root traits in hydroponic-cultured seedlings, including lateral root number (LRN), seminal root number (SRN), root hair length (RHL), root diameter (RD), and total root volume (TRV). A total of seven QTLs were identified for the five root traits, with six possible novel QTLs for LRN, RHL, RD and TRV, accounting for 4.98–12.17% of phenotypic variation. One QTL (QLrn.qau-5D.2), controlling lateral root number, was fine mapped an approximate 5.0-Mb interval harboring 80 annotated genes, including five auxin-related genes. We further validated that QLrn.qau-5D.2 in NIL^TAA10 significantly enhanced yield-related traits, such as plant height, spike length, spike compactness, tiller number per plant and grain yield per plant, as comparison with NIL^XX329. Collectively, these results provide vital insights for fine-mapping QTLs associated with LRN, SRN, RHL, RD and TRV and facilitate the root morphologic designs for enhancing yield performance.

Porcine blood polypeptide (PBP) has been reported to play roles in plant growth. However, its functions in alleviating salt stress in wheat remain unclear. The present study was conducted to investigate the physiological and biochemical mechanisms underlying the effects of PBP on wheat salt tolerance. Morphological analysis showed that PBP-primed seedlings exhibited improved growth performance, significantly greater biomass accumulation, and enhanced root system development. Physiological assessments showed that primed seedlings displayed higher values of Pn, Gs, Tr, Fv/Fm, Fv′/Fm′, Φ_PSII, and NPQ, along with increased contents of total chlorophyll, Pro, TSS, and RWC. In addition, the activities of antioxidant enzymes, including SOD, CAT, POD, and APX, were significantly elevated, whereas the levels of H₂O₂, O₂⁻, MDA, and REC were significantly reduced. PCA indicated that antioxidant enzyme activity, osmotic regulation, and ROS accumulation were the major factors associated with the PBP-mediated salt stress response. Furthermore, qRT-PCR analysis suggested that exogenous PBP might enhance wheat salt tolerance by coordinately modulating multiple molecular mechanisms. Taken together, this study broadens the potential applications of PBP by demonstrating its capacity to improve wheat salt tolerance.

Drought stress is a devastating natural stress that threatens crop productivity and quality. Mitigating the adverse effects of drought stress on wheat is a key object in agriculture. C-repeat binding transcription factor/DROUGHT RESPONSE ELEMENT BINDING FACTOR 1 (CBF/DREB1) transcription factors are well known for their role in cold acclimation. However, the involvement of CBF genes in drought stress and the mechanisms underlying their function remain poorly understood. In this study, 81 CBFs were identified in wheat, which were further clustered into four distinct lineages based on phylogenetic analysis. Chromosomal localization indicated that most CBF genes were dispersed across chromosome 5. We identified three homoeologous genes (TaCBF14A, TaCBF14B, and TaCBF14D) that were simultaneously upregulated under drought stress based on RNA-seq analysis. According to the high expression after drought stress, TaCBF14B was selected for further functional analysis. Subcellular localization and transcriptional activation activity analysis indicated that TaCBF14B likely functions as a transcription factor involved in drought stress tolerance. Overexpression of TaCBF14B in Arabidopsis enhanced the primary root growth by 13.49% (OE1), 12.56% (OE2), and 19.53% (OE3) under 200 mM mannitol treatment, and 21.65% (OE1), 16.63% (OE2), and 28.13% (OE3) under 250 mM mannitol treatment compared to WT. Meanwhile, the water loss rate of transgenic lines was 56% in WT leaves, but only 44%, 50%, and 40% in OE1, OE2, and OE3 lines, respectively. Compared to the wild type, POD activities of OE1, OE2, and OE3 were significantly increased by 42.94%, 29.41%, and 62.52%, respectively. And the Pro activities in OE1, OE2, and OE3 were significantly increased by 16.33%, 5.18%, and 29.09%, respectively, compared to the wild type. Additionally, the MDA content in OE1, OE2, and OE3 was significantly reduced by 40.53%, 15.81%, and 54.36%, respectively. Further analysis showed that the transgenic lines were hypersensitive to abscisic acid (ABA), and exhibited increased expression of AtABI3. We speculate that TaCBF14B plays an important role in enhancing drought tolerance. In summary, our findings provide new insights into the functional roles of CBF genes in drought stress tolerance.