Wheat is one of the most important food crops globally, and understanding the regulation of grain size is crucial for wheat breeding to achieve a higher grain yield. MicroRNAs (miRNAs) play vital roles in plant growth and development. However, the miRNA-mediated mechanism underlying grain size regulation remains largely elusive in wheat. Here, we report the characterization and functional validation of a miRNA, TamiR397a, associated with grain size regulation in wheat. The function of three TaMIR397 homoeologs was determined through histochemical β-glucuronidase-dependent assay. MiRNA expression was detected using quantitative reverse transcription polymerase chain reaction (qRT-PCR), and the function of TamiR397a was validated through its transgenic overexpression and repression in wheat. It was found that TaMIR397-6A and TaMIR397-6B encode active TamiR397a. The expression profiling indicated that TamiR397a was differentially expressed in various tissues and gradually up-regulated during grain filling. The inhibition of TamiR397a perturbed grain development, leading to a decrease in grain size and weight. Conversely, the overexpression of TamiR397a resulted in increased grain size and weight by accelerating the grain filling process. Transcriptome analysis revealed that TamiR397a regulates a set of genes involved in hormone response, desiccation tolerance, regulation of cellular senescence, seed dormancy, and seed maturation biological processes, which are important for grain development. Among the down-regulated genes in the grains of the TamiR397a-overexpressing transgenic plants, 11 putative targets of the miRNA were identified. Taken together, our results demonstrate that TamiR397a is a positive regulator of grain size and weight, offering potential targets for breeding wheat with an increased grain yield.

DNA methylation is known to play a crucial role in regulating plant development and organ or tissue differentiation. Here, we focused on the DNA methylation dynamics during the germination of wheat seeds using the adapted AFLP technique so called methylation-sensitive amplified polymorphism (MSAP). The MSAP profiles of genomic DNA in embryo and endosperm tissues of germinating seeds, as well as dry seeds were characterized and notable changes of cytosine methylation were detected. Comparisons of MSAP profiles in different tissues tested showed that the methylation level in dry seeds is the highest. The alteration analysis of cytosine methylation displayed that the number of demethylation events were three times higher than that of de novo methylation, which indicated that the demethylation was predominant in germinating wheat seeds, though the methylation events occurred as well. Sixteen differentially displayed DNA fragments in MSAP profiles were cloned and the sequencing analysis confirmed that nine of them contained CCGG sites. The further BLAST search showed that four of the cloned sequences were located in coding regions. Interestingly, three of the sixteen candidates were homologous to retrotransposons, which indicated that switches between DNA methylation and demethylation occurred in retrotransposon elements along with the germination of wheat seeds.

Background/Objectives: miR5200 is miRNA unique to Poaceae plants. Induced under short-day conditions, it modulates flowering time by regulating the florigen FT gene expression. However, to date, the genetic locus responsible for mature miR5200 formation remains experimentally unvalidated, and its biological function in abiotic stress responses remains unknown. This has hindered systematic elucidation of miR5200’s physiological role and molecular mechanisms. Methods: This study utilized wheat as the research material. First, through bioinformatics analysis at the genomic level, 13 potential candidate tae-miR5200 gene loci were screened. Subsequently, the authenticity of these gene loci was systematically validated by combining tobacco transient transfection-based GUS staining assay and quantitative real-time PCR (qRT-PCR) to detect expression levels. Building upon this foundation, the expression patterns of tae-miR5200 under abiotic stresses such as low temperature, drought, and salinity, as well as SA, ABA, IAA, GA₃, and MeJA treatments, were further investigated. Results: Experimental validation confirmed that 7 out of 13 potential gene loci are authentic and functional, and tae-miR5200 exhibited specific expression changes under different types of abiotic stress. Conclusions: This study confirms the authenticity of tae-miR5200 gene loci, effectively eliminating interference from bioinformatics-predicted false-positive loci in subsequent functional studies. It provides an experimental foundation for further investigation into the molecular mechanisms of tae-miR5200 in wheat responses to abiotic stress.