The tae-miR164-TaNAC6A Module from Winter Wheat Could Enhance Cold Tolerance in Transgenic Arabidopsis thaliana

Cold stress impedes the growth and development of wheat (<i>Triticum aestivum</i>) and other crops, ultimately reducing both yields and quality. Research indicates that non-coding RNAs (ncRNAs) play a crucial role in regulating plant stress responses and resistance. In a previous study, we observed that the expression of <i>tae-miR164</i> was inversely correlated with the expression of <i>TaNAC6A</i> in Dongnongdongmai 1 (Dn1), a winter wheat variety with high cold resistance, under cold-stress conditions. However, the molecular mechanism governing the cold responsiveness of the <i>tae-miR164-TaNAC6A</i> module was not fully understood. We found <i>that tae-miR164</i> and <i>TaNAC6A</i> were both induced to express in opposite trends, and TaNAC6A was located in the nucleus. We also discovered that the expression of <i>tae-miR164</i> and its target gene, <i>TaNAC6A</i>, was responsive to short-term freezing stress in transgenic Arabidopsis plants. Compared to wild-type (WT) Arabidopsis plants, OE-<i>tae-miR164</i> plants showed decreased cold tolerance, whereas those overexpressing <i>TaNAC6A</i> demonstrated increased tolerance. On average, the OE-<i>TaNAC6A</i> and STTM-<i>tea-miR164</i> plants exhibited fewer morphological abnormalities in response to cold stress, higher antioxidant enzyme activities and gene expression levels, lower levels of reactive oxygen species (ROS) and malondialdehyde (MDA), and higher expressions of <i>AtDREB1</i>, <i>AtDREB2</i>, and <i>AtABI5</i> in the cold-signaling pathway. Thus, the biological functions of <i>tae-miR164</i> and <i>TaNAC6A</i> were initially confirmed through heterologous expression strategies, and we have made the first attempt to elucidate its associated <i>tae-miR164</i>-<i>TaNAC6A</i> module of cold resistance. The findings of this research will support further investigations into the regulation of plant stress resistance by ncRNAs and will inform molecular module breeding strategies aimed at enhancing the cold tolerance of crop plants. Molecular module design breeding, as a significant breakthrough in modern biotechnology, is transforming traditional breeding models. Conventional hybrid breeding relies on empirical screening, which is time-consuming and subject to randomness. In contrast, molecular module breeding directly targets key genes and achieves precise regulation through technologies such as gene editing and synthetic biology.