Qinghua Xu

Cold stress impedes the growth and development of wheat (Triticum aestivum) 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 tae-miR164 was inversely correlated with the expression of TaNAC6A 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 tae-miR164-TaNAC6A module was not fully understood. We found that tae-miR164 and TaNAC6A were both induced to express in opposite trends, and TaNAC6A was located in the nucleus. We also discovered that the expression of tae-miR164 and its target gene, TaNAC6A, was responsive to short-term freezing stress in transgenic Arabidopsis plants. Compared to wild-type (WT) Arabidopsis plants, OE-tae-miR164 plants showed decreased cold tolerance, whereas those overexpressing TaNAC6A demonstrated increased tolerance. On average, the OE-TaNAC6A and STTM-tea-miR164 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 AtDREB1, AtDREB2, and AtABI5 in the cold-signaling pathway. Thus, the biological functions of tae-miR164 and TaNAC6A were initially confirmed through heterologous expression strategies, and we have made the first attempt to elucidate its associated tae-miR164-TaNAC6A 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.

One of the important functions of long non-coding RNA (lncRNA) is to be competing endogenous RNAs (ceRNAs). As miR398 is reported to respond to different stressors, it is necessary to explore its relationship with lncRNA in the cold resistance mechanism of winter wheat. Tae-miR398-precursor sequence was isolated from the winter wheat (Triticum aestivum). RLM-RACE verified that tae-miR398 cleaved its target CSD1. Quantitative detection at 5°C, –10°C and –25°C showed that the expression of tae-miR398 decreased in response to low temperatures, whereas CSD1 showed an opposite expression pattern. LncR9A, lncR117 and lncR616 were predicted and verified to interact with miR398. tae-miR398 and three lncRNAs were transferred into Arabidopsis thaliana respectively. The lncR9A were transferred into Brachypodium distachyom. Transgenic plants were cultivated at –8°C and assessed for the expression of malondialdehyde, chlorophyll, superoxide dismutase and miR398-lncRNA-target mRNA. The results demonstrate that tae-miR398 regulates low temperature tolerance by downregulating its target, CSD1. lncRNA regulates the expression of CSD1 indirectly by competitively binding miR398, which, in turn, affects the resistance of Dn1 to cold. miR398-regulation triggers a regulatory loop that is critical to cold stress tolerance in wheat. Our findings offer an improved strategy to crop plants with enhanced stress tolerance.

‘Dongnong Dongmai1 (Dn1)’ is a cultivar of wheat ( Triticum aestivumL.) which may endure‒30°C in field with a reviving rate of 85%. ABA may enhance plant resistance. Aiming to reveal Dn1...