4.3 CRIR1 regulates cold tolerance through CBF-independent pathways
Initially, we assumed that CRIR1 may function as a cis -acting lncRNA involved in the transcriptional regulation of target genes located in the nearby genomic region. Three genes are located close to CRIR1 . RNA-seq and qRT-PCR results ofCRIR1 transgenic plants showed no difference in the transcript levels of neighboring genes (Figure S3). The results suggested thatCRIR1 functioned as a trans -acting lncRNA. The candidate target genes of CRIR1 were screened using an RNA-seq data set ofCRIR1 OE lines. RNA-seq results demonstrated that the expression of many cold-related genes was increased in OE plants. For example, two transcription factors, MeNAC and MeNFYA , were upregulated in cold-stressed WT plants and CRIR1 overexpressors. NACand NFYA genes are both involved in abiotic stress responses, and overexpression of NAC confers tolerance of cold, salt, and drought stresses in various plant species (Shao, Wang & Tang, 2015). Another two genes that are upregulated in the CRIR1overexpressors are ASR and GOLS , which have been well documented for their positive roles in plant adaption to cold stresses (Li, Li, Zuo, Li & Wei, 2018, Shimosaka & Ozawa, 2016, Taji, Ohsumi, Iuchi, Seki, Kasuga, Kobayashi, Yamaguchi-Shinozaki & Shinozaki, 2002, Wang, Hu, Feng, Yang, Huang, Xiao, Liu, Yang & He, 2016). However, the opposite results were obtained in examining the expression of plant growth-related genes, such as MeYAB2 , auxin-responsive proteinMeIAA18 , and auxin-binding protein MeABP19 . These genes showed a decreased expression pattern during cold stress and inCRIR1 -overexpressing plants. These results suggested a possible interaction of CRIR1 with the auxin signaling pathway. The expression levels of cold stress related marker genes, includingMeCBFs , MeCOR , and MeICE1 , showed no difference between WT and overexpression lines, indicating that CRIR1positively modulated plant cold tolerance through CBF -independent pathways.