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.