3.2 The expression pattern and subcellular localization ofCRIR1
Induction of lncRNAs after cold treatment suggested a role of these lncRNAs in cold stress response. In order to test this hypothesis, we selected a transcript of 329 bp and named it COLD-RESPONSIVE lincRNA 1 (CRIR1 ) for further study. CRIR1 has a short reading frame with 42 amino acids, but it did not encode a protein, showing no homology to any parts of any known proteins. The CRIR1gene was located in chromosome 08 between Manes.08G068600 and Manes.08G068500, and there was no overlap between the CRIR1 and its neighbor genes. Like many lncRNAs, the sequence of cassavaCRIR1 showed no clear homology to other genes and had no homologs in other different plants species. Data from the RNA-seq indicated thatCRIR1 is expressed at a low level in the plant shoot apex. In order to further examine the spatial expression pattern of CRIR1 , total RNA was extracted from different tissues of the wild-type cassava seedlings. The expression level of CRIR1 was determined using qRT-PCR. CRIR1 were preferentially expressed in plant shoots rather than in roots and showed a higher abundance in young leaves (Figure 2a). We further investigated the regulation of CRIR1 by cold stress. Consistent with our RNA-seq data, theĀ CRIR1transcript abundance increased by about 20-fold in cold-stressed seedlings compared with the control seedlings, confirming our result that cold stress strongly induces the expression of CRIR1 (Figure 2b). To investigate the subcellular localization of CRIR1transcripts, fluorescence in situ hybridization (FISH) in leaves and roots was performed on CRIR1 with a Cy3-labeled probe. Fluorescence signal could be seen in the nucleus and cytoplasm of leaf or root cells hybridized with the Cy3-labeled CRIR1 probe (Figure 2c). These data suggest that CRIR1 transcripts are mainly localized in nucleus and cytoplasm.