Figure legends
FIGURE 1 Identification of cold-responsive lncRNAs in cassava. (a) The type and number of predicted lncRNAs. Intergenic lncRNA: lncRNA transcribed from a position
between two annotated protein-coding genes and are at least 1 kb away from the nearest protein-coding genes.  Bidirectional LncRNA: lncRNA is oriented head-to-head with a protein-coding gene within 1kb. Intronic lncRNA: lncRNA overlaps with the intron of annotated coding genes in either sense or antisense orientation. Antisense lncRNA: lncRNA has opposite transcriptional direction and overlap in part with adjacent mRNA. Sense lncRNA: lncRNA has the same transcriptional direction and overlaps with adjacent mRNA. (b) Expression profiles of lncRNA and mRNA under normal conditions. FPKM, fragments per kilobase of exons per million fragments mapped. (c) The abundance of all the expressed lncRNAs in each sample. (d) Volcano plot of differentially expressed genes between normal and cold-treated conditions. (e) The number of differentially expressed lncRNAs in response to cold stress. The red bar represents upregulated lncRNAs, and the blue bar represents down-regulated lncRNAs.
FIGURE 2 The expression pattern and subcellular distribution ofCRIR1 in cassava. (a) Spatial expression pattern analysis ofCRIR1 in different tissues using quantitative RT-PCR. (b) Expression levels of CRIR1  in cassava leaves responding to cold stress. Cassava plants without stress were used as the controls to normalize the expression in the stress treatments. Error bars indicate the sd of three biological replicates. (c) Subcellular distribution ofCRIR1 in cassava leaf cells. CRIR1 transcripts were detected by in situ  hybridizations of Cy3-tagged oligonucleotides (green). U6 (yellow) was used as a control. The location of nuclei is indicated by DAPI staining (blue). Bar=20µm *P<0.05, **P<0.01 by Student’s t -test.
FIGURE 3 Effects of CRIR1 on plant growth, proline accumulation, and cellular membrane stability during cold stress. (a) Relative transcript levels of CRIR1 in the wild-type (WT) and eight overexpressing lines, #1 to #8. (b) Transgene integration pattern in WT and OE lines by Southern analysis. (c) Morphology of pot-grown seedlings before and after cold stress treatment (4℃, 48 h) for one-week recovery under normal growth conditions. Bar = 2 cm. (d, e) Proline concentration (d) and MDA level (e) in the WT and transgenic plants under cold treatment (4℃) for 0 and 24 h. FW, fresh weight. (f) Transmission electron microscopic (TEM) analysis of chloroplasts from WT and OE #1 young leaves under normal and cold-treated conditions (4℃, 48 h). cp, chloroplast. Bar=2µm. Data represent means ± SD, *P<0.05, **P<0.01 by Student’s t -test.
FIGURE 4 Identification of CRIR1 -regulated genes and their corresponding signaling pathways. (a) The numbers of differentially expressed genes (DEGs) across seven comparisons. The red bar represents upregulated DEGs, and the blue bar represents down-regulated DEGs. (b) Venn diagrams show the numbers of theCRIR1 -regulated genes between two OE lines, overlapping and non-overlapping parts with cold-response genes under normal and cold-treated conditions, respectively. (c) Gene Ontology (GO) analysis of CRIR1 -regulated genes under normal and cold-treated conditions, respectively. The top 10 biological processes GO terms are shown.
FIGURE 5 Relative expression levels of selected genes in cold-treated seedlings. (a-c) Relative expression levels ofCRIR1 -regulated genes (a, b) and cold-responsive marker genes (c) are detected by qRT-PCR. Data represent means ± SD, *P<0.05, **P<0.01 by Student’s t -test.
FIGURE 6 CRIR1 interacts with MeCSP5 in cassava and improves translational yield. (a) Silver staining and mass spectrometry analyses following RNA pull-down of CRIR1 sense or CRIR1antisense (AS-CRIR1 ) in cassava leaf cells. (b) Fifteen candidates for CRIR1 -interacted proteins are identified by mass spectrometry analyses. MW, molecular weight. (c) The RNA binding site prediction of CRIR1- MeCSP5 complex using catRAPID. (d) Confirmation of binding of MeCSP5 to CRIR1 with EMSA. NC, negative control. (e) TriFC assay in rice protoplasts. nYFP is fused to MeCSP5, and cYFP is fused to MSCP. 6xMS2 nucleotide sequences are fused to CRIR1 or control lncRNA. Bars = 10 µm. (f) The abundances of mRNAs in WT and transgenic lines under normal and cold-treated conditions are shown. The Density (y-axis) corresponds to the relative number of genes at a given abundance. (g) Total protein abundances in WT and transgenic lines under normal and cold-treated conditions are shown. Data represent means ± SD, *P<0.05 by Student’s t -test.