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.