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\begin{document}

\title{Global identification of full-length cassava lncRNAs unveils the role of
CRIR1 in cold stress response}



\author[1]{shuxia li}%
\author[1]{Zhihao Cheng}%
\author[1]{Shiman Dong}%
\author[1]{Zhibo Li}%
\author[1]{Liangping Zou}%
\author[1]{Pingjuan Zhao}%
\author[1]{Xin Guo}%
\author[2]{Yan Bao}%
\author[3]{Wenquan Wang}%
\author[4]{Ming Peng}%
\affil[1]{Chinese Academy of Tropical Agricultural Sciences}%
\affil[2]{Shanghai Jiao Tong University}%
\affil[3]{Hainan University}%
\affil[4]{Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences}%


\vspace{-1em}



  
  \date{May 24, 2024}


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\selectlanguage{english}
\begin{abstract}
Long non-coding RNAs (lncRNAs) have been considered to be important
regulators of gene expression in a range of biological processes in
plants. A large number of lncRNAs have been identified in plants.
However, most of their biological functions still remain to be
determined. Here, we identified total 3 004 lncRNAs in cassava under
normal or cold-treated conditions from Iso-seq data. We further
characterized a lincRNA, CRIR1, as a novel positive regulator of the
plant response to cold stress. CRIR1 can be significantly induced by
cold treatment. Overexpression of CRIR1 in cassava enhanced the cold
tolerance of transgenic plants. Transcriptome analysis demonstrated that
CRIR1 regulates a range of cold stress-related genes in a
CBF-independent pathway. We further found that CRIR1 RNA can interact
with MeCSP5, a homolog of the cold shock protein that acts as RNA
chaperones, indicating that CRIR1 may recruit MeCSP5 to improve the
translation efficiency of mRNA. In summary, our study greatly extends
the repertoire of lncRNAs in plants as well as its responding to cold
stress. Moreover, it reveals a sophisticated mechanism by which CRIR1
regulates plant cold stress response by modulating the expression of
stress-responsive genes and increasing the translational yield.%
\end{abstract}%



\sloppy


\textbf{1. INTRODUCTION}

Cassava (\emph{Manihot esculenta} ~Crantz) is a starchy root crop that
are widely cultivated throughout the tropics and the subtropics. Over
the last few decades, the demand for cassava has changed rapidly from a
direct food crop to an industrial crop, mainly as human food, animal
feed, biofuels, and other bio-based materials (Malik, Kongsil, Nguyen,
Ou, Sholihin, Srean, Sheela, Becerra Lopez-Lavalle, Utsumi, Lu,
Kittipadakul, Nguyen, Ceballos, Nguyen, Selvaraj Gomez, Aiemnaka,
Labarta, Chen, Amawan, Sok, Youabee, Seki, Tokunaga, Wang, Li, Nguyen,
Nguyen, Ham \& Ishitani, 2020). However, as a tropical and subtropical
crop plant, cassava is very sensitive to cold stress, which drastically
impairs its seedling growth, photosynthesis, and significantly limits
the root weight (E1-Sharkawy, 2004). Severe cold stress is particularly
damaging to its leaves and apical meristems, leading to stunted growth
and even death (An, Yang \& Zhang, 2012). Therefore, cold stress is a
major limiting factor for cassava productivity and spatial distribution.

In order to cope with cold stress, plants have developed a complex
network of pathways to sense and respond to rapid fluctuations in
temperature (Zhu, 2016). During the past two decades, the underlying
mechanisms of the molecular and physiological changes that enable an
adaptive response to cold stress in plants have been extensively
explored. Studies in~\emph{Arabidopsis} lead to the identification of a
set of cold-related transcription factors, such as\emph{C-REPEAT-BINDING
FACTORS} (\emph{CBFs} ), \emph{MYBs} , \emph{NACs} , that can regulate
downstream genes, leading to biochemical and physiological adjustments
at the cellular and plant levels under cold condition (Ding, Shi \&
Yang, 2020, Nuruzzaman, Sharoni \& Kikuchi, 2013, Shi, Ding \& Yang,
2018, Zhao, Zhang, Xie, Si, Li \& Zhu, 2016). The CBF proteins play a
central role in plant cold acclimation by specifically binding to the
CBF responsive element (CRT) motifs presenting in the promoter regions
of \emph{COLD-RESPONSIVE}(\emph{COR} ) genes, such as \emph{RD29a} and
\emph{COR15a} , to activate their transcription (Shi et al., 2018). In
cassava, the \emph{CBF} genes are rapidly and transiently upregulated by
cold stress (An et al., 2012). Overexpression of certain \emph{MeCBF3}
can enhance the cold tolerance of cassava (An, Ma, Wang, Yang, Zhou \&
Zhang, 2017). In addition to basic transcriptional regulation,
post-transcriptional regulation, such as pre-mRNA splicing, mRNA
transport, and mRNA stability, also influence the cold stress response
regulatory network in plants (Chinnusamy, Gong \& Zhu, 2008, Lee,
Kapoor, Zhu \& Zhu, 2006).

Similar to proteins, the functions of many cellular RNAs directly rely
both on their nucleotide sequences and their spatial structures
(Rajkowitsch, Chen, Stampfl, Semrad, Waldsich, Mayer, Jantsch, Konrat,
Blasi \& Schroeder, 2007). Dynamic, environment-mediated structural
changes of RNA molecules can modulate their transcription, maturation,
and translation efficiency. Cold stress causes overstabilization of
incorrectly folded RNA structures, while RNA chaperones can resolve
these structures, maintaing the normal function of the RNAs (Melencion,
Chi, Pham, Paeng, Wi, Lee, Ryu, Koo \& Lee, 2017). \emph{COLD SHOCK
PROTEINS} (\emph{CSPs} ), characterized by the presence of cold shock
domains, are broadly found as nucleic acid chaperones in bacteria,
animals, and plants (Sasaki \& Imai, 2011). In bacteria and humans, the
biological activities of CSPs range from the regulation of transcription
and splicing, to the translation of mRNAs (Lindquist \& Mertens, 2018).
In plants, \emph{CSPs} are involved in various biological processes to
promote normal growth and stress responses. The first functionally
characterized plant \emph{CSP} gene was wheat \emph{WCSP1} with
cold-specific inductions, and the corresponding protein melted
double-stranded nucleic acids, fulfilling various cellular functions
(Karlson, Nakaminami, Toyomasu \& Imai, 2002, Nakaminami, Karlson \&
Imai, 2006). \emph{Arabidopsis} has four CSP proteins (AtCSP1-AtCSP4)
that play distinct roles in plant development and stress responses. For
example, \emph{AtCSP3} can be induced by cold stress, while
overexpression of \emph{AtCSP3} conferred enhanced cold tolerance
without obvious developmental defects (Kim, Sasaki \& Imai, 2009).
However, overexpression of \emph{AtCSP1} or \emph{AtCSP2} did not
enhance cold tolerance in \emph{Arabidopsis} , but complemented the
cold-sensitive phenotypeof \emph{grp7} mutant, and affected seed
germination under salt stress conditions (Kim, Park, Kwak, Kim, Kim,
Song, Jang, Jung \& Kang, 2007, Park, Kwak, Oh, Kim \& Kang, 2009, Yang
\& Karlson, 2013). Therefore, activation of CSP proteins is essential
and may enhance the ability of the plants to cope with cold stress.

Previous studies on stress gene regulation mainly focused on
protein-coding genes. In recent years, long non-coding RNAs (lncRNAs)
have emerged as essential regulators of genomic and phenotypic
diversities (Waititu, Zhang, Liu \& Wang, 2020). LncRNAs are longer than
200 nt and usually have low protein-coding potential. In plants, most
lncRNAs are produced by RNA polymerase II (Pol II), whereas some lncRNAs
are transcribed by RNA Pol III, IV, and V (Wang, Meng, Dobrovolskaya,
Orlov \& Chen, 2017). LncRNAs are involved in various regulatory events
by acting as precursors of small RNAs, such as miRNAs and siRNAs, or as
miRNA target mimics. They are also reported to be involved in gene
expression regulation via multiple ways, including transcriptional
activation/repression, RNA alternative splicing, and chromatin
modification (Csorba, Questa, Sun \& Dean, 2014, Peschansky \&
Wahlestedt, 2014, Rigo, Bazin, Romero-Barrios, Moison, Lucero, Christ,
Benhamed, Blein, Huguet, Charon, Crespi \& Ariel, 2020, Zhao, Li, Lian,
Gu, Li \& Qi, 2018). With RNA sequencing technologies and experimental
approaches, a great number of lncRNAs have been identified and confirmed
to play key roles in plant response and adaptation to abiotic stresses.
For instance, drought and salt stress tolerance of plants can be
enhanced by transgenic \emph{Arabidopsis} overexpressing a
nucleus-localized lncRNA (Qin, Zhao, Cui, Albesher \& Xiong, 2017). A
natural antisense transcript of maize \emph{ZmNAC48} were reported to
interact with sense mRNAs during the drought stress response, resulting
in the formation of nat-siRNA, which could inhibit the expression
of\emph{ZmNAC48} to affect the stomatal closure of maize (Mao, Xu, Wang,
Li, Tang, Liu, Feng, Wu, Li, Xie \& Lu, 2021). In poplar, two
heat-responsive lncRNAs have been shown to modulate target gene
expression via RNA interference and act as RNA scaffolds to enhance heat
tolerance (Song, Chen, Liu, Bu \& Zhang, 2020).

In cassava, several previous studies have identified a great number of
stress-responsive lncRNAs, and their regulatory networks have been
predicted (Ding, Tie, Fu, Yan, Liu, Yan, Li, Wu, Zhang \& Hu, 2019, Li,
Yu, Lei, Cheng, Zhao, He, Wang \& Peng, 2017, Xiao, Shang, Cao, Xie,
Zeng, Lu, Chen \& Yan, 2019). However, only the short-read RNA
sequencing platform was used in these studies, which barely provided
full-length lncRNAs based on the bioinformatic algorithms, and the
number of full-length lncRNAs transcribed from Pol II was unclear. The
experimental validation of their functions remains challenging and has
not yet been reported. Therefore, it is necessary to construct a
complete resource of full-length lncRNAs and elucidate the mechanisms of
their actions for the cassava lncRNAs, which may provide helpful
information for crop breeding. In our previous study, we reported a
large-scale study of alternative splicing dynamics under abiotic stress
conditions in cassava by analyzing~single-molecule long-read isoform
sequencing (Iso-seq) data (Li, Yu, Cheng, Zeng, Li, Zhang \& Peng,
2020). Here, we performed an integrative analysis of long-read and
short-read transcriptome sequencing data from the cassava shoot apex
under cold and normal conditions and identified a total of 3 004
full-length lncRNAs. These data could add into the annotation of
thousands of cassava lncRNA genes and serve as reference sequences for
further in-depth gene function studies. Subsequently, we identified and
characterized the \emph{COLD-RESPONSIVE lincRNA 1} (\emph{CRIR1} ).
Gain-of-function analysis revealed that \emph{CRIR1} ~overexpression
transgenic cassava seedlings conferred tolerance to cold stress. RNA
sequencing (RNA-seq) analysis indicated that a range of cold-responsive
genes was induced in~\emph{CRIR1} -overexpressing transgenic lines.
Furthermore, we found that \emph{CRIR1} interacted directly with the
cold shock protein MeCSP5 in cassava cells, and increased its
translational yield to cope with cold stress. In conclusion, these
results strongly suggest that \emph{CRIR1} has a pivotal role in
manipulating cold-stress tolerance in cassava, possibaly though the
regulation of MeCSP5.

\textbf{2. MATERIALS AND METHODS}

\textbf{2.1 Plant materials, growth conditions, and cold treatment}

Cassava cultivar TMS60444 was used in this study. The uniform stem
cuttings were grown on Murashige and Skoog (MS) medium containing 3.0
g/L Gelrite, 20 g/L sucrose (Fisher), and 2 \selectlanguage{greek}μ\selectlanguage{english}mol/L
CuSO\textsubscript{4}in a growth room at 26\selectlanguage{ngerman}°C under 16 h light/8 h dark.
For cold treatment, 4-week-old seedlings were transplanted into pots and
grown in a greenhouse (16 h days, 26 ± 2 °C). After two weeks, seedlings
with a uniform growth status were transferred to a chamber for cold
treatment at 4°C under light, followed by one week of recovery at 26 °C.
More than 5 plants were treated in each treatment.

\textbf{2.2 Plasmid construction and cassava transformation}

The cDNA sequences of the sense and antisense \emph{CRIR1} were cloned
by PCR using primers covering the full length of transcripts. The PCR
fragment was sequenced and inserted into the binary vector pCAMBIA1301
under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter to
generate the binary vector pC\emph{35S} ::\emph{CRIR1} and pC\emph{35S}
::\emph{anti-CRIR1} , respectively. The plasmid was mobilized into
\emph{Agrobacterium tumefaciens} LBA4404 for cassava transformation
using a friable embryogenic callus of cultivar TMS60444. Cassava
embryogenic callus induction, \emph{Agrobacterium} -mediated genetic
transformation, and hygromycin-resistant calli regeneration were
performed by the methods described previously (Zhang, Potrykus \&
Puonti-Kaerlas, 2000).

\textbf{2.3 Southern blot analysis of transgenic plants of transgenic
plants}

The southern blot assay was performed as described previously (An et
al., 2017). Briefly, mature leaves of wild-type (WT) and transgenic
plant were harvested for genomic DNA isolation. Approximately 20 \selectlanguage{greek}µ\selectlanguage{english}g of
total DNA was digested with EcoRI overnight, followed by separating on a
0.8\% (w/v) agarose gel and subsequently transferred to a positively
charged nylon membrane (Roche, Mannheim, Germany). The blot was
hybridized with the digoxigenin (DIG)-labeled hygromycin
phosphotransferase (HPT) gene probe, which was generated by using the
PCR DIG Probe Synthesis Kit (Roche, Mannheim, Germany). According to the
manufacturer's instructions, hybridization, membrane washing, and
detection were performed using the DIG-High Prime DNA Labeling and
Detection Starter Kit II (Roche, Mannheim, Germany).

\textbf{2.4 Physiological analyses of cold-treated cassava plants}

Malondialdehyde (MDA) content in cassava leaves was determined by using
Lipid oxidation detection~kit (Beyotime) according to the manufacturer's
instructions. MDA was extracted with chilled thiobarbituric acid
reagent, and then the MDA contents were calculated by subtracting the
absorbance at 450 and 600 nm from that at 532 nm. Proline concentrations
were measured by using Proline~Assay Kit (Bioss, Beijing) according to
the manufacturer's instructions. Total protein extraction, purification,
and determination were performed by Sanshu Biotechnology Co., Ltd
(Jiangsu, China). Each treatment had three replications.

\textbf{2.5 RNA isolation and quantitative real-time PCR}

The young leaves and shoot buds were harvested after 24 h treatment at
4\selectlanguage{ngerman}°C, then frozen in liquid nitrogen for RNA extraction. Nonstressed
seedlings were harvested as controls (0 h). More than 6 plants were
harvested and pooled in each treatment. Total RNA was extracted using
the Plant RNA Kit (Omega Bio-Tek). First-strand cDNA was synthesized
using the PrimeScript\selectlanguage{english} RT Reagent Kit (Takara, Otsu, Japan). A
quantitative real-time PCR (qRT-PCR) was conducted using SYBR (r) Premix
Ex Taq\selectlanguage{english} (Takara) and the StepOnePlus Real-Time PCR Detection System
(Bio-Rad, CA, USA). \emph{MeACTIN} was used as a reference gene for
normalizing transcript levels. Details regarding the primers used in
this study are provided in Table S3.

\textbf{2.6 Iso-Seq and ssRNA-Seq data processing and lncRNA
identification}

The RNA preparation, library construction, and sequencing for Iso-Seq
(Li et al., 2020) and ssRNA-Seq (Li et al., 2017) were described
previously. All sequencing data were deposited with NCBI under the
BioProject ID PRJNA198574 and PRJNA377165. For Iso-Seq, total RNA was
extracted using TRIzol reagent (Life technologies) and enriched by Oligo
(dT) magnetic beads. The enriched mRNA was reverse transcribed into cDNA
using Clontech SMARTer PCR cDNA Synthesis Kit. A total of two libraries
(Normal and Cold) were constructed and sequenced on the Pacific
Biosciences (PacBio) Sequel II platform by Gene Denovo Biotechnology
Co., Ltd. (Guangzhou, China). The raw reads were classified and
clustered into transcript consensus using SMRT Link v5.0.1 pipeline
(Gordon, Tseng, Salamov, Zhang, Meng, Zhao, Kang, Underwood, Grigoriev,
Figueroa, Schilling, Chen \& Wang, 2015) supported by PacBio and then
mapped to reference genome using minimap2 (Li, 2018). Long non-coding
RNA identification was performed according to the pipeline described
previously (Li et al., 2017). The intersection of both
non-protein-coding potential results and non-protein annotation results
were chosen as lncRNA candidates. For ssRNA-Seq data processing, clean
reads from two samples (Normal and Cold, 3 replicates per sample) were
mapped to the full-length lncRNA isoforms and cassava reference genome
by HISAT (Kim, Langmead \& Salzberg, 2015). The counts of each lncRNA
were quantified by RSEM (Li \& Dewey, 2011), and the quantitative
estimation of each transcript was achieved using fragments per kilobase
of exon model per million mapped reads (FPKM). Differential expressed
lncRNAs were analyzed by the DESeq2 package (Love, Huber \& Anders,
2014). Significant changes were determined using \textbar{}Log 2
FC\textbar{} \textgreater{} 1 and q-value (false discovery rate, FDR
\textless{} 5\%) from multiple-testing adjustment as cut-off.

\textbf{2.7 Illumina RNA sequencing and data analysis}

Young leaves and shoot tips from four-week-old WT and \emph{CRIR1} OE
lines grown under normal and cold-treated (4degC, 24h) conditions were
harvested. Two biological replicates consisting of six independent
plants for each sample were conducted. The total RNA isolation, whole
transcriptome libraries preparation, and deep sequencing were performed
by Gene Denovo Biotechnology Co., Ltd. (Guangzhou, China). Total RNA
from each sample was extracted using Trizol reagent (Life~Technologies).
According to the manufacturer's instructions, whole transcriptome
libraries were constructed using Next(r) Ultra\selectlanguage{english} RNA Library Prep Kit
(New~England~Biolabs). The libraries were sequenced initially on an
Illumina NovaSeq\textsuperscript{TM}~6000 instrument that generated
paired-end reads of 150 nucleotides. After removing the adapters and
low-quality bases, clean reads were aligned to the cassava genome
assembly with HISAT (Kim et al., 2015), then transcript construction
using Stringtie (Pertea, Pertea, Antonescu, Chang, Mendell \& Salzberg,
2015). The FPKM value was calculated for each unigene using RSEM (Li \&
Dewey, 2011). The differentially expressed genes (DEGs) were identified
by using DESeq2 (Love et al., 2014), with FDR \textless{} 0.05. Gene
Ontology (GO) enrichment analyses were conducted using AgriGO (Tian,
Liu, Yan, You, Yi, Du, Xu \& Su, 2017). The RNA-seq data have been
deposited to the National Center for Biotechnology Information (NCBI)
under the accession number PRJNA522309.

\textbf{2.8 Fluorescence in situ hybridization (FISH) analysis}

The samples for ISH were prepared as described previously (Yang,
Schuster, Prunet, Dong, Landrein, Wightman \& Meyerowitz, 2019). Full
expanded young leaves from three-week-old cassava seedlings were fixed
in 4\% paraformaldehyde for 20 mins, and the leaves were then dehydrated
and embedded in paraffin. Subsequently, the tissue blocks were cut into
7-\selectlanguage{greek}μ\selectlanguage{english}m sections, processed by dewaxing, rehydration, and dehydration. FISH
analysis was performed using an RNA-FISH kit (BersinBio, Guangzhou,
China), a \emph{CRIR1} probe labeled with Cy3 was designed and
constructed by BersinBio. The dehydrated sections were pre-hybridized~in
a 37\selectlanguage{ngerman}°C incubator for 0.5 h. The hybridization solution with
Cy3\selectlanguage{english}-labeled\emph{CRIR1} probes was added, and the slide was allowed to
hybridize overnight at 4degC. The sections were washed once in SSC (2x)
for 10 mins, twice in SSC (1x) for 5 mins each, and once in SSC (0.5x)
for 10 mins. Finally, the samples were stained with (DAPI) dye solution,
incubated in the dark for 8 mins, washed with PBS, and photographed with
a laser confocal microscope (Olympus FluoView FV1100).

\textbf{2.9 TriFC Assay}

In the case of TriFC assay, the full-length coding sequences for MeCSP5
and MSCP were cloned into pSAT4-nEYFP-C1 and pSAT4-cEYFP-C1 vectors,
respectively. The sequence of 6xMS2 was fused with \emph{CRIR1} or
control lncRNA under the control of the 35S CaMV promoter in the
pCAMBIA1301 vector. For the TriFC assay in rice protoplasts, transient
transfection of rice protoplast cultures were performed according to the
protocol described previously (Li, Xiang, Yang, Chen \& Zhang, 2015).
Fluorescence was detected using confocal laser scanning microscope
(Olympus FluoView FV1100) 16-20\selectlanguage{english} h after transfection.

\textbf{2.10 Histological Analysis}

For transmission electron microscopy, cassava leaves were fixed in 2.5\%
glutaraldehyde then transferred to osmium tetraoxide. The fixed samples
were dehydrated in an ethanol gradient then transferred to propylene
oxide solution, after which they were slowly polymerized in acrylic
resin (London Resin Company) for at least 48 h. Thin sections (70 nm)
were obtained using a diamond knife microtome (Ultracut E; Reichert
Technologies). The sections were placed on 100-mesh copper grids,
stained with uranyl acetate for 30 mins, and then stained with lead
citrate for 15 mins. The sections were observed using a Hitachi HT7700
transmission electron microscope (Japan).

\textbf{2.11 Biotin RNA Pull-Down Assay}

RNA pull-down assay was performed as previously described (Hu, Jin, Xu,
Wang, Thorne, Zhang \& Wu, 2018). Biotin-labeled \emph{CRIR1}
or\emph{Anti-CRIR1} RNAs were transcribed \emph{in vitro} using the
Biotin RNA Labeling Mix (Roche) and T7 RNA polymerase (Roche), treated
with RNase-free DNase I (Invitrogen), and purified with RNeasy Mini Kit
(Qiagen). Total protein~was extracted from 3-week-old~cassava~seedlings
using Total Protein Extraction Kit for plant tissues~(Invent
Biotechnologies, Inc) and pre-cleared against streptavidin magnetic
beads (Invitrogen). Biotin-labeled \emph{CRIR1} RNAs adsorbed to
streptavidin magnetic beads were then incubated with pre-cleared protein
extract in the presence of complete protease inhibitors cocktail,
phosphatase inhibitor cocktail, and RNase inhibitors, at 4 degC for 12 h,
followed by washing five times in~wash~buffer (100 mM KCl, 50 mM Tris
(pH 7.4), 0.5 mM dithiothreitol, 0.5\% NP-40, 1mM DTT and RNase
inhibitors)~and elution in 5xSDS sample loading buffer. Eluted proteins
were separated by SDS-PAGE and were then subjected to mass spectrometric
analysis.

\selectlanguage{english}\textbf{2.12 Liquid Chromatography-Mass Spectrometry (LC-MS) analysis}

Proteins purified from RNA pull-down assay were analyzed using LC\selectlanguage{english}-MS by
the Shanghai Applied Protein Technology Co., Ltd (Shanghai, China). Two
libraries (\emph{CRIR1} and \emph{Anti-CRIR1} ), which contained two
replicates for each library, were prepared in this study. Dry pellets of
samples were solubilized in trypsin buffer (Promega) for digestion into
small peptides. The digested peptides were desalted and solubilized in
0.1\% formic acid buffer. The peptide mixture was loaded onto a
reverse-phase trap column (Thermo Scientific Acclaim PepMap100)
connected to the C18 reversed-phase analytical column (Thermo Scientific
Easy Column, 10 cm long, 75 \selectlanguage{greek}μ\selectlanguage{english}m inner diameter, 3\selectlanguage{greek}μ\selectlanguage{english}m resin) in buffer A
(0.1\% Formic acid) and separated with a linear gradient of buffer B
(84\% acetonitrile and 0.1\% Formic acid). LC-MS/MS analysis was
performed on a Q Exactive mass spectrometer (Thermo Scientific) coupled
to Easy nLC (Proxeon Biosystems, now Thermo Fisher Scientific). MS data
were acquired using a data-dependent top10 method dynamically choosing
the most abundant precursor ions from the survey scan (300-1800 m/z) for
HCD fragmentation. Data were analyzed for each sample with MaxQuant
software (Cox \& Mann, 2008).

\textbf{2.13 Electrophoretic mobility shift assay (EMSA)}

The electrophoretic mobility shift assay was conducted using an RNA EMSA
Kit (BersinBio, Guangzhou, China) according to the manufacturer's
instructions. Briefly, the full-length CDS of~\emph{MeCSP5} ~was
amplified by PCR and then cloned into the pCzn1-His to express the
MeCSP5 protein in~\emph{Escherichia coli} ~DE3 strain. The recombinant
MeCSP5 protein was purified using the Ni affinity chromatography. EMSA
was performed in a reaction mixture containing MeCSP5 proteins and
biotin-labeled \emph{CRIR1} RNA probes. Negative control assays were
performed by adding 100-fold excesses of unlabeled RNA probes to the
reaction mixture. After electrophoresis and incubation, signals were
recorded and analyzed.

\textbf{2.14 Accession Numbers}

All sequence data from this article can be found under accession
numbers: \emph{MeCSP5} (Manes.18G074700), \emph{MeNAC}(Manes.01G093200),
\emph{MeNFYA} (Manes.09G025200), \emph{MeGOLS}(Manes.03G208800),
\emph{MeASR} (Manes.18G093100), \emph{MeYAB2}(Manes.04G064600),
\emph{MeIAA18} (Manes.04G134400), \emph{MeABP19}(Manes.04G144900),
\emph{MeGRF5} (Manes.04G144700), \emph{MeCBF3}(Manes.08G096900),
\emph{MeCBF4} (Manes.07G135300), \emph{MeCOR}(Manes.18G037600),
\emph{MeICE1} (Manes.04G092400).

\textbf{3. RESULTS}

\textbf{3.1 Overview of cassava lncRNAs detected by long-read and
short-read transcriptome sequencing}

In this study, through the combined analysis of Iso-Seq and ssRNA-seq
data, a total of 70 146 unique transcripts were obtained, including
95.7\% predicted protein-coding and 4.3\% long non-coding RNA. The
number of predicted lncRNAs based on Iso-seq data is shown in Figure~1a.
A total of 3 004 high-confidence full-length lncRNAs were identified
with an average length of 1 424 bp. To study these lncRNAs in detail, we
classified them into 5 different categories based on their genomic
distribution and potential functions. Among these lncRNAs, a large
proportion (44.6\%) represented sense lncRNAs, 19.5\% of them were
produced from~the~antisense strand~of~protein-coding~genes, while 19.2\%
of them appeared to be intergenic non-coding RNAs.
Like\emph{Arabidopsis} lncRNAs (Liu, Jung, Xu, Wang, Deng, Bernad,
Arenas-Huertero \& Chua, 2012), only a few cassava lncRNAs (11 of 3 004
lncRNAs) acted as precursor transcripts of miRNAs or siRNAs after
aligning miRNA sequences from miRbase to our lncRNAs collection
(Table~S1). We then estimated the expression level of each transcript
using FPKM and found that these lncRNAs had a lower expression level
than mRNAs (Figure~1b), and 13.7\% of them were specifically detected
in cold or normal conditions. We also found that the expression levels
of the sense and intronic lncRNAs were higher than those of other types
but slightly lower than mRNAs. By comparing global expression levels of
lncRNAs between normal and cold-treated conditions (Figure~1c), we
detected 316 lncRNAs that were significantly altered by cold treatment,
of which 139 showed induced expression (Figure 1d-e, Table~S1).

\textbf{3.2 The expression pattern and subcellular localization
of\emph{CRIR1}}

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 \emph{COLD-RESPONSIVE
lincRNA 1} (\emph{CRIR1} ) for further study. \emph{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
\emph{CRIR1}gene was located in chromosome 08 between Manes.08G068600
and Manes.08G068500, and there was no overlap between the \emph{CRIR1}
and its neighbor genes. Like many lncRNAs, the sequence of
cassava\emph{CRIR1} showed no clear homology to other genes and had no
homologs in other different plants species. Data from the RNA-seq
indicated that\emph{CRIR1} is expressed at a low level in the plant
shoot apex. In order to further examine the spatial expression pattern
of \emph{CRIR1} , total RNA was extracted from different tissues of the
wild-type cassava seedlings. The expression level of \emph{CRIR1} was
determined using qRT-PCR. \emph{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
\emph{CRIR1} by cold stress. Consistent with our RNA-seq data,
the~\emph{CRIR1}transcript 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
\emph{CRIR1} (Figure 2b). To investigate the subcellular localization of
\emph{CRIR1}transcripts, fluorescence in situ hybridization (FISH) in
leaves and roots was performed on \emph{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 \emph{CRIR1} probe (Figure
2c). These data suggest that \emph{CRIR1} transcripts are mainly
localized in nucleus and cytoplasm.

\textbf{3.3 \emph{CRIR1} plays a positive role in plant response to cold
stress}

To investigate the physiological function of \emph{CRIR1} \emph{in
planta,} we conducted genetic transformation experiments forgenerating
transgenic cassava lines with knock-down or overexpression
of\emph{CRIR1} . Full-length sequences of antisense or sense
\emph{CRIR1}were transformed into cassava friable embryogenic calli
under the control of the 35S Cauliflower Mosaic Virus (CaMV) promoter
(\emph{35S} ::\emph{anti-CRIR1} and \emph{35S} ::\emph{CRIR1} ).
However, the calli carrying the \emph{35S}
::\emph{anti-CRIR1}construct~failed~to~regenerate into~complete
plantlets, so the basis for this phenomenon still needs to be
investigated. Luckily, eight independent transgenic cassava lines
carrying the\emph{35S} ::\emph{CRIR1} construct were obtained and
numbered from \#1 to \#8. The \emph{CRIR1} mRNA abundance in these
transgenic lines was determined by qRT-PCR analysis. Six independent
lines that showed higher expression levels of~\emph{CRIR1} were
obtained: \#1-3, \#5, \#7, and \#8 (Figure 3a). Then, the Southern
blotting technique was applied to verify the integration events of
\emph{35S} ::\emph{CRIR1} T-DNA in these lines. Two lines, \#1 and \#5,
were detected as single-copy insertion into the genome and therefore
selected for further investigation (Figure 3b).

In order to investigate whether overexpression of \emph{CRIR1} confers
cold stress tolerance, five-week-old wild-type (WT) and
\emph{CRIR1}overexpression (OE) seedlings were incubated in growth
chambers at 4\selectlanguage{ngerman}°C for 2 days and then transferred to 26°C for 7 days.
After the treatment, the WT plants showed withered, dry leaves and could
not be recovered till death, whereas the OE seedlings were only slightly
affected, with fewer leaves wilting and showed a relatively green and
healthy appearance (Figure 3c). Proline, acting as a physico-chemical
indicator during cold exposure, has been demonstrated to play an
important role in protecting cellular enzymes from denaturation (Li,
Yang, Iqbal, Qadri, Shi, Wang, Wu, Fan \& Wu, 2019). Cold stress
enhanced the accumulation of proline in the WT and OE lines, but the
contents of proline in the transgenic lines were significantly higher
than that in the wild type (Figure 3d). Malondialdehyde (MDA), which was
considered to be an important indicator of cell membrane system injuries
and cellular metabolism deterioration, has also been measured as well.
Compared with WT, the MDA contents were consistently lower in OE lines
(Figure 3e). In addition to morphological and physiological changes, a
clear impact on the ultrastructure of chloroplasts membranes was also
observed in leaves of cold-treated cassava plants to further assess the
degree of cold stress-induced membrane injury (Figure 3f). Before cold
treatment, the chloroplasts of leaf mesophyll cells had normal shapes
with the typical arrangement of grana and stroma thylakoids and showed
no significant differences between the WT and OE line. After cold
treatment, the membrane system of chloroplasts in the WT plants showed
disintegration with less organized thylakoids, indicating that
chloroplasts were destroyed. In contrast, there were no significant
alterations in the OE line, except for the appearance of the starch
granules in chloroplasts (Figure 3f). These results indicate that
\emph{CRIR1} plays a positive role in the response of cassava to cold
stress through altering the accumulation of protectant metabolites and
preventing the membrane system from cell damage under cold stress.

\textbf{3.4 \emph{CRIR1} regulates the expression of genes involved in
the stress response}

In order to elucidate the molecular mechanism of \emph{CRIR1} -mediated
cold resistance, we performed RNA-sequencing (RNA-seq) on WT and two OE
lines grown under normal (N) and cold-treated (C) conditions. Among 33
975 detected genes, 6 129, 5 108, and 6 440 differentially expressed
genes (DEGs) were identified after cold treatment in wild-type, OE \#1
and OE \#5 plants, respectively, indicating that low temperature induced
global changes of gene expressions (Figure 4a, Table~S2). To identify
DEGs specifically regulated by \emph{CRIR1} , we compared the
transcriptomes between OE lines and WT plants under normal and
cold-treated conditions. As shown in the venn diagram (Figure 4b), a
total of 285 \emph{CRIR1} -responsive DEGs were identified both in the
OE\#1 and OE\#5 plants under normal conditions. Among them, 30\% of DEGs
were cold-regulated genes. After cold treatment, there were 316 genes
differentially regulated by \emph{CRIR1} , and a strong overlap between
the cold-related genes and \emph{CRIR1} -responsive DEGs were observed.
Subsequently, GO enrichment analysis indicated that these DEGs encode
proteins were significantly enriched in biological processes associated
with stimulus responses, abiotic stimulus responses, and hormone
responses under normal conditions. In cold conditions, a high proportion
of DEGs can be classified as monocarboxylic acid metabolic,
single-organism metabolic, and secondary metabolite biosynthetic process
GO categories (Figure 4c). Furthermore, KEGG pathway analysis showed
their functions were significantly enriched in the carotenoid
biosynthesis pathway, the starch and sucrose metabolism pathway, and the
biosynthesis of secondary metabolites pathway under both normal and
cold-treated conditions (Figure S1a).

\textbf{3.5 \emph{CRIR1} regulates cold stress-related genes in
a~\emph{CBF}-independent pathway}

We next deployed the genes falling into the category of response to the
abiotic stimulus, which included two transcription factors
(\emph{MeNFYA} and \emph{MeNAC} ), \emph{GALACTINOL
SYNTHASE}(\emph{MeGOLS} ), and \emph{ABSCISIC STRESS-RIPENING
PROTEIN}(\emph{MeASR} ). We verified the expression level of these genes
in OE lines with qRT-PCR (Figure 5a). Consistently, transcription levels
of\emph{MeNFYA} and \emph{MeNAC} were increased in OE lines compared to
WT plants under both normal and cold-treated conditions. Quantification
of the \emph{MeGOLS} showed similar trends, whereas we could only
detect\emph{MeASR} after cold stress, suggesting that \emph{MeASR} is
accumulated under low temperature more than under normal conditions. In
our RNA-seq analysis, we also found that several IAA-signaling genes
were expressed at a lower level in cold-treated OE seedlings than in WT
seedlings. These genes include transcription factor \emph{YABBY2}
,\emph{AUXIN-RESPONSIVE PROTEIN} (\emph{IAA18} ), and
\emph{AUXIN-BINDING PROTEIN} (\emph{ABP19} ). Real-time PCR analysis
showed a consistent result with transcriptomic data (Figure 5b).
Additionally, we examined the expression patterns of classical cold
stress markers, including\emph{MeCBFs} , \emph{MeCORs} , and
\emph{MeICE1} . Accumulated evidence has revealed that CBF proteins
function as key regulators of the transcriptomic rearrangement leading
to cold acclimation. In this study, the WT seedlings showed
increased~\emph{MeCBFs} ~and\emph{~MeCOR} ~expression levels under cold
stress. However, the expression levels of~these genes~induced by cold
stress in the OE seedlings were similar to those of the wild type
(Figure 5c). In conclusion, these results indicate that modulation
of~\emph{CRIR1} significantly regulates cold stress-related genes in
a~\emph{CBF} -independent pathway.

\textbf{3.6 \emph{CRIR1} interacts with MeCSP5 protein}

In order to further investigate the biological function of \emph{CRIR1}
, we performed \emph{in vitro} RNA pull-down assays and filtered the
protein interactome of \emph{CRIR1} using mass spectrometry (Figure 6a).
A~total of 15 proteins, represented by two or more unique peptides,
specifically interacted with biotin-labeled sense-\emph{CRIR1} but not
the control antisense-\emph{CRIR1} (Figure 6b).~Among them, three
proteins, including one member of the Pre-rRNA PROCESSING PROTEIN
(RRP5), one 30S ribosomal protein, and one COLD SHOCK DOMAIN CONTAINING
PROTEIN (CSP, named as MeCSP5), were found to be associated
with\emph{CRIR1} RNA function, and the other candidate interactors were
mainly secondary~metabolism\selectlanguage{english}-related~enzymes. CSP has been shown to
regulate RNA processing and function by participating in multiple
complexes to confer cold tolerance in \emph{Arabidopsis} (Sasaki \&
Imai, 2011). The cassava genome contains six \emph{CSPs}
(\emph{MeCSP1}to \emph{6} ) (Figure S2a-b), two of which
(\emph{MeCSP1}and~\emph{MeCSP5} ) were regulated by cold stress
according to the RNA-seq data. Therefore, we verified possible direct
interactions between \emph{CRIR1} and MeCSP5. First, we predicted
regions of\emph{CRIR1} necessary for~interaction with MeCSP5 using the
catRAPID database, and found that MeCSP5 was preferentially bound to the
N-terminal of \emph{CRIR1} (Figure 6c). Further, to check the
specificity of the interaction between \emph{CRIR1} and the MeCSP5
protein, full-length \emph{CRIR1} were separated into three truncated
forms 1-3, and were synthesized by~\emph{in vitro} ~transcription to
evaluate their ability to bind MeCSP5 protein. As presented in~Figure
6d, MeCSP5 co-purified with transcripts of segments 1 and 2 but not
segment 3, demonstrating a sequence-specific binding activity of MeCSP5.
We also checked the interaction of \emph{CRIR1} with MeCSP5 using
trimolecular fluorescence complementation (TriFC) assay, a modified
version of bimolecular fluorescence complementation (BiFC) assay using
an MS2 system as described previously (Seo, Sun, Park, Huang, Yeh, Jung
\& Chua, 2017). We observed that \emph{CRIR1} was strongly associated
with MeCSP5 in both the nucleus and cytoplasm, but the negative control
RNA was not (Figure 6e). Collectively, these results suggest an
association between CSP complexes and \emph{CRIR1} -mediated cold stress
tolerance. Considering that CSP proteins have been confirmed to possess
transcription anti-termination activity and have also been thought to
enhance translation at low temperatures by eliminating stabilized RNA
secondary structures (Sasaki \& Imai, 2011). We thus compared the mRNA
and protein abundance in WT and transgenic plants. According to the
RNA-seq data, transcripts in WT and OE lines have a similar mean
abundance under normal conditions. However, low temperature strongly
reduced cellular mRNA levels in WT or OE lines (Figure 6f). Besides, we
found that the mean protein levels produced from OE lines were
significantly higher than that in WT plants under both normal and
cold-treated conditions based on the mean protein abundance (Figure 6g).
These data indicated that \emph{CRIR1} may potentially coordinatilate
with \emph{MeCSP5} may toincrease translational yield for coping with
cold stress.

\textbf{4. DISCUSSION}

\textbf{4.1 A powerful full-length lncRNA resource in cassava was
collected}

Cold stress is one of the major environmental factors that limit plant
growth, productivity, and geographic distribution. Most tropical plant
species are sensitive to cold stress due to the lack of capacity for
cold acclimation. Over the past decades, genetic and~transcriptomic
screens have identified many genes involved in cold stress response.
Many researchers tried to enhance the cold tolerance of cassava using
the cold-responsive gene and investigated the corresponding mechanism.
However, the regulatory pathways that mediate the cold stress responses
are far from being understood. The lncRNAs are important regulators of
gene expression. The discovery of lncRNAs has filled gaps in
cold-responsive regulatory pathways. In this study, many polys (A)+
lncRNAs were identified systematically, and their expression profile
under normal or cold-treated conditions was analyzed using Iso-seq and
RNA-seq. Many full-length lncRNAs were found for the first time to be
specifically expressed during cold treatment. Therefore, the lncRNAs
identified in this paper represent a valuable addition to
the~cassava~lncRNA collection, providing a rich resource for the
community to investigate the biology of lncRNAs in plants further.

\textbf{4.2 \emph{CRIR1} is a novel lncRNA regulating plant tolerance to
cold stress}

Plant lncRNAs are expected to play important roles in response to
environmental conditions, and thousands of lncRNAs have been identified
in various plant species (Li, Eichten, Shimizu, Petsch, Yeh, Wu,
Chettoor, Givan, Cole, Fowler, Evans, Scanlon, Yu, Schnable, Timmermans,
Springer \& Muehlbauer, 2014, Wang, Chung, Liu, Jang, Kean, Xu \& Chua,
2014, Xiao, Yuan, Guo, Hou, Yin, Zhang \& Li, 2015, Xin, Wang, Yao,
Song, Hu, Qin, Xie, Peng, Ni \& Sun, 2011, Zhang, Han, Guo, Liu, Zheng,
Wu \& Jin, 2014). Besides, there has been a rapid increase in the number
of well-studied lncRNAs involved in abiotic stresses response, such
as\emph{SVALKA} (Kindgren, Ard, Ivanov \& Marquardt, 2018),
\emph{DRIR}(Qin et al., 2017), and \emph{Cis-NAT\textsubscript{ZmNAC48}}
(Mao et al., 2021). However, for revealing the functions of plant lncRNA
in cold stress response is still lagging because most plant lncRNAs are
expressed at low levels and are not evolutionarily conserved across
species. In the present study, an intergenic lncRNA, \emph{CRIR1} , was
identified, whose expression was significantly induced by cold stress.
Compared with WT, overexpression of \emph{CRIR1} in cassava could
significantly accumulate high leve of proline, thus leading to enhanced
survival rates under cold-treated conditions. The cellular morphological
characteristics suggested that the expression of\emph{CRIR1} can protect
the cellular membrane systems from low temperatures. The discovery of
\emph{CRIR1} provides a new avenue to explore a previously unknown
aspect of plant cold stress response.

\textbf{4.3 \emph{CRIR1} regulates cold tolerance
through~\emph{CBF}-independent~pathways}

Initially, we assumed that \emph{CRIR1} may function as a~\emph{cis}
-acting~lncRNA~involved in the transcriptional regulation of target
genes located in the nearby genomic region. Three genes are located
close to~\emph{CRIR1} . RNA-seq and qRT-PCR results of\emph{CRIR1}
transgenic plants showed no difference in the transcript levels of
neighboring genes (Figure S3). The results suggested that\emph{CRIR1}
functioned as a~\emph{trans} -acting~lncRNA. The candidate target genes
of \emph{CRIR1} were screened using an RNA-seq data set of\emph{CRIR1}
OE lines.~RNA-seq results demonstrated that the expression of many
cold-related genes was increased in OE plants. For example, two
transcription factors, \emph{MeNAC} and \emph{MeNFYA} , were upregulated
in cold-stressed WT plants and \emph{CRIR1} overexpressors.
\emph{NAC}and \emph{NFYA} genes are both involved in abiotic stress
responses, and overexpression of \emph{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
\emph{CRIR1}overexpressors are \emph{ASR} and \emph{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 \emph{MeYAB2} ,
auxin-responsive protein\emph{MeIAA18} , and auxin-binding protein
\emph{MeABP19} . These genes showed a decreased expression pattern
during cold stress and in\emph{CRIR1} -overexpressing plants. These
results suggested a possible interaction of \emph{CRIR1} with the auxin
signaling pathway. The expression levels of cold stress related marker
genes, including\emph{MeCBFs} , \emph{MeCOR} , and \emph{MeICE1} ,
showed no difference between WT and overexpression lines, indicating
that \emph{CRIR1}positively modulated plant cold tolerance
through~\emph{CBF} -independent~pathways.

\textbf{4.4 \emph{CRIR1} functions related to \emph{MeCSP5} in cassava}

MeCSPs are nucleic acid-binding proteins that contain both cold shock
domain (CSD) and DNA-binding domains, which have been widely found in
bacteria, animals, and plants. CSPs are important during cold shock
response. Besides, the functions of CSPs were first described in
Escherichia coli (Goldstein, Pollitt \& Inouye, 1990). During cold
stress, cell membrane fluidity and enzyme activity decrease, and
transcription and translation efficiency is reduced to stabilize nucleic
acid secondary structures. CSPs function as RNA chaperones and
counteract these harmful effects by destabilizing secondary structures
in target RNA at low temperatures, thus facilitating the translation
(Keto-Timonen, Hietala, Palonen, Hakakorpi, Lindstrom \& Korkeala,
2016). \emph{Arabidopsis} \emph{AtCSP3} shares an RNA chaperone function
with~\emph{Escherichia coli} ~cold shock proteins and positively
regulates freezing tolerance through a non-\emph{CBF}pathway (Kim et
al., 2009). In this study, \emph{CRIR1} was showed to be able to
interact with MeCSP5, a homology protein of \emph{Arabidopsis}AtCSP2
with 50\% identity.. In addition, MeCSP5 protein exhibited a~similar
cellular distribution pattern with \emph{CRIR1} transcripts (Figure
S2c). Although cold stress dramatically stimulated the\emph{MeCSP5} gene
expression, its transcript level was not significantly altered~in
the~\emph{CRIR1} -overexpressors (Figure S2d), suggesting that
\emph{CRIR1} could regulate \emph{MeCSP5} at protein level. In
\emph{Arabidopsi} s, AtCSP3 protein has been shown to interact with a
range of proteins in the nucleolus, nucleoplasm, and cytoplasm,
including ribosomal proteins, Gar1 RNA-binding protein, and mRNA
decapping protein. The results suggested that AtCSP3 participated in
multiple complexes and possibly regulated RNA processing and translation
(Kim, Sonoda, Sasaki, Kaminaka \& Imai, 2013). One ribosomal protein was
found to interact with \emph{CRIR1} , indicating the possible
involvement of \emph{CRIR1} in ribosome biogenesis and protein
translation. Therefore, it will be interesting to determine if the
complex that forms with \emph{CRIR1} , MeCSP5 and other proteins
regulates cold stress tolerance in cassava by controlling the RNA
unwinding activity or facilitating mRNA translation.

Taken together, in this study, a total of 3 004 full-length lncRNAs in
cassava were identified, and 316 of them were further characterized as
cold-related lncRNAs. Functional analysis shows that \emph{CRIR1} can
confer cold stress tolerance and participate in MeCSP complexes to
enhance translation efficiency at low temperatures. The research has
provided a valuable source for studying full-length lncRNAs in cassava
and their corresponding candidate genes. \emph{CRIR1} , as a case in the
study, will provide applicable genetic resource for breeding cold
tolerant crops without yield penalty.

\textbf{ACKNOWLEDGEMENTS}

This work was supported by the National Key Research and Development
Program of China (2019YFD1001105, 2018YFD1000500), the Central
Public-interest Scientific Institution Basal Research Fund for Chinese
Academy of Tropical Agricultural Sciences (1630052021026), Hainan
Provincial Natural Science Foundation of China (320MS097), and the
Natural Science Foundation of China (31701484). We are grateful to Dr.
Hongwei Guo and Dr. Kang Yan for providing plasmids 6xMS2 and MSCP.

\textbf{CONFLICTS OF INTEREST}

The authors declare that they have no competing interests.

\textbf{AUTHOR CONTRIBUTIONS}

S. Li, W. Wang and M. Peng conceived and designed the experiments; S.
Li, S. Dong, Z. Li, L. Zou and P. Zhao performed most of the experiments
and analyses with helps from X. Guo; S. Li, and Z. Cheng wrote the
original draft; X.Guo, P. Zhao, Y. Bao, W. Wang and M. Peng reviewed and
edited the manuscript. All authors have read and agreed to the published
version of the manuscript.

\textbf{DATA AVAILABILITY STATEMENT}

The data that supports the findings of this study are available in the
supporting materials of this article.

\textbf{ORCID}

Shuxia Li : https://orcid.org/ 0000-0001-7329-9259

Yan Bao : https://orcid.org/ 0000-0002-9870-5943

Ming Peng : //orcid.org/0000-0003-4843-2773

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\textbf{SUPPORTING INFORMATION}

\textbf{FIGURE S1} KEGG pathway enrichment analysis of\emph{CRIR1}
-regulated genes under normal and cold-treated conditions, respectively.

\textbf{FIGURE S2} Phylogenetic and subcellular localization analysis of
cassava \emph{MeCSPs} .

\textbf{FIGURE S3} Expression analysis of the \emph{CRIR1} neighbors.

\textbf{Table S1} Information of lncRNAs identified from Iso-seq data.

\textbf{Table S2} Information of the DEGs in different comparison
groups.

\textbf{Table S3} List of primers used in this study.

\textbf{Figure legends}

\textbf{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.

\textbf{FIGURE 2} The expression pattern and subcellular distribution
of\emph{CRIR1} in cassava. (a) Spatial expression pattern analysis
of\emph{CRIR1} in different tissues using quantitative RT-PCR. (b)
Expression levels of~\emph{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
of\emph{CRIR1} in cassava leaf cells. \emph{CRIR1} transcripts were
detected by~\emph{in situ} ~hybridizations of Cy3-tagged
oligonucleotides (green). \emph{U6} (yellow) was used~as~a control.~The
location of nuclei is indicated by DAPI staining (blue). Bar=20\selectlanguage{greek}µ\selectlanguage{english}m
*P\textless{}0.05,~**P\textless{}0.01 by Student's \emph{t} -test.

\textbf{FIGURE 3} Effects of \emph{CRIR1} on plant growth, proline
accumulation, and cellular membrane stability during cold stress. (a)
Relative transcript levels of \emph{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=2\selectlanguage{greek}µ\selectlanguage{english}m. Data represent means~\selectlanguage{ngerman}± SD,
*P\textless{}0.05,~**P\textless{}0.01 by Student's \emph{t} -test.

\textbf{FIGURE 4} Identification of \emph{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
the\emph{CRIR1} -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 \emph{CRIR1} -regulated genes under normal and cold-treated
conditions, respectively. The top 10 biological processes GO terms are
shown.

\textbf{FIGURE 5} Relative expression levels of selected genes in
cold-treated seedlings. (a-c) Relative expression levels of\emph{CRIR1}
-regulated genes (a, b) and cold-responsive~marker genes (c) are
detected by qRT-PCR. Data represent means~± SD,
*P\textless{}0.05,~**P\textless{}0.01 by Student's \emph{t} -test.

\textbf{FIGURE 6} \emph{CRIR1} interacts with MeCSP5 in cassava and
improves translational yield. (a) Silver staining and mass spectrometry
analyses following RNA pull-down of \emph{CRIR1} sense or
\emph{CRIR1}antisense (\emph{AS-CRIR1} ) in cassava leaf cells. (b)
Fifteen~candidates~for \emph{CRIR1} -interacted~proteins are identified
by mass spectrometry analyses. MW, molecular~weight. (c) The RNA binding
site prediction of \emph{CRIR1-} MeCSP5 complex using catRAPID. (d)
Confirmation of binding of MeCSP5 to \emph{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 \emph{CRIR1} or control lncRNA. Bars = 10 \selectlanguage{greek}µ\selectlanguage{english}m. (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~\selectlanguage{ngerman}± SD, *P\textless{}0.05 by Student's
\emph{t} -test.\selectlanguage{english}
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