1. INTRODUCTION
Cassava (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 Arabidopsis lead to the identification of a
set of cold-related transcription factors, such asC-REPEAT-BINDING FACTORS (CBFs ), MYBs , 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 COLD-RESPONSIVE(COR ) genes, such as RD29a and COR15a , to activate
their transcription (Shi et al., 2018). In cassava, the CBF genes
are rapidly and transiently upregulated by cold stress (An et al.,
2012). Overexpression of certain 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). COLD SHOCK
PROTEINS (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, CSPs are involved in various biological processes to
promote normal growth and stress responses. The first functionally
characterized plant CSP gene was wheat 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). Arabidopsis has four CSP proteins (AtCSP1-AtCSP4)
that play distinct roles in plant development and stress responses. For
example, AtCSP3 can be induced by cold stress, while
overexpression of AtCSP3 conferred enhanced cold tolerance
without obvious developmental defects (Kim, Sasaki & Imai, 2009).
However, overexpression of AtCSP1 or AtCSP2 did not
enhance cold tolerance in Arabidopsis , but complemented the
cold-sensitive phenotypeof 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 Arabidopsis overexpressing a
nucleus-localized lncRNA (Qin, Zhao, Cui, Albesher & Xiong, 2017). A
natural antisense transcript of maize 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 ofZmNAC48 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 COLD-RESPONSIVE lincRNA 1 (CRIR1 ).
Gain-of-function analysis revealed that 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 CRIR1 -overexpressing transgenic lines.
Furthermore, we found that 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 CRIR1 has a pivotal role in
manipulating cold-stress tolerance in cassava, possibaly though the
regulation of MeCSP5.