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.