Genome-wide Systematic Survey and Analysis of the RNA Helicase Gene Family and their Response to Abiotic Stress in Sweetpotato

Genome-wide Systematic Survey and Analysis of the RNA Helicase Gene Family and their Response to Abiotic Stress in Sweetpotato | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genome-wide Systematic Survey and Analysis of the RNA Helicase Gene Family and their Response to Abiotic Stress in Sweetpotato mufangfang Mu, Hao Zheng, Qiaorui Zhao, Mingku Zhu, Tingting Dong, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3407757/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Mar, 2024 Read the published version in BMC Plant Biology → Version 1 posted 4 You are reading this latest preprint version Abstract RNA helicase is a large family that plays a significant role in plant evolution and in the abiotic stress response. Sweetpotato is one of the majority essential foods in the world, and their yields are often contrived by environmental stresses. Previously, the RNA helicase gene family has not been described in sweetpotato, hence we went a comprehensive genome-wide examination of the sweetpotato RNA helicase family, containing chromosome distribution, promoter elements, and motif compositions. All of 300 RNA helicase genes were divided into three subfamilies, including IbDEAD, IbDEAH and IbDExDH, and they are unevenly scattered across 15 chromosomes of the sweetpotato. Furthermore, tandem replication and segmental replication events played a key role in the amplification of sweetpotato RNA helicase genes. The collinear relationship amidst sweetpotato RNA helicase genes and 8 other related homologous genes in plants was deeply explored, which supplied a reliable basis for studying the development of sweetpotato RNA helicase gene family. RNA-seq examination and qRT-PCR recognition showed that the expression of eight RNA helicase genes was significantly diverse under four abiotic stresses (cold, drought, heat, salt). At the same time, the expression of these RNA helicases in different tissues of the 10 sweetpotato varieties also differed significantly. The promoters of the RNA helicase genes contain a great deal of cis-acting elements related to temperature, hormone and light response. The results indicated that sweetpotato RNA helicase genes played a key role in development or the abiotic stress response. abiotic tress genome-wide analysis RNA helicase sweetpotato Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 1. Introduction The RNA helicases are ubiquitous in prokaryotes and eukaryotes, from viruses to humans, catalyzing the unwinding of duplex RNA dependent on the energy of NTP (Vashisht and Tuteja, 2006 ). RNA molecules undergo a series of modifications in the process of RNA metabolism, and their own instability is easy to cause RNA metabolic disorders, which make a difference in the development and different abilities to resist abiotic stress of plants (Nawaz and Kang, 2017 ;Gc et al., 2020 ). The helicases are divided into 6 superfamilies, namely SF-1, -2, -3, -4, -5, and − 6. Among them, the most representative and largest family is SF2. According to the change of DEAD (Asp-Glu-Ala-Asp) motif, the sweetpotato RNA helicase superfamily is DEAD, DEAH and DExD / H, respectively (Rocak and Linder, 2004 ;Xu et al., 2013b ). Almost all the helicase proteins contain nine conserved motifs (Tuteja and Tuteja, 2004 ). Each of these nine motifs plays distinct roles, which are essential for helicase enzymatic activities (Tanner and Linder, 2001 ;Jiang et al., 2020 ). Structurally RNA helicases are very similar, but their functions are very different. The RNA helicase is present during RNA splicing in RNA metabolism, ribosome formation, and nuclear cytoplasmic transport (de la Cruz et al., 1999 ;Lorsch, 2002 ;Sahoo et al., 2022 ). These genes have many functions in RNA metabolism, among which RNA helicase is associated with growth and development and resistance to stress. In Arabidopsis , DEAD-box LOS4 is able to participate in the process of cryogenic stress, flowering, vernalization, and other processes (Gong et al., 2002 ;Gong et al., 2005 ). Among them, in the cold stress response pathway, LOS4-1 and LOS4-2 can regulate the expression of C repeat binding factors and their downstream target genes. LOS RNA helicase plays a key role in target gene output, maturation and reaction to temperature stress. The transcription of STRS1 and STRS2 was inhibited under salt stress. The salt tolerance of mutants strs1 and strs2 was enhanced, and the expression of RD29A, DREB1A and DREB2A was enhanced (Kant et al., 2007 ). The development of the germ and leaf of the Arabidopsis rh7 mutant was seriously delayed under low temperature stress (Liu et al., 2016 ). AtRH3 plays a role in ribosome formation and seedling growth and development, the growth of rh3 mutants was severely inhibited under salt or cold stress (Gu et al., 2014 ). Studies have shown that Arabidopsis TEBICHI is necessary for regulating cell division and meristem (Inagaki et al., 2006 ), ISE2 is involved in the function of the plasmodesmata during Arabidopsis embryogenesis (Kobayashi et al., 2007 ;Carlotto et al., 2016 ). Our previous findings showed that the tolerance of tomato SlDEAD31 was enhanced in salt and drought stress, and the expression of stress genes was increased such as Cat1 , Cat2 , APX2 , and ER5 (Zhu et al., 2015 ). The rice SUV3 protein has DNA and RNA helicase and ATPase activities, and SUV3 expression can be induced by salt stress (Tuteja et al., 2013 ;Sahoo et al., 2015 ). Low temperature and high salt stress can induce the expression of GmRH in soybeans, and GmRH plays a significant in RNA processing (Chung et al., 2009 ). Tobacco P68 can improve plant growth, photosynthesis, stimulate antioxidant mechanism, and enhance tolerance to salt stress (Tuteja et al., 2014 ). AvDH1 increased salt tolerance and played an important role in boll number, boll weight and seed yield (Chen et al., 2015 ). The Arabidopsis RCF1 gene plays an integral role in maintaining normal splicing of mRNA precursors, and some cold stress-induced genes were error spliced in the rcf-1 mutant (Guan et al., 2013 ). Maize DRH1 can interact with the nucleoprotein fiber MA16, which is involved in ribosomal RNA metabolism (Gendra et al., 2004 ). DEVH-box RNA helicase AtHELPS play a key role in K + deprivation in Arabidopsis thaliana (Xu et al., 2011 ). The RNA helicase has been nominated in many plant variety namely Arabidopsis thaliana (Boudet et al., 2001 ), Oryza sativa (Umate et al., 2010 ), Ipomoea trifida (Wan et al., 2020 ), Glycine max (Xu et al., 2013a ), Zea mays (Xu et al., 2013a ), Gossypium spp (Chen et al., 2014 ), soybean (Karthik et al., 2019 ), Gossypium raimondii (Chen et al., 2014 ) and Solanum lycopersicum (Xu et al., 2013b ). A total of 32 DEAD were initially identified in Arabidopsis thaliana (Aubourg et al., 1999 ). Then, 113 and 115 RNA helicase genes were identified in Arabidopsis and Oryza sativa (Umate et al., 2010 ). Studies have shown that RNA helicase genes in Arabidopsis , Oryza sativa , Gossypium spp , Gossypium raimondii and Zea mays are divided into three subfamilies, the numeral of genes in apiece subfamily is as follows: DEAD-box (50, 51, 87, 51, and 57 genes), DEAH-box (40, 33, 48, 52, and 31 genes), and DExDH-box (71, 65, 78, 58, and 50 genes). Sweetpotato ( Ipomoea batatas (L.) Lam.) is an important food source and industrial raw material with high economic value (Katayama et al., 2017 ). Sweetpotato is a hexaploid with 90 chromosomes, high heterozygosity, and a large number of repetitive sequences (Isobe et al., 2017 ;Yan et al., 2022 ), thus hindering gene identification and functional studies. The RNA helicase is a ubiquitous protein that involved in plant growth and abiotic stress. Sweetpotato is susceptible to abiotic stress, which plays a significant in the growth of potato chips (Ramamoorthy et al., 2022 ). At present, however, genome-wide identification of sweetpotato RNA helicase genes has not been communicated. To improve the yield of sweetpotato, genomic assisted breeding technology can be used to develop new or improved sweetpotato varieties. To explore the biological basis of cold resistance in sweetpotato, it is consequence to recognize differentially expressed genes in response to low temperature stress and apply them to production. Therefore, the main molecules are the recognition of proteins and enzymes, it is very important for these molecules to control a large number of metabolic pathways by regulating the occurrence and metabolism of RNA. The RNA helicases are concerned in many molecular functions, including tolerance, and regulation of development. Their identification in sweetpotato and improvement of sweetpotato varieties are of great significance and practical value. Therefore, in order to comprehend the purpose and participation pathway of the RNA helicase genes in sweetpotato, this study aims to conduct genome-wide confirmation of the RNA helicase genes in sweetpotato, and to analyze the molecular mechanism of sweetpotato participation. 2. Materials and methods 2.1 Identification of the RNA helicase genes in sweetpotato genomes The whole sweetpotato genome sequence was derivative by Ipomoea genome Hub ( https://ipomoea-genome.org ) (Yang et al., 2017 ). To identify members of the RNA helicase gene family in Ipomoea batatas , we used BLASTP to search all known Arabidopsis and rice RNA helicase gene sequences in multiple databases (Altschul et al., 1990 ). And all the information about the RNA helicase genes in Arabidopsis ( https://www.arabidopsis.org/ ) and rice ( http://rice.plantbiology.msu.edu/ ) was downloaded (Xu et al., 2020 ). Subsequently, all protein were covered and each member of the RNA helicase gene was verified using the Pfam database ( http://pfam.xfam.org/ ), the CD-search ( https://www.ncbi.nlm.nih.gov/cdd/Structure/cdd/wrpsb.cgi ), and the PROSITE ( https://prosite.expasy.org/ ), and members lacking typical conserved RNA helicase domains were deleted. The sequence information of all sweetpotato RNA helicase proteins can be found in the Supplementary File. 1 . 2.2 Phylogenetic relationships of RNA helicase proteins in sweetpotato The RNA helicase sequence was aligned using the Clustal X program. The MUSCLE program was used for multiple sequence alignment to support Clustal X ( http://www.clustal.org/ ) (Edgar, 2004 ). Phylogenetic trees were constructed employing the maximum likelihood (ML) method (Guindon et al., 2005 ). 2.3 Protein property and conserved domain of helicase genes in sweetpotato The physicochemical properties of the RNA helicase proteins were forecasted by the online ExPASy database ( http://expasy.org/ ). Prediction of subcellular sites ( http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/ ) and phosphorylation sites ( http://www.cbs.dtu.dk/services/NetPhos/ ) of the RNA helicase genes. The gene structure was obtained by comparing the sequence of the RNA helicase genes with the genome sequence. The consequence was realized by Tbtools (Chen et al., 2020 ). The conserved domain was determined using MEME ( https://meme-suite.org/meme/tools/meme ) (Bailey et al., 2015 ). Then, the protein interacting networks were ensured by STRING ( https://string-db.org/ ). 2.4 Chromosomal location and collinearity analysis of the RNA helicase genes in sweetpotato Isoelectric points and molecular weights of these proteins are obtained by ExPASy ( http://expasy.org/ ) (Gasteiger et al., 2003 ). The structural intelligence of these sweetpotato RNA helicase genes was analyzed together with genomic data. To examine the collinearity both RNA helicase gene and other plant genes, The genome sequence information of sweetpotato, Arabidopsis thaliana and rice was downloaded and examined. MCScanX was used to create gene duplication and collinearity relationships through failure parameters (Wang et al., 2012 ), data results are envisaged by TBtools (Krzywinski et al., 2009 ;Chen et al., 2020 ). Default parameters were used in all steps. 2.5 qRT‑PCR ascertain of cold stress The experimental materials were XuShu 18 roots, different cold stress treatment methods have been described in our previous reports (Xie et al., 2019 ). Four abiotic stress treatments and four hormone stress treatments were carried out on XuShu 18, and samples were taken after 0h, 1h, 3h, 6h, 12h, 24h, 48h and 72h, respectively. Ten different varieties of sweetpotato were planted, and their young leaves, leaves, stems and roots were taken respectively. RNA of these samples was extracted for subsequent experiments. The total RNA was excavated using RNA extraction kit (TianGen, Beijing, China), and reverse transcriptions using TransScript® gDNA removal (TransGen, Beijing, China). All the sweetpotato RNA helicase promoter regions were examined by plantCARE ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ). 2.6 Statistical Analysis Statistical reasoning was achieved by Microsoft Excel 2019, Graphpad Prism 5.0 and SPSS statistical. Considering the biological significance, differential gene expression using two times the cut-off value. 3. Results 3.1 Identification of RNA helicase family genes in sweetpotato To recognize the elements of the sweetpotato RNA helicase gene, we used bioinformatics methods to collect details on many sweetpotato RNA helicases. A total of 300 sweetpotato RNA helicase proteins were detected. According to the RNA helicases conserved motifs, 300 sweetpotato RNA helicase genes were divided into subfamilies DEAD-box (53 DEAD genes), DEAH-box (54 DEAH genes) and DExDH-box (193 DExDH genes) subfamilies ( Supplementary File. 1 ). The amount of RNA helicase proteins in Arabidopsis and rice was 113 and 115, respectively. It is predicted that there are 300 RNA helicase proteins in sweetpotato, which is much more than the number of Arabidopsis and rice (Umate et al., 2010 ). Then, the 300 RNA helicase genes on 15 sweetpotato chromosomes were named from top to bottom as IbDEAD1 ~ IbDEAD53 , IbDEAH1 ~ IbDEAH54 and IbDExDH1 ~ IbDExDH193 ( Supplementary Fig. 1 ). Subsequently, the physicochemical properties of 300 sweetpotato RNA helicase proteins were examined. The length and relative molecular mass of the RNA helicase vary greatly. The length of DEAD ranges from 323 aa (IbDEAD34) to 1301 aa (IbDEAD2), the relative molecular mass ranges from 3584.09 to 145261.6 Da, and the isoelectric point ranges from 5.02 (IbDEAD40) to 9.87 (IbDEAD37). The length of DEAH is between 206aa (IbDEAH5) and 2904aa (IbDEAH44), the relative molecular mass is between 23377.03 and 323370.3Da, and the isoelectric point is between 5.16 (IbDEAH22) and 9.25 (IbDEAH36). The length of DExDH ranged from 128aa (IbDExDH7) to 2801aa (IbDExDH30), the relative molecular mass ranged from 13904.77 to 306450.01 Da, and the isoelectric point graded from 4.81 (IbDExDH7) to 9.67 (IbDExDH45). The subcellular location showed that most of the RNA helicase proteins were positioned in the nucleus. Furthermore, the potential phosphorylation sites showed that IbDEAD contains 29 (IbDEAD28) to 141 (IbDEAD2) phosphorylation sites, IbDEAH contains 19 (IbDEAH5) to 280 (IbDEAH44) phosphorylation sites, and IbDExDH contains 18 (IbDExDH141) to 362 (IbDExDH30) phosphorylation sites, all of these sweetpotato RNA helicase proteins restrain more Ser sites than the Tyr and Thr sites ( Supplementary Table. 1 ). 3.2 Phylogenetic analysis of the RNA helicase family proteins in sweetpotato The rootless phylogenetic tree of the identified RNA helicase in sweetpotato and the known RNA helicase in Arabidopsis thaliana was constructed to study the evolution and classification of RNA helicase in sweetpotato (Fig. 1). Appertaining to the classification of the RNA helicase in Arabidopsis , they were separated into 3 subfamilies: DEAD, DEAH and DExDH. 53 IbDEAD proteins were separated into 9 subgroups (except IbDEAD5), 54 IbDEAH proteins were separated into 9 subgroups (except IbDEAH36 ), and 193 IbDExDH proteins were separated into 13 subgroups (except IbDExDH19 and IbDExDH23 ), the V subgroup in IbDEAH and the Ⅻ in IbDExDH are unique to sweetpotato. RNA helicase proteins are widely and unevenly distributed in different subfamilies. IbDEAD and IbDEAH have nine subgroups, and VIII is the largest subgroup. The sweetpotato RNA helicase genes contained in subgroup VI, VIII and Ⅺ of IbDExDH were the least. Interestingly, IbDEAD5 , IbDEAH36 , IbDExDH19 and IbDExDH23 do not exist in any of the above three subfamilies, which suggesting that they may have other functions. 3.3 Gene structure and motif composition analyses in sweetpotato To further understand the structural characteristics of the sweetpotato RNA helicase family, we paralleled the composition of conserved motifs and introns / exons on the basis of the phylogenetic tree of sweetpotato RNA helicase (Figs. 2 and 4). The structures of the IbDEAD, IbDEAH, and IbDExDH subfamily genes are quite different between different subgroups, but the genes in the same side branch are homologous genes, and their genetic structures is also similar, indicating that the exon-intron structure is highly correlated with phylogenetic relationships (Figs. 2 and 4). The genetic structure of the RNA helicase family members is relatively complex, which the IbDEAD and IbDEAH gene members contain multiple exons, the IbDExDH gene members have only five genes with only one exon (2.6%), and other genes contain multiple exons. Next, we analyzed the motifs of each member of these RNA helicases on the MEME website, and found that all sweetpotato RNA helicases contain 15 conserved motifs (Figs. 2 and 4). Motifs 1, 2, 3, 4, 5, 7, 8, 9 and 15 are current in nearly all proteins, and motif 5 contains extremely conserved sequences DEAD, DEAH, and DExD / H. These conserved motifs are the same in sweetpotato RNA helicase, but the domains and amino acid sequences of these maintained motifs are very different (Figs. 2 and 4). In general, phylogenetic tree examination of data shows that the system development and features and the divergence of genetic structure and sequence distribution are closely related. 3.4 Chromosome localization of the RNA helicase family in sweetpotato Physical location detection based on GFF3 genome annotation showed that 53 IbDEAD genes, 54 IbDEAH genes and 193 IbDExDH genes were located on all 15 chromosomes. Among them, Chr6 in IbDEAD contains the most abundant IbDEAD genes, with 7 members. Chr11 in IbDEAH contains the most abundant IbDEAH genes, with 9 members. However, the IbDEAD and IbDEAH genes are not distributed in Chr9. Most chromosomes are rich in IbDExDH genes, but Chr10 contains only four IbDExDH genes (Figs. 5 and Supplementary Fig. 2 ). The distribution of the three subfamilies of RNA helicases on 15 chromosomes is quite different. Description of collinearity analysis manifested that there was a group of tandem duplicated IbDEAH and two groups of tandem duplicated IbDExDHs , which were IbDEAH2/24 , IbDExDH114/168 and IbDExDH162/185 . However, no tandem duplicated gene was found in IbDEAD (Supplementary Table. 7 ). 3.5 Cis element analysis of the RNA helicase family gene promoters To explore the possible regulatory mechanism of sweetpotato RNA helicase on abiotic stress and hormones, we scanned the cis-acting elements in the 2kb promoter upriver of the sweetpotato RNA helicase gene through the PlantCare database (Figs. 6 and 8, Supplementary Table. 2 ). The conclusions showed that each promoter region of RNA helicase had multiple cis-acting elements connected with stress- and / or hormones. Among others, nearly 90% of RNA helicase promoters contain multiple stress cis-elements, for instance stress response elements (TC-rich repeats), low temperature response elements (LTR), MAJA response elements (TGACG-motif), drought response elements (MBS), etc. These cis-acting elements can be associated with expression. For example, the expression of several RNA helicase genes providing IbDEAH-32 / -42 and IbDExDH-36 / -47 / -96 was increased under different stresses. Correspondingly, stress-related repeats of MBS, TC, or LTR cis elements are more numerous in their promoter regions. However, TC-rich repeats, MAJA response elements and LTR elements were found on the promoters of the IbDExDH-25 / -48 and IbDEAH53 genes, their expression was not particularly significant under salt, drought, or cold stress, but there was a certain degree of response under high-temperature stress (Fig. 9). Furthermore, all sweetpotato RNA helicase promoters restrain many hormone elements, such as abscisic acid response element (ABRE), Me-JA response element (CGTCA motif and TGACG motif) or auxin response element (TGA-box) (Figs. 6 and 8, Supplementary Table. 2 ). Nevertheless, the expression of the RNA helicase genes in dissimilar tissues of 10 different sweetpotato varieties was also different ( Figure. 14 ). Most of the RNA helicase genes are communicated in high amounts in sweetpotato stems and root tissues, in particular, the expression levels of IbDExDH36 and IbDExDH48 in the roots were lower than those in other tissues. It shows that RNA helicase is associated to plant development. Among them, the transcription levels of most RNA helicase genes do not respond significantly to hormonal treatment (Fig. 10). These data indicate that the cis-acting elements of sweetpotato RNA helicase can be concerned in both hormonal and abiotic stresses. 3.6 Identification of RNA helicase family cold-response genes in transcriptomes and their expression profiles under multiple abiotic stress and hormonal treatments Many studies have shown that the RNA helicase have an influence in various abiotic stresses (such as cold, drought, and heat) and development. To determine the potential biological function of the sweetpotato RNA helicase gene under adversity stress, based on our previous RNA-seq data, we first studied the expression of XuShu 18 under cold stress (Xie et al., 2019 ). The consequence indicated that eight RNA helicase genes were screened out. Subsequently, we further studied the expression patterns of eight genes ( IbDEAH-32 / -42 / -53 , IbDExDH-25 / -36 / -47 / -48 / -96 ) screened by qRT-PCR under salt, drought, heat and cold stresses, and explore a two-fold cut off value (Zhu et al., 2015 ). The results showed that all selected genes were up-regulated to varying degrees after salt, drought, cold, and heat treatment. Among them, four stresses could up-regulate IbDEAH32 and IbDExDH-36 / -47 transcription, two stresses could up-regulate IbDEAH42 and IbDExDH-48 / -96 expression, and one stress could up-regulate IbDExDH25 and IbDEAH53 transcription. All RNA helicases could be up-regulated under high temperature stress and salt stress. In particular, IbDEAH32 and IbDExDH47 had the highest level of induction after cold treatment, about six times, while the level of induction was relatively low in the transcription of other RNA helicases. Except for IbDExDH48 , the expression of other genes was significantly enhanced under high temperature conditions, which was 2.3 and 19.3-fold that of the control. Under salt stress conditions, the expression of IbDEAH42 and IbDExDH-47 / -48 was enhanced, and the expression of other genes was weakly induced by salt stress. In particular, the response of all genes to drought stress was not very strong, which may be affected by other factors (Fig. 9). Overall, these data suggest that multiple sweetpotato RNA helicase members can take the lead in reaction to abiotic stress. Moreover, qRT-PCR was acclimated to further detect the transcription profiles of eight RNA helicase genes under distinct hormone treatments, encircling ABA, IAA, GA and ZT. Unexpectedly, most of the RNA helicase genes were down-regulated when we used double as the cut-off value for differential expression. Just the stress hormone ZT could prompt the expression of IbDExDH-36 / -47 / -48 / -98 and IbDEAH42 (Fig. 10). It is reported that the response of the RNA helicase to hormone treatment is not obvious, mainly related to development and abiotic stress(Camborde et al., 2022 ;Li et al., 2022b ). Overall, these data propose that multiple members of the sweetpotato RNA helicase can be important players in answer to hormone and / or abiotic stresses. 3.7 Collinearity analysis of the RNA helicase genes between sweetpotato and other plants To furthermore explore the origin and evolutionary mechanism of sweetpotato RNA helicase genes, we compared the homology of 300 RNA helicase genes with 8 representative species-related genes. These species include wild diploid relatives of sweetpotato ( Ipomoea trioba and Ipomoea trifida ), two model plants ( Arabidopsis thaliana and Oryza sativa ), two cruciferous plants ( Brassica rapa and Brassica oleracea ) and two Solanaceae plants ( Solanum lycopersicum and Capsicum annuum ). Among them, 42 (79.2%) and 43 (81.1%) IbDEAD genes were homologous to genes in Ipomoea trioba and Ipomoea trifida , respectively, accompanied by Solanum lycopersicum (8), Capsicum annuum (6), Arabidopsis thaliana (6), Brassica rapa (2), and Brassica oleracea (1), but no homologous genes were found both sweetpotato and rice. 39 (72.2%) and 40 (74.1%) IbDEAH genes were also homologous to the genes in Ipomoea trioba and Ipomoea trifida , respectively, accompanied by Solanum lycopersicum (8), Capsicum annuum (3) and Arabidopsis thaliana (3), nevertheless, no similar homologous genes were noted between sweetpotato and Brassica rapa , cabbage and rice. Similarly, 89 (46.1%) and 87 (45.1%) IbDExDH genes were homologous to the genes in Ipomoea trioba and Ipomoea trifida , followed by tomato (24), pepper (10), Arabidopsis (7), Brassica rapa (3), and cabbage (2), however, no homologous genes were found with rice (Figs. 11 and 13). It should be mentioned that the collinearity of sweetpotato RNA helicase genes between the Ipomoea trioba and Ipomoea trifida genes more than the extra six varieties, which can be associated to the wild diploid relationship of sweetpotato. Moreover, the mass of the RNA helicase genes in our sweetpotato are one-to-one gene pairs with sweetpotato diploids, and a few are one sweetpotato gene corresponding to two or three diploid gene pairs. For example, the sweetpotato genes IbDEAD (10), IbDEAH (2) and IbDExDH (19) have a one-to-many relationship in Ipomoea trioba , and IbDEAD (10), IbDEAH (2) and IbDExDH (17) sweetpotato genes have a one-to-many relationship in Ipomoea trifida ( Supplementary Table. 3 ). Interestingly, we found that IbDEAH did not find some collinear gene pairs between sweetpotato and Oryza sativa / Brassica rapa / Brassica oleracea , and there was no collinear gene pair between sweetpotato RNA helicase and rice. 3.8 Tissue expression patterns of cold-response genes of the RNA helicase family of different sweet potato cultivars Objective to preliminarily understand the role of the RNA helicase gene in the development stage of sweetpotato, the expression contours of the RNA helicase genes in diverse tissues of 10 sweetpotato varieties were analyzed by qRT-PCR, such as young leaves, leaves, stems and roots. The expression contours of eight RNA helicase genes were grouped together on their respective heat maps (Fig. 14). The expression of the RNA helicase in various tissues is different, and the expression in different varieties of sweetpotato is also quite different. This also shows that the RNA helicase is related to growth and development (Tyagi et al., 2020 ;Liu et al., 2023 ). Some the RNA helicase genes were highly communicated in stems and roots of sweetpotato, such as IbDExDH25 , IbDEAH42 , IbDExDH47 , and IbDExDH96 . Similarly, some RNA helicase genes were also highly communicated in sweetpotato leaves, including IbDExDH36 , IbDExDH48 , and IbDEAH53 . This shows that the expression of the RNA helicase in unequal growth stages of sweetpotato is diverse and have an influence in the development stage of plants. This is steady with the results of other plants in RNA helicase. HS3 is located in the DEAD-box RNA helicase 22 in Arabidopsis plastids, which is necessary for proper accumulation of plastid gene mRNA throughout seed germination and plant growth (Kanai et al., 2013 ;Iglesias-Fernández et al., 2019 ). The RNA helicase can also concern the development of plants under chilling stress. AtRH7, one of the RNA helicases in Arabidopsis , is an RNA chaperone involved in cold adaptation (Huang et al., 2016b ). The mutation of rh7 affects the abnormal development of flowers in Arabidopsis thaliana, and makes the leaves of Arabidopsis thaliana smaller in chilling stress (Liu et al., 2016 ). 4. Discussion The RNA helicases are ubiquitous in miscellaneous organisms. It is involved in almost all processes of RNA metabolism, such as transcription, mRNA splicing and output, mRNA translation, etc. It involves almost all aspects of organisms (Vashisht and Tuteja, 2006 ;Sloan and Bohnsack, 2018 ;Donsbach and Klostermeier, 2021 ). In the past, the study of the RNA helicase family in plants mainly focuses on dicotyledonous model plant Arabidopsis thaliana and monocotyledonous rice (Nawaz and Kang, 2019b ;Takagi et al., 2020 ;Banu et al., 2023b ), these play an essential role in plant growth and stress response (Kim et al., 2008 ;Li et al., 2022a ). Sweetpotato is a significant food crop, which is broadly used in food, feed and industrial raw materials. It is the seventh largest food crop in the world, with the strong ability to adapt to the environment, varieties of high yield, strong stress resistance (Yu et al., 2020 ;Banu et al., 2023a ). However, only 17 RNA helicase genes have been nominated in Ipomoea trifida (Wan et al., 2020 ). There is no comprehensive study on RNA helicase in sweetpotato. This study systematically identified RNA helicase genes in sweetpotato, which laid a solid basis for further study on the engagement of the RNA helicase in plant abiotic stress and development. In the present study, we conducted a comprehensive reasoning of the sweetpotato RNA helicase gene family, containing phylogenetic tree, expression profiles of different sweetpotato varieties under common growth circumstances, and expression profiles under diverse abiotic stresses and hormone stresses. Firstly, 300 RNA helicase genes were nominated in sweetpotato genome, which is a very large gene family. A larger family of the RNA helicase gene exists in sweetpotato, suggesting that RNA helicase plays a leading role in regulating environmental responses. According to the family classification of Arabidopsis and rice RNA helicases (Xu et al., 2013a ), they were divided into three subfamilies, including IbDEAD (53), IbDEAH (54) and IbDExDH (193) ( Supplementary File. 1 ). The chromosomal mapping of 300 sweetpotato RNA helicase genes was mapped, and the distribution of the RNA helicase on 15 sweetpotato chromosomes was analyzed. Chromosome localization reasoning proved that sweetpotato RNA helicase gene was scattered on all 15 chromosomes. Among them, IbDEAD was the most distributed on LG4 and LG6 chromosomes (7 genes), and the least distributed on LG2, LG8, LG10, LG12, and LG14 chromosomes (2 genes). IbDEAH was the most distributed on the LG11 chromosome (9 genes), and the least distributed on the LG4, LG5, and LG12 chromosomes (2 genes). IbDExDH was equally scattered on the sweetpotato chromosomes ( Supplementary Fig. 1 ). The sweetpotato RNA helicase gene contains different numbers of exons and different lengths of introns (Figs. 2 and 4). Intriguingly, five genes in the IbDExDH subfamily contain only one exon, while the IbDEAD and IbDEAH subfamilies do not contain this case and contain multiple exons. In fact, the length of RNA helicase family proteins varies greatly. IbDEAD is 323 to 1301 amino acids, IbDEAH is 206 to 2904 amino acids, and IbDExDH is 128 to 2801 amino acids. The highly different amino acid sequences of these sweetpotato RNA helicases induce diverse protein structures and functions in dissimilar developmental and stress-resistant environments. At the same time, the sweetpotato RNA helicase motif was analyzed, the most conserved motif in different species is Asp-Glu-Ala-Asp, which is divided into three subfamilies according to its difference (Tanner and Linder, 2001 ;Linder and Jankowsky, 2011 ;Xu et al., 2023 ). According to the structural characteristics and phylogenetic analysis of the Motif V region, the determined helicases can be moreover divided into these subfamilies, comprising IbDEAD, IbDEAH, and IbDExDH. Phylogenetic analysis showed that IbDEAD, IbDEAH and IbDExDH RNA helicase proteins can be moreover divided into nine, or thirteen great subgroups (Fig. 1). The gene structure results showed that the main RNA helicase genes in Arabidopsis were uniform to the AtRH family genes (Aubourg et al., 1999 ), but the position and length of introns were not fixed. However, this is the first genome-wide examination of the RNA helicase genes family in sweetpotato. The distinct subfamilies and gene structures of sweetpotato RNA helicase genes indirectly indicate the different functions in RNA metabolism, stress resistance and growth and development. In fact, we establish that most RNA helicase gene promoters contain some cis-regulatory elements, such as plant development, abiotic stress, plant hormones, and light response elements(Huang et al., 2016a ;Nawaz and Kang, 2019a ). It is worth noting that IbDEAD and IbDEAH have more cis-regulatory elements associated to low temperature and light response, and IbDExDH has more cis-acting elements associated with abscisic acid and plant hormones (Figs. 6 and 8). According to the study that temperature, abscisic acid and jasmonic acid are involved in abiotic stress processes in plants (Bari and Jones, 2009 ;Nidumukkala et al., 2019 ;Bharath et al., 2021 ). Under hormone and abiotic stress treatments, qRT-PCR data showed that RNA helicase mainly responded to abiotic stress (Figs. 9 and 10). A myriad of cis-regulatory elements in the promoter of sweetpotato RNA helicase genes indicate that these genes given a paramount importance plant stress resistance. Therefore, we found new candidate genes that can regulate the ability of sweetpotato to resist abiotic stress through RNA metabolism. Considering that sweetpotato is the seventh largest food crop in the world, their economic importance in the world and their adaptation to the environment are great challenges. The revelation of stress-related genes laid a solid foundation for promoting the research of molecular basis of sweetpotato resistance and accelerating the breeding of sweetpotato resistant varieties. The RNA helicase given a paramount importance in regulating plant development and responding to environmental stimuli. The expression profiles of the RNA helicase genes were analyzed in young leaves, leaves, stems, and roots of different varieties of sweetpotato, and their response patterns were analyzed under four abiotic stresses and four hormone stresses (Fig. 14). The expression of sweetpotato RNA helicase genes in dissimilar tissues was significantly different, indicating that these genes were related to plant development. Moreover, the RNA helicase genes mainly answer to abiotic stresses in sweetpotato, such as cold, heat and salt, indicating that these genes are related to plant stress resistance. This is consistent with the functional studies of other plant RNA helicases, for instance Arabidopsis (Huang et al., 2016b ), rice (Xiaomei et al., 2020 ), tomato (Capel et al., 2020 ), rapeseed (Zhang et al., 2022b ), chrysanthemum (Zhang et al., 2022a ), Zea mays (Yang et al., 2023 ), barley (Ru et al., 2021 ) and soybean (Wang et al., 2022 ). We hypothesized that RNA helicase protein can be a functional gene related to sweetpotato growth and development, or a regulatory factor under different environmental constraints. Therefore, this study provides new experimental ideas and clues for the above speculation. In conclusion, exploring the reception of the RNA helicase gene in sweetpotato can help transgenic research improve the yield and resistance to stress of sweetpotato. The identification, classification and phylogenetic tree construction of sweetpotato RNA helicase genes were explored through bioinformatics, which produced meaningful information for in-depth study of the biological function of sweetpotato RNA helicase gene. These studies also contribute to understanding the molecular basis of several significant agricultural traits in sweetpotato cultivation. However, the exact regulatory mechanism of sweetpotato RNA helicase gene development and stress response are still unclear and needs to be additional examined. 5. Conclusion In this research, we conducted an exhaustive genome-wide reasoning of the sweetpotato RNA helicase family, containing chromosome distribution, promoter elements, and protein motif analysis. All of 300 RNA helicase genes were detected, containing IbDEAD, IbDEAH and IbDExDH subfamilies. The expression patterns of eight RNA helicase genes in different sweetpotato varieties and their responses to abiotic stress and hormonal stress were analyzed by qRT-PCR. The expression of the RNA helicase genes was significantly distinct in individual tissues of 10 sweetpotato varieties and notably raised under divergent abiotic stresses. The results showed that RNA helicase was complicated in the direction of extension and the resistance to stress of sweetpotato. This study supplies new inspirations into the development and exploration of the RNA helicase gene families. Declarations Contributions Zongyun Li, Mingku Zhu and Tingting Dong conceived and designed the research. 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SlDEAD31, a Putative DEAD-Box RNA Helicase Gene, Regulates Salt and Drought Tolerance and Stress-Related Genes in Tomato. PLoS ONE. 2015;10:e0133849. Additional Declarations No competing interests reported. Supplementary Files Supplementarydata.zip Cite Share Download PDF Status: Published Journal Publication published 16 Mar, 2024 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Major revision 04 Oct, 2023 Editor assigned by journal 04 Oct, 2023 Submission checks completed at journal 04 Oct, 2023 First submitted to journal 03 Oct, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3407757","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":237771572,"identity":"0b9a50d0-2b4a-4125-8036-2102bfd1fbac","order_by":0,"name":"mufangfang Mu","email":"","orcid":"","institution":"Jiangsu Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"mufangfang","middleName":"","lastName":"Mu","suffix":""},{"id":237771573,"identity":"532a340b-e10b-4ff6-ae2b-c4bfde0335e0","order_by":1,"name":"Hao Zheng","email":"","orcid":"","institution":"Jiangsu Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hao","middleName":"","lastName":"Zheng","suffix":""},{"id":237771575,"identity":"650658cd-29bb-4316-a76f-3a02524ba0bd","order_by":2,"name":"Qiaorui Zhao","email":"","orcid":"","institution":"Jiangsu Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qiaorui","middleName":"","lastName":"Zhao","suffix":""},{"id":237771576,"identity":"d0c827e4-fbe1-46e8-83ec-61ded1f102b3","order_by":3,"name":"Mingku Zhu","email":"","orcid":"","institution":"Jiangsu Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mingku","middleName":"","lastName":"Zhu","suffix":""},{"id":237771577,"identity":"d92269b2-227e-4c5a-a11b-e105e441329c","order_by":4,"name":"Tingting Dong","email":"","orcid":"","institution":"Jiangsu Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Tingting","middleName":"","lastName":"Dong","suffix":""},{"id":237771578,"identity":"1a8632de-b36f-4dba-b67b-8e0c0e08fef3","order_by":5,"name":"Lei Kai","email":"","orcid":"","institution":"Jiangsu Normal University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Kai","suffix":""},{"id":237771579,"identity":"aaa142ba-2bee-43ad-ace2-c73e22c1cfa0","order_by":6,"name":"Zongyun Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA50lEQVRIiWNgGAWjYBADGTb2hgQI8wCRWnjYeA6QqoVBAqqDoBb59t7Dr3nb7vDwST54/LmwjUGO70YC4+cCPFoYe86lWfO2PeNhk05Ik57ZxmAseSOBWXoGHi3MEjlmxrxth8FamHnbGBI33EhgY+bBo4UNrkXyQPJnoJZ6glp4JHKMH4O1SDAkSAO1JBgQ0iLBc8aMcc45oBYeoF94zkkYzjzzsFkanxb59h7jD2/KDsvJt59J/sxTZiPPdzz54Gd8WkDekYIo4EkA2QrEjA34NQAD7eMPMM1+gJDKUTAKRsEoGKEAAMXOQzaBAQAZAAAAAElFTkSuQmCC","orcid":"","institution":"Jiangsu Normal University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Zongyun","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2023-10-03 12:59:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3407757/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3407757/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-024-04824-z","type":"published","date":"2024-03-16T15:01:02+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":44388270,"identity":"c50fd2fb-1101-47ea-ab62-0c1157faa961","added_by":"auto","created_at":"2023-10-10 20:09:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":6020284,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree analysis of RNA helicase in Arabidopsis and sweet potato. \u003cstrong\u003e(A)\u003c/strong\u003e The DEAD-box RNA helicase proteins in two species. \u003cstrong\u003e(B)\u003c/strong\u003e The DEAH-box RNA helicase proteins in two species. \u003cstrong\u003e(C)\u003c/strong\u003e The DExD/H-box RNA helicase proteins in two species. These three subfamilies were used to construct the maximum-likelihood (ML) phylogenetic tree by MEGA X with 1,000 bootstrap replicates. Different subgroups are named based on the reports in Arabidopsis and are distinguished with different colors. The gene names are marked at the end of the branch, the red triangle and green circle represent the sweet potato RNA helicase and Arabidopsis RNA helicase, respectively.\u003c/p\u003e","description":"","filename":"Figure.1.png","url":"https://assets-eu.researchsquare.com/files/rs-3407757/v1/b234072de8c51116dade95c4.png"},{"id":44388266,"identity":"8f082b5c-110a-415b-ad2f-1bdaeeead354","added_by":"auto","created_at":"2023-10-10 20:09:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1350531,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree, gene structure and motif distribution analysis of 53 IbDEAD-box members.\u003cstrong\u003e (A)\u003c/strong\u003e Phylogenetic tree of DEAD-box members in sweet potato. A total of 53 IbDEAD-box proteins were used to construct the maximum-likelihood (ML) phylogenetic tree by MEGA X with 1,000 bootstrap replicates. The different groups are marked with different colors. \u003cstrong\u003e(B)\u003c/strong\u003eThe exon-intron structure analysis of 53 IbDEAD-box genes. Exon and introns lengths are displayed proportionally, yellow boxes represent exons, black lines represent introns, and green boxes represent 5’ and 3’ UTR. \u003cstrong\u003e(C)\u003c/strong\u003e Motif distribution of 53 IbDEAD-box proteins. The different colored boxes represent 15 motifs, and the box lengths represent motif lengths.\u003c/p\u003e","description":"","filename":"Figure.2.png","url":"https://assets-eu.researchsquare.com/files/rs-3407757/v1/2d911423d056af9a2301e2f5.png"},{"id":44388267,"identity":"4e74ca1e-7005-4cf1-94c8-91e2071d66f2","added_by":"auto","created_at":"2023-10-10 20:09:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2258786,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree, gene structure and motif distribution analysis of 54 IbDEAH-box members. \u003cstrong\u003e(A)\u003c/strong\u003e Phylogenetic tree of DEAH-box members in sweet potato and the consistent parameters shown in Fig.2(A). The different groups are marked with different colors. \u003cstrong\u003e(B)\u003c/strong\u003e The exon-intron structure analysis of 54 IbDEAH-box genes and the consistent parameters shown in Fig.2(B). \u003cstrong\u003e(C)\u003c/strong\u003e Motif distribution of 54 IbDEAH-box proteins. The different colored boxes represent 15 motifs, and the box lengths represent motif lengths.\u003c/p\u003e","description":"","filename":"Figure.3.png","url":"https://assets-eu.researchsquare.com/files/rs-3407757/v1/93f4184af9665ee12dc2118e.png"},{"id":44391125,"identity":"dfc19985-ee75-413d-b870-ce894e21f92c","added_by":"auto","created_at":"2023-10-10 20:25:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2009565,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree, gene structure and motif distribution analysis of 193 IbDExDH-box members. \u003cstrong\u003e(A)\u003c/strong\u003e Phylogenetic tree of DExDH-box members in sweet potato and the consistent parameters shown in Fig.2(A). The different groups are marked with different colors.\u003cstrong\u003e (B) \u003c/strong\u003eThe exon-intron structure analysis of 193 IbDExDH-box genes and the consistent parameters shown in Fig.2(B). \u003cstrong\u003e(C)\u003c/strong\u003e Motif distribution of 103 IbDExDH-box proteins. The different colored boxes represent 15 motifs, and the box lengths represent motif lengths.\u003c/p\u003e","description":"","filename":"Figure.4.png","url":"https://assets-eu.researchsquare.com/files/rs-3407757/v1/27fa8445b5895012f0d9544d.png"},{"id":44389970,"identity":"587cf1af-9438-4b82-ade2-91f7c9977f93","added_by":"auto","created_at":"2023-10-10 20:17:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2883855,"visible":true,"origin":"","legend":"\u003cp\u003eThe interchromosomal relationship of RNA helicase genes in sweetpotato chromosomes. (A) IbDEAH, (B) IbDExDH. Circular visualizations of RNA helicase genes mapped to the LG1- LG15 chromosomes are indicated by colored rectangles. The colored curves represent duplicated RNA helicase gene pairs. The corresponding RNA helicase genes located in segmental duplications are marked with colors.\u003c/p\u003e","description":"","filename":"Figure.5.png","url":"https://assets-eu.researchsquare.com/files/rs-3407757/v1/1546b9ad3937c32aa6f8bb28.png"},{"id":44388275,"identity":"bec17566-27a1-488c-8360-30ca01927a05","added_by":"auto","created_at":"2023-10-10 20:09:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1281619,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic clustering and predicted stress- and hormone-related cis-elements in the promoters of \u003cem\u003eIbDEAD\u003c/em\u003egenes. \u003cstrong\u003e(A)\u003c/strong\u003e The phylogenetic tree of 53 IbDEADs was constructed by MEGA X based on the consistent parameters used in Fig.2(A). \u003cstrong\u003e(B)\u003c/strong\u003e Predicted cis-elements in the IbDEAD promoters. 2000 bp promoter regions of each \u003cem\u003eIbDEAD\u003c/em\u003egene were detected by PlantCARE database. Different colored rectangles represent different cis-elements that are potentially involved in stress or hormone regulation.\u003c/p\u003e","description":"","filename":"Figure.6.png","url":"https://assets-eu.researchsquare.com/files/rs-3407757/v1/4db84f43cd475f60ae50c465.png"},{"id":44388276,"identity":"7c0285ca-ee99-4e00-aa00-e766eef786d5","added_by":"auto","created_at":"2023-10-10 20:09:30","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1026722,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic clustering and predicted stress- and hormone-related cis-elements in the promoters of \u003cem\u003eIbDEAH\u003c/em\u003egenes. \u003cstrong\u003e(A)\u003c/strong\u003e The phylogenetic tree of 54 species of IbDEAH and the consistent parameters shown in Fig.2(A). \u003cstrong\u003e(B)\u003c/strong\u003e Predicted cis-elements in the IbDEAH promoters. Different colored rectangles represent different cis-elements that are potentially involved in stress or hormone regulation.\u003c/p\u003e","description":"","filename":"Figure.7.png","url":"https://assets-eu.researchsquare.com/files/rs-3407757/v1/2cd77e0f8def580f436dcf51.png"},{"id":44389957,"identity":"1d296c3c-12dc-4a6b-b70e-1eac0c1b9224","added_by":"auto","created_at":"2023-10-10 20:17:30","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2574047,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic clustering and predicted stress- and hormone-related cis-elements in the promoters of \u003cem\u003eIbDExDH\u003c/em\u003egenes. \u003cstrong\u003e(A)\u003c/strong\u003e The phylogenetic tree of 193 species of IbDExDH and the consistent parameters shown in Fig.2(A). \u003cstrong\u003e(B)\u003c/strong\u003e Predicted cis-elements in the IbDExDH promoters. Different colored rectangles represent different cis-elements that are potentially involved in stress or hormone regulation.\u003c/p\u003e","description":"","filename":"Figure.8.png","url":"https://assets-eu.researchsquare.com/files/rs-3407757/v1/2f99de2870cc9b4f06d75e07.png"},{"id":44391126,"identity":"1d9e4374-2ca6-4149-a787-67b6752ce3e8","added_by":"auto","created_at":"2023-10-10 20:25:30","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":597351,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression levels of 8 RNA helicase genes in response to abiotic stresses detected by qRT-PCR. The abiotic stress treatments include cold (4 °C), heat (42 °C), salt (200 mM NaCl) and drought (200 mM PEG4000). The expression levels at 0 h were normalized to 1, and the Y-axis delineates the fold changes of relative expression comparing with 0 h. Bars represent the mean of three biological replicates ± SE. The two-fold threshold is presented by a dotted line.\u003c/p\u003e","description":"","filename":"Figure.9.png","url":"https://assets-eu.researchsquare.com/files/rs-3407757/v1/4c9fca89c6e98429a6a8fbdb.png"},{"id":44391127,"identity":"21f46623-9243-41ac-a538-e2be621edcd0","added_by":"auto","created_at":"2023-10-10 20:25:30","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":627127,"visible":true,"origin":"","legend":"\u003cp\u003eRelative expression levels detected by qRT-PCR under different hormone treatments including GA, IAA, ABA and ZT. The expression levels at 0 h were normalized to 1, and the Y-axis delineates the fold changes of relative expression comparing with 0 h. Bars represent the mean of three biological replicates ± SE. The two-fold threshold is presented by a dotted line.\u003c/p\u003e","description":"","filename":"Figure.10.png","url":"https://assets-eu.researchsquare.com/files/rs-3407757/v1/3f9cfb54567dd471a70f8a64.png"},{"id":44389959,"identity":"a664e19f-ec37-44a0-8cb1-e919a365f1da","added_by":"auto","created_at":"2023-10-10 20:17:30","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":9493290,"visible":true,"origin":"","legend":"\u003cp\u003eSynteny analyses of \u003cem\u003eIbDEAD\u003c/em\u003e genes between sweetpotato and eight representative plant species from \u003cem\u003eIpomoea triloba\u003c/em\u003e and \u003cem\u003eIpomoea trifida \u003c/em\u003e\u003cstrong\u003e(A)\u003c/strong\u003e, \u003cem\u003eSolanum lycopersicum\u003c/em\u003eand \u003cem\u003eCapsicum annuum\u003c/em\u003e \u003cstrong\u003e(B)\u003c/strong\u003e, \u003cem\u003eBrassica rapa\u003c/em\u003e and \u003cem\u003eBrassica oleracea\u003c/em\u003e \u003cstrong\u003e(C)\u003c/strong\u003e, and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eOryza sativa\u003c/em\u003e \u003cstrong\u003e(D)\u003c/strong\u003e. The chromosomes of different plants are distinguished with differential colors. The blue lines connecting two different chromosomes highlight the syntenic \u003cem\u003eIbDEAD\u003c/em\u003e gene pairs within sweetpotato and other plant genomes, respectively.\u003c/p\u003e","description":"","filename":"Figure.11.png","url":"https://assets-eu.researchsquare.com/files/rs-3407757/v1/e1ef564b2e3d03f801d9e43a.png"},{"id":44390005,"identity":"b2bb5035-b67e-4fe4-9dd4-72b6e4e419dd","added_by":"auto","created_at":"2023-10-10 20:17:31","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":21985338,"visible":true,"origin":"","legend":"\u003cp\u003eSynteny analyses of \u003cem\u003eIbDEAH\u003c/em\u003e genes between sweetpotato and eight representative plant species from \u003cem\u003eIpomoea triloba\u003c/em\u003e and \u003cem\u003eIpomoea trifida \u003c/em\u003e\u003cstrong\u003e(A)\u003c/strong\u003e, \u003cem\u003eSolanum lycopersicum\u003c/em\u003eand \u003cem\u003eCapsicum annuum\u003c/em\u003e \u003cstrong\u003e(B)\u003c/strong\u003e, \u003cem\u003eBrassica rapa\u003c/em\u003e and \u003cem\u003eBrassica oleracea\u003c/em\u003e \u003cstrong\u003e(C)\u003c/strong\u003e, and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eOryza sativa\u003c/em\u003e \u003cstrong\u003e(D)\u003c/strong\u003e. The chromosomes of different plants are distinguished with differential colors. The blue lines connecting two different chromosomes highlight the syntenic \u003cem\u003eIbDEAH\u003c/em\u003e gene pairs within sweetpotato and other plant genomes, respectively.\u003c/p\u003e","description":"","filename":"Figure.12.png","url":"https://assets-eu.researchsquare.com/files/rs-3407757/v1/381822f28b42abb7adf73392.png"},{"id":44388280,"identity":"d02755cb-4d65-4839-ac36-fde96a7e1294","added_by":"auto","created_at":"2023-10-10 20:09:31","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":28706821,"visible":true,"origin":"","legend":"\u003cp\u003eSynteny analyses of \u003cem\u003eIbDExDH\u003c/em\u003e genes between sweetpotato and eight representative plant species from \u003cem\u003eIpomoea triloba\u003c/em\u003e and \u003cem\u003eIpomoea trifida \u003c/em\u003e\u003cstrong\u003e(A)\u003c/strong\u003e, \u003cem\u003eSolanum lycopersicum\u003c/em\u003eand \u003cem\u003eCapsicum annuum\u003c/em\u003e \u003cstrong\u003e(B)\u003c/strong\u003e, \u003cem\u003eBrassica rapa\u003c/em\u003e and \u003cem\u003eBrassica oleracea\u003c/em\u003e \u003cstrong\u003e(C)\u003c/strong\u003e, and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eOryza sativa\u003c/em\u003e \u003cstrong\u003e(D)\u003c/strong\u003e. The chromosomes of different plants are distinguished with differential colors. The blue lines connecting two different chromosomes highlight the syntenic \u003cem\u003eIbDExDH\u003c/em\u003e gene pairs within sweetpotato and other plant genomes, respectively.\u003c/p\u003e","description":"","filename":"Figure.13.png","url":"https://assets-eu.researchsquare.com/files/rs-3407757/v1/925d173e28d9b9fe24cc34a9.png"},{"id":44388271,"identity":"9dd785d3-ea57-42dd-ab53-f287ab2c2a54","added_by":"auto","created_at":"2023-10-10 20:09:30","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":87012,"visible":true,"origin":"","legend":"\u003cp\u003eHeat map representation for the organ-specific RNA helicase gene expression profiles in different sweetpotato varieties. YL, Young leaves, L, leaves, S, stems and R, roots. The expression levels of genes are presented using fold-change values transformed to Log\u003csup\u003e2 \u003c/sup\u003eformat. The Log\u003csup\u003e2\u003c/sup\u003e (fold-change values) and the color scale are shown at the bottom of heat map.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"Figure.14.png","url":"https://assets-eu.researchsquare.com/files/rs-3407757/v1/97b10dab532b1a830397a4a0.png"},{"id":44388272,"identity":"9d01536f-cf11-4ec5-95d6-8c5df389ccde","added_by":"auto","created_at":"2023-10-10 20:09:30","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1973105,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.zip","url":"https://assets-eu.researchsquare.com/files/rs-3407757/v1/3ab71794010bd4992797cf77.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide Systematic Survey and Analysis of the RNA Helicase Gene Family and their Response to Abiotic Stress in Sweetpotato","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe RNA helicases are ubiquitous in prokaryotes and eukaryotes, from viruses to humans, catalyzing the unwinding of duplex RNA dependent on the energy of NTP (Vashisht and Tuteja, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). RNA molecules undergo a series of modifications in the process of RNA metabolism, and their own instability is easy to cause RNA metabolic disorders, which make a difference in the development and different abilities to resist abiotic stress of plants (Nawaz and Kang, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2017\u003c/span\u003e;Gc et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe helicases are divided into 6 superfamilies, namely SF-1, -2, -3, -4, -5, and \u0026minus;\u0026thinsp;6. Among them, the most representative and largest family is SF2. According to the change of DEAD (Asp-Glu-Ala-Asp) motif, the sweetpotato RNA helicase superfamily is DEAD, DEAH and DExD / H, respectively (Rocak and Linder, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2004\u003c/span\u003e;Xu et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2013b\u003c/span\u003e). Almost all the helicase proteins contain nine conserved motifs (Tuteja and Tuteja, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Each of these nine motifs plays distinct roles, which are essential for helicase enzymatic activities (Tanner and Linder, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2001\u003c/span\u003e;Jiang et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Structurally RNA helicases are very similar, but their functions are very different. The RNA helicase is present during RNA splicing in RNA metabolism, ribosome formation, and nuclear cytoplasmic transport (de la Cruz et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1999\u003c/span\u003e;Lorsch, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2002\u003c/span\u003e;Sahoo et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These genes have many functions in RNA metabolism, among which RNA helicase is associated with growth and development and resistance to stress.\u003c/p\u003e \u003cp\u003eIn \u003cem\u003eArabidopsis\u003c/em\u003e, DEAD-box LOS4 is able to participate in the process of cryogenic stress, flowering, vernalization, and other processes (Gong et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2002\u003c/span\u003e;Gong et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Among them, in the cold stress response pathway, LOS4-1 and LOS4-2 can regulate the expression of C repeat binding factors and their downstream target genes. LOS RNA helicase plays a key role in target gene output, maturation and reaction to temperature stress. The transcription of STRS1 and STRS2 was inhibited under salt stress. The salt tolerance of mutants strs1 and strs2 was enhanced, and the expression of RD29A, DREB1A and DREB2A was enhanced (Kant et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). The development of the germ and leaf of the \u003cem\u003eArabidopsis\u003c/em\u003e rh7 mutant was seriously delayed under low temperature stress (Liu et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). AtRH3 plays a role in ribosome formation and seedling growth and development, the growth of rh3 mutants was severely inhibited under salt or cold stress (Gu et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Studies have shown that \u003cem\u003eArabidopsis\u003c/em\u003e TEBICHI is necessary for regulating cell division and meristem (Inagaki et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), ISE2 is involved in the function of the plasmodesmata during \u003cem\u003eArabidopsis\u003c/em\u003e embryogenesis (Kobayashi et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2007\u003c/span\u003e;Carlotto et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Our previous findings showed that the tolerance of tomato SlDEAD31 was enhanced in salt and drought stress, and the expression of stress genes was increased such as \u003cem\u003eCat1\u003c/em\u003e, \u003cem\u003eCat2\u003c/em\u003e, \u003cem\u003eAPX2\u003c/em\u003e, and \u003cem\u003eER5\u003c/em\u003e (Zhu et al., \u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The rice SUV3 protein has DNA and RNA helicase and ATPase activities, and \u003cem\u003eSUV3\u003c/em\u003e expression can be induced by salt stress (Tuteja et al., \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2013\u003c/span\u003e;Sahoo et al., \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Low temperature and high salt stress can induce the expression of GmRH in soybeans, and GmRH plays a significant in RNA processing (Chung et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Tobacco P68 can improve plant growth, photosynthesis, stimulate antioxidant mechanism, and enhance tolerance to salt stress (Tuteja et al., \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). AvDH1 increased salt tolerance and played an important role in boll number, boll weight and seed yield (Chen et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The \u003cem\u003eArabidopsis\u003c/em\u003e RCF1 gene plays an integral role in maintaining normal splicing of mRNA precursors, and some cold stress-induced genes were error spliced in the rcf-1 mutant (Guan et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Maize DRH1 can interact with the nucleoprotein fiber MA16, which is involved in ribosomal RNA metabolism (Gendra et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). DEVH-box RNA helicase AtHELPS play a key role in K\u003csup\u003e+\u003c/sup\u003e deprivation in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Xu et al., \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe RNA helicase has been nominated in many plant variety namely \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Boudet et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), \u003cem\u003eOryza sativa\u003c/em\u003e (Umate et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), \u003cem\u003eIpomoea trifida\u003c/em\u003e (Wan et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), \u003cem\u003eGlycine max\u003c/em\u003e (Xu et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2013a\u003c/span\u003e), \u003cem\u003eZea mays\u003c/em\u003e (Xu et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2013a\u003c/span\u003e), \u003cem\u003eGossypium spp\u003c/em\u003e (Chen et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), soybean (Karthik et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), \u003cem\u003eGossypium raimondii\u003c/em\u003e (Chen et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) and \u003cem\u003eSolanum lycopersicum\u003c/em\u003e (Xu et al., \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2013b\u003c/span\u003e). A total of 32 DEAD were initially identified in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Aubourg et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Then, 113 and 115 RNA helicase genes were identified in \u003cem\u003eArabidopsis\u003c/em\u003e and \u003cem\u003eOryza sativa\u003c/em\u003e (Umate et al., \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Studies have shown that RNA helicase genes in \u003cem\u003eArabidopsis\u003c/em\u003e, \u003cem\u003eOryza sativa\u003c/em\u003e, \u003cem\u003eGossypium spp\u003c/em\u003e, \u003cem\u003eGossypium raimondii\u003c/em\u003e and \u003cem\u003eZea mays\u003c/em\u003e are divided into three subfamilies, the numeral of genes in apiece subfamily is as follows: DEAD-box (50, 51, 87, 51, and 57 genes), DEAH-box (40, 33, 48, 52, and 31 genes), and DExDH-box (71, 65, 78, 58, and 50 genes).\u003c/p\u003e \u003cp\u003eSweetpotato (\u003cem\u003eIpomoea batatas\u003c/em\u003e (L.) Lam.) is an important food source and industrial raw material with high economic value (Katayama et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Sweetpotato is a hexaploid with 90 chromosomes, high heterozygosity, and a large number of repetitive sequences (Isobe et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2017\u003c/span\u003e;Yan et al., \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), thus hindering gene identification and functional studies. The RNA helicase is a ubiquitous protein that involved in plant growth and abiotic stress. Sweetpotato is susceptible to abiotic stress, which plays a significant in the growth of potato chips (Ramamoorthy et al., \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). At present, however, genome-wide identification of sweetpotato RNA helicase genes has not been communicated. To improve the yield of sweetpotato, genomic assisted breeding technology can be used to develop new or improved sweetpotato varieties. To explore the biological basis of cold resistance in sweetpotato, it is consequence to recognize differentially expressed genes in response to low temperature stress and apply them to production. Therefore, the main molecules are the recognition of proteins and enzymes, it is very important for these molecules to control a large number of metabolic pathways by regulating the occurrence and metabolism of RNA. The RNA helicases are concerned in many molecular functions, including tolerance, and regulation of development. Their identification in sweetpotato and improvement of sweetpotato varieties are of great significance and practical value.\u003c/p\u003e \u003cp\u003eTherefore, in order to comprehend the purpose and participation pathway of the RNA helicase genes in sweetpotato, this study aims to conduct genome-wide confirmation of the RNA helicase genes in sweetpotato, and to analyze the molecular mechanism of sweetpotato participation.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Identification of the RNA helicase genes in sweetpotato genomes\u003c/h2\u003e \u003cp\u003eThe whole sweetpotato genome sequence was derivative by Ipomoea genome Hub (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ipomoea-genome.org\u003c/span\u003e\u003cspan address=\"https://ipomoea-genome.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Yang et al., \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). To identify members of the RNA helicase gene family in \u003cem\u003eIpomoea batatas\u003c/em\u003e, we used BLASTP to search all known \u003cem\u003eArabidopsis\u003c/em\u003e and rice RNA helicase gene sequences in multiple databases (Altschul et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1990\u003c/span\u003e). And all the information about the RNA helicase genes in \u003cem\u003eArabidopsis\u003c/em\u003e (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.arabidopsis.org/\u003c/span\u003e\u003cspan address=\"https://www.arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and rice (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rice.plantbiology.msu.edu/\u003c/span\u003e\u003cspan address=\"http://rice.plantbiology.msu.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) was downloaded (Xu et al., \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Subsequently, all protein were covered and each member of the RNA helicase gene was verified using the Pfam database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.xfam.org/\u003c/span\u003e\u003cspan address=\"http://pfam.xfam.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), the CD-search (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/cdd/Structure/cdd/wrpsb.cgi\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/cdd/Structure/cdd/wrpsb.cgi\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and the PROSITE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://prosite.expasy.org/\u003c/span\u003e\u003cspan address=\"https://prosite.expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), and members lacking typical conserved RNA helicase domains were deleted. The sequence information of all sweetpotato RNA helicase proteins can be found in the \u003cb\u003eSupplementary File. 1\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Phylogenetic relationships of RNA helicase proteins in sweetpotato\u003c/h2\u003e \u003cp\u003eThe RNA helicase sequence was aligned using the Clustal X program. The MUSCLE program was used for multiple sequence alignment to support Clustal X (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.clustal.org/\u003c/span\u003e\u003cspan address=\"http://www.clustal.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Edgar, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Phylogenetic trees were constructed employing the maximum likelihood (ML) method (Guindon et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Protein property and conserved domain of helicase genes in sweetpotato\u003c/h2\u003e \u003cp\u003eThe physicochemical properties of the RNA helicase proteins were forecasted by the online ExPASy database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://expasy.org/\u003c/span\u003e\u003cspan address=\"http://expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Prediction of subcellular sites (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.csbio.sjtu.edu.cn/bioinf/plant-multi/\u003c/span\u003e\u003cspan address=\"http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and phosphorylation sites (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.cbs.dtu.dk/services/NetPhos/\u003c/span\u003e\u003cspan address=\"http://www.cbs.dtu.dk/services/NetPhos/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) of the RNA helicase genes. The gene structure was obtained by comparing the sequence of the RNA helicase genes with the genome sequence. The consequence was realized by Tbtools (Chen et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The conserved domain was determined using MEME (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://meme-suite.org/meme/tools/meme\u003c/span\u003e\u003cspan address=\"https://meme-suite.org/meme/tools/meme\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Bailey et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Then, the protein interacting networks were ensured by STRING (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Chromosomal location and collinearity analysis of the RNA helicase genes in sweetpotato\u003c/h2\u003e \u003cp\u003eIsoelectric points and molecular weights of these proteins are obtained by ExPASy (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://expasy.org/\u003c/span\u003e\u003cspan address=\"http://expasy.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) (Gasteiger et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The structural intelligence of these sweetpotato RNA helicase genes was analyzed together with genomic data. To examine the collinearity both RNA helicase gene and other plant genes, The genome sequence information of sweetpotato, \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and rice was downloaded and examined. MCScanX was used to create gene duplication and collinearity relationships through failure parameters (Wang et al., \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), data results are envisaged by TBtools (Krzywinski et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e;Chen et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Default parameters were used in all steps.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 qRT‑PCR ascertain of cold stress\u003c/h2\u003e \u003cp\u003eThe experimental materials were XuShu 18 roots, different cold stress treatment methods have been described in our previous reports (Xie et al., \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Four abiotic stress treatments and four hormone stress treatments were carried out on XuShu 18, and samples were taken after 0h, 1h, 3h, 6h, 12h, 24h, 48h and 72h, respectively. Ten different varieties of sweetpotato were planted, and their young leaves, leaves, stems and roots were taken respectively. RNA of these samples was extracted for subsequent experiments.\u003c/p\u003e \u003cp\u003eThe total RNA was excavated using RNA extraction kit (TianGen, Beijing, China), and reverse transcriptions using TransScript\u0026reg; gDNA removal (TransGen, Beijing, China). All the sweetpotato RNA helicase promoter regions were examined by plantCARE (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical reasoning was achieved by Microsoft Excel 2019, Graphpad Prism 5.0 and SPSS statistical. Considering the biological significance, differential gene expression using two times the cut-off value.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 Identification of RNA helicase family genes in sweetpotato\u003c/h2\u003e\n \u003cp\u003eTo recognize the elements of the sweetpotato RNA helicase gene, we used bioinformatics methods to collect details on many sweetpotato RNA helicases. A total of 300 sweetpotato RNA helicase proteins were detected. According to the RNA helicases conserved motifs, 300 sweetpotato RNA helicase genes were divided into subfamilies DEAD-box (53 DEAD genes), DEAH-box (54 DEAH genes) and DExDH-box (193 DExDH genes) subfamilies (\u003cstrong\u003eSupplementary File. 1\u003c/strong\u003e). The amount of RNA helicase proteins in \u003cem\u003eArabidopsis\u003c/em\u003e and rice was 113 and 115, respectively. It is predicted that there are 300 RNA helicase proteins in sweetpotato, which is much more than the number of \u003cem\u003eArabidopsis\u003c/em\u003e and rice (Umate et al., \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). Then, the 300 RNA helicase genes on 15 sweetpotato chromosomes were named from top to bottom as \u003cem\u003eIbDEAD1\u003c/em\u003e\u0026thinsp;~\u0026thinsp;\u003cem\u003eIbDEAD53\u003c/em\u003e, \u003cem\u003eIbDEAH1\u003c/em\u003e\u0026thinsp;~\u0026thinsp;\u003cem\u003eIbDEAH54\u003c/em\u003e and \u003cem\u003eIbDExDH1\u003c/em\u003e\u0026thinsp;~\u0026thinsp;\u003cem\u003eIbDExDH193\u003c/em\u003e (\u003cstrong\u003eSupplementary Fig.\u0026nbsp;1\u003c/strong\u003e). Subsequently, the physicochemical properties of 300 sweetpotato RNA helicase proteins were examined. The length and relative molecular mass of the RNA helicase vary greatly. The length of DEAD ranges from 323 aa (IbDEAD34) to 1301 aa (IbDEAD2), the relative molecular mass ranges from 3584.09 to 145261.6 Da, and the isoelectric point ranges from 5.02 (IbDEAD40) to 9.87 (IbDEAD37). The length of DEAH is between 206aa (IbDEAH5) and 2904aa (IbDEAH44), the relative molecular mass is between 23377.03 and 323370.3Da, and the isoelectric point is between 5.16 (IbDEAH22) and 9.25 (IbDEAH36). The length of DExDH ranged from 128aa (IbDExDH7) to 2801aa (IbDExDH30), the relative molecular mass ranged from 13904.77 to 306450.01 Da, and the isoelectric point graded from 4.81 (IbDExDH7) to 9.67 (IbDExDH45). The subcellular location showed that most of the RNA helicase proteins were positioned in the nucleus. Furthermore, the potential phosphorylation sites showed that IbDEAD contains 29 (IbDEAD28) to 141 (IbDEAD2) phosphorylation sites, IbDEAH contains 19 (IbDEAH5) to 280 (IbDEAH44) phosphorylation sites, and IbDExDH contains 18 (IbDExDH141) to 362 (IbDExDH30) phosphorylation sites, all of these sweetpotato RNA helicase proteins restrain more Ser sites than the Tyr and Thr sites (\u003cstrong\u003eSupplementary Table. 1\u003c/strong\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Phylogenetic analysis of the RNA helicase family proteins in sweetpotato\u003c/h2\u003e\n \u003cp\u003eThe rootless phylogenetic tree of the identified RNA helicase in sweetpotato and the known RNA helicase in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e was constructed to study the evolution and classification of RNA helicase in sweetpotato (Fig.\u0026nbsp;1). Appertaining to the classification of the RNA helicase in \u003cem\u003eArabidopsis\u003c/em\u003e, they were separated into 3 subfamilies: DEAD, DEAH and DExDH. 53 IbDEAD proteins were separated into 9 subgroups (except IbDEAD5), 54 IbDEAH proteins were separated into 9 subgroups (except \u003cem\u003eIbDEAH36\u003c/em\u003e), and 193 IbDExDH proteins were separated into 13 subgroups (except \u003cem\u003eIbDExDH19\u003c/em\u003e and \u003cem\u003eIbDExDH23\u003c/em\u003e), the V subgroup in IbDEAH and the Ⅻ in IbDExDH are unique to sweetpotato. RNA helicase proteins are widely and unevenly distributed in different subfamilies. IbDEAD and IbDEAH have nine subgroups, and VIII is the largest subgroup. The sweetpotato RNA helicase genes contained in subgroup VI, VIII and Ⅺ of IbDExDH were the least. Interestingly, \u003cem\u003eIbDEAD5\u003c/em\u003e, \u003cem\u003eIbDEAH36\u003c/em\u003e, \u003cem\u003eIbDExDH19\u003c/em\u003e and \u003cem\u003eIbDExDH23\u003c/em\u003e do not exist in any of the above three subfamilies, which suggesting that they may have other functions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 Gene structure and motif composition analyses in sweetpotato\u003c/h2\u003e\n \u003cp\u003eTo further understand the structural characteristics of the sweetpotato RNA helicase family, we paralleled the composition of conserved motifs and introns / exons on the basis of the phylogenetic tree of sweetpotato RNA helicase (Figs.\u0026nbsp;2 and 4). The structures of the IbDEAD, IbDEAH, and IbDExDH subfamily genes are quite different between different subgroups, but the genes in the same side branch are homologous genes, and their genetic structures is also similar, indicating that the exon-intron structure is highly correlated with phylogenetic relationships (Figs.\u0026nbsp;2 and 4). The genetic structure of the RNA helicase family members is relatively complex, which the \u003cem\u003eIbDEAD\u003c/em\u003e and \u003cem\u003eIbDEAH\u003c/em\u003e gene members contain multiple exons, the \u003cem\u003eIbDExDH\u003c/em\u003e gene members have only five genes with only one exon (2.6%), and other genes contain multiple exons. Next, we analyzed the motifs of each member of these RNA helicases on the MEME website, and found that all sweetpotato RNA helicases contain 15 conserved motifs (Figs.\u0026nbsp;2 and 4). Motifs 1, 2, 3, 4, 5, 7, 8, 9 and 15 are current in nearly all proteins, and motif 5 contains extremely conserved sequences DEAD, DEAH, and DExD / H. These conserved motifs are the same in sweetpotato RNA helicase, but the domains and amino acid sequences of these maintained motifs are very different (Figs.\u0026nbsp;2 and 4). In general, phylogenetic tree examination of data shows that the system development and features and the divergence of genetic structure and sequence distribution are closely related.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 Chromosome localization of the RNA helicase family in sweetpotato\u003c/h2\u003e\n \u003cp\u003ePhysical location detection based on GFF3 genome annotation showed that 53 \u003cem\u003eIbDEAD\u003c/em\u003e genes, 54 \u003cem\u003eIbDEAH\u003c/em\u003e genes and 193 \u003cem\u003eIbDExDH\u003c/em\u003e genes were located on all 15 chromosomes. Among them, Chr6 in \u003cem\u003eIbDEAD\u003c/em\u003e contains the most abundant \u003cem\u003eIbDEAD\u003c/em\u003e genes, with 7 members. Chr11 in \u003cem\u003eIbDEAH\u003c/em\u003e contains the most abundant \u003cem\u003eIbDEAH\u003c/em\u003e genes, with 9 members. However, the \u003cem\u003eIbDEAD\u003c/em\u003e and \u003cem\u003eIbDEAH\u003c/em\u003e genes are not distributed in Chr9. Most chromosomes are rich in \u003cem\u003eIbDExDH\u003c/em\u003e genes, but Chr10 contains only four \u003cem\u003eIbDExDH\u003c/em\u003e genes (Figs.\u0026nbsp;5 and \u003cstrong\u003eSupplementary Fig.\u0026nbsp;2\u003c/strong\u003e). The distribution of the three subfamilies of RNA helicases on 15 chromosomes is quite different. Description of collinearity analysis manifested that there was a group of tandem duplicated \u003cem\u003eIbDEAH\u003c/em\u003e and two groups of tandem duplicated \u003cem\u003eIbDExDHs\u003c/em\u003e, which were \u003cem\u003eIbDEAH2/24\u003c/em\u003e, \u003cem\u003eIbDExDH114/168\u003c/em\u003e and \u003cem\u003eIbDExDH162/185\u003c/em\u003e. However, no tandem duplicated gene was found in \u003cem\u003eIbDEAD\u003c/em\u003e \u003cstrong\u003e(Supplementary Table. 7\u003c/strong\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 Cis element analysis of the RNA helicase family gene promoters\u003c/h2\u003e\n \u003cp\u003eTo explore the possible regulatory mechanism of sweetpotato RNA helicase on abiotic stress and hormones, we scanned the cis-acting elements in the 2kb promoter upriver of the sweetpotato RNA helicase gene through the PlantCare database (Figs. 6 and 8, \u003cstrong\u003eSupplementary Table. 2\u003c/strong\u003e).\u003c/p\u003e\n \u003cp\u003eThe conclusions showed that each promoter region of RNA helicase had multiple cis-acting elements connected with stress- and / or hormones. Among others, nearly 90% of RNA helicase promoters contain multiple stress cis-elements, for instance stress response elements (TC-rich repeats), low temperature response elements (LTR), MAJA response elements (TGACG-motif), drought response elements (MBS), etc. These cis-acting elements can be associated with expression. For example, the expression of several RNA helicase genes providing \u003cem\u003eIbDEAH-32 / -42\u003c/em\u003e and \u003cem\u003eIbDExDH-36 / -47 / -96\u003c/em\u003e was increased under different stresses. Correspondingly, stress-related repeats of MBS, TC, or LTR cis elements are more numerous in their promoter regions. However, TC-rich repeats, MAJA response elements and LTR elements were found on the promoters of the \u003cem\u003eIbDExDH-25\u003c/em\u003e / \u003cem\u003e-48\u003c/em\u003e and \u003cem\u003eIbDEAH53\u003c/em\u003e genes, their expression was not particularly significant under salt, drought, or cold stress, but there was a certain degree of response under high-temperature stress (Fig. 9). Furthermore, all sweetpotato RNA helicase promoters restrain many hormone elements, such as abscisic acid response element (ABRE), Me-JA response element (CGTCA motif and TGACG motif) or auxin response element (TGA-box) (Figs. 6 and 8, \u003cstrong\u003eSupplementary Table. 2\u003c/strong\u003e). Nevertheless, the expression of the RNA helicase genes in dissimilar tissues of 10 different sweetpotato varieties was also different (\u003cstrong\u003eFigure. 14\u003c/strong\u003e). Most of the RNA helicase genes are communicated in high amounts in sweetpotato stems and root tissues, in particular, the expression levels of \u003cem\u003eIbDExDH36\u003c/em\u003e and \u003cem\u003eIbDExDH48\u003c/em\u003e in the roots were lower than those in other tissues. It shows that RNA helicase is associated to plant development. Among them, the transcription levels of most RNA helicase genes do not respond significantly to hormonal treatment (Fig. 10). These data indicate that the cis-acting elements of sweetpotato RNA helicase can be concerned in both hormonal and abiotic stresses.\u003c/p\u003e\u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003e3.6 Identification of RNA helicase family cold-response genes in transcriptomes and their expression profiles under multiple abiotic stress and hormonal treatments\u003c/strong\u003e\u003c/p\u003e\n \u003c/span\u003e\n \u003cp\u003eMany studies have shown that the RNA helicase have an influence in various abiotic stresses (such as cold, drought, and heat) and development. To determine the potential biological function of the sweetpotato RNA helicase gene under adversity stress, based on our previous RNA-seq data, we first studied the expression of XuShu 18 under cold stress (Xie et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). The consequence indicated that eight RNA helicase genes were screened out. Subsequently, we further studied the expression patterns of eight genes (\u003cem\u003eIbDEAH-32 / -42 / -53\u003c/em\u003e, \u003cem\u003eIbDExDH-25 / -36 / -47 / -48 / -96\u003c/em\u003e) screened by qRT-PCR under salt, drought, heat and cold stresses, and explore a two-fold cut off value (Zhu et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). The results showed that all selected genes were up-regulated to varying degrees after salt, drought, cold, and heat treatment. Among them, four stresses could up-regulate \u003cem\u003eIbDEAH32\u003c/em\u003e and \u003cem\u003eIbDExDH-36 / -47\u003c/em\u003e transcription, two stresses could up-regulate \u003cem\u003eIbDEAH42\u003c/em\u003e and \u003cem\u003eIbDExDH-48 / -96\u003c/em\u003e expression, and one stress could up-regulate \u003cem\u003eIbDExDH25\u003c/em\u003e and \u003cem\u003eIbDEAH53\u003c/em\u003e transcription. All RNA helicases could be up-regulated under high temperature stress and salt stress. In particular, \u003cem\u003eIbDEAH32\u003c/em\u003e and \u003cem\u003eIbDExDH47\u003c/em\u003e had the highest level of induction after cold treatment, about six times, while the level of induction was relatively low in the transcription of other RNA helicases. Except for \u003cem\u003eIbDExDH48\u003c/em\u003e, the expression of other genes was significantly enhanced under high temperature conditions, which was 2.3 and 19.3-fold that of the control. Under salt stress conditions, the expression of \u003cem\u003eIbDEAH42\u003c/em\u003e and \u003cem\u003eIbDExDH-47 / -48\u003c/em\u003e was enhanced, and the expression of other genes was weakly induced by salt stress. In particular, the response of all genes to drought stress was not very strong, which may be affected by other factors (Fig.\u0026nbsp;9). Overall, these data suggest that multiple sweetpotato RNA helicase members can take the lead in reaction to abiotic stress.\u003c/p\u003e\n \u003cp\u003eMoreover, qRT-PCR was acclimated to further detect the transcription profiles of eight RNA helicase genes under distinct hormone treatments, encircling ABA, IAA, GA and ZT. Unexpectedly, most of the RNA helicase genes were down-regulated when we used double as the cut-off value for differential expression. Just the stress hormone ZT could prompt the expression of \u003cem\u003eIbDExDH-36 / -47 / -48 / -98\u003c/em\u003e and \u003cem\u003eIbDEAH42\u003c/em\u003e (Fig.\u0026nbsp;10). It is reported that the response of the RNA helicase to hormone treatment is not obvious, mainly related to development and abiotic stress(Camborde et al., \u003cspan class=\"CitationRef\"\u003e2022\u003c/span\u003e;Li et al., \u003cspan class=\"CitationRef\"\u003e2022b\u003c/span\u003e). Overall, these data propose that multiple members of the sweetpotato RNA helicase can be important players in answer to hormone and / or abiotic stresses.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n \u003ch2\u003e3.7 Collinearity analysis of the RNA helicase genes between sweetpotato and other plants\u003c/h2\u003e\n \u003cp\u003eTo furthermore explore the origin and evolutionary mechanism of sweetpotato RNA helicase genes, we compared the homology of 300 RNA helicase genes with 8 representative species-related genes. These species include wild diploid relatives of sweetpotato (\u003cem\u003eIpomoea trioba\u003c/em\u003e and \u003cem\u003eIpomoea trifida\u003c/em\u003e), two model plants (\u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eOryza sativa\u003c/em\u003e), two cruciferous plants (\u003cem\u003eBrassica rapa\u003c/em\u003e and \u003cem\u003eBrassica oleracea\u003c/em\u003e) and two Solanaceae plants (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e and \u003cem\u003eCapsicum annuum\u003c/em\u003e). Among them, 42 (79.2%) and 43 (81.1%) \u003cem\u003eIbDEAD\u003c/em\u003e genes were homologous to genes in \u003cem\u003eIpomoea trioba\u003c/em\u003e and \u003cem\u003eIpomoea trifida\u003c/em\u003e, respectively, accompanied by \u003cem\u003eSolanum lycopersicum\u003c/em\u003e (8), \u003cem\u003eCapsicum annuum\u003c/em\u003e (6), \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (6), \u003cem\u003eBrassica rapa\u003c/em\u003e (2), and \u003cem\u003eBrassica oleracea\u003c/em\u003e (1), but no homologous genes were found both sweetpotato and rice. 39 (72.2%) and 40 (74.1%) \u003cem\u003eIbDEAH\u003c/em\u003e genes were also homologous to the genes in \u003cem\u003eIpomoea trioba\u003c/em\u003e and \u003cem\u003eIpomoea trifida\u003c/em\u003e, respectively, accompanied by \u003cem\u003eSolanum lycopersicum\u003c/em\u003e (8), \u003cem\u003eCapsicum annuum\u003c/em\u003e (3) and \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (3), nevertheless, no similar homologous genes were noted between sweetpotato and \u003cem\u003eBrassica rapa\u003c/em\u003e, cabbage and rice. Similarly, 89 (46.1%) and 87 (45.1%) \u003cem\u003eIbDExDH\u003c/em\u003e genes were homologous to the genes in \u003cem\u003eIpomoea trioba\u003c/em\u003e and \u003cem\u003eIpomoea trifida\u003c/em\u003e, followed by tomato (24), pepper (10), \u003cem\u003eArabidopsis\u003c/em\u003e (7), \u003cem\u003eBrassica rapa\u003c/em\u003e (3), and cabbage (2), however, no homologous genes were found with rice (Figs. 11 and 13). It should be mentioned that the collinearity of sweetpotato RNA helicase genes between the \u003cem\u003eIpomoea trioba\u003c/em\u003e and \u003cem\u003eIpomoea trifida\u003c/em\u003e genes more than the extra six varieties, which can be associated to the wild diploid relationship of sweetpotato.\u003c/p\u003e\n \u003cp\u003eMoreover, the mass of the RNA helicase genes in our sweetpotato are one-to-one gene pairs with sweetpotato diploids, and a few are one sweetpotato gene corresponding to two or three diploid gene pairs. For example, the sweetpotato genes \u003cem\u003eIbDEAD\u003c/em\u003e (10), \u003cem\u003eIbDEAH\u003c/em\u003e (2) and \u003cem\u003eIbDExDH\u003c/em\u003e (19) have a one-to-many relationship in \u003cem\u003eIpomoea trioba\u003c/em\u003e, and \u003cem\u003eIbDEAD\u003c/em\u003e (10), \u003cem\u003eIbDEAH\u003c/em\u003e (2) and \u003cem\u003eIbDExDH\u003c/em\u003e (17) sweetpotato genes have a one-to-many relationship in \u003cem\u003eIpomoea trifida\u003c/em\u003e (\u003cstrong\u003eSupplementary Table. 3\u003c/strong\u003e). Interestingly, we found that \u003cem\u003eIbDEAH\u003c/em\u003e did not find some collinear gene pairs between sweetpotato and \u003cem\u003eOryza sativa\u003c/em\u003e / \u003cem\u003eBrassica rapa\u003c/em\u003e / \u003cem\u003eBrassica oleracea\u003c/em\u003e, and there was no collinear gene pair between sweetpotato RNA helicase and rice.\u003c/p\u003e\u003cspan\u003e\n \u003cp\u003e\u003cstrong\u003e3.8 Tissue expression patterns of cold-response genes of the RNA helicase family of different sweet potato cultivars\u003c/strong\u003e\u003c/p\u003e\n \u003c/span\u003e\n \u003cp\u003eObjective to preliminarily understand the role of the RNA helicase gene in the development stage of sweetpotato, the expression contours of the RNA helicase genes in diverse tissues of 10 sweetpotato varieties were analyzed by qRT-PCR, such as young leaves, leaves, stems and roots. The expression contours of eight RNA helicase genes were grouped together on their respective heat maps (Fig.\u0026nbsp;14). The expression of the RNA helicase in various tissues is different, and the expression in different varieties of sweetpotato is also quite different. This also shows that the RNA helicase is related to growth and development (Tyagi et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e;Liu et al., \u003cspan class=\"CitationRef\"\u003e2023\u003c/span\u003e). Some the RNA helicase genes were highly communicated in stems and roots of sweetpotato, such as \u003cem\u003eIbDExDH25\u003c/em\u003e, \u003cem\u003eIbDEAH42\u003c/em\u003e, \u003cem\u003eIbDExDH47\u003c/em\u003e, and \u003cem\u003eIbDExDH96\u003c/em\u003e. Similarly, some RNA helicase genes were also highly communicated in sweetpotato leaves, including \u003cem\u003eIbDExDH36\u003c/em\u003e, \u003cem\u003eIbDExDH48\u003c/em\u003e, and \u003cem\u003eIbDEAH53\u003c/em\u003e. This shows that the expression of the RNA helicase in unequal growth stages of sweetpotato is diverse and have an influence in the development stage of plants. This is steady with the results of other plants in RNA helicase. HS3 is located in the DEAD-box RNA helicase 22 in \u003cem\u003eArabidopsis\u003c/em\u003e plastids, which is necessary for proper accumulation of plastid gene mRNA throughout seed germination and plant growth (Kanai et al., \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e;Iglesias-Fern\u0026aacute;ndez et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). The RNA helicase can also concern the development of plants under chilling stress. AtRH7, one of the RNA helicases in \u003cem\u003eArabidopsis\u003c/em\u003e, is an RNA chaperone involved in cold adaptation (Huang et al., \u003cspan class=\"CitationRef\"\u003e2016b\u003c/span\u003e). The mutation of rh7 affects the abnormal development of flowers in \u003cem\u003eArabidopsis\u003c/em\u003e thaliana, and makes the leaves of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e smaller in chilling stress (Liu et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe RNA helicases are ubiquitous in miscellaneous organisms. It is involved in almost all processes of RNA metabolism, such as transcription, mRNA splicing and output, mRNA translation, etc. It involves almost all aspects of organisms (Vashisht and Tuteja, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2006\u003c/span\u003e;Sloan and Bohnsack, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2018\u003c/span\u003e;Donsbach and Klostermeier, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In the past, the study of the RNA helicase family in plants mainly focuses on dicotyledonous model plant Arabidopsis thaliana and monocotyledonous rice (Nawaz and Kang, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019b\u003c/span\u003e;Takagi et al., \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2020\u003c/span\u003e;Banu et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e), these play an essential role in plant growth and stress response (Kim et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2008\u003c/span\u003e;Li et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). Sweetpotato is a significant food crop, which is broadly used in food, feed and industrial raw materials. It is the seventh largest food crop in the world, with the strong ability to adapt to the environment, varieties of high yield, strong stress resistance (Yu et al., \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e2020\u003c/span\u003e;Banu et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e). However, only 17 RNA helicase genes have been nominated in \u003cem\u003eIpomoea trifida\u003c/em\u003e (Wan et al., \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). There is no comprehensive study on RNA helicase in sweetpotato. This study systematically identified RNA helicase genes in sweetpotato, which laid a solid basis for further study on the engagement of the RNA helicase in plant abiotic stress and development.\u003c/p\u003e \u003cp\u003eIn the present study, we conducted a comprehensive reasoning of the sweetpotato RNA helicase gene family, containing phylogenetic tree, expression profiles of different sweetpotato varieties under common growth circumstances, and expression profiles under diverse abiotic stresses and hormone stresses. Firstly, 300 RNA helicase genes were nominated in sweetpotato genome, which is a very large gene family. A larger family of the RNA helicase gene exists in sweetpotato, suggesting that RNA helicase plays a leading role in regulating environmental responses. According to the family classification of \u003cem\u003eArabidopsis\u003c/em\u003e and rice RNA helicases (Xu et al., \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2013a\u003c/span\u003e), they were divided into three subfamilies, including IbDEAD (53), IbDEAH (54) and IbDExDH (193) (\u003cb\u003eSupplementary File. 1\u003c/b\u003e). The chromosomal mapping of 300 sweetpotato RNA helicase genes was mapped, and the distribution of the RNA helicase on 15 sweetpotato chromosomes was analyzed. Chromosome localization reasoning proved that sweetpotato RNA helicase gene was scattered on all 15 chromosomes. Among them, IbDEAD was the most distributed on LG4 and LG6 chromosomes (7 genes), and the least distributed on LG2, LG8, LG10, LG12, and LG14 chromosomes (2 genes). IbDEAH was the most distributed on the LG11 chromosome (9 genes), and the least distributed on the LG4, LG5, and LG12 chromosomes (2 genes). IbDExDH was equally scattered on the sweetpotato chromosomes (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e).\u003c/p\u003e \u003cp\u003eThe sweetpotato RNA helicase gene contains different numbers of exons and different lengths of introns (Figs.\u0026nbsp;2 and 4). Intriguingly, five genes in the IbDExDH subfamily contain only one exon, while the IbDEAD and IbDEAH subfamilies do not contain this case and contain multiple exons. In fact, the length of RNA helicase family proteins varies greatly. IbDEAD is 323 to 1301 amino acids, IbDEAH is 206 to 2904 amino acids, and IbDExDH is 128 to 2801 amino acids. The highly different amino acid sequences of these sweetpotato RNA helicases induce diverse protein structures and functions in dissimilar developmental and stress-resistant environments. At the same time, the sweetpotato RNA helicase motif was analyzed, the most conserved motif in different species is Asp-Glu-Ala-Asp, which is divided into three subfamilies according to its difference (Tanner and Linder, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2001\u003c/span\u003e;Linder and Jankowsky, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2011\u003c/span\u003e;Xu et al., \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). According to the structural characteristics and phylogenetic analysis of the Motif V region, the determined helicases can be moreover divided into these subfamilies, comprising IbDEAD, IbDEAH, and IbDExDH. Phylogenetic analysis showed that IbDEAD, IbDEAH and IbDExDH RNA helicase proteins can be moreover divided into nine, or thirteen great subgroups (Fig.\u0026nbsp;1). The gene structure results showed that the main RNA helicase genes in \u003cem\u003eArabidopsis\u003c/em\u003e were uniform to the AtRH family genes (Aubourg et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), but the position and length of introns were not fixed. However, this is the first genome-wide examination of the RNA helicase genes family in sweetpotato. The distinct subfamilies and gene structures of sweetpotato RNA helicase genes indirectly indicate the different functions in RNA metabolism, stress resistance and growth and development.\u003c/p\u003e \u003cp\u003eIn fact, we establish that most RNA helicase gene promoters contain some cis-regulatory elements, such as plant development, abiotic stress, plant hormones, and light response elements(Huang et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e;Nawaz and Kang, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2019a\u003c/span\u003e). It is worth noting that IbDEAD and IbDEAH have more cis-regulatory elements associated to low temperature and light response, and IbDExDH has more cis-acting elements associated with abscisic acid and plant hormones (Figs.\u0026nbsp;6 and 8). According to the study that temperature, abscisic acid and jasmonic acid are involved in abiotic stress processes in plants (Bari and Jones, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2009\u003c/span\u003e;Nidumukkala et al., \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2019\u003c/span\u003e;Bharath et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Under hormone and abiotic stress treatments, qRT-PCR data showed that RNA helicase mainly responded to abiotic stress (Figs.\u0026nbsp;9 and 10). A myriad of cis-regulatory elements in the promoter of sweetpotato RNA helicase genes indicate that these genes given a paramount importance plant stress resistance. Therefore, we found new candidate genes that can regulate the ability of sweetpotato to resist abiotic stress through RNA metabolism. Considering that sweetpotato is the seventh largest food crop in the world, their economic importance in the world and their adaptation to the environment are great challenges. The revelation of stress-related genes laid a solid foundation for promoting the research of molecular basis of sweetpotato resistance and accelerating the breeding of sweetpotato resistant varieties.\u003c/p\u003e \u003cp\u003eThe RNA helicase given a paramount importance in regulating plant development and responding to environmental stimuli. The expression profiles of the RNA helicase genes were analyzed in young leaves, leaves, stems, and roots of different varieties of sweetpotato, and their response patterns were analyzed under four abiotic stresses and four hormone stresses (Fig.\u0026nbsp;14). The expression of sweetpotato RNA helicase genes in dissimilar tissues was significantly different, indicating that these genes were related to plant development. Moreover, the RNA helicase genes mainly answer to abiotic stresses in sweetpotato, such as cold, heat and salt, indicating that these genes are related to plant stress resistance. This is consistent with the functional studies of other plant RNA helicases, for instance \u003cem\u003eArabidopsis\u003c/em\u003e (Huang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e), rice (Xiaomei et al., \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), tomato (Capel et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), rapeseed (Zhang et al., \u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e), chrysanthemum (Zhang et al., \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e), Zea mays (Yang et al., \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), barley (Ru et al., \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and soybean (Wang et al., \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). We hypothesized that RNA helicase protein can be a functional gene related to sweetpotato growth and development, or a regulatory factor under different environmental constraints. Therefore, this study provides new experimental ideas and clues for the above speculation.\u003c/p\u003e \u003cp\u003eIn conclusion, exploring the reception of the RNA helicase gene in sweetpotato can help transgenic research improve the yield and resistance to stress of sweetpotato. The identification, classification and phylogenetic tree construction of sweetpotato RNA helicase genes were explored through bioinformatics, which produced meaningful information for in-depth study of the biological function of sweetpotato RNA helicase gene. These studies also contribute to understanding the molecular basis of several significant agricultural traits in sweetpotato cultivation. However, the exact regulatory mechanism of sweetpotato RNA helicase gene development and stress response are still unclear and needs to be additional examined.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this research, we conducted an exhaustive genome-wide reasoning of the sweetpotato RNA helicase family, containing chromosome distribution, promoter elements, and protein motif analysis. All of 300 RNA helicase genes were detected, containing IbDEAD, IbDEAH and IbDExDH subfamilies. The expression patterns of eight RNA helicase genes in different sweetpotato varieties and their responses to abiotic stress and hormonal stress were analyzed by qRT-PCR. The expression of the RNA helicase genes was significantly distinct in individual tissues of 10 sweetpotato varieties and notably raised under divergent abiotic stresses. The results showed that RNA helicase was complicated in the direction of extension and the resistance to stress of sweetpotato. This study supplies new inspirations into the development and exploration of the RNA helicase gene families.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZongyun Li, Mingku Zhu and Tingting Dong conceived and designed the research. Fangfang Mu, Hao Zheng and Qiaorui Zhao performed the research and analyzed the data. Fangfang Mu wrote the manuscript. Mingku Zhu, Lei Kai and Zongyun Li helped to revise the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the earmarked fund for CARS-10-Sweetpotato, Postgraduate Research \u0026amp; Practice Innovation Program of Jiangsu Province (KYCX22_2818).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eAltschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAubourg S, Kreis M, Lecharny A. The DEAD box RNA helicase family in Arabidopsis thaliana. 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PLoS ONE. 2015;10:e0133849.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"abiotic tress, genome-wide analysis, RNA helicase, sweetpotato","lastPublishedDoi":"10.21203/rs.3.rs-3407757/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3407757/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRNA helicase is a large family that plays a significant role in plant evolution and in the abiotic stress response. Sweetpotato is one of the majority essential foods in the world, and their yields are often contrived by environmental stresses. Previously, the RNA helicase gene family has not been described in sweetpotato, hence we went a comprehensive genome-wide examination of the sweetpotato RNA helicase family, containing chromosome distribution, promoter elements, and motif compositions. All of 300 RNA helicase genes were divided into three subfamilies, including IbDEAD, IbDEAH and IbDExDH, and they are unevenly scattered across 15 chromosomes of the sweetpotato. Furthermore, tandem replication and segmental replication events played a key role in the amplification of sweetpotato RNA helicase genes. The collinear relationship amidst sweetpotato RNA helicase genes and 8 other related homologous genes in plants was deeply explored, which supplied a reliable basis for studying the development of sweetpotato RNA helicase gene family. RNA-seq examination and qRT-PCR recognition showed that the expression of eight RNA helicase genes was significantly diverse under four abiotic stresses (cold, drought, heat, salt). At the same time, the expression of these RNA helicases in different tissues of the 10 sweetpotato varieties also differed significantly. The promoters of the RNA helicase genes contain a great deal of cis-acting elements related to temperature, hormone and light response. The results indicated that sweetpotato RNA helicase genes played a key role in development or the abiotic stress response.\u003c/p\u003e","manuscriptTitle":"Genome-wide Systematic Survey and Analysis of the RNA Helicase Gene Family and their Response to Abiotic Stress in Sweetpotato","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-10 20:09:25","doi":"10.21203/rs.3.rs-3407757/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-10-04T07:57:02+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-10-04T07:43:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-10-04T07:42:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2023-10-03T12:45:54+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9bd64e20-8c41-4571-9311-c4c64eb81886","owner":[],"postedDate":"October 10th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-03-18T15:08:41+00:00","versionOfRecord":{"articleIdentity":"rs-3407757","link":"https://doi.org/10.1186/s12870-024-04824-z","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2024-03-16 15:01:02","publishedOnDateReadable":"March 16th, 2024"},"versionCreatedAt":"2023-10-10 20:09:25","video":"","vorDoi":"10.1186/s12870-024-04824-z","vorDoiUrl":"https://doi.org/10.1186/s12870-024-04824-z","workflowStages":[]},"version":"v1","identity":"rs-3407757","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3407757","identity":"rs-3407757","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}