Genome-wide identification, expression profiles and phylogenetic analysis of Trihelix transcription factor family genes in sugar beet (Beta vulgaris L.) under abiotic stresses

Genome-wide identification, expression profiles and phylogenetic analysis of Trihelix transcription factor family genes in sugar beet (Beta vulgaris L.) under abiotic stresses | 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 identification, expression profiles and phylogenetic analysis of Trihelix transcription factor family genes in sugar beet (Beta vulgaris L.) under abiotic stresses Shijuan Li, Rui Sun, Yaoyao Du, Haiye Luan, Delai Chen, Muhammad Khurshid, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7657378/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Trihelix transcription factor family genes are known as GT factors, which play vital roles in growth and development process, response to various abiotic stresses in plants. Studies on trihelix gene in several eudicots and monocots have been thoroughly performed, but trihelix family gene in sugar beet ( Beta vulgaris L.), one of the major sugar crops in the world, has not yet been systematic studied. Results We identified 37 non-redundant B. vulgaris trihelix (BvGT) genes named from BvGT1 to BvGT37 in present study and classified them into six clades (SIP1, GTγ, GT1, GT2, GT3 and SH4) by maximum-likelihood phylogeny (IQ-TREE2, 1000 ultrafast bootstraps). The BvGT genes harbour 1–17 exons and ten conserved motifs; motif 1 (core trihelix domain) is present in all members. Chromosomal mapping showed an uneven distribution across eight chromosomes; chromosome 3 lacks any BvGT gene. Only one segmental duplication pair (BvGT30/BvGT37) was detected. Promoter cis-element profiling revealed abundant light-, hormone- and stress-responsive motifs. Expression analysis by qRT-PCR (three biological replicates × three technical replicates) demonstrated tissue- and stress-specific transcription patterns. BvGT10, BvGT23 and BvGT34 were the most highly expressed genes in root, stem and leaf, respectively. Under salt, alkali and osmotic stresses, BvGT4 and BvGT10 showed consistent up-regulation in roots. Conclusions This study identified 37 BvGT genes in sugar beet and further analyzed their evolution and expression pattern. Segmental duplication and purifying selection have shaped the expansion of the trihelix family in sugar beet. The identified stress-responsive BvGT genes provide a theoretical basis for function investigation of BvGT genes and present stress-resistant and high yield candidate genes for molecular breeding toward enhanced abiotic-stress tolerance. Genome-wide identification Beta vulgaris L. Trihelix genes abiotic stresses Gene duplication Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Transcription factors are vital regulators for plants response to biotic and abiotic stresses, they play critical role in regulating the initial stress perception gene expression and triggering stress tolerance [ 1 ]. More than 64 transcription factor families have been discovered in plants [ 2 ]. Trihelix transcription factors (TFs) were first identified in pea in 1980s [ 3 ], which have a specific helix-loop-helix-loop-helix trihelix structure. This TFs bind to a light response element on DNA sequence and were initially named GT factors [ 4 ]. In addition, the trihelix domain of GT factors is similar to MYB family factors [ 5 , 6 ], therefore, the binding target gene sequences were different between GT factors and MYB proteins, and subsequently result in functional differences [ 7 ]. The trihelix gene has been extensively identified by bioinformatic analysis in plants, including Arabidopsis [ 4 ], tomato [ 8 ], rice [ 9 ], soybean [ 10 ], tea [ 11 ], sorghum [ 12 ], quinoa [ 13 ], wheat [ 14 ] and tartary buckwheat [ 15 ], with the number of members 30, 36, 31, 63, 10, 40, 47, 80 and 31 respectively. However, trihelix genes in sugar beet have not been validated. Previous research revealed that trihelix family have functions involving in plant developmental processes, plant morphogenesis, light regulation and respond to biotic and abiotic stresses[ 7 ]. Since then, 30 GT family identified in Arabidopsis and 31 GT family from rice were divided into GT-1, GT-2, GTγ, SH4, and SIP1 [[ 8 , 16 ]. Additionally, 36 GT family were classified into GT-1, GT-2, GTγ, SH4, SIP1 and GTδ [ 8 ]. Although all of the trihelix subfamily contain trihelis domain at the N- or C- terminus, the structures of most trihelix genes vary among plant species, most of them differ at the C- terminal domains. The involvement of trihelix family in complex functions has been performed in a substantial number of plants. Arabidopsis ASIL1, ASIL2 and rice Loc-Os02g3610 genes are involved in regulating embryo development, ASIL1 can affect seedling embryo shape [ 17 – 19 ]. GT-4[ 20 ], AST1[ 21 ] and AtGTL1[ 22 ] in Arabidopsis exhibited improved salt tolerance. In contrast, expression of GmGT-2A and GmGT-2B were induced by high salinity, osmotic and freezing. Furthermore, overexpression OsGT-1 in rice enhanced resistant to salt stress[ 23 , 24 ]. CsGT-3b can be significantly induced by salt stress in cucumber[ 25 , 26 ]. Moreover, a member of GT-1 subfamily gene ShCIGT, overexpressed in tomato showed cold and drought tolerance[ 27 ]. Taken together, trihelix gene family play conserved role in diverse biotic and abiotic stress response. The GT transcription factors in several plants have been systematically identified and assessed, some of them have been used for stress tolerant breeding, therefore, GT factors have not been reported in sugar beet. Sugar beet ( Beta vulgaris L.) is one of the important sugar-yielding economic crop and has been cultivated for hundreds of years. In addition to the conventional nutrients, the sugar beet also contains a variety of physiologically active substances that are beneficial to human health[ 28 ]. In this study, the trihelix gene family members in sugar beet were identified and characterized. We performed system analysis in terms of molecular evolution, chromosomal distributions, gene structures, conserved motifs, protein properties and gene expression patterns. The orthology relations, gene duplication events were also identified, moreover, phylogenetic trees of trihelix genes were constructed. Although trihelix families have been reported in Arabidopsis, rice, tomato, soybean and tartary buckwheat, the sugar beet trihelix family remains uncharacterised. In this study, we present a genome-wide survey of BvGT genes and their expression under eight abiotic stresses, providing resources for stress-resilient sugar beet breeding. Results Identification and physicochemical properties analysis of trihelix family members in sugar beet ( Beta vulgaris L.) In this study, a total of 37 non-redundant trihelix family genes were identified in the sugar beet with reference to genome database. The 37 trihelix genes were designated BvGT1-BvGT37 according to their position on the chromosome. The physical and chemical properties of predicted BvGT genes were listed in Table S1 , including gene ID, protein length, molecular weight (MW), isoelectric point (IP), subcellular localization, domains and chromosome location. Among the 37 members identified, the BvGT gene encoded proteins with lengths ranging from 205 amino acids (BvGT 17) to 886 aa (BvGT13), with an average of 407 aa. The molecular weight of the BvGT proteins ranged from 24.28 KDa to 98.3 KDa, with an average of 6.89. The subcellular localization prediction results showed that 28 BvGT genes were located in the nucleus, 6 in the chloroplast, 2 in the cytosol and 1 in the extracellular. From the results, 19 BvGT genes in 37 (51.4%) contained GT1 domain, 15 BvGT genes (40.5%) contained Myb_DNA-binding domain and 3 BvGT genes (8.1%) contained both GT1 and Myb_DNA-binding domain. Additionally, the amino acid sequences and coding sequences of BvGT genes were provided in Table S1 . Phylogenetic analysis and classification of BvGT genes in Beta vulgaris L. To understand the phylogenetic relationship of trihelix genes, the amino acid sequences of trihelix genes including 37 sugar beet genes and 25 A. thaliana genes were employed to construct a neighbor-joining phylogenetic tree (Fig. 1 ). Trihelix genes shown in phylogenetic tree were clustered into 6 groups ( SIP1, GTγ, GT1, GT2, GT3 and SH4) according to the conserved amino acids of the GT domain and classification method from Kaplan-Levy et al [ 4 , 29 ]. In the phylogenetic tree, the largest subfamily were GT3 and SIP1 (11 members each). GTγ was the smallest subfamily containing 2 BvGT genes. Moreover, GT1, GT2 and SH4 contained 4, 5, and 4 BvGT genes, respectively. Exon-intron organization and conserved motif compositions of BvGT family members The BvGT gene structures and phases were analyzed to characterize the trihelix gene family. Trihelix members in the same clade showed similar exon and intron organization (Fig. 2 a, 2 b). The number and distribution of exons and introns were shown in Fig. 2 b. GT3 genes harbour the most exons (mean = 9.3), whereas SIP1 genes are mostly intronless. Structural characteristics analysis showed that 7 (18.9%) BvGT family members had no intron, 17 (45.9%) BvGT family members had only 1 intron, whereas 1 gene of BvGT13 had the highest number of introns. The number of exons varied from 1 to 17, the BvGT3 in SIP1 subfamily contained the lowest number of exons, whereas the BvGT13 in GT3 subfamily contained the highest. The conserved motif distribution of the trihelix family members in sugar beet were predicted by means of MEME search tool. A total of 10 conserved motifs were shown in Fig. 2 c, the detailed sequence of each motif was provided in Table S2 . The motif arrangement of each BvGT protein were shown with the matching color boxes. Motif 1 existed in all BvGT proteins, indicating motif 1 (trihelix core) is ubiquitous and may play critical role in specific processes. Motif 2, motif 5 and motif 6 exited in all the SIP1 subfamily members except BvGT22. In the GT3 subfamily, BvGT1, BvGT4, BvGT14, BvGT20, BvGT30, BvGT31, BvGT 32 and BvGT36 all possessed motif 3 and motif 4 except BvGT1, BvGT20 and BvGT36. Motif 7 and motif 8 were only found in clade GT3. Motif 9 existed simultaneously in GTI, GT2 and SH4 subfamily. Notably, BvGT19, BvGT16, BvGT2 and BvGT21 possessed two copies of motif 10. Additionally, BvGT16 and BvGT22 contained two copies of motif 6, 9 and 10 at the same time. Furthermore, BvGT29 and BvGT34 possessed the least number of motifs, whereas BvGT2 and BvGT16 possessed the most number of motifs, with 2 and 9 motifs, respectively. Chromosomal localization and collinearity analysis of BvGT genes in Beta vulgaris L. The chromosomal positions of BvGT genes were unevenly distributed on 9 chromosomes of Beta vulgaris L., as shown in Fig. 3 . BvGT genes map to all chromosomes except Chr3, Chromosome 9 contained the highest number of BvGT genes. The number of BvGT genes on chromosomes were as follows, Chr1 (2, 5.4% ), Chr2 (4, 10.8%), Chr4 (3, 8.1%), Chr5 (5, 13.5%), Chr6 (6, 16.2%), Chr7 (6, 16.2%), Chr8 (4, 10.8%), Chr9 (7, 18.9%). Additionally, colinear analysis showed that there was one pair of segmental duplication genes BvGT30 and BvGT37 with Ka/Ks = 0.27 indicates purifying selection, located on chromosome 8 and chromosome 9, respectively. Whereas, there were no duplicated segments located on Chr1-Chr7 (Fig. 4 ). A chromosome area within 200 kb including two more identical genomic regions was defined as a tandem duplication event. Cis-elements in the promoter of BvGT family members Cis-elements are associated with gene transcription through binding to transcription factors. To analyze the putative functions of BvGT family members during plant evolution and response to stresses, a 2000 bp promoter sequences of BvGT genes were extracted and examined by Plant CARE program. Nine cis-element categories with 55 functions were present in the promoter regions, including development, environmental stress, hormone responsive, light responsive, promoter related element, site-binding, wound-responsive related elements and other elements. The distribution of the cis-elements were shown in Table S3 . Light responsive elements (G-box, Box 4) are present in all promoter regions of BvGT genes, with the largest number of 25 in BvGT30 and the least number of 3 in BvGT30. Hormone responsive elements (ABRE, CGTCA-motif) were also identified, including Aauxin, Salicylic acid, MeJA, Estrogen, Ethylene, Gibberellin and Abscisic acid responsive elements. Among all the hormone responsive elements, BvGT11 had the most number with 22, whereas BvGT 30 and BvGT 7 contained the least with only 2 respectively. From the results, BvGT family members contained several abiotic related stress elements, Stress-responsive (MBS, LTR) elements are clade-specific (Table S3 ). TATA boxes are restricted to the plus strand at–30 to–120 bp. Evolutionary relationship of BvGT family and other representative plant species To gain the potential evolutionary relationship of BvGT family and five other plant species ( Arabidopsis , tomato, buckwheat, rice and wheat), an unrooted NJ tree was generated using Geneious R11 based on protein sequences of 37 BvGT genes and trihelix genes of the five other plants. Detailed genetic correspondence was presented in Table S5 . The BvGT proteins were relatively dispersed in the phylogenetic tree. Motif 1 and 7 were shared by most members of the trihelix family from different plant species. Motif 1, 7 and 8 tended to cluster in rice and wheat, notably, motif 1 and 2 tended to cluster in sugar beet, Arabidopsis , tomato and buckwheat, indicating sugar beet may be more closely related to Arabidopsis , tomato and buckwheat than rice and wheat (Fig. 5 ). To gain a closer understanding of the synchronization relation of sugar beet trihelix family, five representative plant species were selected to construct comparative system diagrams, including three eudicots ( Arabidopsis thaliana , tomato, buckwheat) and two monocotyledonous plants (rice and wheat). The results of covariance analysis were shown in Fig. 6 . the number of collinear genes between Beta vulgaris L and Arabidopsis thaliana, Solanum lycopersicum, Fagopyrum tataricum, Oryza sativa Japonica and Triticum aestivum were 20, 22, 18, 5, and 7, respectively. Generally, Solanum lycopersicum was the most similar with Beta vulgaris L and the least similar was Oryza sativa Japonica , indicating the phylogenetic evolutionary relationship among these plant species. To extently understand the evolutionary role of BvGT family, Tajima D neutrality test was performed and the statistic D value was 9.49, indicating the BvGT family was strongly selected in the evolution of sugar beet (Table S6 ). Tissue-specific gene expression patterns of BvGT genes Trihelix genes were reported to play key roles in plant growth and development[ 30 ]. To obtain insight into the physiological role of BvGT genes in sugar beet growth and development. The expression levels of nine selected BvGT genes were examined by qRT-PCR. Histograms were employed to depict the expression profiles of BvGT genes in root, stem and leaf of sugar beet (Fig. 7 a). Notably, the BvGT genes were highly expressed in selected tissues and organs, suggesting that BvGT genes performed multiple function in sugar beet growth and development. Specifically, BvGT10, BvGT23 and BvGT34 were highly expressed in root, BvGT23 and BvGT34 were highly expressed in stem. Additionally, BvGT2, BvGT5, BvGT13 and BvGT34 were relatively highly expressed in leaves. The expression of BvGT10 was the highest in the roots, and BvGT23 expression was the highest in the stem, whereas BvGT34 expression was the highest in leaves. In contrast, the expression patterns of BvGT4 and BvGT23 genes in the leaves were low, as well as BvGT23 expression in root was very low. Furthermore, the correlation between the BvGT expression profiles were examined and found the majority of the BvGT genes were positively correlated with other genes (Fig. 7 b). BvGT11 was positively related with BvGT23, BvGT2, BvGT13, BvGT5 and BvGT34, while BvGT3 was negatively correlated with BvGT23, BvGT11, BvGT13 and BvGT5. Additionally, the pairs of BvGT4 and BvGT13, BvGT10 and BvGT11 were significantly negatively correlated with each other. Expression patterns of BvGT genes in sugar beet under different abiotic stresses Subsequently, the responses of nine selected BvGT genes to different abiotic stresses (osmotic pressure, flooding, acid, alkaline, low temperature, high temperature, dark and salt) were evaluated from RNA-seq data of tissues with various treatments. The BvGT genes exhibited different degrees of responses to these abiotic stresses in specific tissues (Fig. 8 a). Heat-map visualisation shows rapid (2 h) induction under flooding, heat and cold, whereas osmotic, salt and alkaline stresses elicited stronger responses after 24 h. The expression level of BvGT3, 4, 5, 10, 11 and 23 in root were significantly up regulated after 24h under osmotic, acid, alkaline, high temperature, dark and salt stresses, whereas BvGT2 and BvGT34 in root were down regulated after 24h under osmotic pressure. Across the eight different treatments, the nine BvGT genes expressed in specific tissues consistently. For instance, BvGT10 was significantly up regulated in all tissues after exposure to osmotic, acid, alkaline, low temperature, high temperature, and salt for 24h. Moreover, BvGT3 was significantly down regulated in all tissues after exposure to low temperature and dark for 24h. Particularly, BvGT4 and BvGT23 were highly expressed in root after 24h of exposure to osmotic, alkaline and salt. BvGT4 and BvGT10 were consistently up-regulated in roots across all abiotic stresses (> 4-fold), whereas BvGT2 was down-regulated under drought. Correlation networks (Fig. 8 b) imply coordinated transcriptional modules. There was no significant correlation between BvGT2 and the other 8 trihelix genes. In contrast, the relative expression of BvGT5 and BvGT3/BvGT23/BvGT4 were highly positively associated. Additionally, BvGT11 and BvGT4, BvGT34 and BvGT13/BvGT10 were positively correlated. Discussion Sugar beet is an important sugar producing crop in temperate regions. 30% of the world’s annual plant sugar production comes from sugar beet. It is also an important source of bioethanol, natural pigments, edible vegetables, and animal feed. In contrast to the well-known transcription factor of MYB, WORKY, ERF and NAC, trihelix factors had received attention only recently, which was first reported in pea. However, trihelix family in sugar beet genome have not been published. The reference genome of sugar beet ( Beta vulgaris ) was reported in 2014 [ 31 ], which provide an abundant theoretical basis for us to analyze the characteristics and functions of sugar beet trihelix. Within this study, 37 BvGT genes were identified in sugar beet, which was agree with studies on trihelix genes in Arabidopsis , tomato[ 8 ], rice, buckwheat sorghum[ 12 ] and quinoa[ 13 ]. The BvGT genes account 0.13% of the total genes in sugar beet, which was similar to G. max (0.14%) [ 32 ], Arabidopsis (0.11%) [ 33 ] and rice (0.1%) [ 34 ]. Moreover, buckwheat (0.06%) [ 35 ], Chrysanthemum (0.04%) [ 36 ], S. lycopersicum (0.05%) [ 37 ] and wheat (0.08%) [ 38 ] owned a low percent in their genome. Physicochemical properties including protein length, MW and IP were collated by bioinformatic methods. Differences of protein properties demonstrated different functions of the trihelix family members. Additionally, Trihelix family were grouped into three subfamilies previously (GTα, GTβ, and GTγ) [ 23 ], soon afterwards, they were classified into five subfamilies by Kaplan-Levy et al [ 39 ]. With in this study, the sugar beet trihelix genes were classified into six subfamilies (SIP1, GTY, GT1, GT2,GT3, and SH4) according to phylogenetic analysis (Fig. 1 ). Additionally, the functions of BvGT family members presented on the phylogenetic tree will provide potential realistic and reliable resources for gene function exploration in later stages. Studies on the exon and intron architecture of the 37 BvGT genes revealed that the count of exons ranged from 1 to 17 (Fig. 2 a, 2 b). The proportion of BvGT genes without introns was 18.9%, which was close to quinoa (10.6%) and lower than that of rice (43.9%) and sorghum (37.5%). Five of the intro-free genes were distributed in the SIP subfamily and 2 in GTγ subfamily, which were similar to sorghum and Arabidopsis previously reported. Introns can increase the length and complexity of genes, which play key roles in gene expression, protein synthesis, and genome evolution. Moreover, intro-free genes tend to more responsive to environment change [ 40 ]. There were differences in gene exon numbers between subfamilies, further analysis revealed that the GT3 subfamily had the most number of exons whereas the SIP1 subfamily contained the lowest. The results were different from wheat [ 41 ] and buckwheat [ 15 ], which findings of studies showed that GT1subfamily had the most of exons and GTγ family had the least. The results of motif composition of trihelix were consistent with phylogenetic classification results. 19 of the BvGT genes only had the GT domain, most of which belonged to GT1 and GT3 subfamily. 15 of the BvGT genes only had the Myb_DNA-binding domain, most of which belonged to SIP1 subfamily. Whereas 3 BvGT genes had both GT and Myb_DNA-binding domain and attributed to GT2 subfamily. Members in the same subfamily possessed considerable similarities, indicating the conserved motifs may play essential functions of the specific BvGT genes. These results revealed that members in the same subfamily may owns similar roles in growth and development of sugar beet. Results of chromosome localization showed that BvGT genes were unequally distributed on nine chromosomes (Fig. 3 ). Chromosome 3 harbored no BvGT gene, which may experience gene loss during evolution [ 42 ]. Chromosome 9 harbored the most of BvGT genes, with the number of 7, which may be beneficial to gene evolution. In addition, colinear analysis showed that there was one pair of segmental duplication genes in BvGT gene family of sugar beet. Whereas, there were no duplicated segments located on Chr1-Chr7 (Fig. 4 ). Gene duplication is very important for genome evolutionary mechanism, directly influences gene amplification and recombination. Interestingly, the number of BvGT genes in sugar beet is less than soybean (67) [ 43 ], G.max (71) [ 44 ] and B. napus (52). The difference may be due to whole genome replication events from the earliest ancient plants differentiated. From the present results, only one pair of segmental duplication on sugar beet chromosomes was discovered (Table S4 ), indicating that the emergence and the evolution of BvGT genes were probably driven by replication events and some other factors. This is different from research in tartary buckwheat and Populus trichocarpa [ 30 ]. Cis-elements on the promoter may play important roles in the function of BvGT genes. Interestingly, 37 BvGT genes identified in this study are involved in light response and hormone response. Additionally, BvGT 11 had both the most number of light response and hormone response elements, with the number of 16 and 22 respectively, whereas BvGT 30 contained the least light response elements with the number 1, BvGT30 and BvGT 7 contained the least hormone response elements with the number 2 individually. Cis-elements related to drought, low-temperature, wound stress were also identified, including F-box, CGTCA-motif, Myb-binding site and MYC, etc. These results showed that some of the BvGT genes are involved in the regulation of biotic and abiotic stresses, indicating that they may play vital role in response to biotic and abiotic stresses. The trihelix gene family members were involved in plant development in previous studies. The functions of BvGT genes were explored in this research. We determined the relative expression of BvGT genes in roots, stems and leaves of sugar beet by qRT-PCR. The selected nine BvGT genes showed considerable differences in expression patterns (Fig. 7 ). Notably, BvGT23 from GTγ subfamily, homologous to gene HRA1 ( AT3G10040.1 ) had the highest expression level in stems. HRA1 expressed in stems of Arabidopsis thaliana regulating leaf structure and shoot development [ 45 ]. BvGT10 , which is homologous to AT2G38250.1 and Solyc09g008830.3.1 (Table S5 ), was highly expressed in roots. BvGT34 was significantly high expressed in the roots, stems and leaves, indicating multiple different functions. In particular, the expression of BvGT13, BvGT2 and BvGT4 were the lowest in roots, stems and leaves respectively. Therefore, these results suggested that tissue-specific BvGT genes are important in the growth and development of relevant tissues [ 46 ]. However, later experiments are required to verify their functions in the future. In addition, the significantly positive correlation between BvGT genes indicating that they may have spatial and temporal specificity functions. To further elucidate the function of the trihelix gene family in stress adaption, the expression patterns of nine BvGT genes in sugar beet root, stem and leaf were systematically analyzed (Fig. 8 ). Therefore, the expression characteristics of BvGT genes exhibited spatial variation. The roots of plants can perceive drought changes of the soil and trigger physio-chemical reaction of plants to reduce the damage. In present study, under drought treatment, seven BvGT genes ( BvGT3, BvGT4, BvGT5, BvGT10, BvGT11, BvGT13, BvGT23 ) were induced obviously in roots, indicating that they may contribute to the adaption of sugar beet to drought conditions. In addition, there was a substantial decrease in the expression of BvGT2 in roots under drought conditions whereas increased significantly under flooding, which belonged to GT2 subfamily. GT2 subfamily were reported as suppressor related to stomatal density and distribution [ 47 , 48 ]. Therefore, BvGT2 may contribute to water use in sugar beet through regulating stomata number [ 22 ]. Interestingly, BvGT4 and BvGT10 grouped in GT3 and GT1 respectively were highly induced in response to almost all stresses in roots, indicating the two genes may play multiple functions in various stresses, which can be verified by further experiments. Similarly, the expression of BvGT34 from GT3 subfamily in leaves were induced significantly in all stress conditions, suggesting the function of enhancing adaptability of sugar beet to environment. In conclusion, the expression characteristics of BvGT genes from the six subfamilies showed tremendous differences, indicating their unique functions under various stresses. However, functions of BvGT genes still need further validation. Conclusions To sum up, this is the first genome-wide of trihelix family genes in sugar beet. We screened 37 members of trihelix genes across nine chromosomes and clustered them into six groups. Additionally, physicochemical properties, expression profiles and phylogenetic analysis were evaluated. The expression patterns of the BvGT genes in different tissues of sugar beet under eight abiotic stresses were analyzed by qRT-PCR. Based on above research, some stress tolerance candidate genes were screened out. In particular, BvGT23 was highly expressed in stems and responded to drought. BvGT4 and BvGT10 were consistently up-regulated in roots under all abiotic stresses. The present study provide a comprehensive atlas of the 37-member trihelix family in sugar beet. The stress-inducible genes BvGT4, BvGT10 and BvGT23 are promising targets for functional studies and breeding. Materials and Methods B. vulgaris trihelix (BvGT) gene Identification The entire sugar beet genome sequence information was downloaded from ( https://bvseq.boku.ac.at/index . shtml) [ 49 ]. The trihelix genes in sugar beet were identified by BLASTp methods (E ≤ 1e-10, score ≥ 100) using Arabidopsis trihelix proteins as queries. Candidate proteins with score value ≥ 100 and e-value ≤ 1e − 10 were identified from sugar beet genome. The Hidden Markov Model (HMM) files consistent with trihelix domain were downloaded from the PFAM protein family database ( http://pfam.sanger.ac.uk/ ) [ 50 ]. HMMER3.0 with a cutoff of 0.01 ( http://plants.ensembl.org/hmmer/index . html) [ 51 ] and SMART ( http://smart . embl-heidelberg. de/) [ 52 ] were used to determine the candidate BvGT proteins containing the conserved trihelix structural domain. CDS and protein sequences were validated by SMART and InterProScan. In addition, subcellular localization was predicted by Cell-PLoc ( http://chou.med.harvard.edu/bioinf/Cell-PLoc ) and ProtComp 9.0 [ 53 ]. Subsequently, feature information of projected trihelix proteins was computed from the ExPasy website ( http://web.expasy.org/protparam/ ), including CDS length, protein pI and theoretical molecular weights et al [ 52 , 54 ]. In the end, 37 members of trihelix genes from sugar beet were screened out. Characterization and Phylogenetic analyses of BvGT genes The exon-intron structures of BvGT genes were generated from Gene Structure Display Server (GSDS: http://gsds.cbi.pku.edu.cn ) by comparing the CDS of BvGTs with their genomic DNA sequences [ 55 ]. Clustal W with default parameters was employed to create multiple protein sequence alignment of the characterized BvGT proteins [ 56 ]. To characterize the structure and detect the differences of BvGT proteins, the conserved motifs were determined and compared using an online search software Multiple Em for Motif Elicitation MEME ( http://meme-suite . org/ tools /meme) [ 57 ]. The parameters were set as follows: the number of motifs searched with a maximum ten motifs and the motif length was 6-200 amino acid residues [ 58 ]. All motifs were further annotated with InterProScan ( http://www.ebi.ac.uk/interpro/ ) [ 59 ]. Cis-elements on the upstream 2000 bp promoter sequences of BvGT genes were explored on the PlantCARE website ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) [ 60 ]. In addition, multiple sequence alignments of the Arabidopsis trihelix and BvGT proteins were performed to construct a neighbor-joining phylogenetic tree by MEGA 7.0 with a bootstrap value of 1000 replicates and default parameters [ 61 ]. To gain the evolutionary relationship of BvGT family and five other plant species, phylogenetic trees were constructed using the full length amino acid sequences of trihelix proteins from A. thaliana, S. lycopersicum, F. tataricum, O. sativa and T. aestivum. Sequences of trihelix proteins were acquired from UniProt database (UniProt https://www.uniprot.org/ ) [ 62 ]. Gene chromosomal distribution and gene duplication The distribution of all BvGT genes were mapped to B. vulgaris chromosomes based on physical position information obtained from B. vulgaris genome database by Circos software ( http://circos.ca/software/download/ ) [ 63 ]. Gene duplication events of BvGTs were performed by Multiple collinear scanning toolkits (MCScanX) ( https://github.com/wyp1125/MCScanx ) with default settings [ 64 ]. The syntenic relationship between BvGT genes and five representative plants including A. thaliana, S. lycopersicum, F. tataricum, O. sativa and T. aestivum were analyzed by Dual Synteny Plotter ( https://github.com/CJ-Chen/TBtools ) [ 65 ]. Ka/Ks ratios were calculated by KaKs_Calculator 2.0. Plant materials, growth condition and abiotic stress treatment The B. vulgaris variety of “Detian 316” was selected for this study. The sugar beet plants were cultivated in pots filled with 50% soil and 50% vermiculite in greenhouse (12h day/12h night, 25℃ day/18℃ night, the relative humidity was 70%). Twenty one day old seedlings were subjected to eight stresses. Samples of roots, stems and leaves were collected from five good growth plants, then placed in liquid nitrogen for storage at -80℃ for RNA extraction. The healthy plants were exposed to various abiotic stress conditions for 2h and 24h treatment, including 30% PEG6000 [ 66 , 67 ], flooding (root submerged 2 cm), HCI (pH 4.5), NaOH (pH 9.0), low temperature (4℃), high temperature (40℃), darkness and salt stress (400mM NaCl) [ 68 ]. Root (0–2 cm tips), stem (third internode) and Young fully-expanded leaves were harvested. Three biological replicates (five plants each) were snap-frozen in liquid N₂. Subsequently, the expression patterns of nine selected BvGT genes in different parts of sugar beet under stresses were analyzed by qRT-PCR. Expression profiles of BvGT genes by qRT-PCR The expression profiles of nine BvGT genes were analyzed from tissues collected (roots and stress treated roots, stems and stress treated stems, leaves and stress treated leaves) by qRT-PCR analysis. Total RNA (TRIzol) was treated with DNase I (Thermo). First-strand cDNA was synthesised with HiScript III RT SuperMix (Vazyme). qRT-PCR was performed on a CFX96 Touch (Bio-Rad) using SYBR Green (Vazyme). BvGAPDH (accession: XM_010691997) served as the internal reference. Primer sequences shown in Table S7 for qRT-PCR were designed by Primer 5.0 software [ 69 ]. Primers had efficiencies 95–105% (R²≥0.99). Relative expression was calculated by 2 −ΔΔCT method [ 70 ]. Statistical analysis The acquired data in present study were subjected to analysis of variance ANOVA using SPSS software. Mean values were compared by LSD test at the 0.05 significance level. Origin 8.0 was used to construct histograms of the data. Tajima’s D neutrality test was performed by MEGA 7.0 [ 71 ]. Declarations Ethics approval and consent to participate All methods were carried out in accordance with relevant guidelines and regulations. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Funding The Natural Science Foundation of the Jiangsu Higher Education Institutions of China. Author Contribution Shijuan Li: Conceptualization, investigation, data curation, formal analysis, visualization, writing the original draft, project administration. Rui Sun: investigation, data curation. Yaoyao Du: formal analysis, data acquisition. Haiye Luan: formal analysis, project administration. Delai Chen: formal analysis. Muhammad Khurshid: Conceptualization, investigation, formal analysis. Shimei li and Po Li: investigation, formal analysis. Acknowledgements Not applicable. 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Supplementary Files TableS1.Listofthe37BetavulgarisGTgenesidentifiedinthisstudy..xls TableS2.AnalysisanddistributionofconservedmotifsofGTproteinsinsevenspecies...xls TableS3.CisregulatoryelementsinthepromoterregionofBvGTgenes...xlsx TableS4.TheonepairofsegmentalduplicatesinBvGTgenes...xlsx TableS5.OnetooneorthologousgenerelationshipsbetweenBetavulgarisandotherplants...xls TableS6.ResultsofTajimasDneutralitytest...xlsx TableS7.PrimersequencesforqRTPCR...xlsx Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. 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As shown in the figure, the phylogenetic tree is divided into 6 subfamilies, including subfamily SIP1, GTγ, GT1, GT2, GT3 and SH4, different colored branches represent different subfamilies. Trihelix domains of sugar beet and \u003cem\u003eA. thaliana\u003c/em\u003e are marked as blue triangles and green star. Trihelix proteins from Arabidopsis are marked with the prefix ‘At’.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7657378/v1/7e9b9475edd0fc0e333282e6.jpg"},{"id":98423574,"identity":"e4e2e7ec-8cda-4f85-aece-9de5e86a8806","added_by":"auto","created_at":"2025-12-17 16:32:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":348212,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic relationships, gene structures and the conserved protein motifs of the \u003cem\u003eBvGT\u003c/em\u003e genes from sugar beet. (A) Full-length sequences of sugar beet trihelix proteins were used to construct the phylogenetic tree by NJ method with 1000 replicates on each node. (B) Gene structure of exon and intro organization of \u003cem\u003eBvGT \u003c/em\u003egenes are displayed in different colors. Yellow boxes indicate exons of genes, green boxes indicate UTR and the lines refer to introns. (C) The motif composition of the sugar beet trihelix proteins. The motif 1 to 10 are displayed in different colored boxes. The protein length can be estimated by the bottom scale.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7657378/v1/9bb881c39aac89fb7ac78cf5.png"},{"id":98423602,"identity":"98caa495-0443-4edd-a695-736ca0a65266","added_by":"auto","created_at":"2025-12-17 16:32:25","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1327342,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eBvGT\u003c/em\u003echromosome distribution. The chromosome number is indicated to the left of chromosome, the distribution of sugar beet trihelix genes are indicated to the right.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7657378/v1/4082be56b8c19ec630df3fce.jpg"},{"id":97979280,"identity":"eaf24ce0-19b7-4f92-8038-f8afa0c26af1","added_by":"auto","created_at":"2025-12-11 12:34:36","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2075551,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the chromosomal distribution and gene duplication of \u003cem\u003eBvGT\u003c/em\u003e genes. The red lines indicate segmental duplication trihelix gene pairs. Chromosome number is indicated at the bottom of each chromosome.\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7657378/v1/28818acb40c1b18ebf4ed3a8.jpg"},{"id":98423991,"identity":"452bd42e-1d5f-4d31-9545-ab07dd38a1a3","added_by":"auto","created_at":"2025-12-17 16:32:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3185814,"visible":true,"origin":"","legend":"\u003cp\u003eUnrooted phylogenetic tree was derived using the Geneious R11 based on protein sequences of 37 \u003cem\u003eBvGT\u003c/em\u003egenes and trihelix genes of the five other plants including Arabidopsis, tomato, buckwheat, rice and wheat. Motifs numbered 1–10 are displayed in different colored boxes. The abbreviations used for diferent plant.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7657378/v1/b13dc97f75a8f52eb8c5015f.png"},{"id":98423699,"identity":"2b460cf3-efc2-4d5d-a72c-c9fc2dc5a512","added_by":"auto","created_at":"2025-12-17 16:32:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4703035,"visible":true,"origin":"","legend":"\u003cp\u003eSynteny analyses of the trihelix genes between sugar beet and five representative plant species (Arabidopsis thaliana, tomato, buckwheat, rice and wheat). Gray lines in the background indicate the collinear blocks in the genomes of sugar beet and five other plants, while the red lines highlight the syntenic trihelix gene pairs.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-7657378/v1/ab4560cc8bfb927adc450580.png"},{"id":98424298,"identity":"25a2edab-879c-4a48-88f7-703b6b97161b","added_by":"auto","created_at":"2025-12-17 16:33:09","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":754480,"visible":true,"origin":"","legend":"\u003cp\u003eTissue-specifc gene expression patterns of 9 sugar beet trihelix genes and correlation between BvGT gene expression patterns. (A) The expression patterns of 9 BvGT genes in the root, stem and leaf were examined by qPCR. Error bars were obtained from three measurements. Lowercase letter(s) above the bars indicate signifcant diferences (p\u0026lt;0.05, LSD) among the treatments. (B) The correlation between the BvGT gene expression patterns. The green square or positive number: positively correlated; the blue square or negative number: negatively correlated. The signifcance level was 0.01.\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7657378/v1/8433d9bbb3642201d208fb5d.jpg"},{"id":98424833,"identity":"c5eebc09-32b8-473b-98cd-06b066340a32","added_by":"auto","created_at":"2025-12-17 16:33:55","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2928116,"visible":true,"origin":"","legend":"\u003cp\u003eGene expression pattern of 9 BvGT genes in different plant tissues under eight abiotic stresses and correlation analysis. (A) The expression patterns of 9 BvGT genes in the root, stem and leaf treated under different abiotic stresses. Error bars were obtained from three measurements. Lowercase letter(s) above the bars indicate signifcant diferences (p\u0026lt;0.05, LSD) among the treatments. (B) Correlation coefficient analysis of BvGT gene relative expression patterns. The green square or positive number: positively correlated; the blue square or negative number: negatively correlated. The signifcance level was 0.01.\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7657378/v1/5c8bf12c5d44e949f97a06f6.jpg"},{"id":104484779,"identity":"65b49342-471a-46c0-a468-fd735943ef40","added_by":"auto","created_at":"2026-03-12 10:12:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":18011202,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7657378/v1/ac936260-aa63-4c99-97c6-1a73ff26f675.pdf"},{"id":98423441,"identity":"bb89edad-794a-43e3-ae16-b54f7660d9e4","added_by":"auto","created_at":"2025-12-17 16:32:13","extension":"xls","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":156672,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.Listofthe37BetavulgarisGTgenesidentifiedinthisstudy..xls","url":"https://assets-eu.researchsquare.com/files/rs-7657378/v1/1dfe9cfe21db94438ccf98a1.xls"},{"id":97979278,"identity":"d8cc7ff1-0eeb-412b-8177-ca894a963ed5","added_by":"auto","created_at":"2025-12-11 12:34:36","extension":"xls","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23552,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.AnalysisanddistributionofconservedmotifsofGTproteinsinsevenspecies...xls","url":"https://assets-eu.researchsquare.com/files/rs-7657378/v1/25160603af4b21c9a920c279.xls"},{"id":98423694,"identity":"f8faa64a-3d8c-41f9-b7e1-6f3dbc6cebd0","added_by":"auto","created_at":"2025-12-17 16:32:31","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":933676,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.CisregulatoryelementsinthepromoterregionofBvGTgenes...xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7657378/v1/6d64f15c27ef1b759d59a44c.xlsx"},{"id":97979284,"identity":"0e42dfe5-a691-4c76-8d41-10c7c299fdb6","added_by":"auto","created_at":"2025-12-11 12:34:36","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":9877,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.TheonepairofsegmentalduplicatesinBvGTgenes...xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7657378/v1/86b5929ac4ff3b4f37eaa08b.xlsx"},{"id":98424902,"identity":"ace4ec57-5a90-4069-aff2-73c7927b9d56","added_by":"auto","created_at":"2025-12-17 16:34:02","extension":"xls","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":73728,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.OnetooneorthologousgenerelationshipsbetweenBetavulgarisandotherplants...xls","url":"https://assets-eu.researchsquare.com/files/rs-7657378/v1/fd6f99d561d98a798f53d43a.xls"},{"id":98423606,"identity":"051ca1f7-ee52-4f89-9c4a-b49dfd8d1e93","added_by":"auto","created_at":"2025-12-17 16:32:25","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":10371,"visible":true,"origin":"","legend":"","description":"","filename":"TableS6.ResultsofTajimasDneutralitytest...xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7657378/v1/ad036a8349d9987c7614bda1.xlsx"},{"id":98423753,"identity":"15429d57-527c-488b-8f4d-846080b8b7fd","added_by":"auto","created_at":"2025-12-17 16:32:34","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":10083,"visible":true,"origin":"","legend":"","description":"","filename":"TableS7.PrimersequencesforqRTPCR...xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7657378/v1/04ff9e3274fd6dd8deda2dce.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide identification, expression profiles and phylogenetic analysis of Trihelix transcription factor family genes in sugar beet (Beta vulgaris L.) under abiotic stresses","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTranscription factors are vital regulators for plants response to biotic and abiotic stresses, they play critical role in regulating the initial stress perception gene expression and triggering stress tolerance [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. More than 64 transcription factor families have been discovered in plants [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Trihelix transcription factors (TFs) were first identified in pea in 1980s [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], which have a specific helix-loop-helix-loop-helix trihelix structure. This TFs bind to a light response element on DNA sequence and were initially named GT factors [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In addition, the trihelix domain of GT factors is similar to MYB family factors [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], therefore, the binding target gene sequences were different between GT factors and MYB proteins, and subsequently result in functional differences [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe trihelix gene has been extensively identified by bioinformatic analysis in plants, including Arabidopsis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], tomato [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], rice [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], soybean [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], tea [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], sorghum [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], quinoa [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], wheat [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and tartary buckwheat [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], with the number of members 30, 36, 31, 63, 10, 40, 47, 80 and 31 respectively. However, trihelix genes in sugar beet have not been validated. Previous research revealed that trihelix family have functions involving in plant developmental processes, plant morphogenesis, light regulation and respond to biotic and abiotic stresses[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Since then, 30 GT family identified in Arabidopsis and 31 GT family from rice were divided into GT-1, GT-2, GTγ, SH4, and SIP1 [[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Additionally, 36 GT family were classified into GT-1, GT-2, GTγ, SH4, SIP1 and GTδ [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Although all of the trihelix subfamily contain trihelis domain at the N- or C- terminus, the structures of most trihelix genes vary among plant species, most of them differ at the C- terminal domains.\u003c/p\u003e\u003cp\u003eThe involvement of trihelix family in complex functions has been performed in a substantial number of plants. Arabidopsis ASIL1, ASIL2 and rice Loc-Os02g3610 genes are involved in regulating embryo development, ASIL1 can affect seedling embryo shape [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. GT-4[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], AST1[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and AtGTL1[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] in Arabidopsis exhibited improved salt tolerance. In contrast, expression of GmGT-2A and GmGT-2B were induced by high salinity, osmotic and freezing. Furthermore, overexpression OsGT-1 in rice enhanced resistant to salt stress[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. CsGT-3b can be significantly induced by salt stress in cucumber[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Moreover, a member of GT-1 subfamily gene ShCIGT, overexpressed in tomato showed cold and drought tolerance[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Taken together, trihelix gene family play conserved role in diverse biotic and abiotic stress response. The GT transcription factors in several plants have been systematically identified and assessed, some of them have been used for stress tolerant breeding, therefore, GT factors have not been reported in sugar beet.\u003c/p\u003e\u003cp\u003eSugar beet (\u003cem\u003eBeta vulgaris\u003c/em\u003e L.) is one of the important sugar-yielding economic crop and has been cultivated for hundreds of years. In addition to the conventional nutrients, the sugar beet also contains a variety of physiologically active substances that are beneficial to human health[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In this study, the trihelix gene family members in sugar beet were identified and characterized. We performed system analysis in terms of molecular evolution, chromosomal distributions, gene structures, conserved motifs, protein properties and gene expression patterns. The orthology relations, gene duplication events were also identified, moreover, phylogenetic trees of trihelix genes were constructed. Although trihelix families have been reported in Arabidopsis, rice, tomato, soybean and tartary buckwheat, the sugar beet trihelix family remains uncharacterised. In this study, we present a genome-wide survey of \u003cem\u003eBvGT\u003c/em\u003e genes and their expression under eight abiotic stresses, providing resources for stress-resilient sugar beet breeding.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eIdentification and physicochemical properties analysis of trihelix family members in sugar beet (\u003c/b\u003e\u003cb\u003eBeta vulgaris\u003c/b\u003e \u003cb\u003eL.)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn this study, a total of 37 non-redundant trihelix family genes were identified in the sugar beet with reference to genome database. The 37 trihelix genes were designated BvGT1-BvGT37 according to their position on the chromosome. The physical and chemical properties of predicted \u003cem\u003eBvGT\u003c/em\u003e genes were listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, including gene ID, protein length, molecular weight (MW), isoelectric point (IP), subcellular localization, domains and chromosome location. Among the 37 members identified, the BvGT gene encoded proteins with lengths ranging from 205 amino acids (BvGT 17) to 886 aa (BvGT13), with an average of 407 aa. The molecular weight of the BvGT proteins ranged from 24.28 KDa to 98.3 KDa, with an average of 6.89. The subcellular localization prediction results showed that 28 \u003cem\u003eBvGT\u003c/em\u003e genes were located in the nucleus, 6 in the chloroplast, 2 in the cytosol and 1 in the extracellular. From the results, 19 \u003cem\u003eBvGT\u003c/em\u003e genes in 37 (51.4%) contained GT1 domain, 15 \u003cem\u003eBvGT\u003c/em\u003e genes (40.5%) contained Myb_DNA-binding domain and 3 \u003cem\u003eBvGT\u003c/em\u003e genes (8.1%) contained both GT1 and Myb_DNA-binding domain. Additionally, the amino acid sequences and coding sequences of \u003cem\u003eBvGT\u003c/em\u003e genes were provided in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhylogenetic analysis and classification of\u003c/b\u003e \u003cb\u003eBvGT\u003c/b\u003e \u003cb\u003egenes in\u003c/b\u003e \u003cb\u003eBeta vulgaris\u003c/b\u003e \u003cb\u003eL.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo understand the phylogenetic relationship of trihelix genes, the amino acid sequences of trihelix genes including 37 sugar beet genes and 25 \u003cem\u003eA. thaliana\u003c/em\u003e genes were employed to construct a neighbor-joining phylogenetic tree (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Trihelix genes shown in phylogenetic tree were clustered into 6 groups ( SIP1, GTγ, GT1, GT2, GT3 and SH4) according to the conserved amino acids of the GT domain and classification method from Kaplan-Levy et al [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In the phylogenetic tree, the largest subfamily were GT3 and SIP1 (11 members each). GTγ was the smallest subfamily containing 2 \u003cem\u003eBvGT\u003c/em\u003e genes. Moreover, GT1, GT2 and SH4 contained 4, 5, and 4 \u003cem\u003eBvGT\u003c/em\u003e genes, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eExon-intron organization and conserved motif compositions of BvGT family members\u003c/h2\u003e\u003cp\u003eThe BvGT gene structures and phases were analyzed to characterize the trihelix gene family. Trihelix members in the same clade showed similar exon and intron organization (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The number and distribution of exons and introns were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb. GT3 genes harbour the most exons (mean\u0026thinsp;=\u0026thinsp;9.3), whereas SIP1 genes are mostly intronless. Structural characteristics analysis showed that 7 (18.9%) BvGT family members had no intron, 17 (45.9%) BvGT family members had only 1 intron, whereas 1 gene of BvGT13 had the highest number of introns. The number of exons varied from 1 to 17, the BvGT3 in SIP1 subfamily contained the lowest number of exons, whereas the BvGT13 in GT3 subfamily contained the highest.\u003c/p\u003e\u003cp\u003eThe conserved motif distribution of the trihelix family members in sugar beet were predicted by means of MEME search tool. A total of 10 conserved motifs were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec, the detailed sequence of each motif was provided in Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. The motif arrangement of each BvGT protein were shown with the matching color boxes. Motif 1 existed in all BvGT proteins, indicating motif 1 (trihelix core) is ubiquitous and may play critical role in specific processes. Motif 2, motif 5 and motif 6 exited in all the SIP1 subfamily members except BvGT22. In the GT3 subfamily, BvGT1, BvGT4, BvGT14, BvGT20, BvGT30, BvGT31, BvGT 32 and BvGT36 all possessed motif 3 and motif 4 except BvGT1, BvGT20 and BvGT36. Motif 7 and motif 8 were only found in clade GT3. Motif 9 existed simultaneously in GTI, GT2 and SH4 subfamily. Notably, BvGT19, BvGT16, BvGT2 and BvGT21 possessed two copies of motif 10. Additionally, BvGT16 and BvGT22 contained two copies of motif 6, 9 and 10 at the same time. Furthermore, BvGT29 and BvGT34 possessed the least number of motifs, whereas BvGT2 and BvGT16 possessed the most number of motifs, with 2 and 9 motifs, respectively.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eChromosomal localization and collinearity analysis of\u003c/b\u003e \u003cb\u003eBvGT\u003c/b\u003e \u003cb\u003egenes in\u003c/b\u003e \u003cb\u003eBeta vulgaris\u003c/b\u003e \u003cb\u003eL.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe chromosomal positions of \u003cem\u003eBvGT\u003c/em\u003e genes were unevenly distributed on 9 chromosomes of \u003cem\u003eBeta vulgaris\u003c/em\u003e L., as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. \u003cem\u003eBvGT\u003c/em\u003e genes map to all chromosomes except Chr3, Chromosome 9 contained the highest number of \u003cem\u003eBvGT\u003c/em\u003e genes. The number of \u003cem\u003eBvGT\u003c/em\u003e genes on chromosomes were as follows, Chr1 (2, 5.4% ), Chr2 (4, 10.8%), Chr4 (3, 8.1%), Chr5 (5, 13.5%), Chr6 (6, 16.2%), Chr7 (6, 16.2%), Chr8 (4, 10.8%), Chr9 (7, 18.9%). Additionally, colinear analysis showed that there was one pair of segmental duplication genes BvGT30 and BvGT37 with Ka/Ks\u0026thinsp;=\u0026thinsp;0.27 indicates purifying selection, located on chromosome 8 and chromosome 9, respectively. Whereas, there were no duplicated segments located on Chr1-Chr7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). A chromosome area within 200 kb including two more identical genomic regions was defined as a tandem duplication event.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCis-elements in the promoter of BvGT family members\u003c/h3\u003e\n\u003cp\u003eCis-elements are associated with gene transcription through binding to transcription factors. To analyze the putative functions of BvGT family members during plant evolution and response to stresses, a 2000 bp promoter sequences of \u003cem\u003eBvGT\u003c/em\u003e genes were extracted and examined by Plant CARE program. Nine cis-element categories with 55 functions were present in the promoter regions, including development, environmental stress, hormone responsive, light responsive, promoter related element, site-binding, wound-responsive related elements and other elements. The distribution of the cis-elements were shown in Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e. Light responsive elements (G-box, Box 4) are present in all promoter regions of \u003cem\u003eBvGT\u003c/em\u003e genes, with the largest number of 25 in BvGT30 and the least number of 3 in BvGT30. Hormone responsive elements (ABRE, CGTCA-motif) were also identified, including Aauxin, Salicylic acid, MeJA, Estrogen, Ethylene, Gibberellin and Abscisic acid responsive elements. Among all the hormone responsive elements, BvGT11 had the most number with 22, whereas BvGT 30 and BvGT 7 contained the least with only 2 respectively. From the results, BvGT family members contained several abiotic related stress elements, Stress-responsive (MBS, LTR) elements are clade-specific (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). TATA boxes are restricted to the plus strand at\u0026ndash;30 to\u0026ndash;120 bp.\u003c/p\u003e\n\u003ch3\u003eEvolutionary relationship of BvGT family and other representative plant species\u003c/h3\u003e\n\u003cp\u003eTo gain the potential evolutionary relationship of BvGT family and five other plant species (\u003cem\u003eArabidopsis\u003c/em\u003e, tomato, buckwheat, rice and wheat), an unrooted NJ tree was generated using Geneious R11 based on protein sequences of 37 \u003cem\u003eBvGT\u003c/em\u003e genes and trihelix genes of the five other plants. Detailed genetic correspondence was presented in Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e. The BvGT proteins were relatively dispersed in the phylogenetic tree. Motif 1 and 7 were shared by most members of the trihelix family from different plant species. Motif 1, 7 and 8 tended to cluster in rice and wheat, notably, motif 1 and 2 tended to cluster in sugar beet, \u003cem\u003eArabidopsis\u003c/em\u003e, tomato and buckwheat, indicating sugar beet may be more closely related to \u003cem\u003eArabidopsis\u003c/em\u003e, tomato and buckwheat than rice and wheat (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo gain a closer understanding of the synchronization relation of sugar beet trihelix family, five representative plant species were selected to construct comparative system diagrams, including three eudicots (\u003cem\u003eArabidopsis thaliana\u003c/em\u003e, tomato, buckwheat) and two monocotyledonous plants (rice and wheat). The results of covariance analysis were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. the number of collinear genes between \u003cem\u003eBeta vulgaris\u003c/em\u003e L and \u003cem\u003eArabidopsis thaliana, Solanum lycopersicum, Fagopyrum tataricum, Oryza sativa Japonica and Triticum aestivum\u003c/em\u003e were 20, 22, 18, 5, and 7, respectively. Generally, \u003cem\u003eSolanum lycopersicum\u003c/em\u003e was the most similar with \u003cem\u003eBeta vulgaris\u003c/em\u003e L and the least similar was \u003cem\u003eOryza sativa Japonica\u003c/em\u003e, indicating the phylogenetic evolutionary relationship among these plant species. To extently understand the evolutionary role of BvGT family, Tajima D neutrality test was performed and the statistic D value was 9.49, indicating the BvGT family was strongly selected in the evolution of sugar beet (Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTissue-specific gene expression patterns of\u003c/b\u003e \u003cb\u003eBvGT\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTrihelix genes were reported to play key roles in plant growth and development[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. To obtain insight into the physiological role of \u003cem\u003eBvGT\u003c/em\u003e genes in sugar beet growth and development. The expression levels of nine selected \u003cem\u003eBvGT\u003c/em\u003e genes were examined by qRT-PCR. Histograms were employed to depict the expression profiles of \u003cem\u003eBvGT\u003c/em\u003e genes in root, stem and leaf of sugar beet (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). Notably, the \u003cem\u003eBvGT\u003c/em\u003e genes were highly expressed in selected tissues and organs, suggesting that \u003cem\u003eBvGT\u003c/em\u003e genes performed multiple function in sugar beet growth and development. Specifically, BvGT10, BvGT23 and BvGT34 were highly expressed in root, BvGT23 and BvGT34 were highly expressed in stem. Additionally, BvGT2, BvGT5, BvGT13 and BvGT34 were relatively highly expressed in leaves. The expression of BvGT10 was the highest in the roots, and BvGT23 expression was the highest in the stem, whereas BvGT34 expression was the highest in leaves. In contrast, the expression patterns of BvGT4 and BvGT23 genes in the leaves were low, as well as BvGT23 expression in root was very low.\u003c/p\u003e\u003cp\u003eFurthermore, the correlation between the BvGT expression profiles were examined and found the majority of the \u003cem\u003eBvGT\u003c/em\u003e genes were positively correlated with other genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). BvGT11 was positively related with BvGT23, BvGT2, BvGT13, BvGT5 and BvGT34, while BvGT3 was negatively correlated with BvGT23, BvGT11, BvGT13 and BvGT5. Additionally, the pairs of BvGT4 and BvGT13, BvGT10 and BvGT11 were significantly negatively correlated with each other.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression patterns of\u003c/b\u003e \u003cb\u003eBvGT\u003c/b\u003e \u003cb\u003egenes in sugar beet under different abiotic stresses\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSubsequently, the responses of nine selected \u003cem\u003eBvGT\u003c/em\u003e genes to different abiotic stresses (osmotic pressure, flooding, acid, alkaline, low temperature, high temperature, dark and salt) were evaluated from RNA-seq data of tissues with various treatments. The \u003cem\u003eBvGT\u003c/em\u003e genes exhibited different degrees of responses to these abiotic stresses in specific tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Heat-map visualisation shows rapid (2 h) induction under flooding, heat and cold, whereas osmotic, salt and alkaline stresses elicited stronger responses after 24 h. The expression level of BvGT3, 4, 5, 10, 11 and 23 in root were significantly up regulated after 24h under osmotic, acid, alkaline, high temperature, dark and salt stresses, whereas BvGT2 and BvGT34 in root were down regulated after 24h under osmotic pressure. Across the eight different treatments, the nine \u003cem\u003eBvGT\u003c/em\u003e genes expressed in specific tissues consistently. For instance, BvGT10 was significantly up regulated in all tissues after exposure to osmotic, acid, alkaline, low temperature, high temperature, and salt for 24h. Moreover, BvGT3 was significantly down regulated in all tissues after exposure to low temperature and dark for 24h. Particularly, BvGT4 and BvGT23 were highly expressed in root after 24h of exposure to osmotic, alkaline and salt. BvGT4 and BvGT10 were consistently up-regulated in roots across all abiotic stresses (\u0026gt;\u0026thinsp;4-fold), whereas BvGT2 was down-regulated under drought.\u003c/p\u003e\u003cp\u003eCorrelation networks (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb) imply coordinated transcriptional modules. There was no significant correlation between BvGT2 and the other 8 trihelix genes. In contrast, the relative expression of BvGT5 and BvGT3/BvGT23/BvGT4 were highly positively associated. Additionally, BvGT11 and BvGT4, BvGT34 and BvGT13/BvGT10 were positively correlated.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSugar beet is an important sugar producing crop in temperate regions. 30% of the world\u0026rsquo;s annual plant sugar production comes from sugar beet. It is also an important source of bioethanol, natural pigments, edible vegetables, and animal feed. In contrast to the well-known transcription factor of MYB, WORKY, ERF and NAC, trihelix factors had received attention only recently, which was first reported in pea. However, trihelix family in sugar beet genome have not been published. The reference genome of sugar beet (\u003cem\u003eBeta vulgaris\u003c/em\u003e) was reported in 2014 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], which provide an abundant theoretical basis for us to analyze the characteristics and functions of sugar beet trihelix. Within this study, 37 \u003cem\u003eBvGT\u003c/em\u003e genes were identified in sugar beet, which was agree with studies on trihelix genes in \u003cem\u003eArabidopsis\u003c/em\u003e, tomato[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], rice, buckwheat sorghum[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] and quinoa[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The \u003cem\u003eBvGT\u003c/em\u003e genes account 0.13% of the total genes in sugar beet, which was similar to G. max (0.14%) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], \u003cem\u003eArabidopsis\u003c/em\u003e (0.11%) [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and rice (0.1%) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Moreover, buckwheat (0.06%) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], \u003cem\u003eChrysanthemum\u003c/em\u003e (0.04%) [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], \u003cem\u003eS. lycopersicum\u003c/em\u003e (0.05%) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and wheat (0.08%) [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] owned a low percent in their genome. Physicochemical properties including protein length, MW and IP were collated by bioinformatic methods. Differences of protein properties demonstrated different functions of the trihelix family members. Additionally, Trihelix family were grouped into three subfamilies previously (GTα, GTβ, and GTγ) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], soon afterwards, they were classified into five subfamilies by Kaplan-Levy \u003cem\u003eet al\u003c/em\u003e [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. With in this study, the sugar beet trihelix genes were classified into six subfamilies (SIP1, GTY, GT1, GT2,GT3, and SH4) according to phylogenetic analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Additionally, the functions of BvGT family members presented on the phylogenetic tree will provide potential realistic and reliable resources for gene function exploration in later stages.\u003c/p\u003e\u003cp\u003eStudies on the exon and intron architecture of the 37 \u003cem\u003eBvGT\u003c/em\u003e genes revealed that the count of exons ranged from 1 to 17 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). The proportion of \u003cem\u003eBvGT\u003c/em\u003e genes without introns was 18.9%, which was close to quinoa (10.6%) and lower than that of rice (43.9%) and sorghum (37.5%). Five of the intro-free genes were distributed in the SIP subfamily and 2 in GTγ subfamily, which were similar to sorghum and \u003cem\u003eArabidopsis\u003c/em\u003e previously reported. Introns can increase the length and complexity of genes, which play key roles in gene expression, protein synthesis, and genome evolution. Moreover, intro-free genes tend to more responsive to environment change [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. There were differences in gene exon numbers between subfamilies, further analysis revealed that the GT3 subfamily had the most number of exons whereas the SIP1 subfamily contained the lowest. The results were different from wheat [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and buckwheat [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], which findings of studies showed that GT1subfamily had the most of exons and GTγ family had the least.\u003c/p\u003e\u003cp\u003eThe results of motif composition of trihelix were consistent with phylogenetic classification results. 19 of the \u003cem\u003eBvGT\u003c/em\u003e genes only had the GT domain, most of which belonged to GT1 and GT3 subfamily. 15 of the \u003cem\u003eBvGT\u003c/em\u003e genes only had the Myb_DNA-binding domain, most of which belonged to SIP1 subfamily. Whereas 3 \u003cem\u003eBvGT\u003c/em\u003e genes had both GT and Myb_DNA-binding domain and attributed to GT2 subfamily. Members in the same subfamily possessed considerable similarities, indicating the conserved motifs may play essential functions of the specific \u003cem\u003eBvGT\u003c/em\u003e genes. These results revealed that members in the same subfamily may owns similar roles in growth and development of sugar beet.\u003c/p\u003e\u003cp\u003eResults of chromosome localization showed that \u003cem\u003eBvGT\u003c/em\u003e genes were unequally distributed on nine chromosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Chromosome 3 harbored no BvGT gene, which may experience gene loss during evolution [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Chromosome 9 harbored the most of \u003cem\u003eBvGT\u003c/em\u003e genes, with the number of 7, which may be beneficial to gene evolution. In addition, colinear analysis showed that there was one pair of segmental duplication genes in BvGT gene family of sugar beet. Whereas, there were no duplicated segments located on Chr1-Chr7 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Gene duplication is very important for genome evolutionary mechanism, directly influences gene amplification and recombination. Interestingly, the number of \u003cem\u003eBvGT\u003c/em\u003e genes in sugar beet is less than soybean (67) [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], \u003cem\u003eG.max\u003c/em\u003e (71) [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] and \u003cem\u003eB. napus\u003c/em\u003e (52). The difference may be due to whole genome replication events from the earliest ancient plants differentiated. From the present results, only one pair of segmental duplication on sugar beet chromosomes was discovered (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e), indicating that the emergence and the evolution of \u003cem\u003eBvGT\u003c/em\u003e genes were probably driven by replication events and some other factors. This is different from research in tartary buckwheat and \u003cem\u003ePopulus trichocarpa\u003c/em\u003e [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCis-elements on the promoter may play important roles in the function of \u003cem\u003eBvGT\u003c/em\u003e genes. Interestingly, 37 \u003cem\u003eBvGT\u003c/em\u003e genes identified in this study are involved in light response and hormone response. Additionally, BvGT 11 had both the most number of light response and hormone response elements, with the number of 16 and 22 respectively, whereas BvGT 30 contained the least light response elements with the number 1, BvGT30 and BvGT 7 contained the least hormone response elements with the number 2 individually. Cis-elements related to drought, low-temperature, wound stress were also identified, including F-box, CGTCA-motif, Myb-binding site and MYC, etc. These results showed that some of the \u003cem\u003eBvGT\u003c/em\u003e genes are involved in the regulation of biotic and abiotic stresses, indicating that they may play vital role in response to biotic and abiotic stresses.\u003c/p\u003e\u003cp\u003eThe trihelix gene family members were involved in plant development in previous studies. The functions of \u003cem\u003eBvGT\u003c/em\u003e genes were explored in this research. We determined the relative expression of \u003cem\u003eBvGT\u003c/em\u003e genes in roots, stems and leaves of sugar beet by qRT-PCR. The selected nine \u003cem\u003eBvGT\u003c/em\u003e genes showed considerable differences in expression patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Notably, \u003cem\u003eBvGT23\u003c/em\u003e from GTγ subfamily, homologous to gene HRA1 (\u003cem\u003eAT3G10040.1\u003c/em\u003e) had the highest expression level in stems. HRA1 expressed in stems of \u003cem\u003eArabidopsis thaliana\u003c/em\u003e regulating leaf structure and shoot development [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. \u003cem\u003eBvGT10\u003c/em\u003e, which is homologous to \u003cem\u003eAT2G38250.1\u003c/em\u003e and \u003cem\u003eSolyc09g008830.3.1\u003c/em\u003e (Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e), was highly expressed in roots. \u003cem\u003eBvGT34\u003c/em\u003e was significantly high expressed in the roots, stems and leaves, indicating multiple different functions. In particular, the expression of \u003cem\u003eBvGT13, BvGT2\u003c/em\u003e and \u003cem\u003eBvGT4\u003c/em\u003e were the lowest in roots, stems and leaves respectively. Therefore, these results suggested that tissue-specific \u003cem\u003eBvGT\u003c/em\u003e genes are important in the growth and development of relevant tissues [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. However, later experiments are required to verify their functions in the future. In addition, the significantly positive correlation between \u003cem\u003eBvGT\u003c/em\u003e genes indicating that they may have spatial and temporal specificity functions.\u003c/p\u003e\u003cp\u003eTo further elucidate the function of the trihelix gene family in stress adaption, the expression patterns of nine \u003cem\u003eBvGT\u003c/em\u003e genes in sugar beet root, stem and leaf were systematically analyzed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Therefore, the expression characteristics of \u003cem\u003eBvGT\u003c/em\u003e genes exhibited spatial variation. The roots of plants can perceive drought changes of the soil and trigger physio-chemical reaction of plants to reduce the damage. In present study, under drought treatment, seven \u003cem\u003eBvGT\u003c/em\u003e genes (\u003cem\u003eBvGT3, BvGT4, BvGT5, BvGT10, BvGT11, BvGT13, BvGT23\u003c/em\u003e) were induced obviously in roots, indicating that they may contribute to the adaption of sugar beet to drought conditions. In addition, there was a substantial decrease in the expression of \u003cem\u003eBvGT2\u003c/em\u003e in roots under drought conditions whereas increased significantly under flooding, which belonged to GT2 subfamily. GT2 subfamily were reported as suppressor related to stomatal density and distribution [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Therefore, \u003cem\u003eBvGT2\u003c/em\u003e may contribute to water use in sugar beet through regulating stomata number [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Interestingly, \u003cem\u003eBvGT4\u003c/em\u003e and \u003cem\u003eBvGT10\u003c/em\u003e grouped in GT3 and GT1 respectively were highly induced in response to almost all stresses in roots, indicating the two genes may play multiple functions in various stresses, which can be verified by further experiments. Similarly, the expression of \u003cem\u003eBvGT34 from\u003c/em\u003e GT3 subfamily in leaves were induced significantly in all stress conditions, suggesting the function of enhancing adaptability of sugar beet to environment. In conclusion, the expression characteristics of \u003cem\u003eBvGT\u003c/em\u003e genes from the six subfamilies showed tremendous differences, indicating their unique functions under various stresses. However, functions of \u003cem\u003eBvGT\u003c/em\u003e genes still need further validation.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eTo sum up, this is the first genome-wide of trihelix family genes in sugar beet. We screened 37 members of trihelix genes across nine chromosomes and clustered them into six groups. Additionally, physicochemical properties, expression profiles and phylogenetic analysis were evaluated. The expression patterns of the \u003cem\u003eBvGT\u003c/em\u003e genes in different tissues of sugar beet under eight abiotic stresses were analyzed by qRT-PCR. Based on above research, some stress tolerance candidate genes were screened out. In particular, \u003cem\u003eBvGT23\u003c/em\u003e was highly expressed in stems and responded to drought. \u003cem\u003eBvGT4\u003c/em\u003e and \u003cem\u003eBvGT10\u003c/em\u003e were consistently up-regulated in roots under all abiotic stresses. The present study provide a comprehensive atlas of the 37-member trihelix family in sugar beet. The stress-inducible genes BvGT4, BvGT10 and BvGT23 are promising targets for functional studies and breeding.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eB. vulgaris\u003c/b\u003e \u003cb\u003etrihelix (BvGT) gene Identification\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe entire sugar beet genome sequence information was downloaded from (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://bvseq.boku.ac.at/index\u003c/span\u003e\u003cspan address=\"https://bvseq.boku.ac.at/index\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. shtml) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The trihelix genes in sugar beet were identified by BLASTp methods (E\u0026thinsp;\u0026le;\u0026thinsp;1e-10, score\u0026thinsp;\u0026ge;\u0026thinsp;100) using Arabidopsis trihelix proteins as queries. Candidate proteins with score value\u0026thinsp;\u0026ge;\u0026thinsp;100 and e-value\u0026thinsp;\u0026le;\u0026thinsp;1e\u0026thinsp;\u0026minus;\u0026thinsp;10 were identified from sugar beet genome. The Hidden Markov Model (HMM) files consistent with trihelix domain were downloaded from the PFAM protein family database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.sanger.ac.uk/\u003c/span\u003e\u003cspan address=\"http://pfam.sanger.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. HMMER3.0 with a cutoff of 0.01 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://plants.ensembl.org/hmmer/index\u003c/span\u003e\u003cspan address=\"http://plants.ensembl.org/hmmer/index\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. html) [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e] and SMART (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://smart\u003c/span\u003e\u003cspan address=\"http://smart\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. embl-heidelberg. de/) [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e] were used to determine the candidate BvGT proteins containing the conserved trihelix structural domain. CDS and protein sequences were validated by SMART and InterProScan. In addition, subcellular localization was predicted by Cell-PLoc (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://chou.med.harvard.edu/bioinf/Cell-PLoc\u003c/span\u003e\u003cspan address=\"http://chou.med.harvard.edu/bioinf/Cell-PLoc\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and ProtComp 9.0 [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Subsequently, feature information of projected trihelix proteins was computed from the ExPasy website (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://web.expasy.org/protparam/\u003c/span\u003e\u003cspan address=\"http://web.expasy.org/protparam/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), including CDS length, protein pI and theoretical molecular weights \u003cem\u003eet al\u003c/em\u003e [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. In the end, 37 members of trihelix genes from sugar beet were screened out.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCharacterization and Phylogenetic analyses of\u003c/b\u003e \u003cb\u003eBvGT\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe exon-intron structures of \u003cem\u003eBvGT\u003c/em\u003e genes were generated from Gene Structure Display Server (GSDS: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://gsds.cbi.pku.edu.cn\u003c/span\u003e\u003cspan address=\"http://gsds.cbi.pku.edu.cn\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) by comparing the CDS of BvGTs with their genomic DNA sequences [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Clustal W with default parameters was employed to create multiple protein sequence alignment of the characterized BvGT proteins [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. To characterize the structure and detect the differences of BvGT proteins, the conserved motifs were determined and compared using an online search software Multiple Em for Motif Elicitation MEME (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://meme-suite\u003c/span\u003e\u003cspan address=\"http://meme-suite\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. org/ tools /meme) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. The parameters were set as follows: the number of motifs searched with a maximum ten motifs and the motif length was 6-200 amino acid residues [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. All motifs were further annotated with InterProScan (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ebi.ac.uk/interpro/\u003c/span\u003e\u003cspan address=\"http://www.ebi.ac.uk/interpro/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Cis-elements on the upstream 2000 bp promoter sequences of \u003cem\u003eBvGT\u003c/em\u003e genes were explored on the PlantCARE website (\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) [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. In addition, multiple sequence alignments of the \u003cem\u003eArabidopsis\u003c/em\u003e trihelix and BvGT proteins were performed to construct a neighbor-joining phylogenetic tree by MEGA 7.0 with a bootstrap value of 1000 replicates and default parameters [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. To gain the evolutionary relationship of BvGT family and five other plant species, phylogenetic trees were constructed using the full length amino acid sequences of trihelix proteins from \u003cem\u003eA. thaliana, S. lycopersicum, F. tataricum, O. sativa\u003c/em\u003e and \u003cem\u003eT. aestivum.\u003c/em\u003e Sequences of trihelix proteins were acquired from UniProt database (UniProt \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.uniprot.org/\u003c/span\u003e\u003cspan address=\"https://www.uniprot.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eGene chromosomal distribution and gene duplication\u003c/h3\u003e\n\u003cp\u003eThe distribution of all \u003cem\u003eBvGT\u003c/em\u003e genes were mapped to \u003cem\u003eB. vulgaris\u003c/em\u003e chromosomes based on physical position information obtained from \u003cem\u003eB. vulgaris\u003c/em\u003e genome database by Circos software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://circos.ca/software/download/\u003c/span\u003e\u003cspan address=\"http://circos.ca/software/download/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Gene duplication events of \u003cem\u003eBvGTs\u003c/em\u003e were performed by Multiple collinear scanning toolkits (MCScanX) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/wyp1125/MCScanx\u003c/span\u003e\u003cspan address=\"https://github.com/wyp1125/MCScanx\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) with default settings [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. The syntenic relationship between \u003cem\u003eBvGT\u003c/em\u003e genes and five representative plants including \u003cem\u003eA. thaliana, S. lycopersicum, F. tataricum, O. sativa\u003c/em\u003e and \u003cem\u003eT. aestivum\u003c/em\u003e were analyzed by Dual Synteny Plotter (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://github.com/CJ-Chen/TBtools\u003c/span\u003e\u003cspan address=\"https://github.com/CJ-Chen/TBtools\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Ka/Ks ratios were calculated by KaKs_Calculator 2.0.\u003c/p\u003e\n\u003ch3\u003ePlant materials, growth condition and abiotic stress treatment\u003c/h3\u003e\n\u003cp\u003eThe \u003cem\u003eB. vulgaris\u003c/em\u003e variety of \u0026ldquo;Detian 316\u0026rdquo; was selected for this study. The sugar beet plants were cultivated in pots filled with 50% soil and 50% vermiculite in greenhouse (12h day/12h night, 25℃ day/18℃ night, the relative humidity was 70%). Twenty one day old seedlings were subjected to eight stresses. Samples of roots, stems and leaves were collected from five good growth plants, then placed in liquid nitrogen for storage at -80℃ for RNA extraction. The healthy plants were exposed to various abiotic stress conditions for 2h and 24h treatment, including 30% PEG6000 [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e, \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e], flooding (root submerged 2 cm), HCI (pH 4.5), NaOH (pH 9.0), low temperature (4℃), high temperature (40℃), darkness and salt stress (400mM NaCl) [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Root (0\u0026ndash;2 cm tips), stem (third internode) and Young fully-expanded leaves were harvested. Three biological replicates (five plants each) were snap-frozen in liquid N₂. Subsequently, the expression patterns of nine selected \u003cem\u003eBvGT\u003c/em\u003e genes in different parts of sugar beet under stresses were analyzed by qRT-PCR.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eExpression profiles of \u003cem\u003eBvGT\u003c/em\u003e genes by qRT-PCR\u003c/h2\u003e\u003cp\u003eThe expression profiles of nine \u003cem\u003eBvGT\u003c/em\u003e genes were analyzed from tissues collected (roots and stress treated roots, stems and stress treated stems, leaves and stress treated leaves) by qRT-PCR analysis. Total RNA (TRIzol) was treated with DNase I (Thermo). First-strand cDNA was synthesised with HiScript III RT SuperMix (Vazyme). qRT-PCR was performed on a CFX96 Touch (Bio-Rad) using SYBR Green (Vazyme). BvGAPDH (accession: XM_010691997) served as the internal reference. Primer sequences shown in Table \u003cspan refid=\"MOESM7\" class=\"InternalRef\"\u003eS7\u003c/span\u003e for qRT-PCR were designed by Primer 5.0 software [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. Primers had efficiencies 95\u0026ndash;105% (R\u0026sup2;\u0026ge;0.99). Relative expression was calculated by 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe acquired data in present study were subjected to analysis of variance ANOVA using SPSS software. Mean values were compared by LSD test at the 0.05 significance level. Origin 8.0 was used to construct histograms of the data. Tajima\u0026rsquo;s D neutrality test was performed by MEGA 7.0 [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eAll methods were carried out in accordance with relevant guidelines and regulations.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe Natural Science Foundation of the Jiangsu Higher Education Institutions of China.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eShijuan Li: Conceptualization, investigation, data curation, formal analysis, visualization, writing the original draft, project administration. Rui Sun: investigation, data curation. Yaoyao Du: formal analysis, data acquisition. Haiye Luan: formal analysis, project administration. Delai Chen: formal analysis. Muhammad Khurshid: Conceptualization, investigation, formal analysis. Shimei li and Po Li: investigation, formal analysis.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eData availability and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe complete information on sugar beet (\u003cem\u003eBeta vulgaris\u003c/em\u003e L.) genome sequence are available in NCBI taxonomy database (https://www.ncbi.nlm.nih.gov/datasets/taxonomy/3555/). Data analyzed in current study are provided in article and the supplementary information files.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGuo H, Wang L, Yang C, Zhang Y, Zhang C, Wang C. 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Mol Biology Evol 2016(7):1870.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Genome-wide identification, Beta vulgaris L., Trihelix genes, abiotic stresses, Gene duplication","lastPublishedDoi":"10.21203/rs.3.rs-7657378/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7657378/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eTrihelix transcription factor family genes are known as GT factors, which play vital roles in growth and development process, response to various abiotic stresses in plants. Studies on trihelix gene in several eudicots and monocots have been thoroughly performed, but trihelix family gene in sugar beet (\u003cem\u003eBeta vulgaris\u003c/em\u003e L.), one of the major sugar crops in the world, has not yet been systematic studied.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eWe identified 37 non-redundant \u003cem\u003eB. vulgaris\u003c/em\u003e trihelix (BvGT) genes named from \u003cem\u003eBvGT1\u003c/em\u003e to \u003cem\u003eBvGT37\u003c/em\u003e in present study and classified them into six clades (SIP1, GTγ, GT1, GT2, GT3 and SH4) by maximum-likelihood phylogeny (IQ-TREE2, 1000 ultrafast bootstraps). The \u003cem\u003eBvGT\u003c/em\u003e genes harbour 1\u0026ndash;17 exons and ten conserved motifs; motif 1 (core trihelix domain) is present in all members. Chromosomal mapping showed an uneven distribution across eight chromosomes; chromosome 3 lacks any \u003cem\u003eBvGT\u003c/em\u003e gene. Only one segmental duplication pair (BvGT30/BvGT37) was detected. Promoter cis-element profiling revealed abundant light-, hormone- and stress-responsive motifs. Expression analysis by qRT-PCR (three biological replicates \u0026times; three technical replicates) demonstrated tissue- and stress-specific transcription patterns. BvGT10, BvGT23 and BvGT34 were the most highly expressed genes in root, stem and leaf, respectively. Under salt, alkali and osmotic stresses, BvGT4 and BvGT10 showed consistent up-regulation in roots.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis study identified 37 \u003cem\u003eBvGT\u003c/em\u003e genes in sugar beet and further analyzed their evolution and expression pattern. Segmental duplication and purifying selection have shaped the expansion of the trihelix family in sugar beet. The identified stress-responsive \u003cem\u003eBvGT\u003c/em\u003e genes provide a theoretical basis for function investigation of \u003cem\u003eBvGT\u003c/em\u003e genes and present stress-resistant and high yield candidate genes for molecular breeding toward enhanced abiotic-stress tolerance.\u003c/p\u003e","manuscriptTitle":"Genome-wide identification, expression profiles and phylogenetic analysis of Trihelix transcription factor family genes in sugar beet (Beta vulgaris L.) under abiotic stresses","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-11 12:34:31","doi":"10.21203/rs.3.rs-7657378/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5df7ab6f-057f-442f-aa5e-b55de75c88bd","owner":[],"postedDate":"December 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-12T10:11:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-11 12:34:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7657378","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7657378","identity":"rs-7657378","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}