Genome-wide identification and expression profile analysis of the NF-Y transcription factor gene family in Eucalyptus grandis

Genome-wide identification and expression profile analysis of the NF-Y transcription factor gene family in Eucalyptus grandis | 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 and expression profile analysis of the NF-Y transcription factor gene family in Eucalyptus grandis Juan Li, Chaoyan Gong, Li Zhuang, Guangyou Li, Jianmin Xu, Zhaohua Lu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4703272/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 The NF-Y (NUCLEAR FACTOR-Y) transcription factor in plants is composed of NF-YA, NF-YB and NF-YC subunits. It is known to play an important role in plant growth and development and response to stress. Although the NF-Y gene family has been systematically studied in many species, the understanding of the NF-Y gene family in Eucalyptus remains unknown. Results In this study, 31 (7 EgrNF-YA, 16 EgrNF-YB and 8 EgrNF-YC) EgrNF-Y genes were identified in E. grandis using Arabidopsis NF-Y protein sequences as queries and their structural characteristics were comprehensively analyzed. Phylogenetic, conserved domain and exon-intron structure analyzed that the closer relationship in each subfamily. Multiple alignments showed that all EgrNF-Y proteins had conserved core regions. Chromosomal localization of these genes revealed that they were randomly distributed across 11 chromosomes. Cis -element analysis of promoter indicated that EgrNF-Y gene was affected by various hormonal and abiotic stresses. Furthermore, tissue-specific expression showed that all 30 EgrNF-Y genes were widely expressed in various tissues and organs. Additionally, the stress response pattern of EgrNF-Ys was identified under phosphate-starved, and 12 genes and 3 genes were upregulated more than 2-fold in the leaves and roots, respectively. Conclusion Our studies have provided a general understanding of the conservation and characteristics of the EgrNF-Y genes family in E. grandis . And it has been demonstrated that members of the EgrNF-YB1 and EgrNF-YB11 may play important roles in the regulation of floweringin of E. grandis . To provide reference for further study on the role of NF-Y gene in the regulation of flowering in E. grandis . In addition, our also established a theoretical basis for further functional studies on this family. NF-Y Eucalyptus grandis Bioinformatics analysis Expression analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Transcription factors (TFs), a protein that binds to a cis -acting element in the promoter region of a eukaryotic gene and enables the target gene to be expressed in a specific way, play vital roles in numerous in many plants growth, development and abiotic stress. Nuclear factor Y (NF-Y), also famous as heme activator protein (HAP) or CCAAT-binding factor (CBF), is one of the most widespread heterotrimeric transcription factors found in numerous genes in fungi, animal, and plants. In plants, NF-Y contains three unique subunits: NF-YA (HAP2 or CBF-B), NF-YB (HAP3 or CBF-A), and NF-YC (HAP5 or CBF-C) [ 1 ]. NF-Y is highly conserved in all higher eukaryotes [ 2 ]. In yeast and animals, each NF-Y subunit is encoded by a single gene. However, each NF-Y subunit gene is represented by multiple orthologs in plants, indicating that heterotrimer complexes composed of different NF-Y members play different regulatory roles [ 3 ]. To date, NF-Y transcription factors have been identified in several herb species, including 33 NF-Y member genes were identified in Arabidopsis thaliana [ 4 ], 28 NF-Y members in Oryza sativa [ 5 ], 68 NF-Y in Glycine max [ 6 ], 33 members in Brassica napus . L [ 7 ], 60 NF-Y member genes in Medicago sativa [ 8 ], 27 members Petunia hybrida [ 9 ]. Furthermore, NF-Y gene members have also been reported in many species of woody plants. For example, 46 members were identified in Populus [ 10 ], 28 members were identified in Pinus tabuliformis [ 11 ] and 22 members in Citrus [ 12 ]. Recently, the NF-YB family of Eucalyptus has been analyzed and identified. Although many studies have reported the NF-Y gene family in various plant species, the Eucalyptus NF-Y gene family has not been systematically explored. Many studies have shown that NF-Y genes are responsible for regulating diverse physiological processes related to plant growth and development. Among, AtNF-YB9, the first NF-Y gene cloned in a plant, plays a key role in Arabidopsis seed development[ 13 ]. The NF-Y has also been reported to play an important role in flowering time. Such as, NF-Y mediates the effect of photoperiod and GA signaling on SOC1 expression partly through H3K27me3 demethylation [ 14 ]. Also, CmNF-YB8 can influence flowering time through regulating the expression of cmo-MIR156 in the aging pathway in Chrysanthemum [ 15 ]. In addition, NF-Y plays an important role in the process of pollen tube growth [ 16 ], starch biosynthesis [ 17 ], root elongation[ 18 ], fruit ripening [ 19 ]. In addition to plant growth and development, NF-Y participates in stress response. Studies on NF-Y have focused mainly on drought resistance [ 20 – 22 ], salt stress[ 23 ] and cold [ 24 ]. Studies have also shown that NF-Y protein is a key factor in regulating root nodule formation and nutrient absorption in plants. It has been reported in the Medicago truncatula [ 25 ], Lotus japonicus [ 26 ], Phaseolus vulgaris [ 27 ], Triticum aestivum [ 28 ]. However, whether NF-Y plays a role in growth, development and nutrient absorption in Eucalyptus remains unclear. Eucalyptus grandis , a species of Eucalyptus in Myrtaceae, is a significant fast-growing tree species. E. grandis is grown primarily in the sorthern and sorthwestern of China. It has the advantages of short growth cycle, strong stress resistance and strong toughness of wood and high economic value. It is considered one of the three fastest growing trees in the world along with poplar and pine. Because it is mainly distributed in southwest China and South China, the soil in these areas is generally lacking in phosphorus. Furthermore, the lack of nutrients affects the growth of E. grandis . Most previous studies only focused on the involvement of NF-Y transcription factors in plant root growth and development [ 18 ], but the mechanism of how NF-Y regulates root growth and development under nutrient stress is still limited. Therefore, it is of great significance to identify the NF-Y gene family members and clarify the effects of phosphorus deficiency on the growth of E. grandis . In this study, 31 NF-Y genes were identifed from the E. grandis genome and their physicochemical properties, phylogenetic relationships, gene structure, and chromosome localization were comprehensively analyzed. Moreover, we also analyzed the expression levels of EgrNF-Y in six different tissues and organs and in the condition of phosphorus deficiency by qRT-PCR, which is particularly important for identifying candidate genes involved in regulation of the growth and response to phosphorus stress of E. grandis . Overall, our results contribute to a more complete understanding of the function of the NF-Y genes in E. grandis , and the study of other woody plants also has high reference value. Results Isolation and identification of the NF-Y family members in E. grandis To obtain information on the NF-Y genes of E. grandis , we used Arabidopsis NF-Y protein sequences as queries to search for the E. grandis NF-Y genes in Phytozome v13 [4]. Through comprehensive screening, including remove those with improper domains and redundant sequences, a total of 31 EgrNF-Y sequences were identified in the E. grandis genome, including 7 NF-YA, 16 NF-YB and 8 NF-YC genes. Furthermore, the physicochemical properties data of all gene members were estimated by ExPASy server (http://WWW.expasy.org/), and the characteristics of the EgrNF-Y sequences are listed in Table 1. Among them, the identified EgrNF-Y genes encoded peptides ranged from 94 to 339 aa. The molecular weights (MWs) and the isoelectric point (pI) values of these proteins ranged from 10.64 kDa to 37.21 kDa, and from 4.49 to 9.48, respectively (Table1). Gene structure and conserved motifs analysis of the EgrNF-Y genes The analysis of gene structure can understand the evolution of gene families. To investigated the evolutionary conservation and divergence of NF-Ys between E. grandis and Arabidopsis, we analyzed the exon-intron gene structure of 31 identified EgrNF-Ys using the GSDS website. Phylogenetic analysis revealed that the EgrNF-Y genes were divided into three groups: EgrNF-YA, EgrNF-YB and EgrNF-YC. Most of the EgrNF-YAs (except EgrNF-YA5 ) had five or six exons with similar distribution. More than two-thirds of Egr NF-YBs had no introns, and the results showed that members with similar numbers of exons and introns are distributed in the same clade. For the EgrNF-YC subfamily, EgrNF-YC1 and EgrNF-YC4 had two exons. EgrNF-YC5 and EgrNF-YC7 have six and five exons, respectively. In general, the gene structure of NF-Y members was positively correlated with their phylogenetic relationships (Fig. 1). Furthermore, the distributions of conserved motifs were assessed by MEME software. The results showed that all of the genes contained motif 1 except EgrNF-YA6. Interestingly, three EgrNF-Y subunits have a unique motif distribution (Fig. 2). For example, motifs 8 were only present in EgrNF-YA , motif 2 was only observed in EgrNF-Y B , and motif 7 was unique to EgrNF-YC . Chromosomal l ocalization and c ollinearity a nalysis of EgrNF-Y s The 31 EgrNF-Y gene members were widely distributed according to the positions of the annotated chromosomes in the E . grandis genome database (Table 1). To further analyze the distribution of EgrNF-Y family members on each chromosome, we constructed a location map using MapInspect software. The result showed that the distribution of NF-Y gene family on the chromosomes of E . grandis was uneven. Among them, the largest number of members distributed on chromosome 2, while only 1 member distributed on chromosome 3 and chromosome 9. Moreover, the number of genes was not positively correlated with chromosome length. For example, chromosome 3 had the largest length, but only one member is distributed (Fig. 3). To further investigated the homologous genes and their evolutionary relationships between E . grandi s , model plant Arabidopsis and woody model plant poplar, a multicomparative synteny map generated between three species. The 31 EgrNF-Ys were located at 11 scafolds, 46 PtNF-Ys were distributed on 19 chromosomes, and 36 AtNF-Ys were distributed on 5 chromosomes, which have a collinearity relationship with EgNF-Ys. Among them, the relationship between E. grandis and poplar is closer, which indicates that the NF-Y family is a close relationship between woody plants (Fig. 4). Conserved regions and phylogenetic relationships of EgrNF-Ys To further investigate the conserved regions of EgrNF-Ys, the protein sequences of the 31 members were analyzed using ClustalW 2.1 and Genedoc software. Multiple sequence alignment results suggested that each EgrNF-Y family member contains a heterodimerization domain and a DNA-binding domain that recognizes the CCAAT site. This core conserved regions of the EgrNF-YAs proteins were 53AAs, including two highly conserved domains: the NF-YB/C subdomain and the DNA binding, they were separated by a conserved linker with 21 AAs. As shown in Fig. 5 B/C, the central domain of EgrNF-YBs had 91AAs. Among EgrNF-YBs, EgrNF-YB2/4/16 had lower conserved domains. Fig.5C showed that EgrNF-YC subunits were also found to consist of a core histone-like sequence with a central domain about 79 AAs in length. Meanwhile, EgrNF-YC5 and EgrNF-YC7 were slightly different from other NF-YCs. This analysis also suggested that EgrNF-YAs is more evolutionarily conserved in the three subfamilies (Fig. 5). To reveal the evolutionary relationship and potential function of EgrNF-Ys, an phylogenetic tree was constructed using the NF-Y protein sequences of E. grandis , A. thaliana and P. trichocarpa were created by MEGA7 software with the neighbor-joining (NJ) criteria. The phylogenetic analysis revealed that the 107 NF-Y proteins were clustered into three groups: NF-YA (yellow), NF-YB (green), and NF-YC (pinkish red). It is consistent with our subfamily classifications of the EgrNF-Ys (Table 1). Based on the phylogenetic relationship of the evolutionary tree and the reported functions of AtNF-Ys, the functions of EgrNF-Y members can be further predicted. In each group, we found that some pairs of paralogous NF-Y proteins were composed of one EgrNF-Y and one AtNF-Y, such as EgrNF-YA1 and AtNF-YA6, EgrNF-Y5 and AtNF-YA11, and this close evolutionary relationship generally suggested the similarity of their biological functions. We also found EgrNF-YB9, AtNF-YB6/9 and PtNF-YB3/5 clustered in a subgroup, belonging to LEC1 and its homolog LEC1-like. In addition, we also identified three pairs of analogues: EgrNF-YB4 and EgrNF-YB5, EgrNF-YB7 and EgrNF-YB14, EgrNF-YC2 and EgrNF-YC3, while most EgrNF-Ys share low homology with other members, suggesting that they have evolved in diversity. Analysis of cis -elements in the promoter regions of EgrNF-Y genes In order to further explored the potential function of the 31 EgrNF-Y at the transcriptional level, the distribution of cis -elements in the EgrNF-Y promoter regions (2000 bp) was scanned using the PlantCARE software. A total of 19 types of cis -elements were identified in the 31 EgrNF-Y , including light responsive, hormone responsive, stress responsive, growth regulation, and some common and core cis -elements, such as the TATA-box and CAAT-box. The detailed classifcation and sequence information of all the cis -elements are listed in Table S2. Meanwhile, the transcription regulatory cis -elements binding site are also shown in fig.7 except some common and core cis -elements. This result analysis showed that the promoter regions of the EgrNF-Ys contain several phytohormone response cis -elements, including gibberellin, salicylic acid, MeJA, auxin, ethylene, and abscisic acid responsive elements. The results indicated that EgrNF-Ys may play an important role in response to stress and growth regulation. Expression p rofiles of the EgrNF-Ys in d ifferent t issues To further investigate the possible functions of the EgrNF-Ys genes in the developmental processes of E . grandis , we examined their gene expression profiles using quantitative real-time PCR in six different tissues and organs (root, stem, young leaf, mature leaf, xylem and flower). All member genes except EgrNF-YA5 were expressed. Therefore, the tissue-specific expression patterns of 30 EgrNF-Ys member genes were analyzed. The results demonstrated that 30 genes were widely expressed in various tissues and organs of E . grandis , but they exhibited different spatial and temporal expression patterns. For EgrNF-YA subfamilies, EgrNF-YA1 and EgrNF-YA7 were significantly expressed in flowers and roots, respectively. Expression was strongest for EgrNF-YA4 and EgrNF-YA6 in the leaves. For EgrNF-YB subfamilies, EgrNF-YB1 and EgrNF-YB11 was most highly expressed in the flowers, more than 90% of other EgrNF-YB members have high expression levels in young leaves. For EgrNF-YC subfamilies, although all memebers in EgrNF-YC subfamilies were expressed in almost all tissues, their expression levels were highest in young leaves (Fig. 8). The diversity of expression patterns of EgrNF-Y gene indicates that EgrNF-Y gene has different biological functions during the growth and development of eucalyptus, and has a wide range of biological applications. Expression Profiles of the EgrNF-Ys under Low phosphorus environment E . grandis is mainly distributed in southwest and South China where soil is generally deficient in phosphorus. Studies have shown that NF-Y is an important transcriptional regulator and plays an important role in plant stress and growth and development. In order to investigate the response of EgrNF-Y gene expression to phosphate starvation, E . grandis seedlings with the same growth were cultured in normal and phosphate-free nutrient solution for two weeks respectively, and compared their relative expression levels under the above two growth conditions by qRT-PCR. The results analysis showed that 12 genes ( EgrNF-YB3 / B8/B 1 1 / B12 / Bl3 / B14 / B15 , EgrNF-YC1 / C2 / C3 / C5 / C7 ) were upregulated more than 2-fold in the leaves after being phosphate-starved for 14 days. The other genes remained stable or were downregulated. In the root, only EgrNFYB6 / B11 / B13 was upregulated by more than a factor of 2 under low phosphate treatment, while most genes remained stable (Fig. 9). These results suggested that these up-regulated genes may be involved in phosphate uptake when phosphate is limited in the soil. Discussion Multiple evidences showed that NF-Ys plays a variety of important roles in plant growth and response to environmental stress. Because of the duplication of genes, plants usually have large gene families, and have been broadly studied in many herbs. such as A. thaliana [ 4 ], Glycine max [ 6 ], Oryza [ 38 ], Petunia hybrida [ 9 ], Medicago sativa [ 8 ], Brassica napus [ 7 ]. In recent years, NF-Y family gene identification has been gradually identified in woody plants. Such as Populus tomentosa and Pinus tabuliformis . Eucalyptus is recognized by FAO as one of the three fastest growing trees in the world. However, the identification and analysis of the NF-Y gene family in E. grandis have not been reported. Here, we isolated and identified a total of 31 EgrNF-Y genes. Compared with the numbers of NF-Ys in other species, such as 36 in A. thaliana [ 4 ], 28 in Oryz a [ 38 ],46 in Populus trichocarpa [ 10 ]and 28 in P. tabuliformis [ 11 ], Eucalyptus harbored a comparable number of genes. In addition, their gene structures, conserved domains, phylogenetic relationship and expression patterns were systematically analyzed. These findings provide valuable information for a subsequent functional analysis and precision plant breeding of a single EgrNF-Y gene. To explore the function of these EgrNF-Ys , we performed exon-intron structure and motif analysis (Fig. 1 , Fig. 2 ). The number and distribution of exon-intron structures and motifs are similar to previous results [ 11 , 39 ], suggesting that the function of EgrNF-Ys may be similar to that of homologous genes in other species. Studies have shown that Arabidopsis NF-YB subunits can be divided into LEC1 and non-LEC1 categories. Among them, aspartic acid at D55 is considered to be a key protein interaction site in the AtNF-YB subfamily to distinguish LEC1 from non-LEC1 types. Study found that LEC1 plays an important role in plant embryogenesis and seed development. For example, Arabidopsis [ 4 ], castor [ 23 ]. In this study, EgrNF-YB9 changed from Lys (K) to Asp (D) at this binding site. Phylogenetic analysis also showed that EgrNF-YB9 was clustered with AtNF-YB9 (AtLEC1) and AtNF-YB6 (AtLEC1). Therefore, it can be speculated that they play an important role in regulating seed development and embryogenesis. In this study, we constructed a phylogenetic tree to analyze the NF-Y proteins of E. grandis , poplar and Arabidopsis. It was previously reported that the NF-Y family members of Arabidopsis may not contain AtNFYB11/12/13 and AtNF-YC10/11/13, because they do not have the corresponding structure [ 40 ]. Interestingly, the analysis of evolutionary tree results in this study showed that EgrNF-YB2/B16 and EgrNF-YC5/C7 also had distant evolutionary relationships with other EgrNF-YA / B / C gene clusters. Similar to AtNF-YB11/12/13/C11 (Fig. 6 ). In addition, multiple comparison results and our phylogenetic tree support this view, respectively (Fig. 5 ). It has been reported that NF-Y is an important transcriptional regulatory factor, which is widely expressed in the whole life cycle of vegetative and reproductive growth of plants. In this study, the relative expression levels of EgrNF-Y in roots, stems, young leaves, mature leaves, xylem and flowers of E. grandis were analyzed by qRT-PCR. In this study, we found that they are widely expressed in various tissues and organs at different levels. However, the results of this study showed that EgrNF-YC subfamily had high expression levels in young leaves, indicating that they play an important role in leaves. Studies have shown that NF-YC subunits can interact with CONSTANS proteins to regulate transcription levels of the key integrons FT, leading to early flowering in plants. Such as, NF-YC3, NF-YC4, and NF-YC9 are necessary for proper photoperiod-dependent induction of flowering in Arabidopsis, and the function of CO in FT transcriptional activation requires these NF-YCs, whereas synthetic FT proteins are only present in leaves [ 41 , 42 ]. FT is also an important integron regulating plant flowering [ 41 ]. Therefore, high expression of EgrNF-YC subfamily genes in leaves may help activate FT synthesis and promote flowering. In addition to NF-YC members participating in flowering regulation through photoperiodic pathways, studies have shown that NF-YB subfamily members can also participate in flowering regulation through age pathways. For example, chrysanthemum CmNF-YB8 can enhance SPLs transcript accumulation by regulating miR156 expression, and finally regulates the flowering time of chrysanthemum [ 15 ]. Interestingly, in the process of analyzing the tissue-specific expression levels of EgrNF-YB subfamily members, the expression levels of EgrNF-YB1 and EgrNF-YB11 were the highest in flower (Fig. 8 ). Meanwhile, we constructed an evolutionary tree between EgrNF-YB subfamily members and CmNF-YB8 for phylogenetic analysis. The results showed that, EgrNF-YB1, EgrNF-YB11 and CmNF-YB8 are clustered into a subclade (Fig. S2). It is further speculated that EgrNF-YB1 and EgrNF-YB1 1 may have similar functions to CmNF-YB8 in participating in the regulation of flowering. Due to the existence of multiple promoters in the upstream sequence of EgrNF-YB gene, which usually function as dimers or trimers, the mechanism of age-regulation regulation is not clear. Therefore, the exact functional mechanism of the EgrNF-Y gene still needs further investigation. NF-Y is an important transcriptional regulatory factor that plays an important role in response to plant stress. At present, most studies focus on abiotic stress. Such as drought [ 43 ], salt [ 8 ], cold and heat [ 9 , 24 ]. However, there are few studies on how NF-Y affects plant growth and development under nutrient stress conditions. In this study, we found that different subunits of the EgrNF-Y family showed different expression patterns under low phosphorus environment. Among them, the expressions of 12 genes of NF-YBs and NF-YCs in leaves were up-regulated by more than 2 times after 14 days of phosphate starvation treatment. In the root, only three genes were upregulated more than two-fold, while most remained stable. These results suggest that these up-regulated genes may be involved in phosphate uptake when phosphate is limited in the soil. Conclusions In this study, 31 EgrNF-Y genes were identified, including 7 EgrNF-YAs, 16 EgrNF-YBs and 8 EgrNF-YCs, and their conserved domains, gene structure, phylogenetic relationships, c is-elements and expression patterns were analyzed in the E. grandis reference genome. This provides a theoretical basis for researchers to further understand this gene family, and can be used as a candidate functional gene for further study of NF-Y in the growth, development and phosphorus tolerance of E. grandis . In conclusion, our results can provide valuable information for further elucidating the biological function of EgrNF-Ys. Materials and methods Identification of NF-Y family members in E. grandis The NF-Y protein sequences (10 NF-YA, 13 NF-YB and 13 NF-YC genes) in Arabidopsis thaliana from the Arabidopsis Information Resource (TAIR) ( http://www.Arabidopsis.org/ ). These sequences were used to search E. grandis in Phytozome v13 ( https://phytozome-next.jgi.doe.gov/ ) using the BLAST program and selected all sequences cut-off was set as e-value < 10 − 10 . In addition, we used the Pfam and SMART tools to verify whether each candidate gene had NF-Y protein conserved domains [ 29 ]. Further, all protein sequences obtained by BLAST and SMART retrievers were manually screened to remove incomplete and redundant sequences. Finally, 31 candidate members of E. grandis NF-Y were obtained for subsequent analysis. Meanwhile, ProtParam ( http://web.expasy.org/protparam/ ) was used to analyze the information about each EgrNF-Y gene, including protein sequence, molecular weight (MW), isoelectric point (pI). Gene structure, conserved motifs and cis-element analysis of the NF-Y gene family in E. grandis For gene structure analysis, the online service of the Gene Structure Display Server (GSDS) ( http://gsds.cbi.pku.edu.cn/ ) to visualize the number and distribution of exons and introns EgrNF-Y genes [ 30 ]. For conserved motifs analysis, MEME software was used with the default parameters (the maximum number of motifs set to 10) to analyze the conserved motifs in the EgrNF-Y proteins [ 31 ]. For cis -element analysis, the promoter sequences (length, 2 kb) of EgrNF-Ys were collected from the genome of E . grandis as the regulatory promoter region, and were analyzed using the online software PlantCARE database ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) [ 32 ]. Meanwhile, cis -elements present in the promoter regions were also visualized by TBtools [ 33 ]. Chromosome position and cross-species collinearity analysis of the EgrNF-Y genes The chromosomal locations of the EgrNF-Y genes using the MapInspect software [ 34 ]. TBtools was used to extract the location information of NF-Y genes from A. thaliana , P. trichocarpa , E. grandis genome files and gene annotation files to construct the physical map of NF-Ys of three plants on chromosomes. The MCscanX command in TBtools was used to analyze the collinear relationship among the three species and to identify the collinear blocks of NF-Y genes in A. thaliana , P. trichocarpa and E. grandis genome files [ 35 ]. Multiple alignments and phylogenetic analysis of EgrNF-Y proteins The protein sequences of the EgrNF-Y members were obtained using Phytozome v12.1. Multiple sequence alignment analysis of the identified EgrNF-Ys were constructed using using ClustalX2.1 with the default parameters [ 36 ]. For phylogenetic relationship analyse, the phylogenetic tree was constructed using MEGA7.0 with the complete NF-Y protein sequences. The evolutionary relationships were estimated with 1000 bootstrap replications [ 37 ]. The phylogenetic tree constructed by MEGA7 was uploaded to iTOL ( http://itol.embl.de/ ) for further editing. Plant material, growth conditions and abiotic treatment All the seedlings were grown in the Institute of Tropical Forestry, Chinese Academy of Forestry (Guangzhou, China) in this study. The greenhouse was kept at 23 ± 1°C, 60–70% humidity, and 16/ 8 h light/dark photoperiod. In this study, for the phosphate treatments, we designed normal growth condition (KH 2 PO 4 concentrations of 1mM) and phosphorus deficient growth conditions (KH 2 PO 4 concentrations of 0M) for the experiments. Control group and treatment group were replaced with a new nutrient solution every 5 days (800 mL/pot). Here, we used seedling samples grown under normal conditions as controls. Control group and treatment group was applied to18 plants (with three replicate trials) for one month. After P treatments, the roots (the newest and the second lateral roots) and leaves were harvested, respectively. Roots, stems, and leaves tissue were obtained from 6-month-old cultured plantlets. In addition, mature leaves, xylem and flowers were collected from Jiangmen City, Guangdong Province. All samples are immediately frozen in liquid nitrogen after collection and stored at -80℃ until use. RNA extraction and quantitative real-time PCR Total RNAs were isolated from different tissues and organs of E.grandis , as well as from the different phosphate treatment samples. Total RNA from the samples was extracted using the OMEGA plant RNA Kit (Shanghai, China) and following the operating instruction. These RNAs were assessed by agarose gel and NanoDrop 2000 spectrophotometer (Implen, Inc., Westlake Village, CA, USA). cDNA was synthesized using total RNA as the template with the Takara’s PrimeScript Synthesis 1st Strand cDNA Synthesis Kit (Takara, Beijing, China). The obtained cDNA was diluted 10-fold with ddH 2 O and used as the template for qRT-PCR. To measure the expression levels of EgrNF-Y genes, qRT-PCR analysis was performed using SYBR ® Premix Ex Taq™ (TaKaRa). The qRT-PCR primers were listed in Table S1 , and EgrEF2 was used as the internal control. PCR was performed on an Light Cycler 96 Real-Time PCR system following the manufacturer , s instructions. The PCR program was 94°C for 30 s, followed by 40 cycles of 94°C for 5 s and 60°C for 10 s, and a final elongation step of 72°C for 5 min. The program was completed with a melting curve from 70 to 95°C. The relative expression levels of these genes were analyzed using the 2 −ΔΔCT method. Statistical analyses For all experiments, we all performed three independent biological replicates and three technical replicates. Data are statistically described as mean and standard deviation (mean ± SD). SPSS19.0 software was used for data analysis (IBM SPSS, Chicago, IL, USA). Error bars were calculated according to Tukey's multiple range test, and *( p < 0.05) and **( p < 0.01) being used to indicate statistically significant effects. Declarations Acknowledgements We appreciate the contributions and support from the lab members. We extend our sincere gratitude to the editor and reviewers for their thorough evaluation of this manuscript and for providing valuable feedback for its enhancement. Funding This research has been supported by the Young Scientists Fund of the National Natural Science Foundation of China (32201524), the National Key Research and Development Program of China during the 14th five-year plan Period (2022YFD2200203), the Fundamental Research Funds for the Central Non-profit Research Institution of CAF (CAFYBB2022SY017) and the Guangzhou Science and technology plan project (2023A04J0711). Author contribution JL designed and supervised the whole experiments, and wrote the manuscript. JL and GL collected plant materials. JL, LZ and CG analyzed the data and edited manuscript. JL, ZL and JX revised manuscript. All authors approved the manuscript before submission. Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Bucher P, Trifonov EN. CCAAT box revisited: bidirectionality, location and context. J Biomol Struct Dyn. 1988;5(6):1231–6. 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PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002; (1) 1. Chen C, Chen H, Zhang Y, Thomas HR, Frank MH, He Y, et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020;13(8):1194–202. Zhang S, Tong Y, Li Y, Cheng ZM, Zhong Y. Genome-wide identification of the HKT genes in five Rosaceae species and expression analysis of HKT genes in response to salt-stress in Fragaria vesca . Genes Genomics. 2019;41(3):325–36. Wang Y, Tang H, Debarry JD, Tan X, Li J, Wang X et al. MCScanX: a toolkit for detection and evolutionary analysis of gene synteny and collinearity. Nucleic Acids Res. 2012; 40(7), e49. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, et al. Clustal W and Clustal X version 2.0. Bioinformatics. 2007;23(21):2947–8. Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33(7):1870–4. Thirumurugan T, Ito Y, Kubo T, Serizawa A, Kurata N. Identification, characterization and interaction of HAP family genes in rice. Mol Genet Genomics. 2008;279(3):279–89. Yang JY, Yongxue. Genome-wide identification and expression analysis of NF-Y transcription factor Families in watermelon ( Citrullus lanatus ). HortTechnology. 2017; 27(4). Gusmaroli G, Tonelli C, Mantovani R. Regulation of novel members of the Arabidopsis thaliana CCAAT-binding nuclear factor Y subunits. Gene. 2002;283(1–2):41–8. Böhlenius H, Huang T, Charbonnel-Campaa L, Brunner AM, Jansson S, Strauss SH, Nilsson O. CO/FT regulatory module controls timing of flowering and seasonal growth cessation in trees. Science. 2006;312:1040–3. Turck F, Fornara F, Coupland G. Regulation and identity of florigen: FLOWERING LOCUS T moves center stage. Annu Rev Plant Biol. 2008;59(1):573–94. Quach TN, Nguyen HTM, Valliyodan B, Joshi T, Nguyen HT. Genome-wide expression analysis of soybean NF-Y genes reveals potential function in development and drought response. Mol Genet Genomics. 2015;290(3):1095. Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx SupplementaryFile.zip 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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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4703272","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":328837947,"identity":"2249e280-5206-4d84-9d0b-814f43e64ab1","order_by":0,"name":"Juan Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYBACPmaGBCBlA+WyEaGFDaIljRQtEOowKVrYGZ5J/Nxx3t7gdo8Bw4eywwz8sxsIOixNsvfM7cSZc84YMM44d5hB4s4BwlokeNtuJ/BL5Bgw87YdZjCQSCDClr9t5+zZQFr+EqtFmrftAGM/SAsjkVqSrWXbkhNnzkgrONhzLp1H4gYBLfz8ZxJvvm2zsze4kbzxwY8yazn+GQS0MDDwIFQcAHEJqQcC9gNEKBoFo2AUjIIRDQDLyDkqMy1tsAAAAABJRU5ErkJggg==","orcid":"","institution":"Shanxi Normal University","correspondingAuthor":true,"prefix":"","firstName":"Juan","middleName":"","lastName":"Li","suffix":""},{"id":328837949,"identity":"63d0157c-bbb6-4c38-b085-e6b8bfb78624","order_by":1,"name":"Chaoyan Gong","email":"","orcid":"","institution":"Shanxi University of Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Chaoyan","middleName":"","lastName":"Gong","suffix":""},{"id":328837951,"identity":"6c2eed46-4d54-48be-8f22-2342a31fb25f","order_by":2,"name":"Li Zhuang","email":"","orcid":"","institution":"Shanxi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Zhuang","suffix":""},{"id":328837954,"identity":"1db1d5a2-627d-4425-b46a-4b15a9329940","order_by":3,"name":"Guangyou Li","email":"","orcid":"","institution":"Chinese Academy of Forestry","correspondingAuthor":false,"prefix":"","firstName":"Guangyou","middleName":"","lastName":"Li","suffix":""},{"id":328837956,"identity":"9b716f61-c927-4cca-ba85-5f17979a5a88","order_by":4,"name":"Jianmin Xu","email":"","orcid":"","institution":"Chinese Academy of Forestry","correspondingAuthor":false,"prefix":"","firstName":"Jianmin","middleName":"","lastName":"Xu","suffix":""},{"id":328837959,"identity":"e6209781-d118-4b96-a73c-67a140135f72","order_by":5,"name":"Zhaohua Lu","email":"","orcid":"","institution":"Chinese Academy of Forestry","correspondingAuthor":false,"prefix":"","firstName":"Zhaohua","middleName":"","lastName":"Lu","suffix":""}],"badges":[],"createdAt":"2024-07-08 06:59:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4703272/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4703272/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61468448,"identity":"aaf2bb0d-a64a-4afb-8842-62390d6611d9","added_by":"auto","created_at":"2024-07-31 06:14:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":411672,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic and gene structure analyses of the \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEgrNF-Y\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003egenes.\u003c/strong\u003eThe intron is represented by a line and exons are represented by yellow boxes.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4703272/v1/2c7e6a80af4aea17161a4a37.png"},{"id":61468454,"identity":"ca620d2b-c156-4769-9f20-705a0a779132","added_by":"auto","created_at":"2024-07-31 06:14:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":265455,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConserved motif analyses of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. grandis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and Arabidopsis NF-Ys. \u003c/strong\u003eConserved motifs (1-10) are represented by different colored boxes, and nonconserved sequences are indicated by gray line (sequences of conserved motifs are given in Figure S1).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4703272/v1/92963c46b45f9414b9c19c55.png"},{"id":61468457,"identity":"2c2e7d3d-1d05-4114-85c7-f44ee2bc223f","added_by":"auto","created_at":"2024-07-31 06:14:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":225917,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChromosomal location and gene duplication of NF-Y proteins in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. grandis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eChromosome numbers are shown at the top of each bar. Chromosome size is indicated by the vertical scale.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4703272/v1/2d7308b9823f5d9e2cfad964.png"},{"id":61468449,"identity":"4c807368-1be0-4b37-b2a6-01b3fa7978c0","added_by":"auto","created_at":"2024-07-31 06:14:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":531089,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSynteny analyses of NF-Y genes between \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. grandis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand two module plant (\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eA. thaliana \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. trichocarpa\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e). \u003c/strong\u003eGreen lines indicate the collinear blocks with in \u003cem\u003eE. grandis\u003c/em\u003e and other two module plant genomes.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4703272/v1/294e3c0c437405d9a72a3b36.png"},{"id":61468458,"identity":"b46a56fd-9f36-48b7-afe7-0408e2f13fe8","added_by":"auto","created_at":"2024-07-31 06:14:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1296911,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMultiple sequence alignments of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003egrandis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e NF-Y TF members. a. \u003c/strong\u003eEgrNF-YA proteins. \u003cstrong\u003eb.\u003c/strong\u003e EgrNF-YB proteins. \u003cstrong\u003ec.\u003c/strong\u003e EgrNF-YC proteins. The AA numbers of the conserved domains and the functional regions are marked.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4703272/v1/88e0969e7fa3e184a5285bb0.png"},{"id":61469779,"identity":"8cab5fe9-c9fa-480a-9925-0a30842f106e","added_by":"auto","created_at":"2024-07-31 06:30:31","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":441880,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhylogenetic analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE.grandis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e, Arabidopsis and poplarNF-Y proteins.\u003c/strong\u003e Three branches were formed corresponding to genes with different subunits. Pinkish red represents branches of NF-YC, green represents NF-YB and yellow represents NF-YA.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4703272/v1/553f99e4fb2ebb53c9922b25.png"},{"id":61469061,"identity":"baa2fee3-491b-45da-bf51-ee2230535d58","added_by":"auto","created_at":"2024-07-31 06:22:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":493936,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ecis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e-regulatory elements in the promoter region of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEgrNF-Y\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE.grandis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eThe \u003cem\u003ecis\u003c/em\u003e-elements are marked with different-colored boxes in their respective positions.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4703272/v1/6616fada6010f700c58e4fbf.png"},{"id":61468452,"identity":"d98a5dfa-0272-414b-8177-e785050065d6","added_by":"auto","created_at":"2024-07-31 06:14:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":7786827,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTissue-specific expression patterns of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEgrNF-Y\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ein \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. grandis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eR, S, YL, ML, YX and F represent roots, stems, young leaves, mature leaves, xylem and flowers, respectively. Data are mean ± SE of three biological replications. Asterisks on top of the bars indicate statistically significant differences between the stress and counterpart controls (* \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01)\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4703272/v1/7f772f31f3177f1bb77db108.png"},{"id":61468459,"identity":"3fbd7546-5e82-4f84-9fb9-e6f633c91165","added_by":"auto","created_at":"2024-07-31 06:14:32","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":5649671,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression pattern analysis of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEgrNF-Y\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e gene in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. grandis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e seedlings under phosphorus deficiency condition. \u003c/strong\u003eThe black column represents normal growth condition (KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e concentrations of 1mM), and the gray column represents phosphorus deficient growth conditions (KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e concentrations of 0M). The roots and leaves were collected for gene expression analysis, respectively. Error bars show the standard deviation of three biological replicates. Single and double asterisks represent significant differences from the control sample at the 0.05 and 0.01 levels (\u003cem\u003et-test\u003c/em\u003e), respectively.\u003c/p\u003e","description":"","filename":"Figure9.png","url":"https://assets-eu.researchsquare.com/files/rs-4703272/v1/2707737a9c52714c8bb847b7.png"},{"id":66212848,"identity":"77c81fd9-fb7c-426c-99b5-28c19104cf6f","added_by":"auto","created_at":"2024-10-08 18:47:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":16969326,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4703272/v1/14f6cf04-ad17-4d08-aa05-2bfa042f9c7e.pdf"},{"id":61468451,"identity":"33c190f1-cfc3-4724-ac19-47ec8ee9075d","added_by":"auto","created_at":"2024-07-31 06:14:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":25819,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4703272/v1/e673b3f772a4377e1ef2f2c9.docx"},{"id":61469062,"identity":"82335436-70ea-4484-abf9-50d3a211a2bb","added_by":"auto","created_at":"2024-07-31 06:22:31","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3312492,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFile.zip","url":"https://assets-eu.researchsquare.com/files/rs-4703272/v1/6954cf0bea1bf358e029687f.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genome-wide identification and expression profile analysis of the NF-Y transcription factor gene family in Eucalyptus grandis","fulltext":[{"header":"Background","content":"\u003cp\u003eTranscription factors (TFs), a protein that binds to a \u003cem\u003ecis\u003c/em\u003e-acting element in the promoter region of a eukaryotic gene and enables the target gene to be expressed in a specific way, play vital roles in numerous in many plants growth, development and abiotic stress. Nuclear factor Y (NF-Y), also famous as heme activator protein (HAP) or CCAAT-binding factor (CBF), is one of the most widespread heterotrimeric transcription factors found in numerous genes in fungi, animal, and plants. In plants, NF-Y contains three unique subunits: NF-YA (HAP2 or CBF-B), NF-YB (HAP3 or CBF-A), and NF-YC (HAP5 or CBF-C) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNF-Y is highly conserved in all higher eukaryotes [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In yeast and animals, each NF-Y subunit is encoded by a single gene. However, each NF-Y subunit gene is represented by multiple orthologs in plants, indicating that heterotrimer complexes composed of different NF-Y members play different regulatory roles [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. To date, NF-Y transcription factors have been identified in several herb species, including 33 NF-Y member genes were identified in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], 28 NF-Y members in \u003cem\u003eOryza sativa\u003c/em\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], 68 NF-Y in \u003cem\u003eGlycine max\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], 33 members in \u003cem\u003eBrassica napus\u003c/em\u003e. L [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], 60 NF-Y member genes in \u003cem\u003eMedicago sativa\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], 27 members \u003cem\u003ePetunia hybrida\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Furthermore, NF-Y gene members have also been reported in many species of woody plants. For example, 46 members were identified in \u003cem\u003ePopulus\u003c/em\u003e [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], 28 members were identified in \u003cem\u003ePinus tabuliformis\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and 22 members in \u003cem\u003eCitrus\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Recently, the NF-YB family of \u003cem\u003eEucalyptus\u003c/em\u003e has been analyzed and identified. Although many studies have reported the NF-Y gene family in various plant species, the \u003cem\u003eEucalyptus\u003c/em\u003e NF-Y gene family has not been systematically explored.\u003c/p\u003e \u003cp\u003eMany studies have shown that NF-Y genes are responsible for regulating diverse physiological processes related to plant growth and development. Among, AtNF-YB9, the first NF-Y gene cloned in a plant, plays a key role in Arabidopsis seed development[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The NF-Y has also been reported to play an important role in flowering time. Such as, NF-Y mediates the effect of photoperiod and GA signaling on \u003cem\u003eSOC1\u003c/em\u003e expression partly through H3K27me3 demethylation [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Also, \u003cem\u003eCmNF-YB8\u003c/em\u003e can influence flowering time through regulating the expression of cmo-MIR156 in the aging pathway in \u003cem\u003eChrysanthemum\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition, NF-Y plays an important role in the process of pollen tube growth [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], starch biosynthesis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], root elongation[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], fruit ripening [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to plant growth and development, NF-Y participates in stress response. Studies on NF-Y have focused mainly on drought resistance [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], salt stress[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and cold [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Studies have also shown that NF-Y protein is a key factor in regulating root nodule formation and nutrient absorption in plants. It has been reported in the \u003cem\u003eMedicago truncatula\u003c/em\u003e [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], \u003cem\u003eLotus japonicus\u003c/em\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], \u003cem\u003ePhaseolus vulgaris\u003c/em\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], \u003cem\u003eTriticum aestivum\u003c/em\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, whether NF-Y plays a role in growth, development and nutrient absorption in \u003cem\u003eEucalyptus\u003c/em\u003e remains unclear.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEucalyptus grandis\u003c/em\u003e, a species of Eucalyptus in Myrtaceae, is a significant fast-growing tree species. \u003cem\u003eE. grandis\u003c/em\u003e is grown primarily in the sorthern and sorthwestern of China. It has the advantages of short growth cycle, strong stress resistance and strong toughness of wood and high economic value. It is considered one of the three fastest growing trees in the world along with poplar and pine. Because it is mainly distributed in southwest China and South China, the soil in these areas is generally lacking in phosphorus. Furthermore, the lack of nutrients affects the growth of \u003cem\u003eE. grandis\u003c/em\u003e. Most previous studies only focused on the involvement of NF-Y transcription factors in plant root growth and development [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], but the mechanism of how NF-Y regulates root growth and development under nutrient stress is still limited. Therefore, it is of great significance to identify the NF-Y gene family members and clarify the effects of phosphorus deficiency on the growth of \u003cem\u003eE. grandis\u003c/em\u003e. In this study, 31 NF-Y genes were identifed from the \u003cem\u003eE. grandis\u003c/em\u003e genome and their physicochemical properties, phylogenetic relationships, gene structure, and chromosome localization were comprehensively analyzed. Moreover, we also analyzed the expression levels of \u003cem\u003eEgrNF-Y\u003c/em\u003e in six different tissues and organs and in the condition of phosphorus deficiency by qRT-PCR, which is particularly important for identifying candidate genes involved in regulation of the growth and response to phosphorus stress of \u003cem\u003eE. grandis\u003c/em\u003e. Overall, our results contribute to a more complete understanding of the function of the NF-Y genes in \u003cem\u003eE. grandis\u003c/em\u003e, and the study of other woody plants also has high reference value.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eIsolation and identification of the NF-Y family members in \u003cem\u003eE.\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003egrandis\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo obtain information on the NF-Y genes of\u003cem\u003e\u0026nbsp;E.\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003egrandis\u003c/em\u003e, we used Arabidopsis NF-Y protein sequences as queries to search for the \u003cem\u003eE.\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003egrandis\u003c/em\u003e NF-Y genes in Phytozome v13 [4]. Through comprehensive screening, including remove those with improper domains and redundant sequences, a total of 31 EgrNF-Y sequences were identified in the\u003cem\u003e\u0026nbsp;E.\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003egrandis\u003c/em\u003e genome, including 7 NF-YA, 16 NF-YB and 8 NF-YC genes. Furthermore, the physicochemical properties data of all gene members were estimated by ExPASy server (http://WWW.expasy.org/), and the characteristics of the \u003cem\u003eEgrNF-Y\u003c/em\u003e sequences are listed in Table 1. Among them, the identified \u003cem\u003eEgrNF-Y\u003c/em\u003e genes encoded peptides ranged from 94 to 339 aa. The molecular weights (MWs) and the isoelectric point (pI) values of these proteins ranged from 10.64 kDa to 37.21 kDa, and from 4.49 to 9.48, respectively (Table1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene structure and conserved motifs analysis of the \u003cem\u003eEgrNF-Y\u0026nbsp;\u003c/em\u003egenes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe analysis of gene structure can understand the evolution of gene families. To investigated the evolutionary conservation and divergence of NF-Ys between \u003cem\u003eE.\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003egrandis\u003c/em\u003e and Arabidopsis, we analyzed the exon-intron gene structure of 31 identified EgrNF-Ys using the GSDS website. Phylogenetic analysis revealed that the \u003cem\u003eEgrNF-Y\u003c/em\u003e genes were divided into three groups: EgrNF-YA, EgrNF-YB and EgrNF-YC. Most of the EgrNF-YAs (except \u003cem\u003eEgrNF-YA5\u003c/em\u003e) had five or six exons with similar distribution. More than two-thirds of \u003cem\u003eEgr\u003c/em\u003e\u003cem\u003eNF-YBs\u003c/em\u003e had no introns, and the results showed that members with similar numbers of exons and introns are distributed in the same clade. For the EgrNF-YC subfamily, EgrNF-YC1 and EgrNF-YC4 had two exons. EgrNF-YC5 and EgrNF-YC7 have six and five exons, respectively. In general, the gene structure of NF-Y members was positively correlated with their phylogenetic relationships (Fig. 1). Furthermore, the distributions of conserved motifs were assessed by MEME software. The results showed that all of the genes contained motif 1 except EgrNF-YA6. Interestingly, three EgrNF-Y subunits have a unique motif distribution (Fig. 2). For example, motifs 8 were only present in \u003cem\u003eEgrNF-YA\u003c/em\u003e, motif 2 was only observed in \u003cem\u003eEgrNF-Y\u003c/em\u003e\u003cem\u003eB\u003c/em\u003e, and motif 7 was unique to \u003cem\u003eEgrNF-YC\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChromosomal\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003el\u003c/strong\u003e\u003cstrong\u003eocalization and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ec\u003c/strong\u003e\u003cstrong\u003eollinearity\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;a\u003c/strong\u003e\u003cstrong\u003enalysis of \u003cem\u003eEgrNF-Y\u003c/em\u003e\u003cem\u003es\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 31 \u003cem\u003eEgrNF-Y\u003c/em\u003e gene members were widely distributed according to the positions of the annotated chromosomes in the \u003cem\u003eE\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003cem\u003egrandis\u003c/em\u003e genome database (Table 1). To further analyze the distribution of EgrNF-Y family members on each chromosome, we constructed a location map using MapInspect software. The result showed that the distribution of NF-Y gene family on the chromosomes of \u003cem\u003eE\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003cem\u003egrandis\u003c/em\u003e was uneven. Among them, the largest number of members distributed on chromosome 2, while only 1 member distributed on chromosome 3 and chromosome 9. Moreover, the number of genes was not positively correlated with chromosome length. For example, chromosome 3 had the largest length, but only one member is distributed (Fig. 3).\u003c/p\u003e\n\u003cp\u003eTo further investigated the homologous genes and their evolutionary relationships between \u003cem\u003eE\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003cem\u003egrandi\u003c/em\u003e\u003cem\u003es\u003c/em\u003e, model plant Arabidopsis and woody model plant poplar, a multicomparative synteny map generated between three species. The 31 EgrNF-Ys were located at 11 scafolds, 46 PtNF-Ys were distributed on 19 chromosomes, and 36 AtNF-Ys were distributed on 5 chromosomes, which have a collinearity relationship with EgNF-Ys. Among them, the relationship between \u003cem\u003eE. grandis\u003c/em\u003e and poplar is closer, which indicates that the NF-Y family is a close relationship between woody plants (Fig. 4).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConserved regions and phylogenetic relationships of EgrNF-Ys\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the conserved regions of EgrNF-Ys, the protein sequences of the 31 members were analyzed using ClustalW 2.1 and Genedoc software.\u0026nbsp;Multiple sequence alignment results suggested that each EgrNF-Y family member contains a heterodimerization domain and a DNA-binding domain that recognizes the CCAAT site.\u0026nbsp;This\u0026nbsp;core conserved regions of the EgrNF-YAs proteins were 53AAs, including two highly conserved domains: the NF-YB/C subdomain and the DNA binding, they were separated by a conserved linker with 21 AAs. As shown in Fig.\u0026nbsp;5\u0026nbsp;B/C,\u0026nbsp;the central domain of EgrNF-YBs had 91AAs. Among EgrNF-YBs, EgrNF-YB2/4/16 had lower conserved domains. Fig.5C showed that EgrNF-YC subunits were also found to consist of a core histone-like sequence with a central domain about 79 AAs in length. Meanwhile, EgrNF-YC5 and EgrNF-YC7 were slightly different from other NF-YCs. This analysis also suggested that EgrNF-YAs is more evolutionarily conserved in the three subfamilies\u0026nbsp;(Fig.\u0026nbsp;5).\u003c/p\u003e\n\u003cp\u003eTo reveal the evolutionary relationship and potential function of EgrNF-Ys, an phylogenetic tree was constructed using the NF-Y protein sequences of \u003cem\u003eE.\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003egrandis\u003c/em\u003e, \u003cem\u003eA. thaliana\u0026nbsp;\u003c/em\u003eand \u003cem\u003eP. trichocarpa\u003c/em\u003e were created by MEGA7 software with the neighbor-joining (NJ) criteria. The phylogenetic analysis revealed that the 107 NF-Y proteins were clustered into three groups: NF-YA (yellow), NF-YB (green), and NF-YC (pinkish red). It is consistent with our subfamily classifications of the EgrNF-Ys (Table 1). Based on the phylogenetic relationship of the evolutionary tree and the reported functions of AtNF-Ys, the functions of EgrNF-Y members can be further predicted. In each group, we found that some pairs of paralogous NF-Y proteins were composed of one EgrNF-Y and one AtNF-Y, such as EgrNF-YA1 and AtNF-YA6, EgrNF-Y5 and AtNF-YA11, and this close evolutionary relationship generally suggested the similarity of their biological functions. We also found EgrNF-YB9, AtNF-YB6/9 and PtNF-YB3/5 clustered in a subgroup, belonging to LEC1 and its homolog LEC1-like. In addition, we also identified three pairs of analogues: EgrNF-YB4 and EgrNF-YB5, EgrNF-YB7 and EgrNF-YB14, EgrNF-YC2 and EgrNF-YC3, while most EgrNF-Ys share low homology with other members, suggesting that they have evolved in diversity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of \u003cem\u003ecis\u003c/em\u003e-elements in the promoter regions of \u003cem\u003eEgrNF-Y\u003c/em\u003e genes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn order to further explored the potential function of the 31 \u003cem\u003eEgrNF-Y\u003c/em\u003e at the transcriptional level, the distribution of \u003cem\u003ecis\u003c/em\u003e-elements in the \u003cem\u003eEgrNF-Y\u003c/em\u003e promoter regions (2000 bp) was scanned using the PlantCARE software. A total of 19 types of \u003cem\u003ecis\u003c/em\u003e-elements were identified in the 31 \u003cem\u003eEgrNF-Y\u003c/em\u003e, including light responsive, hormone responsive, stress responsive, growth regulation, and some common and core \u003cem\u003ecis\u003c/em\u003e-elements, such as the TATA-box and CAAT-box. The detailed classifcation and sequence information of all the \u003cem\u003ecis\u003c/em\u003e-elements are listed in Table S2. Meanwhile, the transcription regulatory \u003cem\u003ecis\u003c/em\u003e-elements binding site are also shown in fig.7 except some common and core \u003cem\u003ecis\u003c/em\u003e-elements. This result analysis showed that the promoter regions of the EgrNF-Ys contain several phytohormone response \u003cem\u003ecis\u003c/em\u003e-elements, including gibberellin, salicylic acid, MeJA, auxin, ethylene, and abscisic acid responsive elements. The results indicated that \u003cem\u003eEgrNF-Ys\u003c/em\u003e may play an important role in response to stress and growth regulation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ep\u003c/strong\u003e\u003cstrong\u003erofiles of the EgrNF-Ys in\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ed\u003c/strong\u003e\u003cstrong\u003eifferent\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003et\u003c/strong\u003e\u003cstrong\u003eissues\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the possible functions of the \u003cem\u003eEgrNF-Ys\u003c/em\u003e genes in the developmental processes of \u003cem\u003eE\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003egrandis\u003c/em\u003e, we examined their gene expression profiles using quantitative real-time PCR in six different tissues and organs (root, stem, young leaf, mature leaf, xylem and flower). All member genes except \u003cem\u003eEgrNF-YA5\u003c/em\u003e were expressed. Therefore, the tissue-specific expression patterns of 30 \u003cem\u003eEgrNF-Ys\u003c/em\u003e member genes were analyzed. The results demonstrated that 30 genes were widely expressed in various tissues and organs of \u003cem\u003eE\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003cem\u003egrandis\u003c/em\u003e, but they exhibited different spatial and temporal expression patterns. For EgrNF-YA subfamilies, EgrNF-YA1 and EgrNF-YA7 were significantly expressed in flowers and roots, respectively. Expression was strongest for \u003cem\u003eEgrNF-YA4\u003c/em\u003e and \u003cem\u003eEgrNF-YA6\u003c/em\u003e in the leaves. For EgrNF-YB subfamilies, \u003cem\u003eEgrNF-YB1\u003c/em\u003e and \u003cem\u003eEgrNF-YB11\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003ewas most highly expressed in the flowers, more than 90% of other \u003cem\u003eEgrNF-YB\u0026nbsp;\u003c/em\u003emembers have high expression levels in young leaves. For EgrNF-YC subfamilies, although all memebers in EgrNF-YC subfamilies were expressed in almost all tissues, their expression levels were highest in young leaves (Fig. 8). The diversity of expression patterns of \u003cem\u003eEgrNF-Y\u003c/em\u003e gene indicates that \u003cem\u003eEgrNF-Y\u003c/em\u003e gene has different biological functions during the growth and development of eucalyptus, and has a wide range of biological applications.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression Profiles of the \u003cem\u003eEgrNF-Ys\u003c/em\u003e under Low phosphorus environment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eE\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003cem\u003egrandis\u003c/em\u003e is mainly distributed in southwest and South China where soil is generally deficient in phosphorus. Studies have shown that \u003cem\u003eNF-Y\u003c/em\u003e is an important transcriptional regulator and plays an important role in plant stress and growth and development. In order to investigate the response of \u003cem\u003eEgrNF-Y\u003c/em\u003e gene expression to phosphate starvation, \u003cem\u003eE\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003cem\u003egrandis\u003c/em\u003e seedlings with the same growth were cultured in normal and phosphate-free nutrient solution for two weeks respectively, and compared their relative expression levels under the above two growth conditions by qRT-PCR. The results analysis showed that 12 genes (\u003cem\u003eEgrNF-YB3\u003c/em\u003e/\u003cem\u003eB8/B\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e\u003cem\u003e1\u003c/em\u003e/\u003cem\u003eB12\u003c/em\u003e/\u003cem\u003eBl3\u003c/em\u003e/\u003cem\u003eB14\u003c/em\u003e/\u003cem\u003eB15\u003c/em\u003e,\u003cem\u003e\u0026nbsp;EgrNF-YC1\u003c/em\u003e/\u003cem\u003eC2\u003c/em\u003e/\u003cem\u003eC3\u003c/em\u003e/\u003cem\u003eC5\u003c/em\u003e/\u003cem\u003eC7\u003c/em\u003e) were upregulated more than 2-fold in the leaves after being phosphate-starved for 14 days. The other genes remained stable or were downregulated. In the root, only \u003cem\u003eEgrNFYB6\u003c/em\u003e/\u003cem\u003eB11\u003c/em\u003e/\u003cem\u003eB13\u003c/em\u003e was upregulated by more than a factor of 2 under low phosphate treatment, while most genes remained stable (Fig. 9). These results suggested that these up-regulated genes may be involved in phosphate uptake when phosphate is limited in the soil.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eMultiple evidences showed that NF-Ys plays a variety of important roles in plant growth and response to environmental stress. Because of the duplication of genes, plants usually have large gene families, and have been broadly studied in many herbs. such as \u003cem\u003eA. thaliana\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], \u003cem\u003eGlycine max\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], \u003cem\u003eOryza\u003c/em\u003e [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], \u003cem\u003ePetunia hybrida\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], \u003cem\u003eMedicago sativa\u003c/em\u003e [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], \u003cem\u003eBrassica napus\u003c/em\u003e [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In recent years, NF-Y family gene identification has been gradually identified in woody plants. Such as \u003cem\u003ePopulus tomentosa\u003c/em\u003e and \u003cem\u003ePinus tabuliformis\u003c/em\u003e. \u003cem\u003eEucalyptus\u003c/em\u003e is recognized by FAO as one of the three fastest growing trees in the world. However, the identification and analysis of the NF-Y gene family in \u003cem\u003eE. grandis\u003c/em\u003e have not been reported. Here, we isolated and identified a total of 31 EgrNF-Y genes. Compared with the numbers of NF-Ys in other species, such as 36 in \u003cem\u003eA. thaliana\u003c/em\u003e [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], 28 in \u003cem\u003eOryz\u003c/em\u003ea [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e],46 in \u003cem\u003ePopulus trichocarpa\u003c/em\u003e [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]and 28 in \u003cem\u003eP. tabuliformis\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], \u003cem\u003eEucalyptus\u003c/em\u003e harbored a comparable number of genes. In addition, their gene structures, conserved domains, phylogenetic relationship and expression patterns were systematically analyzed. These findings provide valuable information for a subsequent functional analysis and precision plant breeding of a single \u003cem\u003eEgrNF-Y\u003c/em\u003e gene.\u003c/p\u003e \u003cp\u003eTo explore the function of these \u003cem\u003eEgrNF-Ys\u003c/em\u003e, we performed exon-intron structure and motif analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The number and distribution of exon-intron structures and motifs are similar to previous results [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], suggesting that the function of \u003cem\u003eEgrNF-Ys\u003c/em\u003e may be similar to that of homologous genes in other species.\u003c/p\u003e \u003cp\u003eStudies have shown that Arabidopsis NF-YB subunits can be divided into LEC1 and non-LEC1 categories. Among them, aspartic acid at D55 is considered to be a key protein interaction site in the AtNF-YB subfamily to distinguish LEC1 from non-LEC1 types. Study found that \u003cem\u003eLEC1\u003c/em\u003e plays an important role in plant embryogenesis and seed development. For example, Arabidopsis [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], castor [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In this study, EgrNF-YB9 changed from Lys (K) to Asp (D) at this binding site. Phylogenetic analysis also showed that EgrNF-YB9 was clustered with AtNF-YB9 (AtLEC1) and AtNF-YB6 (AtLEC1). Therefore, it can be speculated that they play an important role in regulating seed development and embryogenesis.\u003c/p\u003e \u003cp\u003eIn this study, we constructed a phylogenetic tree to analyze the NF-Y proteins of \u003cem\u003eE. grandis\u003c/em\u003e, poplar and Arabidopsis. It was previously reported that the NF-Y family members of Arabidopsis may not contain AtNFYB11/12/13 and AtNF-YC10/11/13, because they do not have the corresponding structure [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Interestingly, the analysis of evolutionary tree results in this study showed that EgrNF-YB2/B16 and EgrNF-YC5/C7 also had distant evolutionary relationships with other \u003cem\u003eEgrNF-YA\u003c/em\u003e/\u003cem\u003eB\u003c/em\u003e/\u003cem\u003eC\u003c/em\u003e gene clusters. Similar to AtNF-YB11/12/13/C11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In addition, multiple comparison results and our phylogenetic tree support this view, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt has been reported that NF-Y is an important transcriptional regulatory factor, which is widely expressed in the whole life cycle of vegetative and reproductive growth of plants. In this study, the relative expression levels of \u003cem\u003eEgrNF-Y\u003c/em\u003e in roots, stems, young leaves, mature leaves, xylem and flowers of \u003cem\u003eE. grandis\u003c/em\u003e were analyzed by qRT-PCR. In this study, we found that they are widely expressed in various tissues and organs at different levels. However, the results of this study showed that \u003cem\u003eEgrNF-YC\u003c/em\u003e subfamily had high expression levels in young leaves, indicating that they play an important role in leaves. Studies have shown that NF-YC subunits can interact with CONSTANS proteins to regulate transcription levels of the key integrons FT, leading to early flowering in plants. Such as, NF-YC3, NF-YC4, and NF-YC9 are necessary for proper photoperiod-dependent induction of flowering in Arabidopsis, and the function of CO in FT transcriptional activation requires these NF-YCs, whereas synthetic FT proteins are only present in leaves [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. FT is also an important integron regulating plant flowering [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Therefore, high expression of EgrNF-YC subfamily genes in leaves may help activate FT synthesis and promote flowering.\u003c/p\u003e \u003cp\u003eIn addition to NF-YC members participating in flowering regulation through photoperiodic pathways, studies have shown that NF-YB subfamily members can also participate in flowering regulation through age pathways. For example, chrysanthemum \u003cem\u003eCmNF-YB8\u003c/em\u003e can enhance \u003cem\u003eSPLs\u003c/em\u003e transcript accumulation by regulating \u003cem\u003emiR156\u003c/em\u003e expression, and finally regulates the flowering time of chrysanthemum [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Interestingly, in the process of analyzing the tissue-specific expression levels of EgrNF-YB subfamily members, the expression levels of \u003cem\u003eEgrNF-YB1\u003c/em\u003e and \u003cem\u003eEgrNF-YB11\u003c/em\u003e were the highest in flower (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). Meanwhile, we constructed an evolutionary tree between EgrNF-YB subfamily members and CmNF-YB8 for phylogenetic analysis. The results showed that, EgrNF-YB1, EgrNF-YB11 and CmNF-YB8 are clustered into a subclade (Fig. S2). It is further speculated that \u003cem\u003eEgrNF-YB1\u003c/em\u003e and \u003cem\u003eEgrNF-YB1\u003c/em\u003e1 may have similar functions to \u003cem\u003eCmNF-YB8\u003c/em\u003e in participating in the regulation of flowering. Due to the existence of multiple promoters in the upstream sequence of \u003cem\u003eEgrNF-YB\u003c/em\u003e gene, which usually function as dimers or trimers, the mechanism of age-regulation regulation is not clear. Therefore, the exact functional mechanism of the \u003cem\u003eEgrNF-Y\u003c/em\u003e gene still needs further investigation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNF-Y is an important transcriptional regulatory factor that plays an important role in response to plant stress. At present, most studies focus on abiotic stress. Such as drought [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], salt [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], cold and heat [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. However, there are few studies on how \u003cem\u003eNF-Y\u003c/em\u003e affects plant growth and development under nutrient stress conditions. In this study, we found that different subunits of the EgrNF-Y family showed different expression patterns under low phosphorus environment. Among them, the expressions of 12 genes of \u003cem\u003eNF-YBs\u003c/em\u003e and \u003cem\u003eNF-YCs\u003c/em\u003e in leaves were up-regulated by more than 2 times after 14 days of phosphate starvation treatment. In the root, only three genes were upregulated more than two-fold, while most remained stable. These results suggest that these up-regulated genes may be involved in phosphate uptake when phosphate is limited in the soil.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, 31 EgrNF-Y genes were identified, including 7 EgrNF-YAs, 16 EgrNF-YBs and 8 EgrNF-YCs, and their conserved domains, gene structure, phylogenetic relationships, \u003cem\u003ec\u003c/em\u003eis-elements and expression patterns were analyzed in the \u003cem\u003eE. grandis\u003c/em\u003e reference genome. This provides a theoretical basis for researchers to further understand this gene family, and can be used as a candidate functional gene for further study of NF-Y in the growth, development and phosphorus tolerance of \u003cem\u003eE. grandis\u003c/em\u003e. In conclusion, our results can provide valuable information for further elucidating the biological function of EgrNF-Ys.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e \u003cb\u003eIdentification of NF-Y family members in\u003c/b\u003e \u003cb\u003eE. grandis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe NF-Y protein sequences (10 NF-YA, 13 NF-YB and 13 NF-YC genes) in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e from the Arabidopsis Information Resource (TAIR) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.Arabidopsis.org/\u003c/span\u003e\u003cspan address=\"http://www.Arabidopsis.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). These sequences were used to search \u003cem\u003eE. grandis\u003c/em\u003e in Phytozome v13 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://phytozome-next.jgi.doe.gov/\u003c/span\u003e\u003cspan address=\"https://phytozome-next.jgi.doe.gov/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) using the BLAST program and selected all sequences cut-off was set as e-value\u0026thinsp;\u0026lt;\u0026thinsp;10\u003csup\u003e\u0026minus;\u0026thinsp;10\u003c/sup\u003e. In addition, we used the Pfam and SMART tools to verify whether each candidate gene had NF-Y protein conserved domains [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Further, all protein sequences obtained by BLAST and SMART retrievers were manually screened to remove incomplete and redundant sequences. Finally, 31 candidate members of \u003cem\u003eE. grandis\u003c/em\u003e NF-Y were obtained for subsequent analysis. Meanwhile, ProtParam (\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) was used to analyze the information about each \u003cem\u003eEgrNF-Y\u003c/em\u003e gene, including protein sequence, molecular weight (MW), isoelectric point (pI).\u003c/p\u003e \u003cp\u003e \u003cb\u003eGene structure, conserved motifs and cis-element analysis of the\u003c/b\u003e \u003cb\u003eNF-Y\u003c/b\u003e \u003cb\u003egene family in\u003c/b\u003e \u003cb\u003eE. grandis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor gene structure analysis, the online service of the 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) to visualize the number and distribution of exons and introns \u003cem\u003eEgrNF-Y\u003c/em\u003e genes [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. For conserved motifs analysis, MEME software was used with the default parameters (the maximum number of motifs set to 10) to analyze the conserved motifs in the EgrNF-Y proteins [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. For \u003cem\u003ecis\u003c/em\u003e-element analysis, the promoter sequences (length, 2 kb) of \u003cem\u003eEgrNF-Ys\u003c/em\u003e were collected from the genome of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003egrandis\u003c/em\u003e as the regulatory promoter region, and were analyzed using the online software PlantCARE database (\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=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Meanwhile, \u003cem\u003ecis\u003c/em\u003e-elements present in the promoter regions were also visualized by TBtools [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eChromosome position and cross-species collinearity analysis of the\u003c/b\u003e \u003cb\u003eEgrNF-Y\u003c/b\u003e \u003cb\u003egenes\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe chromosomal locations of the \u003cem\u003eEgrNF-Y\u003c/em\u003e genes using the MapInspect software [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. TBtools was used to extract the location information of NF-Y genes from \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eP. trichocarpa\u003c/em\u003e, \u003cem\u003eE. grandis\u003c/em\u003e genome files and gene annotation files to construct the physical map of NF-Ys of three plants on chromosomes. The MCscanX command in TBtools was used to analyze the collinear relationship among the three species and to identify the collinear blocks of NF-Y genes in \u003cem\u003eA. thaliana\u003c/em\u003e, \u003cem\u003eP. trichocarpa\u003c/em\u003e and \u003cem\u003eE. grandis\u003c/em\u003e genome files [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMultiple alignments and phylogenetic analysis of EgrNF-Y proteins\u003c/h2\u003e \u003cp\u003eThe protein sequences of the EgrNF-Y members were obtained using Phytozome v12.1. Multiple sequence alignment analysis of the identified EgrNF-Ys were constructed using using ClustalX2.1 with the default parameters [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. For phylogenetic relationship analyse, the phylogenetic tree was constructed using MEGA7.0 with the complete NF-Y protein sequences. The evolutionary relationships were estimated with 1000 bootstrap replications [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The phylogenetic tree constructed by MEGA7 was uploaded to iTOL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://itol.embl.de/\u003c/span\u003e\u003cspan address=\"http://itol.embl.de/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for further editing.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePlant material, growth conditions and abiotic treatment\u003c/h3\u003e\n\u003cp\u003eAll the seedlings were grown in the Institute of Tropical Forestry, Chinese Academy of Forestry (Guangzhou, China) in this study. The greenhouse was kept at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 60\u0026ndash;70% humidity, and 16/ 8 h light/dark photoperiod. In this study, for the phosphate treatments, we designed normal growth condition (KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e concentrations of 1mM) and phosphorus deficient growth conditions (KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e concentrations of 0M) for the experiments. Control group and treatment group were replaced with a new nutrient solution every 5 days (800 mL/pot). Here, we used seedling samples grown under normal conditions as controls. Control group and treatment group was applied to18 plants (with three replicate trials) for one month. After P treatments, the roots (the newest and the second lateral roots) and leaves were harvested, respectively. Roots, stems, and leaves tissue were obtained from 6-month-old cultured plantlets. In addition, mature leaves, xylem and flowers were collected from Jiangmen City, Guangdong Province. All samples are immediately frozen in liquid nitrogen after collection and stored at -80℃ until use.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eRNA extraction and quantitative real-time PCR\u003c/h2\u003e \u003cp\u003eTotal RNAs were isolated from different tissues and organs of \u003cem\u003eE.grandis\u003c/em\u003e, as well as from the different phosphate treatment samples. Total RNA from the samples was extracted using the OMEGA plant RNA Kit (Shanghai, China) and following the operating instruction. These RNAs were assessed by agarose gel and NanoDrop 2000 spectrophotometer (Implen, Inc., Westlake Village, CA, USA). cDNA was synthesized using total RNA as the template with the Takara\u0026rsquo;s PrimeScript Synthesis 1st Strand cDNA Synthesis Kit (Takara, Beijing, China). The obtained cDNA was diluted 10-fold with ddH\u003csub\u003e2\u003c/sub\u003eO and used as the template for qRT-PCR.\u003c/p\u003e \u003cp\u003eTo measure the expression levels of \u003cem\u003eEgrNF-Y\u003c/em\u003e genes, qRT-PCR analysis was performed using SYBR \u0026reg; Premix Ex Taq\u0026trade; (TaKaRa). The qRT-PCR primers were listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, and \u003cem\u003eEgrEF2\u003c/em\u003e was used as the internal control. PCR was performed on an Light Cycler 96 Real-Time PCR system following the manufacturer\u003csup\u003e,\u003c/sup\u003es instructions. The PCR program was 94\u0026deg;C for 30 s, followed by 40 cycles of 94\u0026deg;C for 5 s and 60\u0026deg;C for 10 s, and a final elongation step of 72\u0026deg;C for 5 min. The program was completed with a melting curve from 70 to 95\u0026deg;C. The relative expression levels of these genes were analyzed using the 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eFor all experiments, we all performed three independent biological replicates and three technical replicates. Data are statistically described as mean and standard deviation (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD). SPSS19.0 software was used for data analysis (IBM SPSS, Chicago, IL, USA). Error bars were calculated according to Tukey's multiple range test, and *(\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and **(\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) being used to indicate statistically significant effects.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe appreciate the contributions and support from the lab members. We extend our sincere gratitude to the editor and reviewers for their thorough evaluation of this manuscript and for providing valuable feedback for its enhancement.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research has been supported by the Young Scientists Fund of the National Natural Science Foundation of China (32201524), the National Key Research and Development Program of China during the 14th five-year plan Period (2022YFD2200203), the Fundamental Research Funds for the Central Non-profit Research Institution of CAF (CAFYBB2022SY017) and the Guangzhou Science and technology plan project (2023A04J0711).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJL designed and supervised the whole experiments, and wrote the manuscript. JL and GL collected plant materials. JL, LZ and CG analyzed the data and edited manuscript. JL, ZL and JX revised manuscript. All authors approved the manuscript before submission.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBucher P, Trifonov EN. CCAAT box revisited: bidirectionality, location and context. J Biomol Struct Dyn. 1988;5(6):1231\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCrombrugghe SNMaBd. Role of the CCAAT-binding protein CBF/NF-Y in transcription. 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Mol Genet Genomics. 2015;290(3):1095.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"NF-Y, Eucalyptus grandis, Bioinformatics analysis, Expression analysis","lastPublishedDoi":"10.21203/rs.3.rs-4703272/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4703272/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe NF-Y (NUCLEAR FACTOR-Y) transcription factor in plants is composed of NF-YA, NF-YB and NF-YC subunits. It is known to play an important role in plant growth and development and response to stress. Although the NF-Y gene family has been systematically studied in many species, the understanding of the NF-Y gene family in \u003cem\u003eEucalyptus\u003c/em\u003e remains unknown.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, 31 (7 EgrNF-YA, 16 EgrNF-YB and 8 EgrNF-YC) \u003cem\u003eEgrNF-Y\u003c/em\u003e genes were identified in \u003cem\u003eE. grandis\u003c/em\u003e using Arabidopsis NF-Y protein sequences as queries and their structural characteristics were comprehensively analyzed. Phylogenetic, conserved domain and exon-intron structure analyzed that the closer relationship in each subfamily. Multiple alignments showed that all EgrNF-Y proteins had conserved core regions. Chromosomal localization of these genes revealed that they were randomly distributed across 11 chromosomes. \u003cem\u003eCis\u003c/em\u003e-element analysis of promoter indicated that \u003cem\u003eEgrNF-Y\u003c/em\u003e gene was affected by various hormonal and abiotic stresses. Furthermore, tissue-specific expression showed that all 30 \u003cem\u003eEgrNF-Y\u003c/em\u003e genes were widely expressed in various tissues and organs. Additionally, the stress response pattern of \u003cem\u003eEgrNF-Ys\u003c/em\u003e was identified under phosphate-starved, and 12 genes and 3 genes were upregulated more than 2-fold in the leaves and roots, respectively.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur studies have provided a general understanding of the conservation and characteristics of the EgrNF-Y genes family in \u003cem\u003eE. grandis\u003c/em\u003e. And it has been demonstrated that members of the EgrNF-YB1 and EgrNF-YB11 may play important roles in the regulation of floweringin of \u003cem\u003eE. grandis\u003c/em\u003e. To provide reference for further study on the role of NF-Y gene in the regulation of flowering in \u003cem\u003eE. grandis\u003c/em\u003e. In addition, our also established a theoretical basis for further functional studies on this family.\u003c/p\u003e","manuscriptTitle":"Genome-wide identification and expression profile analysis of the NF-Y transcription factor gene family in Eucalyptus grandis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-31 06:14:26","doi":"10.21203/rs.3.rs-4703272/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":"6871700f-6de8-47c1-9185-3d68c76d7918","owner":[],"postedDate":"July 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-22T09:53:40+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-31 06:14:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4703272","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4703272","identity":"rs-4703272","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}