Transcriptome-wide isolation and expression profiles of NF-Y gene family in male cone development and hormone treatment of Chinese pine (Pinus tabuliformis)

Transcriptome-wide isolation and expression profiles of NF-Y gene family in male cone development and hormone treatment of Chinese pine (Pinus tabuliformis) | 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 Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Transcriptome-wide isolation and expression profiles of NF-Y gene family in male cone development and hormone treatment of Chinese pine ( Pinus tabuliformis ) Yingtian Guo, Shihui Niu, Wei Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-21552/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 Conifers and angiosperms have difference in reproductive development, especially for flowering. It is known that NUCLEAR FACTOR Y (NF-Y) transcription factor play an important role in flowering, drought stress and GA, ABA signaling, but, little known in auxin, salicylic acid, jasmonic acid, etc. Moreover, the NF-Y genes family has been mainly analyzed in angiosperms, but it has not been comprehensively reported in conifers. Results In this study, we identified 9 NF-YA , 9 NF-YB , and 10 NF-YC genes in Pinus tabuliformis using Arabidopsis NF-Y protein sequences as queries. Besides, by comparing conserved regions and phylogenetic relationships of the PtNF-Ys, we found that the NF-Ys were both conserved and altered during evolution. PtTFL2 , PtCO , PtNF-YC1 and PtNF-YC4 were exploited by expression profile in male cone development and the correlation analysis. In addition, NF-YC1/4 can interact with DPL by yeast two-hybrid assays and BiFC. The multiple types of phytohormones-responsive cis -elements (ABA, JA, IAA, SA) were present and many NF-Y genes responded positively to SA and as opposed to IAA and JA. Conclusions Twenty-eight PtNF-Ys were identified and bioinformatic characterization of NF-Y genes including conserved regions, phylogenetic relationships, gene-motifs, was carried out. Two candidate genes ( NF-YC1 and NF-YC4 ) were found to be involved in the regulation of conifer flowering and gibberellin signalling. The cis -elements and hormone transcriptome analysis revealed that the potential role of NF-Ys in conifers resistance. This study provides the basis for improved understanding of NF - Y genes function in conifers. Epigenetics & Genomics Pinus tabuliformis Conifers NF-Y transcription factor Bioinformatic analysis Expression profiles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Background There are about 630 conifer species on earth, and they dominate many terrestrial ecosystems, especially in the Northern Hemisphere [ 1 ]. At the same time, conifers have great economic and ecological value. Reproductive organ development is an essential feature in the whole life cycle of seed plants. Considerable progress has been made over the past decade in understanding the molecular mechanisms underlying flower development for angiosperms. However, there is still a considerable lack of molecular biology research on the development and regulation of conifers male and female cones. Chinese pine ( Pinus tabuliformis ) of Pinaceae is an indigenous conifer species and widely distributed in 14 provinces and autonomous regions in northern China, with an ecological area of 3 million square kilometers [ 2 , 3 ]. Moreover, Chinese pine has many favored economic and ecological traits, such as wood corrosion resistance, rich in resin and pollen as well as excellent tolerance to drought, wind, barren, pests and diseases, just like Pinus sylvestris var. mongolica , and Pinus thunbergii . NUCLEAR FACTOR Y (NF-Y) transcription factors are almost found in all eukaryotes. NF-Y includes at least three subunits: NF-YA, NF-YB, and NF-YC, all of which can specifically bind to the CCAAT-box in eukaryotic promoters [ 4 ]. The core domain of NF-YA contains the NF-YB/C subdomain and the DNA binding domain. NF-YB and NF-YC which have the highly conserved histone-fold motif (HFM) are structurally similar to histone subunits H2B and H2A [ 5 , 6 ]. NF-Ys should function in the form of a dimer or trimer. Initially, NF-YB and NF-YC dimerize in the cytoplasm, then move into the nucleus, recruit an NF-YA component, and subsequently bind DNA and affect transcription [ 7 , 8 ]. The Nuclear Factor-F (NF-Y) has been reported that play an important role in flowering time [ 9 , 10 ]. NF-YA, in complex with NF-YB/NF-YC proteins, can directly bind the distal CCAAT box and a non-canonical NF-CO complex binding at a proximal CORE site of the FT promoter which are positive regulators of flowering [ 11 – 13 ]. Also, CmNFYB8 can influences flowering time through directly regulating the expression of cmo-MIR156 in the aging pathway in chrysanthemum [ 14 ]. NF-Y proteins of Triticum monococcum can interact with VRN2 in the regulation of flowering initiation by the integration of the vernalization and photoperiod seasonal signals [ 15 ]. NF-Y mediates the effect of photoperiod and GA signaling on SOC1 expression partly through H3K27me3 demethylation [ 16 ]. These results show that NF-Y can participate in regulating flowering time via the aging pathway, the photoperiod, the vernalization and GA pathway. Besides, NF - Y genes have involved in other growth and developmental processes, such as endosperm development [ 17 ], seed germination [ 18 ], pollen tube growth [ 19 ], hypocotyl elongation [ 20 ], starch biosynthesis [ 21 ]. In addition to plant growth and development, NF-Y participates in stress response and hormone signaling. According to the reports, we find studies on NF-Y mainly focus on drought resistance [ 22 – 24 ], ABA response [ 18 , 22 , 25 ] and salt stress [ 23 , 26 ], a small part involving temperature [ 27 ], photoprotective [ 28 , 29 ]. In brief, as an important transcription factor in response to drought stress, NF-Y is mainly regulated by miR169, ABA crossing signal and photosynthesis. However, related to biotic stress, such as pests and diseases and other hormones (auxin, salicylic acid, jasmonic acid, etc.) are also important research directions of plant life activities, and little is known about NF-Y. So, the role of NF-Y in other stresses, and other hormone signals, need to be further explored. Now, the study of NF-Y genes mainly focus on angiosperms, only some studies have been reported on conifers. PwHAP5 and PwNF-YB3 ( Picea wilsonii ) can improve tolerance to salt and drought stress in Arabidopsis [ 30 , 31 ], and PwHAP5 interacts with PwFKBP12 that play a role in pollen tube development and orientation regulation [ 19 ]. In addition, it has been discovered that HAP3A and HAP3B ( Picea abies and Pinus sylvestris ) are necessary to promote embryo maturation during conifer embryogenesis [ 32 ]. But, the study of NF-Y genes family in conifers has not been reported. Besides, one major difference between conifers and angiosperms is their reproductive development, and for example, FLOWERING LOCUS T (FT), the key activators of flowering, has no orthologues genes in conifers [ 1 ]. So, to clarify the role of NF-Y in conifer flowering is of great significance to enrich the evolution of conifer reproductive development. In the research, we took Chinese pine ( Pinus tabuliformis ) as the experimental material to exploit the role of NF-Y in this type of conifer. In our study, we identified 28 NF - Y genes of Pinus tabuliformis and performed a relatively complete bioinformatics analysis, including conserved regions, phylogenetic relationships, gene-motifs analysis. Moreover, we found NF-YC1 and NF-YC4 participating in male cones development and GA signal. Also, PtNF-Ys responded to ABA, JA, IAA and SA signals by the cis -elements and transcriptome analysis. Taken together, the results obtained here provide a comprehensive profile to a more complete understanding of the function of the NF - Y genes in conifers. Results Isolation of the NF‑Y family in Pinus tabuliformis We used Arabidopsis NF-Y protein sequences as queries to search Chinese pine NF-Y genes [ 33 ]. Sixty-nine candidate NF-Y genes were identified in Chinese pine by BLAST (blast-2.6.0+) [ 34 ]. Forty-nine candidate genes were identified by HMMER [ 35 ]. The results of the two search methods were merged resulting in Sixty-nine candidate genes. By removing too long or too short or because they had improper domains and redundant sequences, 28 NF-Y genes were identified, including 9 NF-YA , 9 NF-YB , and 10 NF-YC genes (Table 1 ). The indentified PtNF-Y genes encodes peptides ranging from 136 to 384 aa with the pI value varying from 4.55 to 9.76, and the molecular weight ranging from 14.88 to 41.86 KDa as estimated from ExPASy server ( http://WWW.expasy.org/ ). Table 1 NF-Y transcription factors identified in Pinus tabuliformis Gene name Gene ID Amino acid length (a.a) Protein Mol. Wt.(kDa) Protein PI value PtNF-YA1 Pita_unigene60921 163 17.92 kDa pH 8.25 PtNF-YA2 Pita_unigene63081 170 18.79 kDa pH 9.76 PtNF-YA3 Pita_unigene43998 205 22.40 kDa pH 7.29 PtNF-YA4 Pita_unigene12015 209 22.96 kDa pH 8.79 PtNF-YA5 Pita_unigene11741 287 30.95 kDa pH 8.97 PtNF-YA6 Pita_unigene12516 289 31.25 kDa pH 9.40 PtNF-YA7 Pita_unigene42483 355 38.87 kDa pH 9.51 PtNF-YA8 Pita_unigene41396 359 38.54 kDa pH 8.11 PtNF-YA9 Pita_unigene40560 384 41.86 kDa pH 8.58 PtNF-YB1 Pita_unigene46750 143 16.40 kDa pH 7.91 PtNF-YB2 Pita_unigene17122 164 18.29 kDa pH 6.12 PtNF-YB3 Pita_unigene39726 176 19.36 kDa pH 8.70 PtNF-YB4 Pita_unigene6096 177 20.34 kDa pH 5.31 PtNF-YB5 Pita_unigene27227 184 20.80 kDa pH 4.93 PtNF-YB6 Pita_unigene42866 185 20.13 kDa pH 7.37 PtNF-YB7 Pita_unigene2172 204 22.26 kDa pH 7.62 PtNF-YB8 Pita_unigene41178 220 23.85 kDa pH 8.20 PtNF-YB9 Pita_unigene62067 226 24.51 kDa pH 6.52 PtNF-YC1 Pita_unigene43117 313 35.38 kDa pH 6.93 PtNF-YC2 Pita_unigene59735 305 34.14 kDa pH 4.87 PtNF-YC3 Pita_unigene5387 288 32.10 kDa pH 4.55 PtNF-YC4 Pita_unigene12943 272 30.11 kDa pH 6.40 PtNF-YC5 Pita_unigene43379 268 30.40 kDa pH 7.53 PtNF-YC6 Pita_unigene1846 251 28.04 kDa pH 6.64 PtNF-YC7 Pita_unigene44600 236 27.49 kDa pH 6.56 PtNF-YC8 Pita_unigene15378 202 22.41 kDa pH 6.68 PtNF-YC9 Pita_unigene16781 142 15.81 kDa pH 6.51 PtNF-YC10 Pita_unigene34094 136 14.88 kDa pH 6.79 Conserved Regions And Phylogenetic Relationships Of The PtNF-Ys To further investigate the conserved regions of PtNF-Ys, the protein sequences of 28 members were analyzed using MEGA7 [ 36 ]. Highly conserved domains were found among the members of each subunit as shown in Fig. 1 . The NF-Y family should contain an interaction domain for interacting with other NF-Y subunits and a DNA binding domain for recognizing CCAAT binding sites [ 4 ]. The core conserved regions of the PtNF-YAs proteins were 55AAs but PtNF-YA4 (Fig. 1 A). The NF-YB/C subdomain had 22 AAs and the DNA binding also had 22 AAs. The central domain of PtNF-YBs had 91AAs (Fig. 1 B). Among PtNF-YBs, PtNF-YB1 had lower conserved domains than others. The central domain of PtNF-YCs contained 82 AAs for interactions between subunits, but PtNF-YC9 and PtNF-YC10 had no DNA binding domain (Fig. 1 C). Meanwhile, PtNF-YC2, PtNF-YC3 and PtNF-YC6 were slightly different from other NF-YCs. The above results suggested that PtNF-YAs were more evolutionarily conservative than other types of PtNF-Y subunits. Furthermore, the distributions of conserved motifs were assessed by MEME software. The results showed that the three PtNF-Y subunits have a unique motif distribution (Fig. 2 ). Motif 2 and motif 5 were unique to the PtNF-YAs. Motif 8 and motif 9 were present only in PtNF-YBs. Motif 3 was widely distributed in PtNF-Ys. To predict the functions of PtNF-Y proteins, phylogenetic trees using the NF-Y protein sequences of Arabidopsis and Chinese pine were created by MEGA7 software with the neighbor-joining (NJ) criteria. The phylogenetic analysis showed that the 28 PtNF-Ys were divided into three groups (Fig. 3 ). The phylogenetic tree suggested close relationships among the candidate NF-Ys within each of the three subfamilies. In the three group, we found that there was only a pair of NF-Y orthologue protein, PtNF-YC6 and AtNF-YC13, suggested the similarity biological functions. Moreover, seven pairs of paralogues were identified: PtNF-YB5 and PtNF-YB4; PtNF-B9 and PtNF-YB7; PtNF-A2 and PtNF-YA3; PtNF-A5 and PtNF-YA6; PtNF-A4 and PtNF-YA7; PtNF-C2 and PtNF-YC3; PtNF-C9 and PtNF-YC10. But, the vast majority of PtNF-Ys had lower identities with other members, implying their diversity during the evolutionary process. Analysis of cis -elements in the PtNF-Y promoters To explore the potential function and regulation of PtNF-Y genes, the 2000 bp upstream sequence of PtNF-Y promoters were analyzed. The results were shown except for the core cis -elements, a total of 55 type cis -elements were found, that includes 30 light responsive, 10 phytohormones responsive, 8 plant growth, and 7 stress responsive (Fig. 4 ). Among the cis -elements, light- responsive elements accounted for the largest proportion. G-box, box-4, GT1-motif and TCT-motif that were critical elements of light responsive. In plant growth module, the O 2 -site related to zein metabolism, CAT-box related to meristem expression and GCN4_motif related to endosperm expression were mainly detected. Besides, many stress-responsive elements and phytohormone-responsive were also present. For example, ARE related to anaerobic induction, MBS related to drought-inducibility and LTR related to low-temperature responsive were mainly detected. Analysis phytohormone-responsive elements found that AuxRR-core, TGA-element and TGA-box are involved in auxin-responsive, GARE-motif, TATC-box and P-box are involved in gibberellin-responsive, TGACG-motif and CGTCA-motif are involved in MeJA-responsive, ABRE is involved in ABA- responsive. In conclusion, the above results indicate the potential role of PtNF-Y genes in response to a variety of hormone regulation. Expression patterns of PtNF-Ys in different development stages of male cones The pollen in the male cone is allergenic and potentially harmful to the health of allergic people [ 37 ]. In addition, pollen also has economic value. So, it is of great significance to clarify the male cones development of conifers. Also, the NUCLEAR FACTOR-Y (NF-Y) families of transcription factors are important regulators in flowering time [ 10 ]. To investigate the potential functions of PtNF-Y genes, we examined their gene expression profiles (TPM) using RNA-seq of male cones sampled at six different developmental stages (M1–M6) and vegetative buds (VB). Cluster analysis showed that 4 PtNF-Y genes (i.e., A1 / B1 / B3 / C1 / C4 ) were highly expressed in male cones and 5 PtNF-Y genes (i.e., B4 / B5 / C1 / C7 / C8 ) were high expression in late stages of male. In contrast, 4 genes ( A4 , A7 , B8 and C2 ) were in low expression in male cones and 5 genes ( A5 , A6 , A8 , A9 and B6 ) were low expressed in late stages of male (Fig. 5 A) . Furthermore, studies suggest that NF-Y regulation of FT is mediated through CONSTANS (CO) [ 38 – 40 ]. We selected PtTFL2 (no FT homologs in conifer) and PtCO from the transcriptome sequencing data. The expressions of the PtTFL2 , the PtCO , and the differentially expressed PtNF-Ys were determined by RNA-seq (Fig. 5 A). The expression profile of PtCO was similar to that of PtNF-YA1 , PtNF-YB1 , PtNF-YB3 , PtNF-YC1 and PtNF-YC4 in male cones (Fig. 5 A, red black). However, the expression pattern of PtCO was the opposite of PtTFL2 . Moreover, the correlation of PtTFL2 , PtCO and PtNF-Ys in expression level are shown in Fig. 5 B. PtCO showed positively correlation with PtNF-YC1 (r = 0.596), PtNF-YC4 (r = 0.505) and was negatively correlated with PtTFL2 (r = -0.881). Also, PtTFL2 showed negatively correlation with PtNF-YC1 (r = -0.64) and PtNF-YC4 (r = -0.475) in male cones. But other three PtNF-Ys showed no correlation with PtTFL2 and PtCO . Overall, PtTFL2 , PtCO , PtNF-YC1 and PtNF-YC4 may have regulatory role in conifers flowering that need to be tested. Previous study has shown that NF-Y can regulate flowering by GA signalling, and DELLA proteins are the key transcriptional regulators that regulate plant development by GA mediating [ 16 ]. So transcriptome analysis and yeast two-hybrid assays were performed to confirm if there is a relationship between DPL (DELLA protein-like) and NF-YC1/4. Transcriptome analysis showed no significant changes in the expression of PtNF-YC1 by gibberellins (GA 3 and GA 4 ) and paclobutrazol (PAC) treatments. But, NF-YC4 was down-regulation by PAC treatment (Fig. 6 A). However, we found NF-YC1/4 can interact with DPL via yeast two-hybrid assays (Fig. 6 B). Also, Bimolecular fluorescence complementation (BiFC) assay showed that DPL-cYFP and NF-YC1/4-nYFP interacted in the nuclei of living tobacco cells (Fig. 6 C). These results support that NF-YC1/4 can interact with DPL. Therefore, we can use NF-YC1/4 as candidate genes to study strobil development and GA signal transduction. Gene expression analysis of PtNF-Ys in response to hormone treatments Except for involving in flowering time, the function of NF-Y genes were in response to stress response (drought, salt, light and temperature) and hormone treatments (GA and ABA). But, NF-Y in other stresses, such as pests, diseases, and other hormone signals, such as auxin, salicylic acid, jasmonic acid, etc need to be explored. Promoter analysis showed that PtNF-Y genes had the potential to participate in ABA, IAA, JA and SA signal transduction. Hence, expression of PtNF-Y genes was measured under these hormone treatments. The results showed that PtNF-Y genes response to ABA, IAA, JA and SA vary in strength (Fig. 7 ). The genes that respond positively to SA were in the majority, and in which the NF-YC genes had a large proportion, then ABA was followed by. However, a great part of PtNF-Y genes were negative response to IAA and JA, and the patterns were similar. Moreover, we found that most NF-Ys respond to salicylic acid in contrast to auxin and jasmonic acid. All this suggests that PtNF-Y genes were broadly involved in different hormone-responsive. Discussion Although NF-Y transcription factors have been broadly studied in several plant species, such as Arabidopsis thaliana [ 41 ], Glycine max [ 42 ], Oryza sativa [ 43 ], Zea mays [ 29 ], Triticum aestivum [ 44 ], Populus tomentosa [ 45 ], Picea wilsoni [ 19 ]. However, the identification and characterization of NF-Y gene family in conifers are still no reports. Here we use Pinus tabuliformis , widely distributed in China, to study the characteristics of NF-Y in conifers. Based on the results obtained from NF-Y transcription factors in A. thaliana by using BLAST (blast-2.6.0+) and HMMER, we identified 28 NF-Y genes based on the Chinese pine reference transcriptome (Table 2 ). Compared with the numbers of NF-Ys , such as 36 in Arabidopsis [ 33 ], 25 in castor bean [ 46 ], 59 in tomato [ 47 ], 32 in grape [ 48 ], 33 in walnut [ 49 ] and 46 in Populus trichocarpa [ 45 ] harbored a comparable number of genes. We constructed the phylogenetic tree to analyse NF-Y proteins in Chinese pine and Arabidopsis , and some studies suggest that the Arabidopsis NF-Y family may not consist of AtNF-YB11/12/13 and AtNF-YC10/11/13 because they do not include the proper structure [ 41 ]. Also, PtNF-YC6 , PtNF-YC2 and PtNF-YC3 have a distant evolutionary relationship with the three clusters of NF-YA/B/C which were similar to the six Arabidopsis NF-Ys mentioned above. Multiple alignments and our phylogenetic tree can support this opinion (Fig. 1 and Fig. 3 ). Previous studies reported that the Arabidopsis NF-YB subunits can be divided into two classes, the LEC1-type and the non-LEC1-type [ 50 ]. The LEC1-type (AtNF-YB9 and AtNF-YB6) had Asp (D) residues where Lys (K) is found in mammals and most plants, and the aspartate at D55 site is necessary for LEC1 activity in embryogenesis [ 33 ]. In our study, PtNF-YB5 | Pita unigene27227 and PtNF-YB4 | Pita unigene6096 changed from Lys (K) to Asp (D) at this binding site (Fig. 1 B). In addition, based on analysis of phylogenetic, PtNF-YB5 | Pita unigene27227 and PtNF-YB4 | Pita unigene6096 were most likely to be the two LEC1-type orthologs of AtLEC1(AtNF-YB9) and AtL1L (AtNF-YB6) that might share similar functions in regulating seed development and embryogenesis and like Arabidopsis (Fig. 3 ). According to the expression profiles of NF-Y genes during the developmental process of male cones, six different developmental stages of male from phenotypic recognition to maturation were collected (Table 2 ). PtNF-YB4 and PtNF-YB5 were only up-regulated in later development of male cones which suggested they may be involve in pollen maturation which tube growth and sperm delivery of conifers are fundamentally different from that of angiosperms [ 19 ]. The study found that two conifer LEC1-type HAP3 genes, HAP3A and HAP3B , from Picea abies and Pinus sylvestris were high expression during early embryo development, but decreased during late embryogeny [ 32 ]. Also, we found that the sequence of PtNF-YB4 had high homology (the similarity > 77%) by blast with PsHAP3A and PaHAP3A and PtNF-YB5 was the same gene with PsHAP3A (Additional file 3) which suggested PtNF-YB4 and PtNF-YB5 may also participate embryo development. So, these results gave us the direction to study LEC1-type of NF-YBs in conifers. NF-Y complexes can bind to CO proteins to activate the photoperiodic pathway and regulate FT expression in Arabidopsis [ 10 ]. CO accumulates during the day and its expression peaks at dusk; then CO can replace the NF-YA subunit of Arabidopsis to form a CO/NF-YB/NF-YC trimer (NF-CO) complex that promote the FT peak expression at dusk [ 11 , 12 , 51 , 52 ]. We observed that PtCO expression was positively correlated with PtNF-YC1 and PtNF-YC4 in male cones, but was negatively correlated with PtTFL2 (Fig. 5 ). Because there are no FT orthologous gene members in conifer, and the FT/TFL1-like members are functionally repressors, meanwhile perennial characteristics of conifers [ 53 ]. The PtCO , NF-Ys ( PtNF-YC1 and PtNF-YC4 ) and PtTFL2 may be have different regulatory mechanisms that compared with Arabidopsis. In addition, the discovery of the Arabidopsis NF-Y complex that can regulate flowering time by developmental signals, such as gibberellin pathway. So, we want to know PtNF-YC1and PtNF-YC4, that participating in flowering, whether respond to gibberellin. Via yeast two-hybrid assays and bimolecular fluorescence complementation assay, we found that NF-YC1/4 can interact with DPL. But, PtNF-YC1 was no significant changes by gibberellins (GA 3 and GA 4 ) and paclobutrazol (PAC) treatments, and maybe it depends on the sampling time. In a word, we found PtNF-YC1and PtNF-YC4 participate in flowering and gibberellin pathway, but further research is needed. Moreover, it provides a good direction whether NF-YC1/4 can regulate flowering time by gibberellin pathway in conifers to be tested. NF-Y genes are not only involved in flowering time, early seedling development but also have roles in stress responses and hormone signaling [ 54 ]. The AtNF-YC (3/4/9) [ 55 ], and CdtNF-YC1 ( Cynodon dactylon × Cynodon transvaalensis ) [ 23 ], SlNF-YA-L1 ( Solanum pimpinellifolium ) [ 26 ] and ZmNF-YA3 ( Zea mays ) [ 29 ] can result in enhanced drought tolerance. Over-expressing AtNF-YA2 or AtNF-YC1 displayed enhance tolerance against freezing stress [ 27 , 56 ]. According to our analysis of cis -elements in the PtNF-Y promoters (Fig. 4 ), the promoter regions containing MBS related to drought-inducibility and LTR related to low-temperature responsive implied that PtNF-Ys can be involved in the drought and low-temperature pathway. Moreover, we found multiple types of phytohormones-responsive cis -elements (ABA, JA, IAA, SA) suggested that PtNF-Ys might be involved in drought tolerance, pathogen and pest resistance and it conformed to the resistance characteristics of some conifers, such as Pinus tabuliformis , Pinus sylvestris var. mongolica , Pinus thunbergii . Besides, researches show that salicylic acid can inhibit pathogen growth through repression of the auxin signaling pathway [ 57 ], while JA and SA interact with each other in an antagonistic manner [ 58 ]. Based on an analysis of gene expression under hormone treatments, NF-Y genes can be in response to salicylic acid (SA), abscisic acid (ABA) and jasmonic acid (JA) treatment which were consistent with our analysis of cis -elements inference (Fig. 7 ). Also, many NF-Y genes responded positively to salicylic acid, as opposed to auxin and jasmonic acid. Our data suggest that PtNF-Y may control disease resistance by dynamically regulating SA, IAA and JA signaling. This study provided the possibility for further study of novel resistant pathways related to NF-Y genes in Pinus tabuliformis even for kinds of conifers. Conclusions NF-Y transcription factors have been extensively recognized and classified in several plants. Although there have been some studies on NF-Y in conifer trees, most studies have focused on single genes and most family studies have focused on angiosperms. So, this research trend of NF-Y should been extended to conifer trees. Our study, carried out in Pinus tabuliformis , a conifer widely distributed in China can pay close attention to current research priorities. 28 PtNF-Ys were first identified in conifer trees and their evolutionary, structural were analyzed. Comparison of NF-Ys in Chinese pine and Arabidopsis can provide rudimentary understanding on the function between less studied PtNF-Ys and its known homologs. Moreover, by analyzing transcriptome data of male development and experimental verification, two candidate genes ( NF-YC1 and NF-YC4 ) were found to be involved in the regulation of conifer flowering and gibberellin. Furthermore, analysis the cis -elements combined with the hormone treatment transcriptome indicated the potential role of NF-Y in a class of conifer resistance. According these results, we found ome special scientific problems that may contribute to further functional investigation of NF-Y family in conifers. Methods ldentification of NF-Y family members in Pinus tabuliformis The protein sequences of NF-Y genes (10 NF-YA genes, 13 NF-YB genes, and 13 NF-YC genes) in A.thaliana were retrieved from the TAIR ( http://www.arabidopsis.org/ ) (Additional file 1). These sequences were used to search our Pinus tabuliformis transcriptome database (unpublished) with the blastx program in BLAST (blast-2.6.0+) and the E-value cut-off was set as 1e-10. In addition, hidden Markov model (HMM) for the NF-Y genes was constructed using HMMER package version 3.0. The results of the BLAST and HMMER searchers were merged, resulting in 69 candidate NF-Y genes in Pinus tabuliformis . The incomplete and redundant sequences were omitted. Finally, 28 unigenes were identified (Additional file 2). Multiple Alignments And Phylogenetic Analysis Multiple sequence alignments of identified NF-Ys in Pinus tabuliformis were constructed using ClustalX. The Neighbor-Joining tree was constructed using MEGA7.0.21 software with 1000 bootstrap replications. The phylogenetic tree constructed by MEGA was uploaded to iTOL ( http://itol.embl.de/ ) for further editing. Motifs were predicted using MEME software ( http://meme-suite.org/tools/meme ). Cis -elements of the PtNF-Y promoter The promoter sequences (length, 2 kb) of PtNF-Ys were collected from the Genome Database of Pinus tabuliformis (has yet to genome annotation and obtained the promoter sequences based on the CDS alignment by blast). The cis -elements were analyzed in the PlantCARE program ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ). Transcriptome Plant Materials And Data Analysis The seeds of Pinus tabuliformis were obtained from a primary clonal seed orchard located in Pingquan City, Hebei Province, China (40°99’ N, 118°45’ E, 560 m above sea level). The seeds of Pinus tabuliformis were sown on sphagnum moss soaked with water and then germinated for 14 days in a growth chamber under conditions of 22 °C 14 h light/10 h dark photoperiod. Then transferred in plastic pots and irrigated weekly according to the hormone types and concentrations in Table 2 . After 50 days of treatment, needles were collected, liquid nitrogen quick-frozen, and stored at -80 °C. Furthermore, the male cones development samples for RNA-Seq analysis were collected from individual trees at the botanical gardens in Beijing, China (116°33.91160’E, 40°00.08610’N and 44 m above sea level) and datas were deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRA 056887. The RNA-seq datas (Additional file 4) were shown as heat map by TBtools toolkit [ 59 ]. Hierarchical clustering displays the expression profiles and the color scale indicating Normalize expression values. Also all the samples used for RNA-Seq analysis is this study are provided in Table 2 . Table 2 Pinus tabuliformis sampling for gene expression analysis Developmental stages Analysis Time point Repetitions Vegetative buds(VB) RNA-seq 26 September 2012 3 Male cones(M) RNA-seq M1 (26 September 2012); M2–M6 (16 March 2013-every 9 d) 3 Hormone treatments Analysis Hormone concentration Repetitions Needles RNA-seq Water + ethyl alcohol (CK) 6 RNA-seq Abscisic acid (ABA) 10 µM 6 RNA-seq Auxin (IAA) 10 µM 6 RNA-seq Jasmonic acid (JA) 30 µM 6 RNA-seq Salicylic acid (SA) 100 µM 6 Yeast Two-hybrid Assay The coding regions of NF-YC1 , NF-YC4 and DPL were amplified and cloned into pGBKT7 and pGADT7 (Clontech). Yeast two-hybrid assays were performed using the Yeastmaker Yeast Transformation System 2 (Clontech). Yeast AH109 cells were co-transformed with the specific bait and prey constructs. All yeast transformants were grown on SD/-Trp/-Leu or SD/-Trp/-Leu/-His/-Ade medium for selection or interaction test. BiFC Analysis For the bimolecular fluorescence complementation (BiFC) assay, DPL gene was cloned into the pSPYCE vector and NF-YC1 / 4 were cloned into the pSPYNE [ 60 ]. All expression vectors were introduced into A. tumefaciens LBA4404. Agrobacteria were incubated, harvested, and resuspended in agroinfiltration buffer (0.2 mM acetosyringone, 10 mM MgCl 2 , and 10 mM MES). Agroinfiltration buffer was mixed with an equal volume of the protein mixture and injected into tobacco leaves using a syringe. Seventy two hours after infiltration, images were taken using a Leica TCS SP8 confocal microscope. Statistical Analysis The data are statistically described as mean ± standard deviation (± SD) and visualization through GraphPad Prism 7.0. The correlation were analyzed by R 3.6.2. Abbreviations ABA: abscisic acid; IAA: auxin; JA: jasmonic acid; SA: salicylic acid; CO: CONSTANS; TFL2: TERMINAL FLOWER2; HMMs: Hidden Markov Models Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials All data analyzed during this study are included in this published article and its additional files. Competing interests The authors declare no conflict of interest. Funding This work was supported by grant from the National Natural Science Foundation of China (31770713). The funding bodies had no role in the design of the study, collection, analysis, or interpretation of data or in the writing of the manuscript. Authors’ contributions YTG analyzed the data and wrote the manuscript, SHN collected data and samples in the field, WL modified the manuscript. All authors have read and agreed to the published version of the manuscript. 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CURR BIOL. 2007;17(20):1784–90. Han G. Evolution of jasmonate biosynthesis and signaling mechanisms. J EXP BOT. 2016:w470. Chen C, Xia R, Chen H. TBtools, a Toolkit for Biologists integrating various HTS-data handling tools with a user-friendly interface. bioRxiv. 289660. Walter M, Chaban C, Schütze K, Batistic O, Weckermann K, Näke C, et al. Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 2004;40(3):428–38. Additional Files Additional file 1: Full length sequences of the Arabidopsis. Additional file 2 : CDS sequences and translated amino acid sequences of 28 Chinese pine NF-Ys. Additional file 3: Sequence alignment of PtNF-YB4, PtNF-YB5, PsHAP3A and PaHAP3A proteins. Additional file 4: Transcriptome data (TPM) of PtNF-Ys in different development stages of male cones and hormone treatments. 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Also discoverable on Platform About Our Team In Review Editorial Policies 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-21552","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research article","associatedPublications":[],"authors":[{"id":496154,"identity":"67222c40-81e8-4c9a-95ee-67f2f07abb1b","order_by":1,"name":"Yingtian Guo","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yingtian","middleName":"","lastName":"Guo","suffix":""},{"id":496155,"identity":"86bade73-19c4-475c-9154-22ed56b51f7e","order_by":2,"name":"Shihui Niu","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Shihui","middleName":"","lastName":"Niu","suffix":""},{"id":496156,"identity":"85f2aa4d-0e5d-4fda-872d-7b0a7d725940","order_by":3,"name":"Wei Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA9ElEQVRIie3RsWrDMBAG4DMCdTHuKmOIX+GCByeQh7lQ6BSKp9AtgYC69AGSt8gjyAjSxU1WQxb3AQIpWQOJ5KHdZI+F6l90SPo4HQLw8fmDQSgbaBCAP7wDvtot1Uk0AlkSVoBVP8IMsZWYAfQieaC5oEIPovikilLCIKopuBQOMl5agjrjyQuhIVlcE0vWroepw86Q41QmM8y+JUy3NXEWOknb5biQcYW2y6I3IS7ClhB2ETMLGxHehjI0s6i9GG6qr1XiIrnQQX2+Pqfp2+cO1XySRh9P5cVFQKifkpsfFWYNli4A8Ph7zhr3VR8fH5//mjsMS09WkA2SSgAAAABJRU5ErkJggg==","orcid":"","institution":"","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2020-04-06 11:35:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-21552/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-21552/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":931420,"identity":"2422d676-30a0-4240-9a0c-c27cc85939ca","added_by":"auto","created_at":"2020-04-20 21:13:41","extension":"tif","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1862902,"visible":true,"origin":"","legend":"Multiple alignments of Pinus tabuliformis NF-Y family members. Multiple alignment of (A) PtNF-YA proteins, (B) PtNF-YB proteins, and (C) PtNF-YC proteins. Amino acids critical for distinguishing between LEC1 and non-LEC1 are indicated by black box.","description":"","filename":"Figure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-21552/v1/Figure1.tif"},{"id":931422,"identity":"0fd76310-f87f-487d-bc71-2a5587d23764","added_by":"auto","created_at":"2020-04-20 21:13:42","extension":"tif","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":715962,"visible":true,"origin":"","legend":"Motif distributions of NF-YA, NF-YB, NF-YC subfamilies in Chinese pine proteins. Ten motifs were identified through MEME tool search and indicated with different colors.","description":"","filename":"Figure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-21552/v1/Figure2.tif"},{"id":931424,"identity":"fd813e8d-5f81-4515-bac4-7881a7b2bfb3","added_by":"auto","created_at":"2020-04-20 21:13:42","extension":"tif","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1070488,"visible":true,"origin":"","legend":"Phylogenetic analysis of NF-Y proteins in Chinses pine and Arabidopsis. The phylogenetic tree was constructed by protein sequences of 28 NF-Ys in Chinese pine and36 NF-Ys in Arabidopsis. Among, AtNF-YB6 and AtNF-YB9, which are marked by red circles, represent AtL1L and AtLEC1, respectively. Purple, orange and green indicate the NF-YA, NF-YB, and NF-YC subfamilies, respectively. The bootstrap values are shown on branches.","description":"","filename":"Figure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-21552/v1/Figure3.tif"},{"id":931426,"identity":"011a3cf9-78fa-412c-8ca5-ad039a1180da","added_by":"auto","created_at":"2020-04-20 21:13:42","extension":"tif","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1172042,"visible":true,"origin":"","legend":"Heat map of cis-elements in the promoter region of PtNF-Ys. Color bars and circle sizes indicate the numbers of cis-elements. Yellow box indicates MeJA-responsive, black box indicates gibberellin-responsive, red box indicates ABA-responsive, gray box indicates SA-responsive and blue box indicates auxin-responsive in phytohormone-responsive module. Red box indicates drought-responsive and blue box indicates low temperature-responsive in stress-responsive module.","description":"","filename":"Figure4.tif","url":"https://assets-eu.researchsquare.com/files/rs-21552/v1/Figure4.tif"},{"id":931427,"identity":"e96aaaee-5a3a-40b5-84d2-2ada2614e3df","added_by":"auto","created_at":"2020-04-20 21:13:42","extension":"tif","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":987303,"visible":true,"origin":"","legend":"Expression patterns of PtNF-Ys and correlations in expression levels between PtCO, PtTFL2 with PtNF-Ys in different development stages of male cones. (A) Expression patterns of PtNF-Ys. (B) Correlations in expression levels. VB represents vegetative bud, M1-M6 represent male cones sampled at consequential developmental stages. The color scale indicates fold-change values (normalize expression values of TPM) with red representing increased transcript abundance and blue indicating decreased transcript abundance.","description":"","filename":"Figure5.tif","url":"https://assets-eu.researchsquare.com/files/rs-21552/v1/Figure5.tif"},{"id":931428,"identity":"1ad780e2-bdd5-4b8c-9a4b-7495df793b7c","added_by":"auto","created_at":"2020-04-20 21:13:42","extension":"tif","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2447764,"visible":true,"origin":"","legend":"NF-YC1/4 physically interact with DPL in vivo. (A) Transcriptomic analysis of NF-YC1/4 in response to GA and PAC. Red histogram represents NF-YC4 expression level, and blue histogram represents NF-YC1 expression level. (B) Yeast two-hybrid assays show the interactions between NF-YC1/4 and DPL. Transformed yeast cells were grown on SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu medium. (C) BiFC analysis of the interactions a between NF-YC1/4 and DPL in tobacco epidermal cells. YFP, fluorescence of yellow fluorescent protein; BF, bright field; Merge, merge of YFP and bright field. Scale bar, 50 μm.","description":"","filename":"Figure6.tif","url":"https://assets-eu.researchsquare.com/files/rs-21552/v1/Figure6.tif"},{"id":931429,"identity":"4c63dfc9-9a1e-4c2e-b2f5-3660a73384b5","added_by":"auto","created_at":"2020-04-20 21:13:43","extension":"tif","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":771139,"visible":true,"origin":"","legend":"Expression patterns of PtNF-Ys in response to hormone treatments. ABA, IAA, JA, SA treatments of needles in Chinese pine are shown. The color scale indicates fold-change values (normalize expression values of TPM) with red representing increased transcript abundance and blue indicating decreased transcript abundance.","description":"","filename":"Figure7.tif","url":"https://assets-eu.researchsquare.com/files/rs-21552/v1/Figure7.tif"},{"id":15666963,"identity":"67ca1aa9-964b-4d04-9446-a068ac935955","added_by":"auto","created_at":"2021-11-18 13:39:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9531928,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-21552/v1/6e62424e-1995-4448-a1fb-8b5e911997a9.pdf"},{"id":931425,"identity":"06d2efe9-88a4-41e9-a957-a1434549d019","added_by":"auto","created_at":"2020-04-20 21:13:42","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":15644,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfiles3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-21552/v1/Additionalfiles3.xlsx"},{"id":931423,"identity":"407a33c2-5797-4d2c-8771-fbce863f4a3a","added_by":"auto","created_at":"2020-04-20 21:13:42","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":18603,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfiles1.docx","url":"https://assets-eu.researchsquare.com/files/rs-21552/v1/Additionalfiles1.docx"},{"id":931419,"identity":"d0e87de5-d6c0-423c-8451-44bf18339985","added_by":"auto","created_at":"2020-04-20 21:13:41","extension":"xlsx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":39094,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfiles4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-21552/v1/Additionalfiles4.xlsx"},{"id":931421,"identity":"c745c106-8195-485d-b102-5fa62630677e","added_by":"auto","created_at":"2020-04-20 21:13:41","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":28048,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfiles2.docx","url":"https://assets-eu.researchsquare.com/files/rs-21552/v1/Additionalfiles2.docx"}],"financialInterests":"","formattedTitle":"\u003cp\u003eTranscriptome-wide isolation and expression profiles of \u003cem\u003eNF-Y\u003c/em\u003e gene family in male cone development and hormone treatment of Chinese pine (\u003cem\u003ePinus tabuliformis\u003c/em\u003e)\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eThere are about 630 conifer species on earth, and they dominate many terrestrial ecosystems, especially in the Northern Hemisphere [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. At the same time, conifers have great economic and ecological value. Reproductive organ development is an essential feature in the whole life cycle of seed plants. Considerable progress has been made over the past decade in understanding the molecular mechanisms underlying flower development for angiosperms. However, there is still a considerable lack of molecular biology research on the development and regulation of conifers male and female cones. Chinese pine (\u003cem\u003ePinus tabuliformis\u003c/em\u003e) of Pinaceae is an indigenous conifer species and widely distributed in 14 provinces and autonomous regions in northern China, with an ecological area of 3\u0026nbsp;million square kilometers [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Moreover, Chinese pine has many favored economic and ecological traits, such as wood corrosion resistance, rich in resin and pollen as well as excellent tolerance to drought, wind, barren, pests and diseases, just like \u003cem\u003ePinus sylvestris\u003c/em\u003e var. \u003cem\u003emongolica\u003c/em\u003e, and \u003cem\u003ePinus thunbergii\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eNUCLEAR FACTOR Y (NF-Y) transcription factors are almost found in all eukaryotes. NF-Y includes at least three subunits: NF-YA, NF-YB, and NF-YC, all of which can specifically bind to the CCAAT-box in eukaryotic promoters [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The core domain of NF-YA contains the NF-YB/C subdomain and the DNA binding domain. NF-YB and NF-YC which have the highly conserved histone-fold motif (HFM) are structurally similar to histone subunits H2B and H2A [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. NF-Ys should function in the form of a dimer or trimer. Initially, NF-YB and NF-YC dimerize in the cytoplasm, then move into the nucleus, recruit an NF-YA component, and subsequently bind DNA and affect transcription [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe Nuclear Factor-F (NF-Y) has been reported that play an important role in flowering time [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. NF-YA, in complex with NF-YB/NF-YC proteins, can directly bind the distal \u003cem\u003eCCAAT\u003c/em\u003e box and a non-canonical NF-CO complex binding at a proximal CORE site of the \u003cem\u003eFT\u003c/em\u003e promoter which are positive regulators of flowering [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Also, CmNFYB8 can influences flowering time through directly regulating the expression of \u003cem\u003ecmo-MIR156\u003c/em\u003e in the aging pathway in chrysanthemum [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. NF-Y proteins of \u003cem\u003eTriticum monococcum\u003c/em\u003e can interact with VRN2 in the regulation of flowering initiation by the integration of the vernalization and photoperiod seasonal signals [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. NF-Y mediates the effect of photoperiod and GA signaling on \u003cem\u003eSOC1\u003c/em\u003e expression partly through H3K27me3 demethylation [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These results show that NF-Y can participate in regulating flowering time via the aging pathway, the photoperiod, the vernalization and GA pathway. Besides, \u003cem\u003eNF\u003c/em\u003e-\u003cem\u003eY\u003c/em\u003e genes have involved in other growth and developmental processes, such as endosperm development [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], seed germination [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], pollen tube growth [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], hypocotyl elongation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], starch biosynthesis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn addition to plant growth and development, NF-Y participates in stress response and hormone signaling. According to the reports, we find studies on NF-Y mainly focus on drought resistance [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], ABA response [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and salt stress [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], a small part involving temperature [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], photoprotective [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In brief, as an important transcription factor in response to drought stress, NF-Y is mainly regulated by miR169, ABA crossing signal and photosynthesis. However, related to biotic stress, such as pests and diseases and other hormones (auxin, salicylic acid, jasmonic acid, etc.) are also important research directions of plant life activities, and little is known about NF-Y. So, the role of NF-Y in other stresses, and other hormone signals, need to be further explored.\u003c/p\u003e \u003cp\u003eNow, the study of \u003cem\u003eNF-Y\u003c/em\u003e genes mainly focus on angiosperms, only some studies have been reported on conifers. \u003cem\u003ePwHAP5\u003c/em\u003e and \u003cem\u003ePwNF-YB3\u003c/em\u003e (\u003cem\u003ePicea wilsonii\u003c/em\u003e) can improve tolerance to salt and drought stress in Arabidopsis [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], and PwHAP5 interacts with PwFKBP12 that play a role in pollen tube development and orientation regulation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In addition, it has been discovered that \u003cem\u003eHAP3A\u003c/em\u003e and \u003cem\u003eHAP3B\u003c/em\u003e (\u003cem\u003ePicea abies\u003c/em\u003e and \u003cem\u003ePinus sylvestris\u003c/em\u003e) are necessary to promote embryo maturation during conifer embryogenesis [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. But, the study of \u003cem\u003eNF-Y\u003c/em\u003e genes family in conifers has not been reported. Besides, one major difference between conifers and angiosperms is their reproductive development, and for example, FLOWERING LOCUS T (FT), the key activators of flowering, has no orthologues genes in conifers [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. So, to clarify the role of NF-Y in conifer flowering is of great significance to enrich the evolution of conifer reproductive development. In the research, we took Chinese pine (\u003cem\u003ePinus tabuliformis\u003c/em\u003e) as the experimental material to exploit the role of NF-Y in this type of conifer. In our study, we identified 28 \u003cem\u003eNF\u003c/em\u003e-\u003cem\u003eY\u003c/em\u003e genes of \u003cem\u003ePinus tabuliformis\u003c/em\u003e and performed a relatively complete bioinformatics analysis, including conserved regions, phylogenetic relationships, gene-motifs analysis. Moreover, we found NF-YC1 and NF-YC4 participating in male cones development and GA signal. Also, \u003cem\u003ePtNF-Ys\u003c/em\u003e responded to ABA, JA, IAA and SA signals by the \u003cem\u003ecis\u003c/em\u003e-elements and transcriptome analysis. Taken together, the results obtained here provide a comprehensive profile to a more complete understanding of the function of the \u003cem\u003eNF\u003c/em\u003e-\u003cem\u003eY\u003c/em\u003e genes in conifers.\u003c/p\u003e "},{"header":"Results","content":" \u003cp\u003e \u003cb\u003eIsolation of the NF‑Y family in\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ePinus tabuliformis\u003c/span\u003e\u003c/p\u003e \u003cp\u003eWe used Arabidopsis NF-Y protein sequences as queries to search Chinese pine \u003cem\u003eNF-Y\u003c/em\u003e genes [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Sixty-nine candidate \u003cem\u003eNF-Y\u003c/em\u003e genes were identified in Chinese pine by BLAST (blast-2.6.0+) [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Forty-nine candidate genes were identified by HMMER [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The results of the two search methods were merged resulting in Sixty-nine candidate genes. By removing too long or too short or because they had improper domains and redundant sequences, 28 \u003cem\u003eNF-Y\u003c/em\u003e genes were identified, including 9 \u003cem\u003eNF-YA\u003c/em\u003e, 9 \u003cem\u003eNF-YB\u003c/em\u003e, and 10 \u003cem\u003eNF-YC\u003c/em\u003e genes (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The indentified \u003cem\u003ePtNF-Y\u003c/em\u003e genes encodes peptides ranging from 136 to 384 aa with the pI value varying from 4.55 to 9.76, and the molecular weight ranging from 14.88 to 41.86 KDa as estimated from ExPASy server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://WWW.expasy.org/\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eNF-Y transcription factors identified in \u003cem\u003ePinus tabuliformis\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003cp\u003ename\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003cp\u003eID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAmino acid\u003c/p\u003e \u003cp\u003elength (a.a)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eProtein Mol.\u003c/p\u003e \u003cp\u003eWt.(kDa)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProtein\u003c/p\u003e \u003cp\u003ePI value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YA1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene60921\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e163\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e17.92\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 8.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YA2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene63081\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e170\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.79\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 9.76\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YA3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene43998\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e205\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.40\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 7.29\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YA4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene12015\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e209\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.96\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 8.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YA5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene11741\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e287\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.95\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 8.97\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YA6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene12516\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e289\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e31.25\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 9.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YA7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene42483\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e355\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38.87\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 9.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YA8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene41396\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e359\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e38.54\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 8.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YA9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene40560\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e384\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e41.86\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 8.58\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YB1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene46750\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e143\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16.40\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 7.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YB2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene17122\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e164\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e18.29\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 6.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YB3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene39726\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e176\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e19.36\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 8.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YB4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene6096\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e177\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.34\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 5.31\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YB5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene27227\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e184\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.80\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 4.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YB6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene42866\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e185\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e20.13\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 7.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YB7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene2172\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e204\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.26\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 7.62\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YB8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene41178\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e220\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e23.85\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 8.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YB9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene62067\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e226\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24.51\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 6.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YC1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene43117\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e313\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e35.38\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 6.93\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YC2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene59735\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e305\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e34.14\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 4.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YC3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene5387\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e288\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32.10\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 4.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YC4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene12943\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e272\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.11\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 6.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YC5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene43379\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e268\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.40\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 7.53\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YC6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene1846\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e251\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.04\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 6.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YC7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene44600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e236\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e27.49\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 6.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YC8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene15378\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e202\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e22.41\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 6.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YC9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene16781\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15.81\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 6.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePtNF-YC10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePita_unigene34094\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e136\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e14.88\u0026nbsp;kDa\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003epH 6.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \n\u003cp\u003e\u003cb\u003eConserved Regions And Phylogenetic Relationships Of The PtNF-Ys\u003c/b\u003e\u003c/p\u003e\n \u003cp\u003eTo further investigate the conserved regions of PtNF-Ys, the protein sequences of 28 members were analyzed using MEGA7 [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Highly conserved domains were found among the members of each subunit as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The NF-Y family should contain an interaction domain for interacting with other NF-Y subunits and a DNA binding domain for recognizing CCAAT binding sites [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The core conserved regions of the PtNF-YAs proteins were 55AAs but PtNF-YA4 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The NF-YB/C subdomain had 22 AAs and the DNA binding also had 22 AAs. The central domain of PtNF-YBs had 91AAs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Among PtNF-YBs, PtNF-YB1 had lower conserved domains than others. The central domain of PtNF-YCs contained 82 AAs for interactions between subunits, but PtNF-YC9 and PtNF-YC10 had no DNA binding domain (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Meanwhile, PtNF-YC2, PtNF-YC3 and PtNF-YC6 were slightly different from other NF-YCs. The above results suggested that PtNF-YAs were more evolutionarily conservative than other types of PtNF-Y subunits. Furthermore, the distributions of conserved motifs were assessed by MEME software. The results showed that the three PtNF-Y subunits have a unique motif distribution (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Motif 2 and motif 5 were unique to the PtNF-YAs. Motif 8 and motif 9 were present only in PtNF-YBs. Motif 3 was widely distributed in PtNF-Ys.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo predict the functions of PtNF-Y proteins, phylogenetic trees using the NF-Y protein sequences of Arabidopsis and Chinese pine were created by MEGA7 software with the neighbor-joining (NJ) criteria. The phylogenetic analysis showed that the 28 PtNF-Ys were divided into three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The phylogenetic tree suggested close relationships among the candidate NF-Ys within each of the three subfamilies. In the three group, we found that there was only a pair of NF-Y orthologue protein, PtNF-YC6 and AtNF-YC13, suggested the similarity biological functions. Moreover, seven pairs of paralogues were identified: PtNF-YB5 and PtNF-YB4; PtNF-B9 and PtNF-YB7; PtNF-A2 and PtNF-YA3; PtNF-A5 and PtNF-YA6; PtNF-A4 and PtNF-YA7; PtNF-C2 and PtNF-YC3; PtNF-C9 and PtNF-YC10. But, the vast majority of PtNF-Ys had lower identities with other members, implying their diversity during the evolutionary process.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAnalysis of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ecis\u003c/span\u003e\u003cb\u003e-elements in the\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ePtNF-Y\u003c/span\u003e \u003cb\u003epromoters\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo explore the potential function and regulation of \u003cem\u003ePtNF-Y\u003c/em\u003e genes, the 2000\u0026nbsp;bp upstream sequence of \u003cem\u003ePtNF-Y\u003c/em\u003e promoters were analyzed. The results were shown except for the core \u003cem\u003ecis\u003c/em\u003e-elements, a total of 55 type \u003cem\u003ecis\u003c/em\u003e-elements were found, that includes 30 light responsive, 10 phytohormones responsive, 8 plant growth, and 7 stress responsive (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Among the \u003cem\u003ecis\u003c/em\u003e-elements, light- responsive elements accounted for the largest proportion. G-box, box-4, GT1-motif and TCT-motif that were critical elements of light responsive. In plant growth module, the O\u003csub\u003e2\u003c/sub\u003e-site related to zein metabolism, CAT-box related to meristem expression and GCN4_motif related to endosperm expression were mainly detected. Besides, many stress-responsive elements and phytohormone-responsive were also present. For example, ARE related to anaerobic induction, MBS related to drought-inducibility and LTR related to low-temperature responsive were mainly detected. Analysis phytohormone-responsive elements found that AuxRR-core, TGA-element and TGA-box are involved in auxin-responsive, GARE-motif, TATC-box and P-box are involved in gibberellin-responsive, TGACG-motif and CGTCA-motif are involved in MeJA-responsive, ABRE is involved in ABA- responsive. In conclusion, the above results indicate the potential role of \u003cem\u003ePtNF-Y\u003c/em\u003e genes in response to a variety of hormone regulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression patterns of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ePtNF-Ys\u003c/span\u003e \u003cb\u003ein different development stages of male cones\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe pollen in the male cone is allergenic and potentially harmful to the health of allergic people [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In addition, pollen also has economic value. So, it is of great significance to clarify the male cones development of conifers. Also, the NUCLEAR FACTOR-Y (NF-Y) families of transcription factors are important regulators in flowering time [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. To investigate the potential functions of \u003cem\u003ePtNF-Y\u003c/em\u003e genes, we examined their gene expression profiles (TPM) using RNA-seq of male cones sampled at six different developmental stages (M1\u0026ndash;M6) and vegetative buds (VB). Cluster analysis showed that 4 \u003cem\u003ePtNF-Y\u003c/em\u003e genes (i.e., \u003cem\u003eA1\u003c/em\u003e/\u003cem\u003eB1\u003c/em\u003e/\u003cem\u003eB3\u003c/em\u003e/\u003cem\u003eC1\u003c/em\u003e/\u003cem\u003eC4\u003c/em\u003e) were highly expressed in male cones and 5 \u003cem\u003ePtNF-Y\u003c/em\u003e genes (i.e., \u003cem\u003eB4\u003c/em\u003e/\u003cem\u003eB5\u003c/em\u003e/\u003cem\u003eC1\u003c/em\u003e/\u003cem\u003eC7\u003c/em\u003e/\u003cem\u003eC8\u003c/em\u003e) were high expression in late stages of male. In contrast, 4 genes (\u003cem\u003eA4\u003c/em\u003e, \u003cem\u003eA7\u003c/em\u003e, \u003cem\u003eB8\u003c/em\u003e and \u003cem\u003eC2\u003c/em\u003e) were in low expression in male cones and 5 genes (\u003cem\u003eA5\u003c/em\u003e, \u003cem\u003eA6\u003c/em\u003e, \u003cem\u003eA8\u003c/em\u003e, \u003cem\u003eA9\u003c/em\u003e and \u003cem\u003eB6\u003c/em\u003e) were low expressed in late stages of male (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) .\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, studies suggest that NF-Y regulation of \u003cem\u003eFT\u003c/em\u003e is mediated through CONSTANS (CO) [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. We selected \u003cem\u003ePtTFL2\u003c/em\u003e (no \u003cem\u003eFT\u003c/em\u003e homologs in conifer) and \u003cem\u003ePtCO\u003c/em\u003e from the transcriptome sequencing data. The expressions of the \u003cem\u003ePtTFL2\u003c/em\u003e, the \u003cem\u003ePtCO\u003c/em\u003e, and the differentially expressed \u003cem\u003ePtNF-Ys\u003c/em\u003e were determined by RNA-seq (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The expression profile of \u003cem\u003ePtCO\u003c/em\u003e was similar to that of \u003cem\u003ePtNF-YA1\u003c/em\u003e, \u003cem\u003ePtNF-YB1\u003c/em\u003e, \u003cem\u003ePtNF-YB3\u003c/em\u003e, \u003cem\u003ePtNF-YC1\u003c/em\u003e and \u003cem\u003ePtNF-YC4\u003c/em\u003e in male cones (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, red black). However, the expression pattern of \u003cem\u003ePtCO\u003c/em\u003e was the opposite of \u003cem\u003ePtTFL2\u003c/em\u003e. Moreover, the correlation of \u003cem\u003ePtTFL2\u003c/em\u003e, \u003cem\u003ePtCO\u003c/em\u003e and \u003cem\u003ePtNF-Ys\u003c/em\u003e in expression level are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB. \u003cem\u003ePtCO\u003c/em\u003e showed positively correlation with \u003cem\u003ePtNF-YC1\u003c/em\u003e (r\u0026thinsp;=\u0026thinsp;0.596), \u003cem\u003ePtNF-YC4\u003c/em\u003e (r\u0026thinsp;=\u0026thinsp;0.505) and was negatively correlated with \u003cem\u003ePtTFL2\u003c/em\u003e (r = -0.881). Also, \u003cem\u003ePtTFL2\u003c/em\u003e showed negatively correlation with \u003cem\u003ePtNF-YC1\u003c/em\u003e (r = -0.64) and \u003cem\u003ePtNF-YC4\u003c/em\u003e (r = -0.475) in male cones. But other three \u003cem\u003ePtNF-Ys\u003c/em\u003e showed no correlation with \u003cem\u003ePtTFL2\u003c/em\u003e and \u003cem\u003ePtCO\u003c/em\u003e. Overall, \u003cem\u003ePtTFL2\u003c/em\u003e, \u003cem\u003ePtCO\u003c/em\u003e, \u003cem\u003ePtNF-YC1\u003c/em\u003e and \u003cem\u003ePtNF-YC4\u003c/em\u003e may have regulatory role in conifers flowering that need to be tested.\u003c/p\u003e \u003cp\u003ePrevious study has shown that NF-Y can regulate flowering by GA signalling, and DELLA proteins are the key transcriptional regulators that regulate plant development by GA mediating [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. So transcriptome analysis and yeast two-hybrid assays were performed to confirm if there is a relationship between DPL (DELLA protein-like) and NF-YC1/4. Transcriptome analysis showed no significant changes in the expression of \u003cem\u003ePtNF-YC1\u003c/em\u003e by gibberellins (GA\u003csub\u003e3\u003c/sub\u003e and GA\u003csub\u003e4\u003c/sub\u003e) and paclobutrazol (PAC) treatments. But, \u003cem\u003eNF-YC4\u003c/em\u003e was down-regulation by PAC treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). However, we found NF-YC1/4 can interact with DPL via yeast two-hybrid assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). Also, Bimolecular fluorescence complementation (BiFC) assay showed that DPL-cYFP and NF-YC1/4-nYFP interacted in the nuclei of living tobacco cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). These results support that NF-YC1/4 can interact with DPL. Therefore, we can use NF-YC1/4 as candidate genes to study strobil development and GA signal transduction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGene expression analysis of\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ePtNF-Ys\u003c/span\u003e \u003cb\u003ein response to hormone treatments\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExcept for involving in flowering time, the function of \u003cem\u003eNF-Y\u003c/em\u003e genes were in response to stress response (drought, salt, light and temperature) and hormone treatments (GA and ABA). But, NF-Y in other stresses, such as pests, diseases, and other hormone signals, such as auxin, salicylic acid, jasmonic acid, etc need to be explored. Promoter analysis showed that \u003cem\u003ePtNF-Y\u003c/em\u003e genes had the potential to participate in ABA, IAA, JA and SA signal transduction. Hence, expression of \u003cem\u003ePtNF-Y\u003c/em\u003e genes was measured under these hormone treatments. The results showed that \u003cem\u003ePtNF-Y\u003c/em\u003e genes response to ABA, IAA, JA and SA vary in strength (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The genes that respond positively to SA were in the majority, and in which the \u003cem\u003eNF-YC\u003c/em\u003e genes had a large proportion, then ABA was followed by. However, a great part of \u003cem\u003ePtNF-Y\u003c/em\u003e genes were negative response to IAA and JA, and the patterns were similar. Moreover, we found that most \u003cem\u003eNF-Ys\u003c/em\u003e respond to salicylic acid in contrast to auxin and jasmonic acid. All this suggests that \u003cem\u003ePtNF-Y\u003c/em\u003e genes were broadly involved in different hormone-responsive.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e "},{"header":"Discussion","content":" \u003cp\u003eAlthough NF-Y transcription factors have been broadly studied in several plant species, such as \u003cem\u003eArabidopsis thaliana\u003c/em\u003e [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], \u003cem\u003eGlycine max\u003c/em\u003e [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], \u003cem\u003eOryza sativa\u003c/em\u003e [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e], \u003cem\u003eZea mays\u003c/em\u003e [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], \u003cem\u003eTriticum aestivum\u003c/em\u003e [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], \u003cem\u003ePopulus tomentosa\u003c/em\u003e [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], \u003cem\u003ePicea wilsoni\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, the identification and characterization of \u003cem\u003eNF-Y\u003c/em\u003e gene family in conifers are still no reports. Here we use \u003cem\u003ePinus tabuliformis\u003c/em\u003e, widely distributed in China, to study the characteristics of NF-Y in conifers. Based on the results obtained from NF-Y transcription factors in \u003cem\u003eA. thaliana\u003c/em\u003e by using BLAST (blast-2.6.0+) and HMMER, we identified 28 \u003cem\u003eNF-Y\u003c/em\u003e genes based on the Chinese pine reference transcriptome (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Compared with the numbers of \u003cem\u003eNF-Ys\u003c/em\u003e, such as 36 in Arabidopsis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], 25 in castor bean [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], 59 in tomato [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], 32 in grape [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], 33 in walnut [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] and 46 in \u003cem\u003ePopulus trichocarpa\u003c/em\u003e [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e] harbored a comparable number of genes. We constructed the phylogenetic tree to analyse NF-Y proteins in Chinese pine and \u003cem\u003eArabidopsis\u003c/em\u003e, and some studies suggest that the Arabidopsis NF-Y family may not consist of AtNF-YB11/12/13 and AtNF-YC10/11/13 because they do not include the proper structure [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Also, \u003cem\u003ePtNF-YC6\u003c/em\u003e, \u003cem\u003ePtNF-YC2\u003c/em\u003e and \u003cem\u003ePtNF-YC3\u003c/em\u003e have a distant evolutionary relationship with the three clusters of NF-YA/B/C which were similar to the six Arabidopsis NF-Ys mentioned above. Multiple alignments and our phylogenetic tree can support this opinion (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePrevious studies reported that the Arabidopsis NF-YB subunits can be divided into two classes, the LEC1-type and the non-LEC1-type [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The LEC1-type (AtNF-YB9 and AtNF-YB6) had Asp (D) residues where Lys (K) is found in mammals and most plants, and the aspartate at D55 site is necessary for LEC1 activity in embryogenesis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In our study, \u003cem\u003ePtNF-YB5\u003c/em\u003e|\u003cem\u003ePita unigene27227\u003c/em\u003e and \u003cem\u003ePtNF-YB4\u003c/em\u003e|\u003cem\u003ePita unigene6096\u003c/em\u003e changed from Lys (K) to Asp (D) at this binding site (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In addition, based on analysis of phylogenetic, \u003cem\u003ePtNF-YB5\u003c/em\u003e|\u003cem\u003ePita unigene27227\u003c/em\u003e and \u003cem\u003ePtNF-YB4\u003c/em\u003e|\u003cem\u003ePita unigene6096\u003c/em\u003e were most likely to be the two LEC1-type orthologs of AtLEC1(AtNF-YB9) and AtL1L (AtNF-YB6) that might share similar functions in regulating seed development and embryogenesis and like Arabidopsis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). According to the expression profiles of \u003cem\u003eNF-Y\u003c/em\u003e genes during the developmental process of male cones, six different developmental stages of male from phenotypic recognition to maturation were collected (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). \u003cem\u003ePtNF-YB4\u003c/em\u003e and \u003cem\u003ePtNF-YB5\u003c/em\u003e were only up-regulated in later development of male cones which suggested they may be involve in pollen maturation which tube growth and sperm delivery of conifers are fundamentally different from that of angiosperms [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The study found that two conifer LEC1-type \u003cem\u003eHAP3\u003c/em\u003e genes, \u003cem\u003eHAP3A\u003c/em\u003e and \u003cem\u003eHAP3B\u003c/em\u003e, from \u003cem\u003ePicea abies\u003c/em\u003e and \u003cem\u003ePinus sylvestris\u003c/em\u003e were high expression during early embryo development, but decreased during late embryogeny [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Also, we found that the sequence of PtNF-YB4 had high homology (the similarity\u0026thinsp;\u0026gt;\u0026thinsp;77%) by blast with PsHAP3A and PaHAP3A and \u003cem\u003ePtNF-YB5\u003c/em\u003e was the same gene with \u003cem\u003ePsHAP3A\u003c/em\u003e (Additional file 3) which suggested \u003cem\u003ePtNF-YB4\u003c/em\u003e and \u003cem\u003ePtNF-YB5\u003c/em\u003e may also participate embryo development. So, these results gave us the direction to study LEC1-type of NF-YBs in conifers.\u003c/p\u003e \u003cp\u003eNF-Y complexes can bind to CO proteins to activate the photoperiodic pathway and regulate \u003cem\u003eFT\u003c/em\u003e expression in Arabidopsis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. CO accumulates during the day and its expression peaks at dusk; then CO can replace the NF-YA subunit of \u003cem\u003eArabidopsis\u003c/em\u003e to form a CO/NF-YB/NF-YC trimer (NF-CO) complex that promote the \u003cem\u003eFT\u003c/em\u003e peak expression at dusk [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. We observed that \u003cem\u003ePtCO\u003c/em\u003e expression was positively correlated with \u003cem\u003ePtNF-YC1\u003c/em\u003e and \u003cem\u003ePtNF-YC4\u003c/em\u003e in male cones, but was negatively correlated with \u003cem\u003ePtTFL2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Because there are no \u003cem\u003eFT\u003c/em\u003e orthologous gene members in conifer, and the \u003cem\u003eFT/TFL1-like\u003c/em\u003e members are functionally repressors, meanwhile perennial characteristics of conifers [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The \u003cem\u003ePtCO\u003c/em\u003e, \u003cem\u003eNF-Ys\u003c/em\u003e (\u003cem\u003ePtNF-YC1\u003c/em\u003eand \u003cem\u003ePtNF-YC4\u003c/em\u003e) and \u003cem\u003ePtTFL2\u003c/em\u003e may be have different regulatory mechanisms that compared with Arabidopsis. In addition, the discovery of the Arabidopsis NF-Y complex that can regulate flowering time by developmental signals, such as gibberellin pathway. So, we want to know PtNF-YC1and PtNF-YC4, that participating in flowering, whether respond to gibberellin. Via yeast two-hybrid assays and bimolecular fluorescence complementation assay, we found that NF-YC1/4 can interact with DPL. But, \u003cem\u003ePtNF-YC1\u003c/em\u003e was no significant changes by gibberellins (GA\u003csub\u003e3\u003c/sub\u003e and GA\u003csub\u003e4\u003c/sub\u003e) and paclobutrazol (PAC) treatments, and maybe it depends on the sampling time. In a word, we found PtNF-YC1and PtNF-YC4 participate in flowering and gibberellin pathway, but further research is needed. Moreover, it provides a good direction whether NF-YC1/4 can regulate flowering time by gibberellin pathway in conifers to be tested.\u003c/p\u003e \u003cp\u003e \u003cem\u003eNF-Y\u003c/em\u003e genes are not only involved in flowering time, early seedling development but also have roles in stress responses and hormone signaling [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The AtNF-YC (3/4/9) [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e], and \u003cem\u003eCdtNF-YC1\u003c/em\u003e (\u003cem\u003eCynodon dactylon\u003c/em\u003e\u0026thinsp;\u0026times;\u0026thinsp;\u003cem\u003eCynodon transvaalensis\u003c/em\u003e) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], \u003cem\u003eSlNF-YA-L1\u003c/em\u003e(\u003cem\u003eSolanum pimpinellifolium\u003c/em\u003e) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and \u003cem\u003eZmNF-YA3\u003c/em\u003e (\u003cem\u003eZea mays\u003c/em\u003e) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] can result in enhanced drought tolerance. Over-expressing \u003cem\u003eAtNF-YA2\u003c/em\u003e or \u003cem\u003eAtNF-YC1\u003c/em\u003e displayed enhance tolerance against freezing stress [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. According to our analysis of \u003cem\u003ecis\u003c/em\u003e-elements in the \u003cem\u003ePtNF-Y\u003c/em\u003e promoters (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), the promoter regions containing MBS related to drought-inducibility and LTR related to low-temperature responsive implied that \u003cem\u003ePtNF-Ys\u003c/em\u003e can be involved in the drought and low-temperature pathway. Moreover, we found multiple types of phytohormones-responsive \u003cem\u003ecis\u003c/em\u003e-elements (ABA, JA, IAA, SA) suggested that \u003cem\u003ePtNF-Ys\u003c/em\u003e might be involved in drought tolerance, pathogen and pest resistance and it conformed to the resistance characteristics of some conifers, such as \u003cem\u003ePinus tabuliformis\u003c/em\u003e, \u003cem\u003ePinus sylvestris\u003c/em\u003e var. \u003cem\u003emongolica\u003c/em\u003e, \u003cem\u003ePinus thunbergii\u003c/em\u003e. Besides, researches show that salicylic acid can inhibit pathogen growth through repression of the auxin signaling pathway [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], while JA and SA interact with each other in an antagonistic manner [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Based on an analysis of gene expression under hormone treatments, \u003cem\u003eNF-Y\u003c/em\u003e genes can be in response to salicylic acid (SA), abscisic acid (ABA) and jasmonic acid (JA) treatment which were consistent with our analysis of \u003cem\u003ecis\u003c/em\u003e-elements inference (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Also, many \u003cem\u003eNF-Y\u003c/em\u003e genes responded positively to salicylic acid, as opposed to auxin and jasmonic acid. Our data suggest that PtNF-Y may control disease resistance by dynamically regulating SA, IAA and JA signaling. This study provided the possibility for further study of novel resistant pathways related to \u003cem\u003eNF-Y\u003c/em\u003e genes in \u003cem\u003ePinus tabuliformis\u003c/em\u003e even for kinds of conifers.\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eNF-Y transcription factors have been extensively recognized and classified in several plants. Although there have been some studies on NF-Y in conifer trees, most studies have focused on single genes and most family studies have focused on angiosperms. So, this research trend of NF-Y should been extended to conifer trees. Our study, carried out in \u003cem\u003ePinus tabuliformis\u003c/em\u003e, a conifer widely distributed in China can pay close attention to current research priorities. 28 \u003cem\u003ePtNF-Ys\u003c/em\u003e were first identified in conifer trees and their evolutionary, structural were analyzed. Comparison of NF-Ys in Chinese pine and \u003cem\u003eArabidopsis\u003c/em\u003e can provide rudimentary understanding on the function between less studied \u003cem\u003ePtNF-Ys\u003c/em\u003e and its known homologs. Moreover, by analyzing transcriptome data of male development and experimental verification, two candidate genes (\u003cem\u003eNF-YC1\u003c/em\u003e and \u003cem\u003eNF-YC4\u003c/em\u003e) were found to be involved in the regulation of conifer flowering and gibberellin. Furthermore, analysis the \u003cem\u003ecis\u003c/em\u003e-elements combined with the hormone treatment transcriptome indicated the potential role of NF-Y in a class of conifer resistance. According these results, we found ome special scientific problems that may contribute to further functional investigation of NF-Y family in conifers.\u003c/p\u003e "},{"header":"Methods","content":" \u003cp\u003e \u003cb\u003eldentification of NF-Y family members in\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ePinus\u003c/span\u003e \u003cb\u003etabuliformis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe protein sequences of \u003cem\u003eNF-Y\u003c/em\u003e genes (10 \u003cem\u003eNF-YA\u003c/em\u003e genes, 13 \u003cem\u003eNF-YB\u003c/em\u003e genes, and 13 \u003cem\u003eNF-YC\u003c/em\u003e genes) in \u003cem\u003eA.thaliana\u003c/em\u003e were retrieved from the TAIR (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.arabidopsis.org/\u003c/span\u003e\u003c/span\u003e ) (Additional file 1). These sequences were used to search our \u003cem\u003ePinus tabuliformis\u003c/em\u003e transcriptome database (unpublished) with the blastx program in BLAST (blast-2.6.0+) and the E-value cut-off was set as 1e-10. In addition, hidden Markov model (HMM) for the NF-Y genes was constructed using HMMER package version 3.0. The results of the BLAST and HMMER searchers were merged, resulting in 69 candidate \u003cem\u003eNF-Y\u003c/em\u003e genes in \u003cem\u003ePinus tabuliformis\u003c/em\u003e. The incomplete and redundant sequences were omitted. Finally, 28 unigenes were identified (Additional file 2).\u003c/p\u003e \n\u003cp\u003e\u003cb\u003eMultiple Alignments And Phylogenetic Analysis\u003c/b\u003e\u003c/p\u003e\n \u003cp\u003eMultiple sequence alignments of identified \u003cem\u003eNF-Ys\u003c/em\u003e in \u003cem\u003ePinus tabuliformis\u003c/em\u003e were constructed using ClustalX. The Neighbor-Joining tree was constructed using MEGA7.0.21 software with 1000 bootstrap replications. The phylogenetic tree constructed by MEGA was uploaded to iTOL (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://itol.embl.de/\u003c/span\u003e\u003c/span\u003e) for further editing. Motifs were predicted using MEME software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://meme-suite.org/tools/meme\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003eCis\u003c/span\u003e \u003cb\u003e-elements of the\u003c/b\u003e \u003cspan type=\"BoldItalic\" class=\"BoldItalic\" name=\"Emphasis\"\u003ePtNF-Y\u003c/span\u003e \u003cb\u003epromoter\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe promoter sequences (length, 2\u0026nbsp;kb) of \u003cem\u003ePtNF-Ys\u003c/em\u003e were collected from the Genome Database of\u003c/p\u003e \u003cp\u003e \u003cem\u003ePinus tabuliformis\u003c/em\u003e (has yet to genome annotation and obtained the promoter sequences based on the CDS alignment by blast). The \u003cem\u003ecis\u003c/em\u003e-elements were analyzed in the PlantCARE program (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \n\u003cp\u003e\u003cb\u003eTranscriptome Plant Materials And Data Analysis\u003c/b\u003e\u003c/p\u003e\n \u003cp\u003eThe seeds of \u003cem\u003ePinus tabuliformis\u003c/em\u003e were obtained from a primary clonal seed orchard located in Pingquan City, Hebei Province, China (40\u0026deg;99\u0026rsquo; N, 118\u0026deg;45\u0026rsquo; E, 560\u0026nbsp;m above sea level). The seeds of \u003cem\u003ePinus tabuliformis\u003c/em\u003e were sown on sphagnum moss soaked with water and then germinated for 14 days in a growth chamber under conditions of 22\u0026nbsp;\u0026deg;C 14\u0026nbsp;h light/10\u0026nbsp;h dark photoperiod. Then transferred in plastic pots and irrigated weekly according to the hormone types and concentrations in Table \u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. After 50 days of treatment, needles were collected, liquid nitrogen quick-frozen, and stored at -80\u0026nbsp;\u0026deg;C. Furthermore, the male cones development samples for RNA-Seq analysis were collected from individual trees at the botanical gardens in Beijing, China (116\u0026deg;33.91160\u0026rsquo;E, 40\u0026deg;00.08610\u0026rsquo;N and 44\u0026nbsp;m above sea level) and datas were deposited in the NCBI Sequence Read Archive (SRA) under the accession number SRA 056887. The RNA-seq datas (Additional file 4) were shown as heat map by TBtools toolkit [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Hierarchical clustering displays the expression profiles and the color scale indicating Normalize expression values. Also all the samples used for RNA-Seq analysis is this study are provided in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cem\u003ePinus tabuliformis\u003c/em\u003e sampling for gene expression analysis\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003eDevelopmental stages\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnalysis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eTime point\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRepetitions\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVegetative buds(VB)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRNA-seq\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003e26 September 2012\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale cones(M)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRNA-seq\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eM1 (26 September 2012); M2\u0026ndash;M6 (16 March 2013-every 9 d)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"5\" nameend=\"c6\" namest=\"c2\"\u003e \u003cp\u003eHormone treatments\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAnalysis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eHormone concentration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRepetitions\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003eNeedles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRNA-seq\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eWater\u0026thinsp;+\u0026thinsp;ethyl alcohol (CK)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRNA-seq\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eAbscisic acid (ABA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u0026nbsp;\u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRNA-seq\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eAuxin (IAA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10\u0026nbsp;\u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRNA-seq\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eJasmonic acid (JA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30\u0026nbsp;\u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRNA-seq\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eSalicylic acid (SA)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e100\u0026nbsp;\u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \n\u003cp\u003e\u003cb\u003eYeast Two-hybrid Assay\u003c/b\u003e\u003c/p\u003e\n \u003cp\u003eThe coding regions of \u003cem\u003eNF-YC1\u003c/em\u003e, \u003cem\u003eNF-YC4\u003c/em\u003e and \u003cem\u003eDPL\u003c/em\u003e were amplified and cloned into pGBKT7 and pGADT7 (Clontech). Yeast two-hybrid assays were performed using the Yeastmaker Yeast Transformation System 2 (Clontech). Yeast AH109 cells were co-transformed with the specific bait and prey constructs. All yeast transformants were grown on SD/-Trp/-Leu or SD/-Trp/-Leu/-His/-Ade medium for selection or interaction test.\u003c/p\u003e \n\u003cp\u003e\u003cb\u003eBiFC Analysis\u003c/b\u003e\u003c/p\u003e\n \u003cp\u003eFor the bimolecular fluorescence complementation (BiFC) assay, \u003cem\u003eDPL\u003c/em\u003e gene was cloned into the\u003c/p\u003e \u003cp\u003epSPYCE vector and \u003cem\u003eNF-YC1\u003c/em\u003e/\u003cem\u003e4\u003c/em\u003e were cloned into the pSPYNE [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. All expression vectors were introduced into \u003cem\u003eA. tumefaciens\u003c/em\u003e LBA4404. Agrobacteria were incubated, harvested, and resuspended in agroinfiltration buffer (0.2\u0026nbsp;mM acetosyringone, 10\u0026nbsp;mM MgCl\u003csub\u003e2\u003c/sub\u003e, and 10\u0026nbsp;mM MES). Agroinfiltration buffer was mixed with an equal volume of the protein mixture and injected into tobacco leaves using a syringe. Seventy two hours after infiltration, images were taken using a Leica TCS SP8 confocal microscope.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe data are statistically described as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (\u0026plusmn;\u0026thinsp;SD) and visualization through GraphPad Prism 7.0. The correlation were analyzed by R 3.6.2.\u003c/p\u003e \u003c/div\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eABA: abscisic acid; IAA: auxin; JA: jasmonic acid; SA: salicylic acid; CO: CONSTANS; TFL2: TERMINAL FLOWER2; HMMs: Hidden Markov Models\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data analyzed during this study are included in this published article and its additional files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grant from the National Natural Science Foundation of China (31770713). The funding bodies had no role in the design of the study, collection, analysis, or interpretation of data or in the writing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYTG analyzed the data and wrote the manuscript, SHN collected data and samples in the field, WL modified the manuscript. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWare are thankful to a primary clonal seed orchard located in Pingquan City, Hebei Province, China (40\u0026deg;99\u0026rsquo; N, 118\u0026deg;45\u0026rsquo; E, 560 m above sea level) for providing the seed material.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e \u003cspan\u003eNystedt B, Street NR, Wetterbom A, Zuccolo A, Lin Y, Scofield DG, et al. 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Plant J. 2004;40(3):428\u0026ndash;38.\u003c/span\u003e \u003c/li\u003e\u003c/ol\u003e"},{"header":"Additional Files","content":"\u003cp\u003e\u003cstrong\u003eAdditional file\u003c/strong\u003e\u003cstrong\u003e 1: \u003c/strong\u003eFull length sequences of the Arabidopsis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file 2\u003c/strong\u003e\u003cstrong\u003e:\u003c/strong\u003e CDS sequences and translated amino acid sequences of 28 Chinese pine NF-Ys.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file\u003c/strong\u003e\u003cstrong\u003e 3:\u003c/strong\u003e Sequence alignment of PtNF-YB4, PtNF-YB5, PsHAP3A and PaHAP3A proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional file\u003c/strong\u003e\u003cstrong\u003e 4: \u003c/strong\u003eTranscriptome data (TPM) of \u003cem\u003ePtNF-Ys\u003c/em\u003e in different development stages of male cones and hormone treatments.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pinus tabuliformis, Conifers, NF-Y transcription factor, Bioinformatic analysis, Expression profiles","lastPublishedDoi":"10.21203/rs.3.rs-21552/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-21552/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eConifers and angiosperms have difference in reproductive development, especially for flowering. It is known that NUCLEAR FACTOR Y (NF-Y) transcription factor play an important role in flowering, drought stress and GA, ABA signaling, but, little known in auxin, salicylic acid, jasmonic acid, etc. Moreover, the \u003cem\u003eNF-Y\u003c/em\u003e genes family has been mainly analyzed in angiosperms, but it has not been comprehensively reported in conifers.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this study, we identified 9 \u003cem\u003eNF-YA\u003c/em\u003e, 9 \u003cem\u003eNF-YB\u003c/em\u003e, and 10 \u003cem\u003eNF-YC\u003c/em\u003e genes in \u003cem\u003ePinus tabuliformis\u003c/em\u003e using \u003cem\u003eArabidopsis\u003c/em\u003e NF-Y protein sequences as queries. Besides, by comparing conserved regions and phylogenetic relationships of the PtNF-Ys, we found that the NF-Ys were both conserved and altered during evolution. \u003cem\u003ePtTFL2\u003c/em\u003e, \u003cem\u003ePtCO\u003c/em\u003e, \u003cem\u003ePtNF-YC1\u003c/em\u003e and \u003cem\u003ePtNF-YC4\u003c/em\u003e were exploited by expression profile in male cone development and the correlation analysis. In addition, NF-YC1/4 can interact with DPL by yeast two-hybrid assays and BiFC. The multiple types of phytohormones-responsive \u003cem\u003ecis\u003c/em\u003e-elements (ABA, JA, IAA, SA) were present and many \u003cem\u003eNF-Y\u003c/em\u003e genes responded positively to SA and as opposed to IAA and JA.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eTwenty-eight \u003cem\u003ePtNF-Ys\u003c/em\u003e were identified and bioinformatic characterization of \u003cem\u003eNF-Y\u003c/em\u003e genes including conserved regions, phylogenetic relationships, gene-motifs, was carried out. Two candidate genes (\u003cem\u003eNF-YC1\u003c/em\u003e and \u003cem\u003eNF-YC4\u003c/em\u003e) were found to be involved in the regulation of conifer flowering and gibberellin signalling. The \u003cem\u003ecis\u003c/em\u003e-elements and hormone transcriptome analysis revealed that the potential role of \u003cem\u003eNF-Ys\u003c/em\u003e in conifers resistance. This study provides the basis for improved understanding of \u003cem\u003eNF\u003c/em\u003e-\u003cem\u003eY\u003c/em\u003e genes function in conifers.\u003c/p\u003e","manuscriptTitle":"Transcriptome-wide isolation and expression profiles of NF-Y gene family in male cone development and hormone treatment of Chinese pine (Pinus tabuliformis)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-04-20 21:13:36","doi":"10.21203/rs.3.rs-21552/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":"4d27d6a9-4bfb-423a-b582-8b840fb867e0","owner":[],"postedDate":"April 20th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":86083,"name":"Epigenetics \u0026 Genomics"}],"tags":[],"updatedAt":"2020-04-29T14:11:36+00:00","versionOfRecord":[],"versionCreatedAt":"2020-04-20 21:13:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-21552","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-21552","identity":"rs-21552","version":["v1"]},"buildId":"cTy_lsJlmDsVRNrSptgXS","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}