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Article

Genome-Wide Identification and Expression Pattern Analysis of Nuclear Factor Y B/C Genes in Pinus koraiensis, and Functional Identification of LEAFY COTYLEDON 1

by
Xiuyue Xu
1,†,
Xin He
1,†,
Qun Zhang
1 and
Ling Yang
1,2,*
1
State Key Laboratory of Tree Genetics and Breeding, Northeast Forestry University, Harbin 150040, China
2
College of Forestry, Beijing Forestry University, Beijing 100091, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2025, 14(3), 438; https://doi.org/10.3390/plants14030438
Submission received: 20 December 2024 / Revised: 27 January 2025 / Accepted: 30 January 2025 / Published: 2 February 2025
(This article belongs to the Special Issue Advances in Forest Tree Genetics and Breeding)
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Abstract

:
The nuclear factor Y (NF-Y) transcription factor is widely involved in various plant biological processes, such as embryogenesis, abscisic acid signaling, and abiotic stress responses. This study presents a comprehensive genome-wide identification and expression profile of transcription factors NF-YB and NF-YC in Pinus koraiensis. Eight NF-YB and seven NF-YC transcription factors were identified through bioinformatics analysis, including sequence alignment, phylogenetic tree construction, and conserved motif analysis. We evaluate the expression patterns of NF-YB/C genes in various tissues and somatic embryo maturation processes through the transcriptomics of ABA-treated tissues from multiple nutritional tissues, reproductive tissues, and somatic embryo maturation processes. The Leafy cotyledon1 (LEC1) gene belongs to the LEC1-type gene in the NF-YB family, numbered PkNF-YB7. In this study, we characterized the function of PkLEC1 during somatic embryonic development using genetic transformation techniques. The results indicate that PkNF-YB/C transcription factors are involved in the growth and development of nutritional tissues and reproductive organs, with specific high expression in PkNF-YB7 embryogenic callus, somatic embryos, zygotic embryos, and macropores. Most PkNF YB/C genes do not respond to ABA treatment during the maturation culture process. Compared with the absence of ABA, PkNF-YB8 was up-regulated in ABA treatment for one week (4.1 times) and two weeks (11.6 times). However, PkNF-YC5 was down-regulated in both one week (0.6 times) and two weeks (0.36 times) of culture, but the down-regulation trend was weakened in tissues treated with ABA (0.72–0.83 times). In addition, the promoter of PkNF YB/Cs was rich in elements that respond to various plant hormones, indicating their critical role in hormone pathways. The overexpression of PkLEC1 stimulated the generation of early somatic embryos from callus tissue with no potential for embryogenesis, enhancing the somatic embryogenesis ability of P. koraiensis callus tissue.

1. Introduction

Nuclear factor Y (NF-Y) is widespread in plants, animals, and other eukaryotes, and is also termed the CCAAT Binding Factor (CBF) or Heme Activator Protein (HAP) [1]. The individual subunits of NF-Y cannot regulate transcription independently; they must function in heterodimers or heterotrimers [2]. The NF-Y complex consists of the subunits NF-YA (CBF-B/HAP2), NF-YB (CBF-A/HAP3), and NF-YC (CBF-C/HAP5), all of which are necessary for binding to the CCAAT box [2,3]. The structural units of the NF-YA, NF-YB, and NF-YC are dissimilar. The NF-YA subunits are located at the nucleus and predominantly bind to the CCAAT box present in the target gene promoters [4]. NF-YB and NF-YC possess histone-fold motifs that allow them to form a tight dimer. This dimer can then interact with NF-YA. NF-YA [5], NF-YB, and NF-Y-C are unable to form homodimer (indicated through yeast two hybrid assays) but have been known to form many heteromeric complexes (NF-YA-C or NF-YC-B) and differentially affect target genes and downstream mechanisms [6]. The NF-Y complex can thus act either as a transcriptional activator or as a repressor, and its activity can be modulated by interaction with other transcription factors (TFs) or regulatory proteins [7,8].
In plants, NF-Y is involved in various biological processes, such as embryogenesis and endosperm formation, seed germination, root morphogenesis, flowering, chloroplast biogenesis and photosynthesis, starch biosynthesis, and fruit ripening [9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28]. NF-Ys have also been involved in abiotic and biotic stress responses like drought stress, temperature salt stress, and microbial stress [29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46]. bZIP67 interacts with NF-YB9, and NF-YC2 directly binds to the ABA response elements (ABRE) in the SUS2 and CRC promoter region to activate gene expression for seed development in A. thaliana [47]. LEC1 (LEAFY COTYLEDON 1) was the first identified NF-Y subunit actively participating in embryogenesis and post-embryo development in A. thaliana [48].
The non-LEC1 members of the NF-YB family of plant regulators have also been studied. Arabidopsis plants overexpressing AtNF-YB1 showed increased tolerance to drought stress compared with wild type [29]. When the orthologous maize gene ZmNF-YB2 was overexpressed, drought tolerance was also observed, leading to a significant increase in grain yield under water-limiting conditions [49]. In rice, an RNAi construct silencing OsHAP3A, OsHAP3B, and OsHAP3C resulted in the reduced accumulation of nuclear-encoded photosynthesis transcript [24]. In wheat (Triticum aestivum), the overexpression of TaNF-YB3 led to a significant increase in leaf chlorophyll content and photosynthesis. Multiple NF-YC subunits (C1, C3, C4, and C9) and NF-YB subunits (B2 and B3) can interact with CONSTANS (CO) to regulate flowering time [50]. REPRESSOR OF ga1-3 (RGA) and RGA-LIKE2 (RGL2) interact with NF-YA2, NF-YB2/B3, or NF-YC3/C4/C9 to regulate gibberellin (GA) and photoperiod-mediated flowering time, or abscisic acid (ABA) and GA signaling pathways during seed germination, respectively [15,21].
P. koraiensis is an important nut economic forest and timber tree species in China, as well as an important greening tree species for landscaping and urban green spaces, with significant economic and ecological value [51]. Due to issues such as the long production cycle and unstable seed yield of P. koraiensis, the widespread application of its improved varieties in production is limited, making large-scale propagation challenging [52]. Somatic embryogenesis of plants is currently one of the widely used biotechnologies [53,54,55]. Somatic embryogenesis has the advantages of genetic stability, fast propagation rate, and high survival rate, and is one of the effective means of plant regeneration using biotechnology methods [56,57,58]. At the same time, the pathway of somatic embryogenesis is also a model system for studying the physiological processes, metabolic pathways, gene functions, and molecular regulatory mechanisms of embryonic development [59].
LEC1 belongs to the NUCLEAR FACTOR-Y B family, a key participant in the regulation of somatic and zygotic embryonic development in plants. LEC1 is crucial for the development of zygotic and somatic embryos [9]. LEC1 can activate the LEC1-ABI3-FUS3-LEC2 network in the embryo, thereby regulating embryonic maturation [47]. In Scots pine, the LEC1 gene is relatively highly expressed in the proliferation and late-maturation stages during somatic embryogenesis, while the expression pattern in zygotic embryos is different, with higher expression levels in the proliferation stage followed by a gradual decrease in expression [60].
In this study, a total of eight PkNF-YB and seven PkNF-YC transcription factors were identified in P. koraiensis through the whole genome. We analyzed their protein sequences, chromosome locations, evolutionary relationships, basic physiochemical properties, and gene structures. The expression patterns of these genes in different plant tissues upon nutritional growth, reproductive growth stages, and ABA treatment were also investigated. This study provides a reference for future research on PkNF-YB/C transcription factors and plant stress response. By integrating the somatic embryogenesis and genetic transformation systems of P. koraiensis, we investigated the function of the PkLEC1. This research complements the molecular level studies on somatic embryogenesis in P. koraiensis and offers theoretical and technical support for molecular design breeding aimed at enhancing the yield and quality of somatic embryos in this species.

2. Results

2.1. Identification and Sequence Analysis of PkNF-YB/C Genes in P. koraiensis

Based on the CBFD_NFYB_HMF (PF00808) conserved domain specific to the NF-YB and NF-YC family members in plant, eight members of the NF-YB and seven members of the NF-YC family members were identified from P. koraiensis, and they were named PkNF-YB 1–8 and PkNF-YC 1–7, respectively, according to their locations on chromosomes I to XI. The details of these 15 PkNF-YB/C genes, including locus name, chromosome location, number of amino acids, molecular weight, isoelectric point, aliphatic index, and GRAVY score are listed in Table 1. The putative PkNF-YB protein sequences contained 78–225 amino acids and had isoelectric points ranging between 4.96 and 7.76, while putative PkNF-YC protein sequences contained 202–311 amino acids and had isoelectric points ranging between 5.96 and 8.90. All were determined to be hydrophilic proteins.

2.2. Phylogenetic Analysis of Multiple Species, Protein Sequence Alignment, and Conservative Motif Analysis of PkNF-YB/Cs

The NF-YB/C amino acid sequences of multiple species were aligned and a phylogenetic tree was constructed (Figure 1A,B). Phylogenetic trees were constructed, and intron and exon structures were compared to further understand the relationship between the members of the PkNF YB/C gene families. The NF-YB family members were divided into five subfamilies, with the pink subfamily branch being the LEC-type NF-YBs, and the other branches (green, blue, yellow, and pinky purple) were non-LEC-type NF-YBs. The NF-YC family members were divided into five branches, with seven members of P. koraiensis concentrated in the green and purple branches. Multiple alignment of protein sequences shows that NF-YB members contain a conserved NF-YA interacting region, a NF-YC interacting region, DNA-binding region, and HFM (H2B) conserved domains (Figure 1C). NF-YC members contain conserved NF-YA interacting regions, NF-YB interacting regions, DNA-binding regions, and HFM (H2A) conserved domains (Figure 1D).
The conserved motifs of the PkNF-YB/C family were analyzed using the online tool MEME and a schematic map was constructed to represent the structure of the proteins (Figure 2). The PkNF-YB family members have predicted four conserved motifs, each containing these four motifs with the same arrangement. However, NF-YB1, 2, 3, 4, 7, and 8 members do not contain introns, while NF-YB5 and 6 have ultra-long introns (Figure 2A). The PkNF-YC family members have predicted six conserved motifs, each containing motif 1, 2, 3, and 4. And PkNF-YC2, 4, 5, and 6 members contain motif 3; PkNF-YC4 and 5 members contain motif 5; PkNF-YC4, 5, and 6 members contain motif 6. Similarly, NF-YC1, 2, 4, 5, 6, and 7 members do not contain introns, while NF-YB3 has ultra-long introns (Figure 2B). The sequences of motifs are shown in Table S3.

2.3. Chromosomal Location, Collinearity Analysis, and Ka/Ks Calculation of PkNF-YB/C Genes

To determine the distribution of the PkNF-YB/C genes, we mapped their positions on the chromosomes. The eight PkNF-YB genes were distributed on seven chromosomes: Chr 01, Chr 02, Chr 03, Chr 06, Chr 08, Chr 10, and Chr 11. The seven PkNF-YC genes were distributed on five chromosomes: Chr 02, Chr 06, Chr 08, Chr 09, and Chr 10 (Figure 3).
We first conducted collinearity analysis within P. koraiensis and found that there was no collinearity between the NF-YB and NF-YC family members (Figure 3). Then, we downloaded the genome data of Arabidopsis thaliana and Pinus tabulaeformis and conducted collinearity analysis between the genomes of P. koraiensis, A. thaliana, and P. tabulaeformis. The NF-YB/C family members of P. koraiensis do not have collinear relationships in Arabidopsis, but PkNF-YB2,4,5,6 and PkNF-YC1,3,7 have collinear members in P. koraiensis (Figure 4). The gene pairs with collinearity between different species are listed in Table S4.
To better understand the evolutionary constraints on the PkNF-YB/C gene families, the Ka/Ks ratios of the PkNF-YB/C gene pairs were calculated (Table S5). The selection stress analysis showed that the duplicated gene pairs were mainly under purifying selection (Ka/Ks < 1.0).

2.4. Analysis of Cis-Elements of PkNF-YB/C Promoters

PkNF-YB/C promoters contain a large number of hormone response elements, such as abscisic acid responsive, auxin responsive, gibberellin responsive, MeJA responsive, and salicylic acid responsive. There are also many abiotic stress response elements, such as low-temperature responsive, drought inducibility, defense and stress responsive, and anaerobic induction. In addition, the largest number was the light-responsive element. In addition, there are also some specific elements related to tissue development and metabolism, such as the meristem expression element, endosperm expression element, seed-specific regulation element, wound-responsive element, cycle regulation element, and zein metabolism regulation element (Figure 5). Thus, we speculate that PkNF-YB/C genes may also play an important role in plant growth and development, hormone response, and stress resistance, which adds a new direction to the future research of PkNF-YB/C. The information of the cis-acting elements are listed in Table S6.

2.5. GO Enrichment and KEGG Enrichment

In order to further evaluate the biological function of the PkNF-YB/C genes, we conducted GO enrichment and KEGG enrichment analysis on them (Figure 6). In GO enrichment, eight members of PkNF-YB were annotated with two molecular functional entries, one cellular component entry, and forty-two biological process entries (Figure 6A). The seven members of NF-YC were annotated with two molecular functional entries, one cellular component entry, and sixty-six biological process entries (Figure 6B). In KEGG enrichment, eight members of NF-YB were annotated into transcription factors, protein families: genetic information processes, and Brite Hierarchies metabolic pathways (Figure 6C). The seven members of NF-YC were annotated into the transcription factor and protein families: genetic information processes (Figure 6D). This indicates that NF-YB and NF-YC members were widely involved in the metabolic processes of plant growth and development.

2.6. Tissue Expression Specificity and Abiotic Stress Expression Pattern

As shown in Figure 7, the expression patterns of PkNF-YB and PkNF-YC members in embryonic callus, non-embryonic callus, somatic embryo, zygotic embryo, seed, external bark, phloem, cambium and xylem, roots, leaves, and branches of different parts were displayed. PkNF-YB1, 3, 4, 5, and 6 were expressed in all tissues, while PkNF-YB2 was up-regulated in the root and almost no expression in other parts. PkNF-YB7 and 8 were mainly up-regulated during seed development and somatic embryonic development. PkNF-YC1, 2, 3, and 7 were expressed in all tissues, while PkNF-YC6 was down-regulated in all tissues. PkNF-YC4 and 5 were mainly up-regulated during seed and somatic embryonic development, as well as in roots and leaves.
As shown in Figure 8, the expression patterns of PkNF-YB and PkNF-YC members in microsporophyll, megasporophyll, and ovules at different developmental stages are displayed. PkNF-YB1, 3, 4, 5, and 6 were up-regulated in microsporophyll and megasporophylls, while PkNF-YB7 and 8 were mainly up-regulated in the middle and late stages of microsporophyll development and ovule development. PkNF-YB2 and 6 were down-regulated at all stages. PkNF-YC1 and 7 were up-regulated in all tissues, PkNF-YC3 and 4 were mainly up-regulated during the development of microspores and ovules, while PkNF-YC2 was mainly up-regulated during the development of microsporophyll and megasporophylls. PkNF-YC5 was mainly up-regulated in the early stage of megasporophyll development and the middle to late stage of ovule development, while PkNF-YC6 was down-regulated only in the early stage of ovule development.
As shown in Figure 9, PkNF-YB 2 showed almost no expression in both the control and ABA treatments. Compared with the control group, the expression levels of PkNF-YB 7, PkNF-YC 3, and 4 increased and maintained high levels in the treatment with added ABA, while in the treatment without added ABA, the expression levels decreased in the second week of culture. However, the expression levels of PkNF-YC 1, 5, and PkNF-YB 4 showed a decreasing trend in both the treatment with and without ABA, but the decreasing trend was faster in the treatment without ABA, reaching almost undetectable levels in the second week. On the contrary, PkNF YB 1 and 5 showed a rapid downward trend in the treatment with added ABA and dropped to almost undetectable levels in the second week of treatment. The expression levels of PkNF-YB 3, 6, PkNF-YC 2, and 7 were higher in the control and non-ABA treated groups.

2.7. Subcellular Localization and Transcriptional Self-Activation Analysis of Transcription Factor

Based on the expression pattern of the PkNF-YB/C genes, all genes were selected for subsequent experiments, except for PkNF-YB 2 and PkNF-YC 6, which were not expressed during the development of somatic and zygotic embryos. Instantaneous transformation was performed by injecting tobacco leaves into the lower epidermis to detect the subcellular localization of the NF-YB/C proteins.
As shown in Figure 10, similar to empty vector, PkNF-YB 1, 3, 5, and 6 were located in the nucleus and cell membrane, while PkNF-YB 4, 7, and 8 are co-located in the nucleus with DAPI staining. Among the PkNF-YC proteins, except for PkNF-YC2, which was located in the nucleus and nucleus, all other proteins were located in the nucleus. As shown in Figure 11, analyze the transcriptional activation activity of PkNF-YB/C proteins by using the yeast hybridization system (Y2H). The Y2H yeast cells corresponding to the PkNF-YB/C proteins fused with the GAL4 DNA-binding domain (GALDA4DB) can grow normally on SD/-Trp medium but cannot grow on SD/-Trp/-His/-Ade/x-α-gal medium. This is consistent with the growth status of the negative control, indicating that the PkNF-YB/C proteins do not possess transcriptional autoactivation activity.

2.8. Predicting Protein Interaction Networks for PkNF-YB and PkNF-YC Genes

We further identified putative interacting proteins for PkNF-YB/C (Figure 12). Among them, PkNF-YB 2, 3, 5, 6, and 7 predicted 54, 54, 43, 1, and 39 interacting proteins, respectively. PkNF-YC 2, 3, 4, 5, and 7 predicted 7, 25 (including PkNF-YB 2 and 3), 2, 5, and 17 interacting proteins, respectively. The description of the interacting proteins is shown in Table S7.

2.9. Screening and Mature Phenotype of PkNF-YB 7 Transgenic Cell Lines

PkNF-YB 7 was named PkLEC1 because it is homologous to LEC1 in Arabidopsis. To characterize its function during somatic embryogenesis, a plant overexpression vector was constructed and genetically transformed into P. koraiensis callus tissue to obtain multiple transgenic callus cell lines.
QRT-PCR was used to quantitatively analyze the expression levels of PkLEC1 in these transgenic lines. Three transgenic strains (T31, T33, T34) were selected for subsequent experiments because their PkLEC1 expression levels were higher than other strains (Figure 13A).
According to the method described by Peng et al. [61], the somatic embryogenesis was tested. The wild-type callus cells were previously induced from immature seeds by the research group and had completely lost their ability of somatic embryogenesis (L-SE) before genetic transformation began.
As shown in Figure 13B, there were no observable mid- or late-stage somatic embryos in the wild type cultured for 2 months. However, transgenic cell lines that have been cultured for 2 months have observable mid- or late-stage somatic embryos, some of which have even developed into cotyledons (Figure S1). The results indicate that overexpression of PkLEC1 enhances the somatic embryogenesis ability of L-SE to a certain extent.

3. Discussion

Previous studies have shown that NF-YB and NF-YC family transcription factors are involved in various biological processes in plants [13,23,28,62,63,64,65]. The NF-YB/C family has been characterized and identified in multiple species, such as Arabidopsis [66], rice [67], wheat [68], soybeans [69], and Populus [70]. However, there is limited research in gymnosperms, especially in P. koraiensis, which has not yet been characterized.
This article identifies the NF-YB and NF-YC gene families for the first time in P. koraiensis, with high economic and ecological value in the Northeast region in China. Gene family identification and phylogenetic analysis indicate that the NF-YB gene family of P. koraiensis includes eight gene members, and the NF-YC gene family includes seven members, which have close evolutionary relationships with the gene families of P. taeda, P. koraiensis s and P. abies (Figure 1). The motif analysis shows that NF-YBs have highly conserved HFM (H2B) motifs, while NF-YCs have highly conserved HFM (H2A) motifs (Figure 1). Collinearity analysis showed that four and four collinearity relationships were identified within the NF-YB and NF-YC families of P. koraiensis and P. taeda. This indicates that these two families are relatively conservative in their evolutionary relationship, and research on the NF-YB/C family in other gymnosperms is of great reference value in P. koraiensis. And Ka/Ks analysis showed that the repetitive gene pairs were mainly in purification selection (Ka/Ks < 1.0).
Research has shown that many plant transcription factor promoters have highly conserved cis-elements that play a crucial role in transcriptional regulatory signaling pathways when plants are subjected to both biological and abiotic stresses [71,72]. Therefore, the cis-acting elements in the promoter region of the PkNF-YB/C genes were analyzed, and it was found that each gene contains at least three elements related to hormones or biological stress. Thus, we speculate that PkNF-YB/C genes may also play an important role in plant hormone response and stress resistance. This provides important ideas for the subsequent study of gene function. This is also consistent with previous studies in other species [73,74].
Tissue expression specificity analysis showed that PkNF-YB and PkNF-YC members were differentially expressed in different tissue parts, indicating their involvement in different developmental processes. The differential functional roles of NF-YB/C family members in different stages of plant growth and development are explained, with less redundancy in co-expression, which is different from angiosperms. Subcellular localization analysis shows that both PkNF-YB and PkNF-YC proteins have nuclear localization, indicating that they are transcription factors. Transcription activation experiments have shown that both PkNF-YB and PkNF-YC do not possess transcriptional activation activity. Due to the fact that NF-YBs generally form heterodimers with NF-YC proteins, and then form oligomers with NF-YA proteins or other transcription factors to regulate downstream target genes. In contrast, most previously reported NF-YB and NF-YC transcription factors are exclusively localized in the nucleus. This may also be attributed to the scarcity of research on NF-YB and NF-YC.
Then, we further predicted the protein interaction network of PkNF-YB/C members. A large number of interacting genes were found in PkNF-YB 2, 3, 5, 7, PkNF-YC 3, and 7. The interaction genes between PkNF-YB 2 and PkNF-YB3 overlap, and both interact with PkNF-YC 3. In addition, PkNF-YB 5 and PkNF-YB 7 also share many common interacting genes. This further confirms that NF-YB/C family proteins function in the form of oligomers. Among them, PkNF YB2 and PkNF YB3, as well as PkNF YB5 and PkNF YB7, share multiple potential interacting proteins, indicating that they are functionally similar. And PkNF YC 2, 3, 4, 5, and 7 may be the more core genes in the NF-YC family, especially PkNF YC 3, because it has a close relationship with the NF-YB family and has multiple predicted potential interacting proteins. These genes that interact with multiple PkNF-YB/C members deserve our further attention and research.
The overexpression of PkLEC1 can enhance the somatic embryogenesis ability of L-SE, extending callus that can only develop to the early stage of somatic embryogenesis to the middle or even late stage of somatic embryogenesis, and improving the quality of somatic embryogenesis. So, we speculate that the overexpression of PkLEC1 can improve the quality of somatic embryogenesis in callus with poor or good somatic embryogenesis ability, consistent with studies on other species.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

The experimental material was the embryogenic callus of P. koraiensis preserved in our laboratory, which consisted of Modified Litvay medium (MLV) (Coolaber, Beijing, China) [61] supplemented with 0.5 mg·L−1 of 2,4-D (Phytotech, Kansas, America), 0.1 mg·L−1 of 6-BA (Phytotech, Kansas, America), 30 g·L−1 of sucrose, 0.5 g·L−1 of L-glutamine (Sinopharm Chemical, Shanghai, China), and 0.5 g·L−1 of CH (Sinopharm Chemical, Shanghai, China). The pH of the medium was then adjusted to 5.8 before sterilization. Embryonic callus (EC) was subcultured at intervals of 2 weeks to maintain and proliferate, with the culture placed in darkness at 24 ± 1°C. Embryogenic callus tissue cultured for 7 days was transferred to mature medium for cultivation. The mature medium was MLV with 80 μmol·L−1 of ABA, 1.2% gellan gum, and 0.2 mol·L−1 sucrose, pH 5.8. They were cultivated in the dark for 8 weeks to count the number of somatic embryos [61]. Nicotiana tabacum seedlings were propagated and planted in a mixture of turfy peat and sand (1:1 v/v) and grown under a 16 h/8 h day/night photoperiod at 25 ± 1°C.

4.2. Identification of PkNF-YB/C Family Genes in P. koraiensis and Physicochemical Property Analysis

In order to identify NF-YB and NF-YC proteins in P. koraiensis, according to the P. koraiensis protein database (not yet published) and Hidden Markov Model (HMM), File CBFD_NFYB_HMF (PF00808) was downloaded from the Pfam database (https://pfam.xfam.org/, accessed on 16 June 2023). Using the hmmsearch command in HMMER (v3.1) software, the P. koraiensis protein database was searched with PF00808.hmm file, and the identified sequences were integrated [75]. After screening and identification, 8 NF-YB amino acid sequences and 7 NF-YC amino acid sequences of P. koraiensis were obtained. The characteristics of these amino acid sequences, including predicted molecular weight, isoelectric point, number of amino acids, and grand average of hydropathicity (GRAVY) score were analyzed using the online ExPASy program (https://www.expasy.org/, accessed on 17 September 2024).

4.3. Multiple Sequence Alignment and Phylogenetic Analysis

The NF-YB and NF-YC amino acid sequences were aligned using MUSCLE and checked for the presence of the conserved HFM (H2B) and HFM (H2A) sites, respectively. The maximum likelihood method was used to construct phylogenetic trees (1000 bootstrap replications) using MEGA (accessed on 17 January 2025) [76].

4.4. Exon/Intron Structure and Conserved Motifs Analysis

The conserved domains of PkNF-YBs and PkNF-YCs were predicted and analyzed by the online tool MEME (https://meme-suite.org/meme/tools/meme, accessed on 19 November 2023) [77]. The Motif Number was set to 4, and the remaining parameters were set to Default. The gene structure information was extracted and visualized by TBTools v2.003 [78].

4.5. Chromosomal Location, Collinearity Analysis, and Ka/Ks Calculation

Extract the location information of NF-YB and NF-YC family members from the P. koraiensis gff file. TBtools was used to construct the circular chromosome distribution map and interspecies collinearity analysis of genes. We downloaded chromosome files and gff3 files of Pinus tabulaeformis and Arabidopsis thaliana from NCBI, and TBtools was used for multispecies collinearity alignment and visualization [78]. TBtools v2.102 was used to calculate the Ka and Ks values (accessed on 18 March 2024) (Table S1).

4.6. Cis-Element Analysis of the PkNF-YB/C Promoters

The upstream 2000 bp of transcriptional initiation of family members was defined as the promoter region of PkNF-YB/C genes for analysis. The elements in the promoter region were predicted using the PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 12 November 2023) online website [79] and visualized with TBtools.

4.7. GO and KEGG Enrichment Analysis of PkNF-YB/C Genes

TBtools was used to visualize the GO enrichment and KEGG enrichment analysis of PkNF-YB/C genes.

4.8. Expression Patterns of PkNF-YB/C Genes

Based on the transcriptome data of roots, stems, leaves, seeds, embryonic callus, non-embryonic callus, somatic embryos, zygotic embryos from different tissue parts of P. koraiensis in our research, as well as transcriptome data of megasporophylls, microspores, flower buds, and ovules at different developmental stages, the gene expression levels of PkNF-YB/C genes were analyzed and gene expression calorimeter maps were drawn. The expression levels of PkNF-YB/C genes were analyzed based on transcriptome data from 80 μmol·L−1 ABA-treated callus of P. koraiensis in our research.

4.9. Cloning PkNF-YB/C Genes from P. koraiensis

The full PkNF-YB/C genes were amplified with gene-specific primers (Table S2) by using the published method, and then transformed into Escherichia coli cells (DH5α, ANGYUBIO) for validation by Sanger sequencing.

4.10. Transcriptional Repression in Yeast

The transcriptional activation of PkNF-YB/C genes and the GAL4BD/UAS/LacZ transient assays were performed in yeast cells. Yeast GaL4BD expression vectors were obtained from Clontech. The complete CDS sequences of PkNF-YB/C genes were amplified using specifically designed primers (Table S2). The sequences encoding PkNF-YB/C genes were amplified by PCR and cloned into BamH I sites of the pGBKT7 vector (Clontech). The amplified fragments were fused in-frame to the pGBKT7 vector to generate the pGBKT7-PkNF-YB/Cs construct. The pGBKT7-PkNF-YB/Cs and the pGBKT7 blank vector (as negative control) were transformed into Y2H yeast cells independently. The transformed Y2H yeast cells were plated onto SD/-Trp (growth control), SD/-Trp/-His/-Ade, and X-α-Gal media and incubated at 30 °C for three to five days to identify the transcriptional activation.

4.11. Subcellular Localization of PkNF-YB/C Genes

The full-length coding regions of PkNF-YB/Cs without termination codon were amplified using specific primers (Table S2) and then fused to the N-terminal of GFP driven by a CaMV 35S promoter in the pFGC-eGFP vector. The two fusion constructs (pFGC-eGFP and pFGC- PkNF-YB/Cs-eGFP) were injected into Nicotiana tabacum epidermal cells. The GFP fluorescent images were photographed with confocal microscopy (ZEISS LSM 800, Shanghai, China) at 48 h after injected.

4.12. Genetic Transformation of P. koraiensis Callus for Generating PkNF-YB 7 Transgenic Lines

The PkNF-YB7 was amplified with specific primers, and then inserted into the pCambia1301 vector at the position immediately downstream of the CaMV 35S promoter, and transferred into Agrobacterium tumefaciens GV3101 using the freeze–thaw method. We performed genetic transformation based on published patents [80]. Genomic DNA of all hygromycin-resistant shoots was amplified by regular PCR using the p1301 sequencing primers listed in Table S1 to verify whether PkNF-YB 7 was integrated into the P. koraiensis genome. We performed real-time RT-qPCR on various transgenic cell lines, using the Pk18S gene from red pine as the internal reference gene. The forward primer sequence for detecting the Pk18S was 5′-GGGTGGTTTATGTTTG-3′, and the reverse primer sequence was 5′-GAAAGGGTTGAGGAAG-3. The forward primer sequence for detecting the PkLEC1 was 5′-TGTCCGAGAGTGGCAGCC-3′, and the reverse primer sequence was 5′-GCATGGGTGGGCAGAACT-3′.

4.13. Observation of Phenotypic Characteristics of Transgenic Strains

Phenotypic differences between wild-type and transgenic callus lines during the proliferation and somatic embryo maturation stages were observed.

5. Conclusions

This article identifies the NF-YB and NF-YC gene families for the first time in P. koraiensis with high economic and ecological value in the Northeast region in China. Gene family identification and phylogenetic analysis indicate that the NF-YB gene family of P. koraiensis includes eight gene members, and the NF-YC gene family includes seven members, which have close evolutionary relationships with the gene families of P. taeda, P. koraiensis s and P. abies (Figure 1). The motif analysis shows that NF-YBs have highly conserved HFM (H2B) motifs, while NF-YCs have highly conserved HFM (H2A) motifs (Figure 1). Collinearity analysis showed that four and four collinearity relationships were identified within the NF-YB and NF-YC families of P. koraiensis and P. taeda. And Ka/Ks analysis showed that the repetitive gene pairs were mainly in purification selection (Ka/Ks < 1.0).
The cis elements in the promoter region of the PkNF-YB/C genes were analyzed, and it was found that each gene contains at least three elements related to hormones or biological stress. A tissue expression specificity analysis showed that PkNF-YB and PkNF-YC members were differentially expressed in different tissue parts, indicating their involvement in different developmental processes. Subcellular localization analysis shows that both PkNF-YB and PkNF-YC proteins have nuclear localization, indicating that they are transcription factors. Transcription activation experiments have shown that both PkNF-YB and PkNF-YC do not possess transcriptional activation activity. A large number of interacting genes were found in PkNF-YB 2, 3, 5, 7, PkNF-YC 3, and 7. The interacting genes between PkNF-YB 2 and PkNF-YB3 overlap, and both interact with PkNF-YC 3. In addition, PkNF-YB 5 and PkNF-YB 7 also share many common interacting genes.
The overexpression of PkLEC1 can enhance the somatic embryogenesis ability of L-SE, extending callus that can only develop to the early stage of somatic embryogenesis to the middle or even late stage of somatic embryogenesis, and improving the quality of somatic embryogenesis.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14030438/s1, Table S1: The Ka/Ks ratios of duplication for PkNF-YB/Cs; Table S2: The prime list of PkNF-YB/Cs; Table S3: The prime list of PkNF-YB/Cs for analysis of transcriptional activation activity and subcellular localization; Table S4: Motif sequences of PkNF-YBs predicted in P. koraiensis using MEME tools; Table S5: Motif sequences of PkNF-YCs predicted in P. koraiensis using MEME tools; Table S6: The Synlinea of PkNF-YB/Cs between Pinus tabulaeformis and Pinus koraiensis; Table S7: Analysis of cis-acting elements in the promoters of PkNF-YB/Cs; Table S8: The annotation of the interacting proteins; Table S9: The node ID of the interacting proteins; Figure S1: Early cotyledon-type embryo from transgenic somatic embryo; Figure S2: GFP fluorescence observation of wild-type and transgenic embryonic callus tissues.

Author Contributions

X.X. and L.Y.: conceptualization. X.X., Q.Z. and X.H.: experiment. X.X. and X.H.: data processing and graphing. X.X. and X.H.: writing the original draft. X.X. and L.Y.: review and editing. All authors contributed to the article and approved the submitted version. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Key R&D Projects of China, grant number: 2023YFD2200103.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The genome and transcriptome provided in this study are not yet published; therefore, login numbers cannot be provided. We apologize for any inconvenience caused. The datasets supporting the conclusions of this article are included within the article and its Supplementary Files.

Acknowledgments

We thank the editors and reviewers for their careful reading and valuable comments.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Zhao, H.; Wu, D.; Kong, F.; Lin, K.; Zhang, H.; Li, G. The Arabidopsis thaliana Nuclear Factor Y Transcription Factors. Front. Plant Sci. 2017, 7, 2045. [Google Scholar] [CrossRef]
  2. Nardini, M.; Gnesutta, N.; Donati, G.; Gatta, R.; Forni, C.; Fossati, A.; Vonrhein, C.; Moras, D.; Romier, C.; Bolognesi, M.; et al. Sequence-Specific Transcription Factor NF-Y Displays Histone-like DNA Binding and H2B-like Ubiquitination. Cell 2013, 152, 132–143. [Google Scholar] [CrossRef]
  3. Mantovani, R. The molecular biology of the CCAAT-binding factor NF-Y. Gene 1999, 239, 15–27. [Google Scholar] [CrossRef]
  4. Petroni, K.; Kumimoto, R.W.; Gnesutta, N.; Calvenzani, V.; Fornari, M.; Tonelli, C.; Holt, B.F., III; Mantovani, R. The Promiscuous Life of Plant NUCLEAR FACTOR Y Transcription Factors. Plant Cell 2012, 24, 4777–4792. [Google Scholar] [CrossRef] [PubMed]
  5. Xing, Y.; Fikes, J.D.; Guarente, L. Mutations in yeast HAP2/HAP3 define a hybrid CCAAT box binding domain. EMBO J. 1993, 12, 4647–4655. [Google Scholar] [CrossRef]
  6. Coustry, F.; Maity, S.N.; Sinha, S.; de Crombrugghe, B. The Transcriptional Activity of the CCAAT-binding Factor CBF Is Mediated by Two Distinct Activation Domains, One in the CBF-B Subunit and the Other in the CBF-C Subunit. J. Biol. Chem. 1996, 271, 14485–14491. [Google Scholar] [CrossRef]
  7. Hackenberg, D.; Wu, Y.; Voigt, A.; Adams, R.; Schramm, P.; Grimm, B. Studies on Differential Nuclear Translocation Mechanism and Assembly of the Three Subunits of the Arabidopsis thaliana Transcription Factor NF-Y. Mol. Plant 2012, 5, 876–888. [Google Scholar] [CrossRef] [PubMed]
  8. Gusmaroli, G.; Tonelli, C.; Mantovani, R. Regulation of the CCAAT-Binding NF-Y subunits in Arabidopsis thaliana. Gene 2001, 264, 173–185. [Google Scholar] [CrossRef]
  9. Kwong, R.W.; Bui, A.Q.; Lee, H.; Kwong, L.W.; Fischer, R.L.; Goldberg, R.B.; Harada, J.J. LEAFY COTYLEDON1-LIKE Defines a Class of Regulators Essential for Embryo Development. Plant Cell 2003, 15, 5–18. [Google Scholar] [CrossRef]
  10. Sun, X.; Ling, S.; Lu, Z.; Ouyang, Y.D.; Liu, S.; Yao, J. OsNF-YB1, a rice endosperm-specific gene, is essential for cell proliferation in endosperm development. Gene 2014, 551, 214–221. [Google Scholar] [CrossRef]
  11. Xu, J.J.; Zhang, X.F.; Xue, H.W. Rice aleurone layer specific OsNF-YB1 regulates grain filling and endosperm development by interacting with an ERF transcription factor. J. Exp. Bot. 2016, 67, 6399–6411. [Google Scholar] [CrossRef]
  12. Xiong, Y.; Ren, Y.; Li, W.; Wu, F.; Yang, W.; Huang, X.; Yao, J. NF-YC12 is a key multi-functional regulator of accumulation of seed storage substances in rice. J. Exp. Bot. 2019, 70, 3765–3780. [Google Scholar] [CrossRef] [PubMed]
  13. Niu, B.; Deng, H.; Li, T.; Sharma, S.; Yun, Q.; Li, Q.; E, Z.; Chen, C. OsbZIP76 interacts with OsNF-YBs and regulates endosperm cellularization in rice (Oryza sativa). J. Integr. Plant Biol. 2020, 62, 1983–1996. [Google Scholar] [CrossRef]
  14. Siriwardana, C.L.; Kumimoto, R.W.; Jones, D.S.; Holt, B.F. Gene Family Analysis of the Arabidopsis NF-YA Transcription Factors Reveals Opposing Abscisic Acid Responses During Seed Germination. Plant Mol. Biol. Report. 2014, 32, 971–986. [Google Scholar] [CrossRef] [PubMed]
  15. Liu, X.; Hu, P.; Huang, M.; Tang, Y.; Li, Y.; Li, L.; Hou, X. The NF-YC–RGL2 module integrates GA and ABA signalling to regulate seed germination in Arabidopsis. Nat. Commun. 2016, 7, 12768. [Google Scholar] [CrossRef] [PubMed]
  16. Sun, X.; Ren, Y.; Zhang, X.; Lian, H.; Zhou, S.; Liu, S. Overexpression of a garlic nuclear factor Y (NF-Y) B gene, AsNF-YB3, affects seed germination and plant growth in transgenic tobacco. Plant Cell Tissue Organ. Cult. 2016, 127, 513–523. [Google Scholar] [CrossRef]
  17. Ballif, J.; Endo, S.; Kotani, M.; MacAdam, J.; Wu, Y. Over-expression of HAP3b enhances primary root elongation in Arabidopsis. Plant Physiol. Biochem. 2011, 49, 579–583. [Google Scholar] [CrossRef] [PubMed]
  18. Sorin, C.; Declerck, M.; Christ, A.; Blein, T.; Ma, L.; Lelandais-Brière, C.; Njo, M.F.; Beeckman, T.; Crespi, M.; Hartmann, C. A miR169 isoform regulates specific NF-YA targets and root architecture in Arabidopsis. New Phytol. 2014, 202, 1197–1211. [Google Scholar] [CrossRef]
  19. Zhou, Y.; Zhang, Y.; Wang, X.; Han, X.; An, Y.; Lin, S.; Shen, C.; Wen, J.; Liu, C.; Yin, W.; et al. Root-specific NF-Y family transcription factor, PdNF-YB21, positively regulates root growth and drought resistance by abscisic acid-mediated indoylacetic acid transport in Populus. New Phytol. 2020, 227, 407–426. [Google Scholar] [CrossRef] [PubMed]
  20. Cao, S.; Kumimoto, R.W.; Gnesutta, N.; Calogero, A.M.; Mantovani, R.; Holt, B.F., III. A Distal CCAAT/NUCLEAR FACTOR Y Complex Promotes Chromatin Looping at the FLOWERING LOCUS T Promoter and Regulates the Timing of Flowering in Arabidopsis. Plant Cell 2014, 26, 1009–1017. [Google Scholar] [CrossRef]
  21. Hou, X.; Zhou, J.; Liu, C.; Liu, L.; Shen, L.; Yu, H. Nuclear factor Y-mediated H3K27me3 demethylation of the SOC1 locus orchestrates flowering responses of Arabidopsis. Nat. Commun. 2014, 5, 4601. [Google Scholar] [CrossRef] [PubMed]
  22. Brambilla, V.; Fornara, F. Y flowering? Regulation and activity of CONSTANS and CCT-domain proteins in Arabidopsis and crop species. Biochim. Biophys. Acta 2017, 1860, 655–660. [Google Scholar] [CrossRef]
  23. Lv, X.; Zeng, X.; Hu, H.; Chen, L.; Zhang, F.; Liu, R.; Liu, Y.; Zhou, X.; Wang, C.; Wu, Z.; et al. Structural insights into the multivalent binding of the Arabidopsis FLOWERING LOCUS T promoter by the CO–NF–Y master transcription factor complex. Plant Cell 2021, 33, 1182–1195. [Google Scholar] [CrossRef] [PubMed]
  24. Miyoshi, K.; Ito, Y.; Serizawa, A.; Kurata, N. OsHAP3 genes regulate chloroplast biogenesis in rice. Plant J. 2003, 36, 532–540. [Google Scholar] [CrossRef] [PubMed]
  25. Stephenson, T.J.; McIntyre, C.L.; Collet, C.; Xue, G.P. TaNF-YC11, one of the light-upregulated NF-YC members in Triticum aestivum, is co-regulated with photosynthesis-related genes. Funct. Integr. Genom. 2010, 10, 265–276. [Google Scholar] [CrossRef]
  26. Xuanyuan, G.; Lu, C.; Zhang, R.; Jiang, J. Overexpression of StNF-YB3.1 reduces photosynthetic capacity and tuber production, and promotes ABA-mediated stomatal closure in potato (Solanum tuberosum L.). Plant Sci. 2017, 261, 50–59. [Google Scholar] [CrossRef] [PubMed]
  27. Bai, A.; Lu, X.D.; Li, D.Q.; Liu, J.X.; Liu, C.M. NF-YB1-regulated expression of sucrose transporters in aleurone facilitates sugar loading to rice endosperm. Cell Res. 2016, 26, 384–388. [Google Scholar] [CrossRef]
  28. Li, S.; Li, K.; Ju, Z.; Cao, D.; Fu, D.; Zhu, H.; Zhu, B.; Luo, Y. Genome-wide analysis of tomato NF-Y factors and their role in fruit ripening. BMC Genom. 2016, 17, 36. [Google Scholar] [CrossRef] [PubMed]
  29. Nelson, D.E.; Repetti, P.P.; Adams, T.R.; Creelman, R.A.; Wu, J.; Warner, D.C.; Anstrom, D.C.; Bensen, R.J.; Castiglioni, P.P.; Donnarummo, M.G.; et al. Plant nuclear factor Y (NF-Y) B subunits confer drought tolerance and lead to improved corn yields on water-limited acres. Proc. Natl. Acad. Sci. USA 2007, 104, 16450–16455. [Google Scholar] [CrossRef] [PubMed]
  30. Li, W.X.; Oono, Y.; Zhu, J.; He, X.J.; Wu, J.M.; Iida, K.; Lu, X.Y.; Cui, X.; Jin, H.; Zhu, J.K. The Arabidopsis NFYA5 Transcription Factor Is Regulated Transcriptionally and Posttranscriptionally to Promote Drought Resistance. Plant Cell 2008, 20, 2238–2251. [Google Scholar] [CrossRef]
  31. Lee, D.K.; Kim, H.I.; Jang, G.; Chung, P.J.; Jeong, J.S.; Kim, Y.S.; Bang, S.W.; Jung, H.; Choi, Y.D.; Kim, J.K. The NF-YA transcription factor OsNF-YA7 confers drought stress tolerance of rice in an abscisic acid independent manner. Plant Sci. 2015, 241, 199–210. [Google Scholar] [CrossRef] [PubMed]
  32. Palmeros-Suárez, P.A.; Massange-Sánchez, J.A.; Martínez-Gallardo, N.A.; Montero-Vargas, J.M.; Gómez-Leyva, J.F.; Délano-Frier, J.P. The overexpression of an Amaranthus hypochondriacus NF-YC gene modifies growth and confers water deficit stress resistance in Arabidopsis. Plant Sci. 2015, 240, 25–40. [Google Scholar] [CrossRef]
  33. Zhang, T.; Zhang, D.; Liu, Y.; Luo, C.; Zhou, Y.; Zhang, L. Overexpression of a NF-YB3 transcription factor from Picea wilsonii confers tolerance to salinity and drought stress in transformed Arabidopsis thaliana. Plant Physiol. Biochem. 2015, 94, 153–164. [Google Scholar] [CrossRef] [PubMed]
  34. Wu, X.; Shi, H.; Guo, Z. Overexpression of a NF-YC Gene Results in Enhanced Drought and Salt Tolerance in Transgenic Seashore Paspalum. Front. Plant Sci. 2018, 9, 1355. [Google Scholar] [CrossRef]
  35. Sato, H.; Suzuki, T.; Takahashi, F.; Shinozaki, K.; Yamaguchi-Shinozaki, K. NF-YB2 and NF-YB3 Have Functionally Diverged and Differentially Induce Drought and Heat Stress-Specific Genes. Plant Physiol. 2019, 180, 1677–1690. [Google Scholar] [CrossRef] [PubMed]
  36. Sato, H.; Todaka, D.; Kudo, M.; Mizoi, J.; Kidokoro, S.; Zhao, Y.; Shinozaki, K.; Yamaguchi-Shinozaki, K. The Arabidopsis transcriptional regulator DPB3-1 enhances heat stress tolerance without growth retardation in rice. Plant Biotechnol. J. 2016, 14, 1756–1767. [Google Scholar] [CrossRef]
  37. Gyula, P.; Baksa, I.; Tóth, T.; Mohorianu, I.; Dalmay, T.; Szittya, G. Ambient temperature regulates the expression of a small set of sRNAs influencing plant development through NF-YA2 and YUC2. Plant Cell Environ. 2018, 41, 2404–2417. [Google Scholar] [CrossRef]
  38. Chen, M.; Zhao, Y.; Zhuo, C.; Lu, S.; Guo, Z. Overexpression of a NF-YC transcription factor from bermudagrass confers tolerance to drought and salinity in transgenic rice. Plant Biotechnol. J. 2015, 13, 482–491. [Google Scholar] [CrossRef] [PubMed]
  39. Feng, Z.J.; He, G.H.; Zheng, W.J.; Lu, P.P.; Chen, M.; Gong, Y.M.; Ma, Y.Z.; Xu, Z.S. Foxtail Millet NF-Y Families: Genome-Wide Survey and Evolution Analyses Identified Two Functional Genes Important in Abiotic Stresses. Front. Plant Sci. 2015, 6, 1142. [Google Scholar] [CrossRef]
  40. Ma, X.; Zhu, X.; Li, C.; Song, Y.; Zhang, W.; Xia, G.; Wang, M. Overexpression of wheat NF-YA10 gene regulates the salinity stress response in Arabidopsis thaliana. Plant Physiol. Biochem. 2015, 86, 34–43. [Google Scholar] [CrossRef]
  41. Combier, J.P.; Frugier, F.; de Billy, F.; Boualem, A.; El-Yahyaoui, F.; Moreau, S.; Vernié, T.; Ott, T.; Gamas, P.; Crespi, M.; et al. MtHAP2-1 is a key transcriptional regulator of symbiotic nodule development regulated by microRNA169 in Medicago truncatula. Genes Dev. 2006, 20, 3084–3088. [Google Scholar] [CrossRef] [PubMed]
  42. Combier, J.P.; de Billy, F.; Gamas, P.; Niebel, A.; Rivas, S. Trans-regulation of the expression of the transcription factor MtHAP2-1 by a uORF controls root nodule development. Genes Dev. 2008, 22, 1549–1559. [Google Scholar] [CrossRef]
  43. Hogekamp, C.; Arndt, D.; Pereira, P.A.; Becker, J.D.; Hohnjec, N.; Küster, H. Laser Microdissection Unravels Cell-Type-Specific Transcription in Arbuscular Mycorrhizal Roots, Including CAAT-Box Transcription Factor Gene Expression Correlating with Fungal Contact and Spread. Plant Physiol. 2011, 157, 2023–2043. [Google Scholar] [CrossRef]
  44. Schaarschmidt, S.; Gresshoff, P.M.; Hause, B. Analyzing the soybean transcriptome during autoregulation of mycorrhization identifies the transcription factors GmNF-YA1a/b as positive regulators of arbuscular mycorrhization. Genome Biol. 2013, 14, R62. [Google Scholar] [CrossRef] [PubMed]
  45. Battaglia, M.; Rípodas, C.; Clúa, J.; Baudin, M.; Aguilar, O.M.; Niebel, A.; Zanetti, M.E.; Blanco, F.A. A Nuclear Factor Y Interacting Protein of the GRAS Family Is Required for Nodule Organogenesis, Infection Thread Progression, and Lateral Root Growth. Plant Physiol. 2014, 164, 1430–1442. [Google Scholar] [CrossRef]
  46. Laporte, P.; Lepage, A.; Fournier, J.; Catrice, O.; Moreau, S.; Jardinaud, M.F.; Mun, J.H.; Larrainzar, E.; Cook, D.R.; Gamas, P.; et al. The CCAAT box-binding transcription factor NF-YA1 controls rhizobial infection. J. Exp. Bot. 2014, 65, 481–494. [Google Scholar] [CrossRef]
  47. Boulard, C.; Thévenin, J.; Tranquet, O.; Laporte, V.; Lepiniec, L.; Dubreucq, B. LEC1 (NF-YB9) directly interacts with LEC2 to control gene expression in seed. Biochimica et biophysica acta. Gene Regul. Mech. 2018, 1861, 443–450. [Google Scholar] [CrossRef]
  48. Cagliari, A.; Turchetto-Zolet, A.C.; Korbes, A.P.; Maraschin, F.d.S.; Margis, R.; Margis-Pinheiro, M. New insights on the evolution of Leafy cotyledon1 (LEC1) type genes in vascular plants. Genomics 2014, 103, 380–387. [Google Scholar] [CrossRef]
  49. Wang, B.; Li, Z.; Ran, Q.; Li, P.; Peng, Z.; Zhang, J. ZmNF-YB16 Overexpression Improves Drought Resistance and Yield by Enhancing Photosynthesis and the Antioxidant Capacity of Maize Plants. Front. Plant Sci. 2018, 9, 709. [Google Scholar] [CrossRef]
  50. Stephenson, T.J.; McIntyre, C.L.; Collet, C.; Xue, G.P. TaNF-YB3 is involved in the regulation of photosynthesis genes in Triticum aestivum. Funct. Integr. Genom. 2011, 11, 327–340. [Google Scholar] [CrossRef]
  51. Li, X.; Zhao, M.; Xu, Y.; Li, Y.; Tigabu, M.; Zhao, X. Genetic Diversity and Population Differentiation of Pinus koraiensis in China. Horticulturae 2021, 7, 104. [Google Scholar] [CrossRef]
  52. Yang, K.; Gu, H.; Luan, H.; Yang, F. Method and standard for selecting elite nut trees of Korean pine. J. For. Res. 2001, 12, 84–88. [Google Scholar] [CrossRef]
  53. Aronen, T.; Pehkonen, T.; Malabadi, R. Somatic embryogenesis of Scots pine—Advances in pine tissue culture at Metla. In Proceedings of the Nordic Meeting at Punkaharju, Finland; Punkaharju Research Unit, Finnish Forest Research Institute: Punkaharju, Finland, 2008. [Google Scholar]
  54. Uma, S.; Kumaravel, M.; Backiyarani, S.; Saraswathi, M.S.; Durai, P.; Karthic, R. Somatic embryogenesis as a tool for reproduction of genetically stable plants in banana and confirmatory field trials. Plant Cell Tissue Organ Cult. 2021, 147, 181–188. [Google Scholar] [CrossRef]
  55. Varis, S.; Tikkinen, M.; Välimäki, S.; Aronen, T. Light Spectra during Somatic Embryogenesis of Norway Spruce-Impact on Growth, Embryo Productivity, and Embling Survival. Forests 2021, 12, 301. [Google Scholar] [CrossRef]
  56. Gongora-Castillo, E.; Nic-Can, G.I.; Galaz-Avalos, R.M.; Loyola-Vargas, V.M. Elaboration of Transcriptome During the Induction of Somatic Embryogenesis. Methods Mol. Biol. 2018, 1815, 411–427. [Google Scholar] [CrossRef] [PubMed]
  57. Karami, O.; Saidi, A. The molecular basis for stress-induced acquisition of somatic embryogenesis. Mol. Biol. Rep. 2010, 37, 2493–2507. [Google Scholar] [CrossRef]
  58. Zhang, C.X.; Li, Q.; Kong, L.S. Induction, development and maturation of somatic embryos in Bunge’s pine (Zucc. ex Endl.). Plant Cell Tissue Organ Cult. 2007, 91, 273–280. [Google Scholar] [CrossRef]
  59. Zimmerman, J.L. Somatic Embryogenesis: A Model for Early Development in Higher Plants. Plant Cell 1993, 5, 1411–1423. [Google Scholar] [CrossRef]
  60. Uddenberg, D.; Valladares, S.; Abrahamsson, M.; Sundstrom, J.F.; Sundas-Larsson, A.; von Arnold, S. Embryogenic potential and expression of embryogenesis-related genes in conifers are affected by treatment with a histone deacetylase inhibitor. Planta 2011, 234, 527–539. [Google Scholar] [CrossRef]
  61. Gao, F.; Peng, C.; Wang, H.; Tretyakova, I.N.; Nosov, A.M.; Shen, H.; Yang, L. Key Techniques for Somatic Embryogenesis and Plant Regeneration of Pinus koraiensis. Forests 2020, 11, 912. [Google Scholar] [CrossRef]
  62. Zhang, T.H.; Wang, X.M.; Zhou, C.C.; Chen, Y.P.; Wang, X.; Wang, S.X.; He, S.C.; Guo, Y.; Liu, Z.J.; Chen, M.X. Transcription factor DIVARICATA1 positively modulates seed germination in response to salinity stress. Plant Physiol. 2024, 195, 2997–3009. [Google Scholar] [CrossRef]
  63. Li, X.; Li, C.; Shi, L.; Lv, G.; Li, X.; Liu, Y.; Jia, X.; Liu, J.; Chen, Y.; Zhu, L.; et al. Jasmonate signaling pathway confers salt tolerance through a NUCLEAR FACTOR-Y trimeric transcription factor complex in Arabidopsis. Cell Rep. 2024, 43, 113825. [Google Scholar] [CrossRef] [PubMed]
  64. Gaudinier, A.; Rodriguez-Medina, J.; Zhang, L.; Olson, A.; Liseron-Monfils, C.; Bågman, A.M.; Foret, J.; Abbitt, S.; Tang, M.; Li, B.; et al. Transcriptional regulation of nitrogen-associated metabolism and growth. Nature 2018, 563, 259–264. [Google Scholar] [CrossRef] [PubMed]
  65. Kim, I.; Lee, K.R.; Park, M.E.; Kim, H.U. The seed-specific transcription factor DPBF2 modulates the fatty acid composition in seeds. Plant Direct 2022, 6, e395. [Google Scholar] [CrossRef] [PubMed]
  66. Siefers, N.; Dang, K.K.; Kumimoto, R.W.; Bynum, W.E.; Tayrose, G.; Holt, B.F. Tissue-specific expression patterns of Arabidopsis NF-Y transcription factors suggest potential for extensive combinatorial complexity. Plant Physiol. 2009, 149, 625–641. [Google Scholar] [CrossRef] [PubMed]
  67. Yang, W.J.; Lu, Z.H.; Xiong, Y.F.; Yao, J.L. Genome-wide identification and co-expression network analysis of the OsNF-Y gene family in rice. Crop J. 2017, 5, 21–31. [Google Scholar] [CrossRef]
  68. Stephenson, T.J.; McIntyre, C.L.; Collet, C.; Xue, G.P. Genome-wide identification and expression analysis of the NF-Y family of transcription factors in Triticum aestivum. Plant Mol. Biol. 2007, 65, 77–92. [Google Scholar] [CrossRef]
  69. Quach, T.N.; Nguyen, H.T.; Valliyodan, B.; Joshi, T.; Xu, D.; Nguyen, H.T. Genome-wide expression analysis of soybean NF-Y genes reveals potential function in development and drought response. Mol. Genet. Genom. 2015, 290, 1095–1115. [Google Scholar] [CrossRef] [PubMed]
  70. Wang, R.; Zhu, L.; Zhang, Y.; Fan, J.; Li, L. Genome-wide analysis of poplar NF-YB gene family and identified PtNF-YB1 important in regulate flowering timing in transgenic plants. BMC Plant Biol. 2019, 19, 251. [Google Scholar] [CrossRef]
  71. Liu, J.H.; Teng, T.; Dai, W. Critical cis-Acting Elements and Interacting Transcription Factors: Key Players Associated with Abiotic Stress Responses in Plants. Plant Mol. Biol. Rep. 2014, 32, 303–317. [Google Scholar] [CrossRef]
  72. Roopali, B.; Olive, O.; Tanushree, H.; Manisha, S.; Darshan, S. Transcription factors—Insights into abiotic and biotic stress resilience and crop improvement. Curr. Plant Biol. 2025, 41, 100434. [Google Scholar] [CrossRef]
  73. Litvay, J.D.; Verma, D.C.; Johnson, M.A. Influence of a loblolly pine (Pinus taeda L.). Culture medium and its components on growth and somatic embryogenesis of the wild carrot (Daucus carota L.). Plant Cell Rep. 1985, 4, 325–328. [Google Scholar] [CrossRef] [PubMed]
  74. Bhattacharjee, B.; Hallan, V. NF-YB family transcription factors in Arabidopsis: Structure, phylogeny, and expression analysis in biotic and abiotic stresses. Front. Microbiol. 2023, 13, 1067427. [Google Scholar] [CrossRef] [PubMed]
  75. Potter, S.C.; Luciani, A.; Eddy, S.R.; Park, Y.; Lopez, R.; Finn, R.D. HMMER web server: 2018 update. Nucleic Acids Res. 2018, 46, W200–W204. [Google Scholar] [CrossRef] [PubMed]
  76. Kumar, S.; Stecher, G.; Suleski, M.; Sanderford, M.; Sharma, S.; Tamura, K. MEGA12: Molecular Evolutionary Genetic Analysis version 12 for adaptive and green computing. Mol. Biol. Evol. 2024, 41, msae263. [Google Scholar] [CrossRef]
  77. Bailey, T.L.; Boden, M.; Buske, F.A.; Frith, M.; Grant, C.E.; Clementi, L.; Ren, J.; Li, W.W.; Noble, W.S. MEME SUITE: Tools for motif discovery and searching. Nucleic Acids Res. 2009, 37, W202–W208. [Google Scholar] [CrossRef]
  78. Chen, C.; Wu, Y.; Li, J.; Wang, X.; Zeng, Z.; Xu, J.; Liu, Y.; Feng, J.; Chen, H.; He, Y.; et al. TBtools-II: A “one for all, all for one” bioinformatics platform for biological big-data mining. Mol. Plant 2023, 16, 1733–1742. [Google Scholar] [CrossRef] [PubMed]
  79. Lescot, M.; Déhais, P.; Thijs, G.; Marchal, K.; Moreau, Y.; Van de Peer, Y.; Rouzé, P.; Rombauts, S. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res. 2002, 30, 325–327. [Google Scholar] [CrossRef] [PubMed]
  80. Li, S.J.; Yang, L.; Shen, H.L.; Hou, H.; Yang, Y.N.; Dong, H.; An, Y.; Zhao, W.N. A Genetic Transformation Method for Embryogenic Callus Tissue of Pinus koraiensis. CN113736821B. 2021-12-03. Available online: https://patents.google.com/patent/CN113736821B/en (accessed on 3 December 2021).
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Figure 1. Phylogenetic analysis and multiple alignment of amino acid sequences. Multispecies phylogenetic analysis of NF-YB (A) and NF-YC (B) proteins, including Arabidopsis thalian, Pinus koraiensis, Pinus taeda, Pinus tabuliformis, Populus trichocarpa, Picea abies, Oryza sativa, and Triticum aestivum. Amino acid alignment of conserved domains of NF-YB (C) and NF-YC (D) proteins from different organisms. The sequence under the red line represents the region that interacts with NF-YA, while the sequence under the blue line represents the region that interacts with NF-YB; the sequence above the green line represents the region that interacts with NF-YC, while the sequence below the cyan line represents the region that binds to DNA. Gray lines include segments with conserved HFM regions.
Figure 1. Phylogenetic analysis and multiple alignment of amino acid sequences. Multispecies phylogenetic analysis of NF-YB (A) and NF-YC (B) proteins, including Arabidopsis thalian, Pinus koraiensis, Pinus taeda, Pinus tabuliformis, Populus trichocarpa, Picea abies, Oryza sativa, and Triticum aestivum. Amino acid alignment of conserved domains of NF-YB (C) and NF-YC (D) proteins from different organisms. The sequence under the red line represents the region that interacts with NF-YA, while the sequence under the blue line represents the region that interacts with NF-YB; the sequence above the green line represents the region that interacts with NF-YC, while the sequence below the cyan line represents the region that binds to DNA. Gray lines include segments with conserved HFM regions.
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Figure 2. Structural analysis of proteins and conserved structures encoding corresponding proteins. Predicted conserved motifs and structures of PkNF-YBs (A) and PkNF-YCs (B). Different colors represent different conserved motifs. Yellow represents the protein-coding sequence (exon); green represents upstream/downstream sequences; the black line represents introns.
Figure 2. Structural analysis of proteins and conserved structures encoding corresponding proteins. Predicted conserved motifs and structures of PkNF-YBs (A) and PkNF-YCs (B). Different colors represent different conserved motifs. Yellow represents the protein-coding sequence (exon); green represents upstream/downstream sequences; the black line represents introns.
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Figure 3. Chromosomal location and intra species collinearity analysis of PKNF-YB/C. Thick red lines indicate duplicated gene pairs. The red font represents members of the NF-YB family, while the green font represents members of the NF-YC family. Chromosome numbers were displayed in the outer ring of each chromosome. There is no collinearity between NF-YB and NF-YC family members within this species. Scale bars marked on each chromosome indicate chromosome length (Mb).
Figure 3. Chromosomal location and intra species collinearity analysis of PKNF-YB/C. Thick red lines indicate duplicated gene pairs. The red font represents members of the NF-YB family, while the green font represents members of the NF-YC family. Chromosome numbers were displayed in the outer ring of each chromosome. There is no collinearity between NF-YB and NF-YC family members within this species. Scale bars marked on each chromosome indicate chromosome length (Mb).
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Figure 4. The genomes of P. koraiensis, A. thaliana, and P. tabulaeformis were subjected to collinearity analysis. The highlighted red line represents the NF-YB/C gene pairs with collinearity.
Figure 4. The genomes of P. koraiensis, A. thaliana, and P. tabulaeformis were subjected to collinearity analysis. The highlighted red line represents the NF-YB/C gene pairs with collinearity.
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Figure 5. Analysis of cis-elements of PKNF-YB/C promoters.
Figure 5. Analysis of cis-elements of PKNF-YB/C promoters.
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Figure 6. GO enrichment analysis of NF-YB (A) and NF-YC (B) family members. KEGG enrichment analysis of NF-YB (C) and NF-YC (D) family members.
Figure 6. GO enrichment analysis of NF-YB (A) and NF-YC (B) family members. KEGG enrichment analysis of NF-YB (C) and NF-YC (D) family members.
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Figure 7. Tissue-specific expression patterns of PkNF-YB/C genes in callus, embryo, root, stem, and leaf. EC: embryogenic callus; NEC: non-embryogenic callus; SE: somatic embryogenesis; embryo: zygotic embryo; seed: mature seed. The depth of color and the size of the circle area represent the level of expression. The transcriptome expression data underwent log2(FPKM) transformation and were normalized using a 0–1 approach. The circle on the right serves as the scale bar. The larger the area, the redder the color, indicating a higher relative expression level.
Figure 7. Tissue-specific expression patterns of PkNF-YB/C genes in callus, embryo, root, stem, and leaf. EC: embryogenic callus; NEC: non-embryogenic callus; SE: somatic embryogenesis; embryo: zygotic embryo; seed: mature seed. The depth of color and the size of the circle area represent the level of expression. The transcriptome expression data underwent log2(FPKM) transformation and were normalized using a 0–1 approach. The circle on the right serves as the scale bar. The larger the area, the redder the color, indicating a higher relative expression level.
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Figure 8. Expression patterns of the PkNF YB/C genes at different stages of reproductive organ development. FA–FD indicate different stages of microsporophyll: tender branches without differentiated microsporophyll, immature microsporophyll, mature microsporophyll, and dried and withered microspore leaves after pollen dispersion. MA–ME indicate different stages of megasporophylls: tender branches without differentiated megasporophyll, female flower bud (megasporophyll), closed megasporophylls before pollination, macrosporophyll unfolding during pollination, and closed megasporophylls after pollination. ZA–ZD: ovules during the female gametophyte stage, ovules during pollen tube extension, fertilized ovules, and an ovule that begins cell division and development. The color and the size of the circle area represent the level of expression. The transcriptome expression data underwent log2(FPKM) transformation and were normalized using a 0–1 approach. The circle on the right serves as the scale bar. The larger the area, the redder the color, indicating a higher relative expression level.
Figure 8. Expression patterns of the PkNF YB/C genes at different stages of reproductive organ development. FA–FD indicate different stages of microsporophyll: tender branches without differentiated microsporophyll, immature microsporophyll, mature microsporophyll, and dried and withered microspore leaves after pollen dispersion. MA–ME indicate different stages of megasporophylls: tender branches without differentiated megasporophyll, female flower bud (megasporophyll), closed megasporophylls before pollination, macrosporophyll unfolding during pollination, and closed megasporophylls after pollination. ZA–ZD: ovules during the female gametophyte stage, ovules during pollen tube extension, fertilized ovules, and an ovule that begins cell division and development. The color and the size of the circle area represent the level of expression. The transcriptome expression data underwent log2(FPKM) transformation and were normalized using a 0–1 approach. The circle on the right serves as the scale bar. The larger the area, the redder the color, indicating a higher relative expression level.
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Figure 9. Expression patterns of PkNF YB/C genes in ABA treatment. CK: white space handling; +ABA: somatic embryos grown on mature medium supplemented with 80 μmol∙L−1 ABA; −ABA: somatic embryos grown on mature medium supplemented without ABA. The transcriptome expression data were normalized using CK as the control standard, while the data from other treatment groups were normalized using log2FPKM (treatment)/FPKM (CK) as values.
Figure 9. Expression patterns of PkNF YB/C genes in ABA treatment. CK: white space handling; +ABA: somatic embryos grown on mature medium supplemented with 80 μmol∙L−1 ABA; −ABA: somatic embryos grown on mature medium supplemented without ABA. The transcriptome expression data were normalized using CK as the control standard, while the data from other treatment groups were normalized using log2FPKM (treatment)/FPKM (CK) as values.
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Figure 10. Subcellular localization of PkNF-YB/C proteins. DAPI: a nuclear staining dye; BF: bright field; merge: The merged images of BF, eGFP, and DAPI staining. White arrow indicates the location of the cell nucleus.
Figure 10. Subcellular localization of PkNF-YB/C proteins. DAPI: a nuclear staining dye; BF: bright field; merge: The merged images of BF, eGFP, and DAPI staining. White arrow indicates the location of the cell nucleus.
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Figure 11. Analysis of the transcriptional activation activity of PkNF-YB/C proteins. pGBKT7: negative control.
Figure 11. Analysis of the transcriptional activation activity of PkNF-YB/C proteins. pGBKT7: negative control.
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Figure 12. The protein interaction network of PkNF-YB and PkNF-YC. The red nodes represent the PkNF-YB and PkNF-YC proteins, while the green nodes represent the predicted interaction proteins. The connecting lines represent the interacting relationships.
Figure 12. The protein interaction network of PkNF-YB and PkNF-YC. The red nodes represent the PkNF-YB and PkNF-YC proteins, while the green nodes represent the predicted interaction proteins. The connecting lines represent the interacting relationships.
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Figure 13. Identification and transgenic lines of PkLEC1 in P. koraiensis. qRTPCR detection of PkLEC1 transgenic lines (A). Pk18S was used as a control. Each error bar represents the standard deviation of three biological replicates. Asterisks indicate levels of significance (Dunnett’s test; *, p < 0.05). Mature phenotype of PkNF-YB 7 transgenic cell lines (B). WT and PkNF-YB 7 transgenic lines under 8-week mature culturation (T31, T33, and T34).
Figure 13. Identification and transgenic lines of PkLEC1 in P. koraiensis. qRTPCR detection of PkLEC1 transgenic lines (A). Pk18S was used as a control. Each error bar represents the standard deviation of three biological replicates. Asterisks indicate levels of significance (Dunnett’s test; *, p < 0.05). Mature phenotype of PkNF-YB 7 transgenic cell lines (B). WT and PkNF-YB 7 transgenic lines under 8-week mature culturation (T31, T33, and T34).
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Table 1. Parameters for the ten identified PkNF-YB/Cs and deduced polypeptide sequences present in the P. koraiensis genome.
Table 1. Parameters for the ten identified PkNF-YB/Cs and deduced polypeptide sequences present in the P. koraiensis genome.
Gene NameLocus NameAmino Acid No.Molecular Weight (Da)Isoelectric PointsGRAVYChromosome Location
PkNF-YB1Pkor01G0177722524,436.996.08−0.866Chr01:1563581603..1563582280
PkNF-YB2Pkor02G0071122023,832.197.76−0.931Chr02:805110131..805110793
PkNF-YB3Pkor03G0185719421,331.176.65−0.646Chr03:1580969493..1580970077
PkNF-YB4Pkor06G0091318920,187.226.75−0.877Chr06:823725832..823726401
PkNF-YB5Pkor08G0086716318,176.365.67−0.798Chr08:877008868..877021563
PkNF-YB6Pkor08G02153788531.374.96−0.738Chr08:1957384587..1957384823
PkNF-YB7Pkor10G0125418420,743.255.15−0.694Chr10:1036699488..1036700042
PkNF-YB8Pkor11G0154217720,291.515.36−0.801Chr11:1222883089..1222883622
PkNF-YC1Pkor02G0100520222,438.706.20−0.409Chr02:1063370683..1063371291
PkNF-YC2Pkor06G0111927230,067.305.96−0.440Chr06:993282521..993283339
PkNF-YC3Pkor08G0170823627,453.396.13−0.687Chr08:1604161452..1604167962
PkNF-YC4Pkor09G0016526530,137.926.89−0.634Chr09:166805975..166806772
PkNF-YC5Pkor09G0016626129,105.978.90−0.452Chr09:167008318..167009103
PkNF-YC6Pkor09G0016725027,833.708.42−0.301Chr09:168737804..168738556
PkNF-YC7Pkor10G0223231135,237.536.49−0.917Chr10:1969147323..1969148258
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Xu, X.; He, X.; Zhang, Q.; Yang, L. Genome-Wide Identification and Expression Pattern Analysis of Nuclear Factor Y B/C Genes in Pinus koraiensis, and Functional Identification of LEAFY COTYLEDON 1. Plants 2025, 14, 438. https://doi.org/10.3390/plants14030438

AMA Style

Xu X, He X, Zhang Q, Yang L. Genome-Wide Identification and Expression Pattern Analysis of Nuclear Factor Y B/C Genes in Pinus koraiensis, and Functional Identification of LEAFY COTYLEDON 1. Plants. 2025; 14(3):438. https://doi.org/10.3390/plants14030438

Chicago/Turabian Style

Xu, Xiuyue, Xin He, Qun Zhang, and Ling Yang. 2025. "Genome-Wide Identification and Expression Pattern Analysis of Nuclear Factor Y B/C Genes in Pinus koraiensis, and Functional Identification of LEAFY COTYLEDON 1" Plants 14, no. 3: 438. https://doi.org/10.3390/plants14030438

APA Style

Xu, X., He, X., Zhang, Q., & Yang, L. (2025). Genome-Wide Identification and Expression Pattern Analysis of Nuclear Factor Y B/C Genes in Pinus koraiensis, and Functional Identification of LEAFY COTYLEDON 1. Plants, 14(3), 438. https://doi.org/10.3390/plants14030438

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