Genomic Organization, Phylogenetic Comparison, and Differential Expression of the Nuclear Factor-Y Gene Family in Apple (Malus Domestica)

The nuclear factor Y (NF-Y) as a transcription factor plays an important role in plants growth and development, and response to stress. However, few genome-wide analyzes and functional research of the NF-Y family has been undertaken in apple (Malus domestica Borkh.) so far. In this study, we comprehensively identified the 43 MdNF-Y genes in apple, which dispersedly distributed among the three subgroups based on their sequence alignment analysis, including 11 MdNF-YAs, 22 MdNF-YBs and 10 MdNF-YCs. The members in the same subgroups had similar evolution relationships, gene structures, and conserved motifs. The gene duplication analysis suggested that all the genes were dispersed followed by 27 segmental duplication. Moreover, based on synteny analysis of MdNF-Ys with eight plant species results suggested that some ortholog genes were preserved during the evolution of these species. Cis-element analysis showed potential functions of MdNF-Ys in apple growth and development and responded to abiotic stress. Furthermore, the interaction among MdNF-Ys protein were investigated in yeast two-hybrid assays. The expression patterns of MdNF-Ys in tissue-specific response reveled divergence and might play important role in apple growth and development. Subsequently, whole MdNF-Y genes family was carried out for RT-PCR in response to five abiotic stress (ABA, drought, heat, cold, and salinity) to identify their expression patterns. Taken together, our study will provide a foundation for the further study to the molecular mechanism of apple in growing development and response to abiotic stresses.


Introduction
Transcription factors (TFs) control the transcription or expression of downstream target genes by interacting with cis-elements through covalent binding to the DNA binding domain. Nuclear factor Y (NF-Y) TFs, known as CCAAT-binding factors (CBFs) or heme activator proteins (HAPs), play a critical regulatory role in plant vital movement by binding to the CCAAT element. NF-Y consists of three distinct subunits including NF-YA (also known as CBF-B/HAP2), NF-YB (CBF-A/HAP3), and NF-YC (CBF-C/HAP5) [1]. NF-Y is ubiquitously expressed in most eukaryotes and in mammals and yeast, each subunit is encoded by only one or two NF-Y genes. However, in plants, each NF-Y subunit has evolutionarily formed relatively large gene families expressed from multiple NF-Y genes. For example, in the model plant Arabidopsis thaliana, a total of 30 AtNF-Y genes exist including 10 AtNF-YAs, 10 AtNF-YBs, and 10 AtNF-YCs [2,3]. In addition, the three subunits each possess conserved DNA-binding domains and mutual interaction domains to form heterotrimeric complexes. It is also worth noting that NF-YA and NF-YC family members analyzed the cis-regulatory elements in the promoters and their transcriptional expression in different tissues. Yeast two-hybrid (Y2H) assays were conducted to study the interaction between various MdNF-Y subunits. The transcription profiles of the MdNF-Y genes were detected under various abiotic stresses. Our results provide a foundation for further study of the functional and regulatory mechanisms controlled by the MdNF-Y gene family.

Identification and Characterization of NF-Y Family Genes in Apple
Following the removal of redundant sequences, we initially identified 11 MdNF-YAs, 26 MdNF-YBs, and 12 MdNF-YCs through Hidden Markov Model (HMM) analysis of the M. domestica genome (https://www.rosaceae.org/). Four putative MdNF-YBs (MD03G1280100, MD06G1209300, MD11G1164600, and MD14G1219800) and two putative MdNF-YCs (MD02G1273400 and MD07G1042300) did not contain the core structure of the NF-Y domain, so we removed them from further analysis. In order to facilitate further research, these genes were renamed according to their locations in chromosomes and the Arabidopsis nomenclature [2,36] (Table 1) Table S1.
In addition, the three NF-Y subunits contained highly conserved domains among the previously reported species such as Arabidopsis (A. thaliana), grape (V. vinifera), and orange (C. sinensis) [2,12,15]. Similarly, multiple alignments indicated that apple NF-Y subunits also contained the same highly conserved regions as Arabidopsis, grape, and orange NF-Y subunits (Figure 1). The conserved central domain of the MdNF-YA, MdNF-YB, and MdNF-YC subunits, respectively, consisted of about 54, 90, and 81 aas, and three MdNF-Y subunits all contained a DNA binding domain. Moreover, the MdNF-YA contained an NF-YB/C interaction domain, and the MdNF-YB and MdNF-YC both contained an NF-YA interaction domain ( Figure 1). These domains were necessary for NF-YA, NF-YB, and NF-YC to form a heterotrimeric complex to bind to CCAAT boxes [37]. Furthermore, the MdNF-YB and MdNF-YC both contained a histone-fold motif (HFM) resembling the core of H2B and H2A, respectively.

Phylogenetic Tree, Conserved Motifs, and Gene Structure Analysis of MdNF-Y Family
To deduce the potential evolutionary diversity and relationship of MdNF-Y subunit genes, we constructed a phylogenetic tree and analyzed the conserved motifs and gene structure for each MdNF-Y subunit ( Figure 2). For each MdNF-Y subunit the corresponding genes had close phylogenetic relationships and consistently displayed conserved motifs and gene structures. For example, MdNF-YA9 and MdNF-YA10 were closely related evolutionarily and they presented similar motifs type (motif 3, 4, 7, 12, and 19) ( Figure  2A). On the other hand, differences between various MdNF-Y subunits were evident in terms of major motifs. For example, motifs 3, 4, 7, 8, and 12 were contained in most MdNF-YA genes whereas MdNF-YA9 and MdNF-YA10 lacked motif 8. Most of the MdNF-YB

Phylogenetic Tree, Conserved Motifs, and Gene Structure Analysis of MdNF-Y Family
To deduce the potential evolutionary diversity and relationship of MdNF-Y subunit genes, we constructed a phylogenetic tree and analyzed the conserved motifs and gene structure for each MdNF-Y subunit ( Figure 2). For each MdNF-Y subunit the corresponding genes had close phylogenetic relationships and consistently displayed conserved motifs and gene structures. For example, MdNF-YA9 and MdNF-YA10 were closely related evolutionarily and they presented similar motifs type (motif 3, 4, 7, 12, and 19) (Figure 2A). On the other hand, differences between various MdNF-Y subunits were evident in terms of major motifs. For example, motifs 3, 4, 7, 8, and 12 were contained in most MdNF-YA genes whereas MdNF-YA9 and MdNF-YA10 lacked motif 8. Most of the MdNF-YB genes contained motif 1, 2, and 3 except MdNF-YB4, MdNF-YB14, and MdNF-YB17. Most of the MdNF-YC genes contained seven types of motifs (motif 1, 4, 5, 6, 9, 10, and 14). However, MdNF-YC3 and MdNF-YC9 presented different motif types compared with the other MdNF-YC genes. In addition, the gene structures of the various MdNF-Y subunits also displayed differences in the distribution of exons and introns, whereas the gene structures within each MdNF-Y subunit were relatively similar ( Figure 2B). For example, in the MdNF-YA subfamily, the CDS were separated by four introns except MdNF-YA4, and all MdNF-YCs contained a long CDS region which is not separated by introns.

Chromosome Distribution and Synteny Analysis of MdNF-Y Family Genes
To understand the chromosomal distribution of the different MdNF-Ys, chromosomal location map was created ( Figure 3). All MdNF-Y genes were unevenly distributed on 16 of the 17 apple chromosomes except chr08. The number of genes on the chromosomes that contained MdNF-Y genes varied from one to four. Six chromosomes with four MdNF-Y genes, including chr02, chr03, chr05, chr11, chr12, and chr15, while only MdNF-YB1 was located on the chr01. In addition, two MdNF-Ys were located on chr06, chr07, chr09, chr13, and chr14, and three MdNF-Ys were located on the terminus of chr04 and chr10, respec-  To investigate the gene family expansion mechanism of the MdNF-Y genes, we an lyzed their synteny relationships in apple genomes ( Figure 4). The results showed that tandem duplication event had occurred but that 27 pairs of gene segmental duplicati events could be identified ( Figure 4, Table S2). Overall, 11 MdNF-YA, 15 MdNF-YB, and MdNF-YC genes were mapped to the 16 chromosomes except for chr 08, while the pa of paralogous genes were 7, 14, and 6, respectively. These results suggested that segmen duplications were the cause of MdNF-Y genes amplification. Interestingly, we found th there was respectively one triangular relationship in the  To investigate the gene family expansion mechanism of the MdNF-Y genes, we analyzed their synteny relationships in apple genomes ( Figure 4). The results showed that no tandem duplication event had occurred but that 27 pairs of gene segmental duplication events could be identified ( Figure 4, Table S2). Overall, 11 MdNF-YA, 15 MdNF-YB, and 8 MdNF-YC genes were mapped to the 16 chromosomes except for chr 08, while the pairs of paralogous genes were 7, 14, and 6, respectively. These results suggested that segmental duplications were the cause of MdNF-Y genes amplification. Interestingly, we found that there was respectively one triangular relationship in the MdNF-YA (MdNF-YA1, MdNF-YA6, and MdNF-YA11) and MdNF-YC subunits (MdNF-YC1, MdNF-YC6, and MdNF-YC10). There were four triangular relationships in MdNF-YB subunit including MdNF-YB1, MdNF-YB11, and MdNF-YB17; MdNF-YB6, MdNF-YB16, and MdNF-YB19; MdNF-YB7, MdNF-YB11, and MdNF-YB17; and MdNF-YB8, MdNF-YB18, and MdNF-YB19. In addition, more gene pairs are listed in Table S2.  To further study the gene expansion relationship and evolution of NF-Y subunits ( Figure 5), we chose eight representative plant models with widely ranging homologies to analyze the synteny of NF-Y genes. The eight species contained six dicots, including three Rosacea species (Pyrus betulifolia, Prunus persica, and Fragaria vesca), Vitis vinifera, A. thaliana, and Brassica rapa and two monocots (Oryza sativa L. and Zea Mays L). The results suggested that many NF-Y genes in apple have homology to reference plants. It is well known that apples also belong to the Rosaceae, like F. vesca, P. betulifolia, and P. persica. Furtherly, many ortholog pairs of MdNF-Y genes were found among P. betulifolia (59 orthologous gene pairs distributed on all apple chr except chr8), P. persica (37 orthologous gene pairs distributed on all apple chr except chr8 and chr17), and F. vesca (37 orthologous gene pairs distributed on all apple chr except chr8), respectively. Further, with the exception of MdNF-YA1 and MdNF-YB10, the majority of MdNF-Y subunits had orthologous pairs in pears, which indicated that the NF-Y transcription factor families are highly homologous in apples and pears. However, fewer homologous gene pairs were observed between apple and O. sativa L. (only seven) and between apple and Z. Mays. (only one) (Table S3). In addition, we found some highly homologous genes were preserved during To further study the gene expansion relationship and evolution of NF-Y subunits ( Figure 5), we chose eight representative plant models with widely ranging homologies to analyze the synteny of NF-Y genes. The eight species contained six dicots, including three Rosacea species (Pyrus betulifolia, Prunus persica, and Fragaria vesca), Vitis vinifera, A. thaliana, and Brassica rapa and two monocots (Oryza sativa L. and Zea Mays L). The results suggested that many NF-Y genes in apple have homology to reference plants. It is well known that apples also belong to the Rosaceae, like F. vesca, P. betulifolia, and P. persica. Furtherly, many ortholog pairs of MdNF-Y genes were found among P. betulifolia (59 orthologous gene pairs distributed on all apple chr except chr8), P. persica (37 orthologous gene pairs distributed on all apple chr except chr8 and chr17), and F. vesca (37 orthologous gene pairs distributed on all apple chr except chr8), respectively. Further, with the exception of MdNF-YA1 and MdNF-YB10, the majority of MdNF-Y subunits had orthologous pairs in pears, which indicated that the NF-Y transcription factor families are highly homologous in apples and pears. However, fewer homologous gene pairs were observed between apple and O. sativa L. (only seven) and between apple and Z. Mays. (only one) (Table S3). In addition, we found some highly homologous genes were preserved during the species evolution. For example, MdNF-YA6 had homologous pairs in seven species excepted maize, and MdNF-YB19 had homologous pairs in six species excepted Arabidopsis and B. rapa. Likewise, MdNF-YC1, MdNF-YC6, and MdNF-YC8 had homologous pairs in five species, respectively.

The Cis-Acting Regulatory Members in the Promoter of MdNF-Y Family Genes
To predict the potential function of MdNF-Y transcription factors, we chose the 1500 bp upstream sequences using plantCARE to analyze the type of cis-elements in the promoter ( Figure 6 and Table S5). We found a preponderant number of TATA-boxes and CAAT-boxes, which have been analyzed in terms of their roles in transcription [38]. The results also revealed the presence of a very large number of light cis-elements. Therefore, we classified the results into four broad categories: Those participated in plant development, phytohormones, abiotic, and biotic stress-responsive, and light-responsive. The ciselements participated in light responsiveness included a 3-AF1 binding site, G-Box, GAmotif, and a GATA-motif. The cis-elements involved in plant development included flavonoid biosynthetic genes regulation (MBSI), endosperm expression (GCN4), meristem expression (CAT-box), circadian control (Circadian), Seed (RY-element), and root-specific (motif I). The cis-elements partake in abiotic and biotic stress included anaerobic induction (ARE), heat stress responsiveness (HSE), low-temperature responsiveness (LTR), anoxic specific inducibility (GC), drought-inducibility (MBS), defense and stress responsiveness (TC-rich repeats), auxin-responsive element (TGA-element), pathogen (W-box), and wound-responsive element (WUN-motif). The cis-elements involved in phytohormone responsive included abscisic acid (ABA) responsiveness (ABRE) MeJA-responsiveness (CGTCA-motif), ethylene (ERE), and salicylic acid (SA) responsiveness (TCA-element).

The Cis-Acting Regulatory Members in the Promoter of MdNF-Y Family Genes
To predict the potential function of MdNF-Y transcription factors, we chose the 1500 bp upstream sequences using plantCARE to analyze the type of cis-elements in the promoter ( Figure 6 and Table S5). We found a preponderant number of TATA-boxes and CAATboxes, which have been analyzed in terms of their roles in transcription [38]. The results also revealed the presence of a very large number of light cis-elements. Therefore, we classified the results into four broad categories: Those participated in plant development, phytohormones, abiotic, and biotic stress-responsive, and light-responsive. The cis-elements participated in light responsiveness included a 3-AF1 binding site, G-Box, GA-motif, and a GATA-motif. The cis-elements involved in plant development included flavonoid biosynthetic genes regulation (MBSI), endosperm expression (GCN4), meristem expression (CAT-box), circadian control (Circadian), Seed (RY-element), and root-specific (motif I). The cis-elements partake in abiotic and biotic stress included anaerobic induction (ARE), heat stress responsiveness (HSE), low-temperature responsiveness (LTR), anoxic specific inducibility (GC), drought-inducibility (MBS), defense and stress responsiveness (TC-rich repeats), auxin-responsive element (TGA-element), pathogen (W-box), and woundresponsive element (WUN-motif). The cis-elements involved in phytohormone responsive included abscisic acid (ABA) responsiveness (ABRE) MeJA-responsiveness (CGTCA-motif), ethylene (ERE), and salicylic acid (SA) responsiveness (TCA-element).
In addition, most of the MdNF-Ys had ABRE cis-acting regulatory elements with a total of 169 elements, indicating that MdNF-Y genes play an important role in ABA response. All MdNF-Y genes contained more or less different types of light-responsive elements, although all MdNF-YBs possessed a G-Box element. A number of cis-acting regulatory elements involved in plant development were found in some of the MdNF-Y genes. For example, motif I, which was involved in plant root development [39], was only found in MdNF-YC6. A circadian cis-acting element, involved in circadian control [40], was only discovered in MdNF-YC4. An RY-element, participated in plant seed-specific regulation [41], was only detected in MdNF-YB13. Therefore, these MdNF-Y genes need to be further explored since they may play a critical role in apple growth. The specific cis-element analysis of the MdNF-Y genes is shown in Table S5. Actually, some reports in other plants have indicated that NF-Ys participated in response to abiotic stresses (cold [42], heat [25], drought [43], and salinity [28], root development [44], seed-specific regulation [44] and photoperioddependent flowering [29]. So, we further detected the expression levels of MdNF-Y genes in different tissues and under various abiotic stresses in subsequent experiments. In addition, most of the MdNF-Ys had ABRE cis-acting regulatory elements with a total of 169 elements, indicating that MdNF-Y genes play an important role in ABA response. All MdNF-Y genes contained more or less different types of light-responsive elements, although all MdNF-YBs possessed a G-Box element. A number of cis-acting regulatory elements involved in plant development were found in some of the MdNF-Y genes. For example, motif I, which was involved in plant root development [39], was only found in MdNF-YC6. A circadian cis-acting element, involved in circadian control [40], was only discovered in MdNF-YC4. An RY-element, participated in plant seed-specific regulation [41], was only detected in MdNF-YB13. Therefore, these MdNF-Y genes need to be further explored since they may play a critical role in apple growth. The specific cis-element analysis of the MdNF-Y genes is shown in Table S5. Actually, some reports in other plants have indicated that NF-Ys participated in response to abiotic stresses (cold [42], heat [25], drought [43], and salinity [28], root development [44], seed-specific regulation [44] and photoperiod-dependent flowering [29]. So, we further detected the expression levels of Figure 6. The cis-acting regulatory elements of promoters in apple MdNF-Ys genes. The blue, green, and orange blocks represent phytohormone and abiotic stress, plant development, and light responsive cis-elements, respectively. The number of cis-acting elements was indicated by different colors and circle sizes. The size of green to yellow circle represented the number of cis-acting elements.

Protein Interaction Analysis of MdNF-Y Genes
NF-YB, which does not contain a nuclear localization signal, needs to form a tight dimer with NF-YC in order to translocate from the cytoplasm to the nucleus, and subsequently binds to NF-YA to form a heterotrimeric complex. Then, the complex can interact with other regulatory factors to activate or repress the expression of downstream genes [45,46]. To determine the potential interactions between MdNF-YB and MdNF-YC, or between MdNF-YB and MdNF-YA members, we detected a physical interaction of some MdNF-Y gene products including five MdNF-YBs, three MdNF-YCs, and one MdNF-YA in the Y2H system (Figure 7). The full CDS of five MdNF-YBs were fused with the activation domain (AD) of the pGADT7 vector (expressing the "prey"), while the CDS of three MdNF-YCs and one MdNF-YA were fused to the DNA-binding domain (BD) of the pGBKT7 vector (expressing the "bait"). After pairwise co-transforming AD-and BD-expressing vectors into the Y2H Gold yeast strain, almost all yeast cells bearing both the MdNF-YB and MdNF-YC-expressing plasmids, or both MdNF-YB and MdNF-YA-expressing plasmids (except yeast cells bearing both MdNF-YB11 and MdNF-YA5) were capable of growth on SD-Leu/-Trp/-His medium indicating an interaction between prey and bait. Then, the surviving yeast cells were transferred to SD-Leu/-Trp/-His/-Ade medium. MdNF-YC5 showed strong interaction signals with MdNF-YB7, 11, and 17. MdNF-YC8 showed strong interaction signals with MdNF-YB1 and MdNF-YB8. MdNF-YB1 showed weak interaction signals with MdNF-YA5 and MdNF-YC4, and 5. MdNF-YC4 showed no interaction signals with MdNF-YB8, 11, or 17. MdNF-YA5 showed no interaction signals with MdNF-YB11 and 17. Taken together, these results suggested that many MdNF-YBs could interact with MdNF-YAs or three MdNF-YC genes on SD-Leu/-Trp/-His/-Ade medium.
with other regulatory factors to activate or repress the expression of downstream genes [45,46]. To determine the potential interactions between MdNF-YB and MdNF-YC, or between MdNF-YB and MdNF-YA members, we detected a physical interaction of some MdNF-Y gene products including five MdNF-YBs, three MdNF-YCs, and one MdNF-YA in the Y2H system (Figure 7). The full CDS of five MdNF-YBs were fused with the activation domain (AD) of the pGADT7 vector (expressing the "prey"), while the CDS of three MdNF-YCs and one MdNF-YA were fused to the DNA-binding domain (BD) of the pGBKT7 vector (expressing the "bait"). After pairwise co-transforming AD-and BD-expressing vectors into the Y2H Gold yeast strain, almost all yeast cells bearing both the MdNF-YB and MdNF-YC-expressing plasmids, or both MdNF-YB and MdNF-YA-expressing plasmids (except yeast cells bearing both MdNF-YB11 and MdNF-YA5) were capable of growth on SD-Leu/-Trp/-His medium indicating an interaction between prey and bait. Then, the surviving yeast cells were transferred to SD-Leu/-Trp/-His/-Ade medium. MdNF-YC5 showed strong interaction signals with MdNF-YB7, 11, and 17. MdNF-YC8 showed strong interaction signals with MdNF-YB1 and MdNF-YB8. MdNF-YB1 showed weak interaction signals with MdNF-YA5 and MdNF-YC4, and 5. MdNF-YC4 showed no interaction signals with MdNF-YB8, 11, or 17. MdNF-YA5 showed no interaction signals with MdNF-YB11 and 17. Taken together, these results suggested that many MdNF-YBs could interact with MdNF-YAs or three MdNF-YC genes on SD-Leu/-Trp/-His/-Ade medium.

Transcript Profiles Analysis of MdNF-Y Family Genes in Different Apple Tissues
To initially understand the function of the apple MdNF-Y genes, we analyzed the transcript profiles of the MdNF-Y gene family in roots, stems, leaves, receptacles, peel, sarcocarps, young fruits, and seeds using qRT-PCR. In general, the 43 MdNF-Y genes show diverse tissue-specific expression patterns and spatiotemporal expression characteristics (Figure 8). For the MdNF-YA subfamily, many genes had higher expression levels in vegetative and reproductive organs. For example, MdNF-YA3, 4, 7, 8 were strongly expressed in both young fruit and leaves. Moreover, MdNF-YA6 was highly expressed in roots, stem, and leaves, and MdNF-YA9 were highly expressed in roots. For the MdNF-YB subfamily, MdNF-YB1, 9,11,12,17,18 was highly expressed in sarcocarps, and MdNF-YB2, 4, 6, 13, 14, 16 were highly expressed in peel. MdNF-YB8, 18, 19 were highly expressed in root tissue. In addition, all MdNF-YC genes had higher expression levels in at least one reproductive organ, such as receptacles, peel, sarcocarps, young fruits, and seeds. However, it is worth noting that MdNF-Y genes sharing very high sequence and exon-intron structure similarity in duplicated genomic regions (Figure 2), exhibited similar expression patterns (Figure 8). For example, MdNF-YB1, MdNF-YB11, and MdNF-YB17 located in the duplicated genomic regions, were all highly expressed in sarcocarp. On the other hand, MdNF-YB8, MdNF-YB18, and MdNF-YB19 were highly expressed in root tissue. Overall, the overlapping but distinct expression patterns of MdNF-Y genes indicated that the MdNF-Y family plays a critical role in different growth and development stages of apples. seeds. However, it is worth noting that MdNF-Y genes sharing very high sequence and exon-intron structure similarity in duplicated genomic regions (Figure 2), exhibited similar expression patterns (Figure 8). For example, MdNF-YB1, MdNF-YB11, and MdNF-YB17 located in the duplicated genomic regions, were all highly expressed in sarcocarp. On the other hand, MdNF-YB8, MdNF-YB18, and MdNF-YB19 were highly expressed in root tissue. Overall, the overlapping but distinct expression patterns of MdNF-Y genes indicated that the MdNF-Y family plays a critical role in different growth and development stages of apples. Figure 8. Expression profiles of MdNF-Ys in various apple tissues including roots, stems, leaves, receptacles, sarcocarps, young fruits, and seeds. In the heat map, values were transformed to log2 (value). Green, low expression; black, medium expression; red, high expression. MdActin was used as an internal control. The results were based on three biological replicates and three technical replicates. Figure 8. Expression profiles of MdNF-Ys in various apple tissues including roots, stems, leaves, receptacles, sarcocarps, young fruits, and seeds. In the heat map, values were transformed to log2 (value). Green, low expression; black, medium expression; red, high expression. MdActin was used as an internal control. The results were based on three biological replicates and three technical replicates.

Expression Levels of MdNF-Y Genes under Different Abiotic Stresses
Previous studies have indicated that NF-Y not only regulates plant growing development but also responds to abiotic stresses [47]. Similarly, in our study, a very high number of abiotic and biotic stress-responsive elements were detected in the upstream promoter of MdNF-Y genes ( Figure 6). Therefore, we used qRT-PCR to investigate the response of the MdNF-Y gene family to abiotic stress. The transcriptional profiles of MdNF-Y genes under abiotic stresses from 0 to 24 h were monitored in this study (Figure 9). Under lowtemperature treatment, the expression of all MdNF-YAs and MdNF-YCs was upregulated and the expression levels of MdNF-YA genes were higher than those of MdNF-YC genes as a whole (Figure 9). In MdNF-YB gene family, MdNF-YB3, MdNF-YB5, and MdNF-YB9 gene expression levels reached their peak at 24 h whereas the expression of the rest of the MdNF-YB genes reached their peak at 12 h. The expression of MdNF-YB3 decreased at first but had recovered at 24 h. Under high-temperature treatment, the expression levels of most MdNF-Y genes were upregulated except MdNF-YC7 and MdNF-YC10 that were downregulated ( Figure 9C). Interestingly, of the MdNF-YA subfamily, MdNF-YA3, MdNF-YA5, and MdNF-YA8 were highly expressed under low-or high-temperature treatment. Under drought treatment, the MdNF-Y gene family members were all upregulated showing a strong response to water deficit (Figure 9). In MdNF-YB gene family, MdNF-YB10 and while the rest of MdNF-YB genes was upregulated. The expression of 59% of the MdNF-YB genes reached their peak at 24 h. However, the expression of all MdNF-YC genes reached its peak at 24 h. With the exception of the MdNF-YC4 and MdNF-YC6 genes, the expression of other genes was first downregulated but then upregulated.

Conservation, Evolutionary and Divergence of the MdNF-Y Gene Family in Apple
Studies on the NF-Y genes in plant species have been accumulating since the function and regulatory mechanism of the first plant NF-Y gene was identified [47][48][49]. To date, NF-Y genes have been identified from simple model plants to more complex plants, and from monocotyledons to dicotyledons [2,15,16,50,51], such as Arabidopsis, maize, rice, grapes, rubber trees, and so on. However, there is few reports concerning the NF-Y gene family in apple. The number of NF-Y genes identified from plants varied from 13 in tomato [52] to 50 in maize [53]. In this study, we first systematically identified and analyzed the NF-Y gene family in apple at the genomic level and discovered 43 MdNF-Y genes in the apple genome, which was a greater number than for most of the other plants investigated so far.
Duplications at gene, chromosomal, or entire genomic level have been considered a major source of evolution, contributing to the origin of new gene functions and expression patterns [54]. Therefore, we further identified 27 paralogous pairs from segmental duplication events in apple, including 7 MdNF-YAs, 14 MdNF-YBs, and 6 MdNF-YCs (Figure 4 and Table S2). However, only 11 or 12 paralogous segmental duplication events had happened in S. bicolor L. or maize, while a total of 42 and 50 NF-Y proteins were respectively identified in those species [53,55]. Moreover, the genome size of plant NF-Y members studied greatly varied, from 265 Mb in peach to 742 Mb in apple, and only five pairs of paralogous events were found in peach [16]. These results all suggested that a potential correlation between MdNF-Y gene duplications and genome expansion existed during the species evolution.
In addition, the majority NF-Y genes from M. domestica were located in syntenic regions of other eight species genomes (P. betulifolia, P. persica, F. vesca, V. vinifera, A. thaliana, B. rapa, O. sativa and Z. Mays) ( Figure 5 and Table S3). From the evolutionary data on NF-Y genes, we found that the number of ortholog pairs of apple and other species were related to their evolutionary relationship. It is widely known that M. domestica, F. vesca, P. betulifolia, and P. persica all belong to the Rosaceae and have a closer relationship than other species selected. As expected, they have significantly more ortholog pairs with apples than other species. Therefore, we concluded that the conservation of gene duplication during species evolution also supports the great differentiation of genome evolution. Meanwhile, the ortholog pairs from other plants can provide references to determine the function and mechanisms of apple NF-Y transcription factors.
Gene structural analysis of MdNF-Y subfamilies showed that there were many similarities in each subfamily with the corresponding subfamily in other species. For example, the genes in the MdNF-YA subunit, were interrupted by at least four introns. While, many MdNF-YB genes lacked introns. Intriguingly, MdNF-YB17 and CsNF-YB16, as the longest apple or tea NF-YB gene, both have five introns [56]. The result was consistent with the gene structure of PpNF-Y and CsNF-Y families [14,16].

Differentially Expression Pattern of MdNF-Y Genes in Apple Tissues
To date, NF-Y genes have been found to play critical roles in regulating flower and fruit development, as well as various other physiological processes; however, their roles in apple have remained unclear. Therefore, in this study, we predicted the functions of the apple NF-Y genes based on their other species ortholog pairs in syntenic regions of the two genomes ( Figure 5).
In general, the NF-Y genes exhibited distinct spatiotemporal expression patterns in apple tissues and organs. However, NF-Y genes with very high sequence and exon-intron structure similarity have similar expression patterns and gene function in different apple tissues. For example, MdNF-YB1 and MdNF-YB11, located in the duplicated genomic regions, were all highly expressed in the sarcocarp and receptacle (Figures 4 and 8). Apple fruits are considered 'false fruits', since the sarcocarp has developed from the receptacle. Therefore, we hypothesized that these three homologous genes may play a synergistic role in the development of the receptacle into the fruit. In addition, the interactions between MdNF-YB17 and MdNF-YC5 or MdNF-YC8 proteins were identified by the Y2H experiments (Figure 7). Interestingly, MdNF-YC5 and MdNF-YC8 were also highly expressed in the sarcocarp or peel (Figure 8), indicating that they may play an important role in apple reproductive growth. Actually, many studies of NF-Ys in other plants have supported this hypothesis. In Arabidopsis, NF-YB and NF-YC subunits could interact with CONSTANS (CO) to form complexes, and further affected FLOWERING LOCUS T (FT) expression to induce the floral transition [2,23,57]. Analogously, ZmNF-YA3, in complex with CO and flowering promoting factor1 (FPF1) could bind to the FT-like12 promoter to promote early flowering in maize [29]. In addition, overexpression of the TaNF-YB4 gene significantly improved transgenic wheat grain yield [58].
LEAFY COTYLEDON1 (LEC1), also known as NF-YB9 is a key regulator that controls the complex process of seed development in Arabidopsis [44]. AtNF-YB9 (LEC1) and AtNF-YB6 (LEC1-Like) were both expressed in seeds [59]. Previous research has suggested that the gene expression patterns are related to the complex process of seed development which is highly coordinated both temporally and spatially in cellular processes [60]. In this study, we found that MdNF-YB13 gene promoter contained an RY-element, which is involved in plant seed-specific regulation ( Figure 6). Interestingly, MdNF-YB13 and MdNF-YB10, located in the duplicated genomic regions, are ortholog genes of AtNF-YB6 (LEC1-Like) and AtNF-YB9 (LEC1) (Figureas 4 and 5 and Table S3), and also show high expression levels in seeds compared with other tissues (Figure 8). Recent studies have demonstrated that LEC1 combinates with other TFs, such as ABA-RESPONSIVE ELEMENT BINDING PROTEIN3 (AREB3), bZIP67, and ABI3 to regulate the diverse stages of seed development [61]. Moreover, previous studies have shown that LEC1 acts as a regulon to regulate hormone synthesis genes and as an integrator for light-and hormone signals to play an important role in the development of plant embryo [62]. The result of cisacting element analysis also showed that the promoter region of MdNF-YB13 contained those important plant endogenous hormone and light elements during seed development, such as ABRE (ABA), TGA-element (auxin), and G-box (light). Taken together, our study provides a direction for future research of the analysis of the specific regulatory mechanism of MdNF-YB10 and MdNF-YB13 during apple seed development.

Function of MdNF-Y Genes in Abiotic Stress
It has been reported that TFs, including MYB, WRKY, and NAC participated in the fight against abiotic stress to help plants resist or optimize the changes in the environment [63][64][65][66]. To date, numerous studies have demonstrated that NF-Y genes also played an important role in abiotic stress response. For instance, overexpression of AtNF-YB2 and AtNF-YB3 in Arabidopsis specifically conferred tolerance to drought and heat stress, respectively [25]. Allogenic overexpression of the CdtNF-YC1 transcription factor from bermudagrass enhanced the transgenic rice tolerance under the drought and salt treatment, through ABA-dependent and ABA-independent pathways [28]. Another recent study has shown that PdNF-YB21 positively regulates the tolerance to drought stress by ABA-mediated IAA transport in Populus [30].
In addition, it is widely known that root is the major organ to respond to osmotic stress caused by drought and salt [43]. In this study, MdNF-YA6, MdNF-YA9, MdNF-YB8, MdNF-YB9, MdNF-YB18, MdNF-YB19, MdNF-YC7, and MdNF-YC8 are mainly or specifically expressed in roots ( Figure 8). Further, we found the promoter region of MdNF-YB8, MdNF-YB9, MdNF-YB18, and MdNF-YC7 contains drought cis-acting element MBS (CAACTG), and MdNF-YA6 contains defence cis-element TC-rich repeats (ATTCTC-TAAC/GTTTTCTTAC) ( Figure 6). Meanwhile, the RT-PCR results also showed that these genes were highly expressed under drought and salinity stress ( Figure 9). Therefore, we hypothesized that they may response to abiotic stress by regulating root growth. Existing research has also verified this hypothesis. For example, overexpression of the transcription factor AtNF-YB3 increased the length of primary root [67], and improved drought and heat tolerance in A. thaliana [25]. Overexpression of PdNF-YB21 in poplar promoted root growth with highly lignified and enlarged xylem vessels, resulting in increased drought resistance [30]. TaNF-YB4 plays an important role in root development and in nitrogen and phosphorus uptake in wheat [58]. Higher levels CdtNF-YC1 was detected in roots of bermudagrass and overexpression of CdtNF-YC1 elevated tolerance to drought and salt stress in transgenic rice [28].
In summary, the MdNF-Y genes displayed different degrees of responses to abiotic stress. The specific mechanisms were studied in model plants. Although the detailed molecular mechanisms of the responses of MdNF-Y genes to abiotic stress remain unclear, this study points to a number of genes that deserve further exploration in future studies.

Plant Materials and Treatments
The apple Xinjiang No. 1 tissue culture seedlings used for stress treatment was cultured in a specific-medium with Murashigeand Skoog (MS) medium, 0.8% agar, 0.5 mmol L −1 indole-3-butytric acid (IBA), and 0.7 mmol L −1 6-benzylaminopurine (6-BA). Apple tissue culture seedlings were first grown in specific-medium formulation under 16 h light/8 h dark at 25 • C environmental conditions for thirty days. Thirty days later, 10% polyethylene glycol (PEG) 6000, 100 mmol L −1 NaCl, and 100 mmol L −1 Abscisic acid (ABA) were added to the specific medium, respectively, to induce the stress response of apple tissue culture seedlings and cultivated under the above environmental conditions. Besides, apple tissue culture seedlings were cultured in a specific medium, and the environmental temperature was adjusted to 4 • C and 40 • C, respectively. After the above five stress treatments, samples of apple tissue culture seedlings were collected at 0, 6, 12, and 24 h respectively, frozen rapidly with liquid nitrogen, and stored in −80 • C refrigerator for succedent experiments [68]. For all treatments, three biological replicates were collected.
The different apple tissues, including root, stem, leaves, peel, receptacle, young fruits (20 days after flowering), sarcocarp, and seed was obtained from seven-year-old 'Xinjiang No.1' apple trees at the experimental station (longitude 120 • 39 E, latitude 36 • 27 N) of Qing Dao Agricultural University in 2018. These tissues materials were frozen rapidly with liquid nitrogen and stored in −80 • C refrigerator for subsequent experiments. For all sample, three biological replicates were collected.

Identification of Apple MdNF-Y Genes
The amino acid sequences of Arabidopsis 30 AtNF-Y family were obtained from the TAIR database (https://www.arabidopsis.org/). After that, The NF-Y domains were searched from BlastP (https://www.rosaceae.org/blast/protein/protein) in the NCBI database in the apple genome in the NCBI database. The Hidden Markov Models (HMM) profile of the conserved domains of NF-YA (PF02045) and NF-YB (PF00808) were downloaded from the Pfam database (http://pfam.xfam.org/). Then, they were used to search for protein sequences in the apple genome. Apple candidate NF-Y gene family members obtained by the above two methods. To ensure accuracy, SMART (http://smart. embl-heidelberg.de/) and CCD programs were used to verify the existence of conserved Pfam and complete NF-Y domains. The amino acid number, PI, and molecular weight of the identified NF-Y protein sequence was obtained from the ExPASy website (http: //web.expasy.org/). The location of NF-Y gene on chromosome was determined by Gene Structure Display Server (GSDS, http://gsds.cbi.pku.edu.cn) [69].

Phylogenetic, Conversed Motifs, and Gene Structure Analysis of MdNF-Ys
The CDS and DNA sequences of MdNF-Y were obtained from the Apple Genome Browser. Using MEGA 7 software, the Neighbor-Joining (NJ) method was used to construct the phylogenetic tree of apple and Arabidopsis NF-Y protein sequences. Bootstrap was set to 1000 replicates. The protein sequence of the candidate MdNF-Y gene was analyzed using MEME (http://meme-suite.org/) software [72]. Set to look for 20 motifs. Phylogenetic tree, conserved motifs, and gene structure of apple MdNF-Y gene family was visualized on the TBtools toolkit [73].

Prediction of Cis-Acting Elements in Promoters of MdNF-Ys
The Plant CARE database was employed to predict the potential cis-acting elements in the 1500 bp promoters upstream region of the apple MdNF-Ys gene family [74], and visualized on the TBtools toolkit [73]. Details information for the promoters used were listed in Table S4.

Yeast Two Hybrid Assays
The CDS of MdNF-YB, MdNF-YA, or MdNF-YC genes were cloned into prey vector pGADT7 with the activating domain (AD). The CDS of MdNF-YA or MdNF-YC genes were cloned into bait vector pGBKT7 with the DNA binding domain (BD). The primers were listed in Table S6. Two constructs were co-transformed into the Y2H Gold yeast strain according to the protocol (www.weidibio.com). Then, these yeast strains were cultured on SD(-Leu/-Trp) growth medium (Clontech) and selected on SD(-Leu/-Trp/-His) and SD(-Leu/-Trp/-His/-Ade) screening medium (Clontech).

Quantitative Real-Time RT-PCR Analysis
RNA from various stress treatments (ABA, drought, salinity, heat, and cold) or different apple tissues (root, stem, leaves, peel, receptacle, young fruits (20 days after flowering), sarcocarp, and seed) was extracted with the EASYspin Plant RNA Rapid Extraction Kit (YPHBIO, Beijing, China), and the extracted RNA concentration was determined by the instrument Nano Drop 2000 (Gene Company Limited, Hong Kong, China). The cDNA was obtained by using the reverse transcription kit (Takara, Dalian, China) according to the manufacturer's methods. Quantitative real-time PCR (qRT-PCR) was performed with ChamQ SYBR Color qPCR Master Mix (Without ROX) (Vazyme, Nanjing, China) in Roche machine (Roche, Shanghai, China). The PCR program was as followed: 95 • C for 2 min, 40 cycles of 95 • C for 30 s, 56 • C for 30 s and 72 • C for 30 s with a final dissociation stage. The internal reference gene, MdActin (nucleotide, CN938023), was used to normalize the expression levels of the tested genes. The primers for qRT-PCR experiment were designed on NCBI-BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) and listed in Table S6. MdActin was used as an internal control. The relative expression of MdNF-Y genes was calculated with 2 −∆∆Ct methods [75] and figured by GraphPad.Prism.5.0 software. Three biological replicates with three technical replicates each were measured. Transcripts profiles from different apple tissues were visualized heat-mapped using the TBtools toolbox [73].

Conclusions
In this work, 43 MdNF-Y genes (11 MdNF-YAs, 22 MdNF-YBs, and 10 MdNF-YCs) were identified and their evolutionary, structure, biological function, and expression pattern were analyzed. Based on prediction and experimental data, MdNF-Ys might play an important role in apple development and response to five abiotic stress (ABA, drought, heat, cold, and salinity). Our findings will contribute a foundation for further study of the functional and regulatory mechanisms controlled by the NF-Y gene family in apple.