The Arabidopsis thaliana Nuclear Factor Y Transcription Factors

1Nuclear factor Y (NF-Y) is widespread in plants, animals, and other eukaryotes, and is also termed CCAAT Binding Factor (CBF) or Heme Activator Protein (HAP). 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. Individual subunits of NF-Y cannot regulate transcription independently; instead, they must function in heterodimers or heterotrimers. In yeast and mammals, each subunit of NF-Y is encoded by a single gene, but these genes have multiple splicing forms and undergo various post-translational modifications. In mammals, the physiological function and underlying molecular mechanism of the NF-Y complex has been extensively characterized in different cellular processes, such as endoplasmic reticulum stress, DNA damage, and cell cycle regulation. In plants, each subunit of NF-Y is encoded by multiple members, which further form different sub-families. A previous classification system described 36 NF-Y members (10 NF-YA, 13 NF-YB, and 13 NF-YC) in Arabidopsis. This system considered the plant homologs of Negative Cofactors (NC2), DNA Polymerase II Subunit B3 (Dpb3), and Dpb4 as members of the NF-Y family; however, NC2, Dpb3p, and Dpb4p do not functionally overlap with NF-Y. NC2 associates with TATA-binding protein (TBP) to bind TATA boxes, whereas Dpb3p and Dpb4p participate in the complex with DNA polymerase and the complex of chromatin remodeling. Therefore, the current consensus on Arabidopsis NF-Y members excludes AtNF-YC11/B12/B13 (NC2 subfamily) and AtNF-YC10/C13/B11 (Dpb3/4 subfamily), but does include AtNF-YC12. Thus, in the updated scheme, Arabidopsis has 30 members of the NF-Y family, 10 from each family.

2The initial reports of NF-Y genes in plants date back to the 1990s. In the past decades, many studies have shed light on the biological functions of individual NF-Y subunits in Arabidopsis and other plant species, showing that this complex acts in gametogenesis, embryogenesis, seed development, flowering time regulation, primary root elongation, abscisic acid signaling, drought resistance, the endoplasmic reticulum stress response, hypocotyl elongation, and so on (;; and references therein). All these studies suggest that the NF-Y gene family is a powerful and mysterious gene family and important for many aspects of plant life.

3The A subunits of NF-Y generally localize to the nucleus and most NF-YA proteins can bind to the CCAAT cis-element in the promoter regions of target genes, but with different affinities. Protein structure analysis indicated that two conserved α-helix domains (A1 and A2) are present in the core regions of NF-YA subunits (Figure 1). The 20-amino-acid α helix A1 is in the N-terminal of the core region and functions in the interaction with NF-YB and NF-YC subunits, whereas the 21-amino-acid α helix A2 is in the C-terminal and provides sequence-specificity for recognition and binding of the CCAAT cis-element.

4The NF-YB and NF-YC subunits contain the highly conserved Histone Fold Domain (HFD, also termed as Histone Fold Motif, HFM) (Figure 1), which functions in protein-DNA and protein-protein interactions. The HFD domains of NF-YB and NF-YC subunits are more closely related to the core histone H2B and H2A, respectively. Typically, the HFD domain of NF-YB/YC is formed by a minimum of three α-helices (α1, α2, and α3) separated by two loops, similar to the HFD domains of histones H2B and H2A, and even similar to the HFD domain of the β and α subunits of NC2. In NF-YB, the α1 helices contain the putative DNA-binding domain. In NF-YC, the α1 helices contain a few conserved amino acids which supposed to be the putative DNA-binding domains. Indeed, the α1 helix makes essential stabilizing contacts with the DNA backbone flanking the central CCAAT pentamer. NF-YB and NF-YC form a heterodimer via their HFDs, specifically via α2 and α3. The fourth helix of the conserved domain of NF-YB and NF-YC (called αC) has been proposed to function in interactions with other protein in mammals. Formation of NF-YB/YC heterodimers is necessary for the translocation of NF-YB from the cytoplasm to the nucleus since NF-YB family members lack a nuclear localization signal (NLS), in contrast to the NF-YA and NF-YC subunits. The HFD domains of NF-YB and NF-YC subunits assemble on a head-to-tail fashion to form a dimer, thus producing a structural scaffold for DNA-binding and interaction with NF-YA subunits.

5Crystal structure analysis of the NF-Y/CCAAT complex in mammals indicated that NF-YA binds to NF-YB/YC and inserts an α-helix into the minor groove of DNA, thus providing a sequence-specific contact to the CCAAT box. By contrast, NF-YB/NF-YC subunits form heterodimers that bind to the DNA sugar-phosphate backbone, mimicking the nucleosome H2A/H2B-DNA assembly. NF-YB/NF-YC interacts with DNA through non-specific HFD-DNA contact, which is similar to those of the core histone H2A/H2B. NF-YB/NF-YC also interact with NF-YA to produce the NF-Y complex with its sequence-specific recognition properties and nucleosome-like capabilities of non-specific DNA-binding. Thus combinations of subunits lead to stable DNA-binding activity of the NF-Y complex. Recently, the first crystal structure of the Arabidopsis NF-YB6/NF-YC3 dimer has been solved, which revealed that AtNF-YB6 and AtNF-YC3 subunits interact in a head-to-tail fashion and form a classical histone-like pair. The structural information on the NF-Y/CCAAT complex has given crucial insight into the molecular mechanism responsible for the architecture of the NF-YB/NF-YC heterodimer and the NF-Y heterotrimer, and also for the capacity of recognition and binding to DNA of NF-Y complexes.

6There are 30 predicted NF-Y members in the Arabidopsis genome; in theory, this could result in about 1000 heterotrimeric combinations. To define the molecular mechanism of NF-Y in plants, two fundamental questions about NF-Y complexes should be considered. First, how many heterotrimeric combinations actually exist in vivo and, second, what determines the specificity of those interactions and of DNA-binding. To define the unique NF-Y complexes, tissue- and development-specific expression patterns for all the subunits of Arabidopsis NF-Y have been investigated using stable promoter:beta-glucuronidase (GUS) fusion reporter lines. The different subunits of NF-Y are specifically expressed in different tissues and organs during special developmental stages, or in response to environmental changes, suggesting that only some combinations of NF-Y subunits can be assembled and act in different developmental stages or under certain stimuli or conditions. In addition, yeast two-hybrid and three-hybrid systems have also been used to detect protein-protein interactions and formation of heterotrimers between Arabidopsis NF-Y subunits. Yeast two-hybrid assays indicated that most of the NF-Y subunits of the same type (A-A, B-B, or C-C) could not form homodimers or heterodimers, and NF-YA and NF-YB show little or no interaction. However, NF-YB and NF-YC subunits could interact in many combinations and form different heterodimers. These systematic studies of the protein interactions between NF-Y subunits have facilitated the discovery of complete NF-Y complexes, but tissue, development, and even time-dependent specific combinations should also be considered.

7Research based on yeast three-hybrid and co-immunoprecipitation assays showed that NF-Y could function as a NF-YA-YB-YC heterotrimeric complex in vivo (Table 1). In theory, Arabidopsis NF-Y subunits can form 1000 different heterotrimeric combinations; however, only a few NF-Y complexes have been verified through molecular and biochemical studies (Table 1). One possible reason is that some or many heterotrimeric of NF-Y may be transient and highly dynamic in vivo, and thus hard to detect. Nevertheless, with the emergence of new experimental methods and in-depth studies of NF-Y family members, more and more NF-Y complexes are being identified and explored.

8In the past few years, a growing number of studies have reported that subunits of NF-Y not only form heterodimeric and heterotrimeric complexes, but also interact with other proteins in various kinds of complexes. For example, 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. In addition, basic region/leucine zipper motif (bZIP) type transcription factors bZIP28 and bZIP67 interact with NF-Y subunits to regulate endoplasmic reticulum (ER) stress and seed development, respectively. Phytochrome interacting factor 4 (PIF4) interacts with LEAFY COTYLEDON1 (LEC1/NF-YB9) to regulate hypocotyl elongation. 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. Studies of the interaction of NF-Y with other proteins regulating target gene expression have largely improved our understanding of the physiological roles of NF-Y in many biological activities (Table 1).

9Since NF-Y regulates gene expression mainly as a protein complex, it is important to understand its specific regulatory mechanisms. Previous reports have proposed that NF-Y modulates expression of downstream target genes mainly by two mechanisms (Figure 2). In the first mechanism, the NF-YB-YC heterodimer assembles in the cytoplasm, then translocates into the nucleus, where it interacts with NF-YA to form an active heterotrimer (Figure 2A). The NF-YA-YB-YC complex mainly binds to the CCAAT box in the promoter regions of the downstream target genes through NF-YA, and regulates the expression of target genes, which is a highly conserved transcriptional regulation mechanism for NF-Y complexes in yeast, mammals, and plants. For example, the AtNF-YA4-YC2-YB3 complex binds to the CCAAT cis-element in the promoter region of BINDING PROTEIN 3 (BiP3) through AtNF-YA4 and up-regulates the expression of ER stress-induced genes. In addition, the AtNF-YA6 subunit interacts with the AtNF-YB6/YC3 dimer and the resulting trimer can directly bind CCAAT cis-elements, even a probe containing the CCAAT box derived from the human Heat Shock Protein70 (HSP70) promoter. Most NF-Y complexes bind to the CCAAT box mainly through NF-YA; however, in plants, a recent study in rice reported that OsNF-YB1 might bind to the CCAAT box of Sucrose Transporter1 (SUT1), SUT3, and SUT4. However, the in vitro and in vivo data are not sufficient to prove OsNF-YB1 binding to CCAAT box directly, since some sequences flanking the CCAAT box could bind via NF-YB.

10In the second molecular mechanism, the NF-YB/NF-YC heterodimer associates with specific transcription factors to form a complex (NF-YB-YC-transcription factor), and regulates the expression of their target genes through the binding of transcription factors to specific cis-elements in the promoters of various target genes (Figure 2B). In this mechanism, NF-YA probably could suppress the formation of the NF-YB-YC-transcription factor trimeric complex by preventing the combination of transcription factors with NF-YB-YC. Although this kind of transcriptional regulation has been proposed by many studies, while the direct molecular evidences are still required since the opinion of compete of YA with TF derived from supposes upon some opposite observations. For example, AtB9-C2-bZIP67 directly binds to the ABA-response elements (ABREs) through bZIP67 in the promoter regions of SUCROSE SYNTHASE 2 (SUS2) and CRUCIFERIN C (CRC), then activates the expression of SUS2 and CRC and promotes seed development. However, NF-YA subunits strongly inhibited the expression of CRC by competing with bZIP67 to form an NF-YA-YB9-YC2 complex, which indicated different members of the NF-Y subunits play distinct roles in plant development and growth. In mammals, genome-wide association with selected transcription factors indicated that the NF-Y complex directly binds to the CCAAT box and associates with other cis-elements by interacting with different transcription factors. Recently, a ChIP-sequencing approach in rice identified genome-wide downstream targets of OsNF-YB1, indicating that OsNF-YB1 directly binds to the CCAAT box and associates with the GCC box by interacting with an ERF-type transcription factor.

11Some special transcriptional regulatory mechanisms do not fit these two major regulatory models in Arabidopsis. For instance, AtNF-YA2 can directly bind to the NFYBE cis-element (not the CCAAT box) of the SUPPRESOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) promoter to regulate the expression of SOC1. In addition, a recent study indicated that NF-YC subunits (C1, C3, C4, and C9) directly interact with HISTONE DEACETYLASE 15 (HDA15), recognize the promoter of hypocotyl elongation-related genes, and repress their expression through the deacetylation of H4 in a light-dependent manner. A previous study also showed that NF-Y directly activates the expression of SOC1 partly through the H3K27 demethylase RELATIVE OF EARLY FLOWERING 6 (REF6; ). Therefore, the NF-Y complex may function together with various epigenetic factors to regulate the transcription of downstream target genes. Certainly, deeper research will discover more and more novel mechanisms.

12NF-Y transcription factors play crucial roles in embryogenesis. The first identified and extensively studied NF-Y subunit acting in embryogenesis and seed maturation is NF-YB9, which was identified as LEAFY COTYLEDON1 (LEC1;;; ). NF-YB9/LEC1 plays multiple essential roles in embryogenesis and post-embryonic development in Arabidopsis, where it is required to maintain the fate of embryonic cells and prevent immature seeds from germinating prematurely. NF-YB9 and LEC1-LIKE (L1L/NF-YB6) affect embryo development through induction of genes related to embryogenesis and cellular differentiation.

13Based on the tissue-specific expression patterns and mutant phenotypes, many Arabidopsis NF-Y genes participate in embryo development. For example, NF-YA1, YA2, A3, A4, A6, A7, A8, and A9 are expressed in the embryo and may affect embryo development. Moreover, the transgenic plants overexpressing NF-YA1, A5, A6, or A9 show hypersensitivity to ABA during seed germination and promotion of the vegetative-to-embryonic transition. However, single or double mutants of the four NF-YA genes do not have detectable phenotypes. NF-YA3 and A8 show their highest expression in the embryo from the globular to torpedo stages. The nf-ya3 nf-ya8 double mutants are embryo lethal, but the nf-ya3 and nf-ya8 single mutants do not display an obvious phenotype, indicating that NF-YA3 and NF-YA8 function redundantly in early embryogenesis of Arabidopsis. Therefore, multiple individual NF-YA subunits likely play redundant roles in embryo development and seed germination.

14Recent research indicates that NF-YC subunits are also involved in seed germination through ABA responses and different NF-YC subunits have both unique and opposing functions in ABA-mediated seed germination. For example, Arabidopsis nf-yc4 single mutants are hypersensitive to ABA during seed germination, but nf-yc3 nf-yc9 double mutants are hyposensitive to ABA. Although extensive genetic evidence indicated that many individual NF-Y subunits function in embryonic and seed development, and seed germination, most studies focus on single subunits; therefore, how different subunits act in the NF-Y complexes to regulate these process remains elusive.

15NF-Y also functions in plant photomorphogenesis, especially in hypocotyl elongation. Previous genetic evidence showed that overexpression of AtNF-YA1 and NF-YA9 changed cell identity, and led to the hypocotyl becoming greener and swollen. In addition, overexpression of most NF-Y A subunits significantly reduced hypocotyl elongation. However, the underlying molecular mechanism by which different A-type subunits of NF-Y regulate hypocotyl elongation requires further investigation.

16After seed imbibition, induced expression of NF-YB9/LEC1 resulted in longer hypocotyls, compared with non-induced seedlings, and mutants of NF-YB9 had short hypocotyls. In addition, genome-wide chromatin immunoprecipitation (ChIP) microarray (ChIP-chip) analysis revealed that a number of auxin and cell elongation-related genes (such as YUC10, IAA5, and IAA19) are putative target genes of NF-YB9/LEC1. Moreover, previous studies reported that the evening complex (EC, ELF4-ELF3-LUX) binds to the promoter regions of PIF4/PIF5 through the transcription factor LUX to repress the expression of PIF4 and PIF5 in the evening. Further study found that PIF4 could interact with NF-YB9 to coordinately regulate the expression of IAA19 and thus control the elongation of the hypocotyl in the dark (Figure 3). At dawn, PIF4 protein interacts with photoactivated phytochrome and then is degraded through the 26S proteasome pathway. With prolonged illumination, the ELF4-ELF3-LUX complex accumulates, and reaches a peak at dusk. The accumulated ELF4-ELF3-LUX complex can repress the transcription of PIF4, thus suppressing the binding of the PIF4-NF-YB9 complex to the promoter region of IAA19 and inhibiting its expression. Therefore, Arabidopsis hypocotyl elongation is inhibited at this stage. At midnight, the levels of the ELF4-ELF3-LUX EC decrease, allowing the restoration of PIF4 mRNA levels and accumulation of PIF4 protein. So, PIF4 can recruit NF-YB9 to form a complex and target the promoter region of IAA19, positively regulating IAA19 expression. Thus, Arabidopsis hypocotyl elongation gradually increases at this stage (Figure 3).

17Recent reports demonstrated that five NF-YC subunits (C1, C3, C4, C6, and C9) also function in hypocotyl elongation by interacting with HDA15. The triple mutant of nf-yc3 nf-yc4 nf-yc9 displayed longer hypocotyls under blue, and red light conditions; therefore these NF-YC subunits positively regulate photomorphogenesis. Although all three types of NF-Y subunits function in hypocotyl elongation, the underlying molecular mechanism by which NF-Y complexes regulate photomorphogenesis and hypocotyl elongation remains elusive.

18The timing of the transition from vegetative to reproductive development is crucial for reproductive success of flowering plants. In Arabidopsis, the major flowering pathways include photoperiod and GA pathways, which promote flowering in response to seasonal changes in day length and the endogenous content of GA, respectively. The interactions of these flowering pathways regulate the expression of two floral pathway integrators, FT and SOC1, which in turn activate the genes involved in the formation of floral meristems. Overexpression of many individual NF-Y subunits (such as NF-YA1, YA4, NF-YB1, B2, B3, NF-YC1, C2, C3, C4, and C9) altered flowering time, indicating that NF-Y complexes regulate flowering time by a highly redundant and complicated mechanism. Multiple individual subunits of NF-Y can interact with CO and affect the transcript levels of FT and SOC1, two key integrators in the flowering time pathway, thus resulting in early or late flowering (Figure 4A).

19In the photoperiod-dependent pathway, AtNF-YB2 and B3 have additive roles in the positive regulation of FT expression and flowering time. Further studies indicate that the NF-YC subunits AtNF-YC3, C4, and C9 interact with AtNF-YB2, B3, and CO to form NF-YB-YC-CO complexes, and NF-YB and NF-YC subunits are required for the transcriptional activation of FT mediated by CO under long-day (LD) conditions (Figure 4B). In addition, two CO-Responsive Elements (CORE1 and 2) present in the proximal region and the CCAAT box present in the distal region of the FT promoter are crucial for regulation of FT expression. CO directly binds to CORE2, interacts with NF-Y complexes, which can bind to the CCAAT box, resulting in chromatin loops, and positively regulates the expression of FT. Whether CO associates with the CORE2 in the NF-YB-YC-CO complex and how protein binding to the CORE can drive FT expression remain to be investigated. Conversely, some NF-Y subunits can delay flowering by repressing the transcription of FT. For example, the overexpression of AtNF-YA1, A4, and AtNF-YB1 reduced the transcript level of FT and postponed flowering. Further studies have shown that NF-YA might delay flowering by preventing the interaction between CO and NF-YB/NF-YC. For instance, overexpression of NF-YA1 and A4 prevents the interaction between CO and the NF-YB/NF-YC heterodimer, finally reducing the expression of FT and postponing flowering. Thus, different subunits of NF-Y might act in different complexes and have opposite effects on the final phenotypic output.

20In the regulation of flowering time, both the NF-YB-YC-CO complexes and NF-YA-YB-YC complexes can bind to FT promoter. Indeed, both the transcript and protein levels of CO and NF-Y undergo rhythmic changes during the day; thus the complexes of NF-YA-YB-YC and the complexes of NF-YB-YC-CO likely change dynamically over time (Figure 4C). The protein level of CO decreases gradually from night to dawn. Meanwhile, increasing NF-YA1-YB-YC forms a trimer and binds to the FT promoter to repress FT expression. In addition, CO protein accumulates gradually during the day and its expression peaks at dusk; thus CO can compete with NF-YA1 to form the trimeric complex NF-YB-YC-CO and promote the transcription of FT leading to peak expression at dusk.

21NF-Y also affects flowering time by interacting with DELLA protein in the GA-dependent pathway (Figure 4). A study using ChIP showed that the NF-YA2/B2/C9 complex could interact with CO and DELLA and bind to the NFYBE cis-element in the promoter of SOC1 through NF-YA2. The NF-Y complex then recruits REF6 to demethylate H3K27me3, thus promoting SOC1 expression and leading to early flowering. Under LD conditions, RGA (a member of the DELLA protein family) not only interacts with NF-YB2 and CO but also represses the interaction of CO with NF-YB2. This interaction was tested through in vitro pull-down assays, but in vivo tests need further investigate. In addition, a previous study reported that NF-YB2 and CO form a complex with part of NF-YC family members to affect FT expression. Therefore, the effect of RGA on CO-NF-Y binding to the promoter of SOC1 very likely represses the activity of CO in activating FT expression.

22Arabidopsis NF-Y also has an important role in the responses to abiotic stresses, such as drought, salt, cold, and heat (Table 2). Many NF-YA, NF-YB, and NF-YC subunits play key roles in the drought response. Overexpressing NF-YA5 reduced leaf water loss and increased resistance to drought stress compared with the wild type, whereas nf-ya5 mutants were more sensitive to drought stress, which suggests that NF-YA5 positively regulates drought stress responses. Beside NF-YA5, overexpression of other NF-YA genes such as YA2, A3, A7, and A10 also increased the tolerance to drought, which suggests that NF-Y subunits share overlapping functions in stress responses. In addition to NF-YA, NF-YB is also involved in Arabidopsis drought stress tolerance. Overexpression of NF-YB1 enhanced plant drought resistance. However, this did not affect the expression of genes involved in ABA signaling, indicating that NF-YB1 might regulate drought stress responses independent of ABA signaling.

23A previous study demonstrated that overexpression of NF-YC1 improved freezing resistance, whereas nf-yc1 mutants exhibited decreased freezing resistance, which suggests that NF-YC1 positively regulates freezing responses. Further research found that NF-YC1 improved freezing resistance by binding to the CCAAT cis-elements in the promoter region of Xyloglucan Endotransglucosylase/Hydrolase 21 (XTH21) in Arabidopsis. Further, overexpression of NF-YA1 enhanced plant resistance to salt stress by increasing the expression of Abscisic Acid Insensitive3 (ABI3) and Abscisic Acid Insensitive5 (ABI5; ). In addition, the NF-YA2-B3-C10 ternary complex enhanced the expression of the heat stress-inducible gene HEAT SHOCK FACTOR A3 (HsfA3) during heat stress responses in cooperation with DREB2A.

24In addition to these above functions, NF-Y also plays roles in other plant processes. For example, NF-YA2, A3, and A5 participate in nitrogen nutrition; NF-YA2, NF-YA10, and NF-YB2 function in the control of primary root growth; NF-YB1 affects shoot apical meristem growth; NF-YC4 is involved in starch and protein metabolism; and NF-YA5/B9/C9 associates with chlorophyll biosynthesis.

25In plant, the different individual subunits of NF-Y have attracted extensive attention in recent years, but the many functions of the NF-Y complex remain only partially defined. In contrast to yeast and mammals, plants have a large NF-Y family, generally having multi-gene families encoding each subunit. This provides more combinations of NF-YA-YB-YC complexes in different developmental stages or under certain conditions, and increases functional complexity. This indicates that NF-Y is widely involved in the intricate regulatory processes in plants, compared with its more narrow roles in other organisms.

26The downstream target genes and upstream regulators of NF-Y, and its functional redundancy and specificity are largely unknown. The diversity of NF-Y subunits and their many potential combinations, as well as likely redundant and divergent functions, provide a substantial challenge for work aiming to tease apart the functions of the different subunits. In mammals, NF-Y is a pioneer factor that binds to the CCAAT box in the core promoter and enhancer region, but its targets remain elusive in plants. In addition, other fundamental questions about NF-Y, such its transcriptional regulation, and post-translational modification, need to be investigated. NF-Y complexes definitely function as essential regulatory hubs for many processes in plant, but its functional redundancy remains a problem for further investigation. Therefore, future studies on NF-Y will play an indispensable part in plant science.

27HZ and GL conceived the manuscript; HZ and DW drafted the manuscript. HZ, GL, FK, HZ, and KL edited the draft, and all authors approved the final version of manuscript.

28The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer JPL and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.