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Os02g0725900 is the gene loci name called HAP3 subunit of the CCAAT-box-binding transcription factor.Gene symbol is OsNF-YB1 locating at the second chromosome in rice.Now,rice has been identified 11 HAP3 genes in its genome.
A HAP complex, which consists of three subunits, namely HAP2 (also called NF-YA or CBF-B), HAP3 (NF-YB/CBF-A) and HAP5 (NF-YC/CBF-C), binds to CCAAT sequences in a promoter to control the expression of target genes. We identified 10 HAP2 genes, 11 HAP3 genes and 7 HAP5 genes in the rice genome. All the three HAP family genes encode a protein with a conserved domain in each family and various non-conserved regions in both length and amino acid sequence. These genes showed various expression patterns depending on genes, and various combinations of overlapped expression of the HAP2, HAP3 and HAP5genes were observed. Furthermore, protein interaction analyses showed interaction of OsHAP3A, a ubiquitously expressed HAP3 subunit of rice, with specific members of HAP5. These results indicate that the formation of specific complex with various HAP subunits combinations can be achieved by both tissue specific expression of three subunit genes and specific interaction of three subunit proteins. This may suggest that the HAP complexes may control various aspects of rice growth and development through tissue specific expression and complex formation of three subunit members. In rice OsHAP3A and possibly its close homologues OsHAP3B andOsHAP3C were shown to control chloroplast biogenesis. These studies indicate that the functions of the HAP3 genes are diverged from embryogenesis and chloroplast biogenesis, and suggest that each HAP3 gene has a specific role.
Rice has 11 HAP3 genes in its genome. OsHAP3A, OsHAP3B and OsHAP3C showed ubiquitous expression . They were expressed in all organs examined such as shoot apices, roots, young panicles, flowering panicles, young leaves, developing embryos and their surrounding tissues at 0, 2, 4, 6 and 8 DAP, and regenerating calli at 0, 2, 4 and 6 days after regeneration (DAR). The expression was also observed in developing young inflorescence with a panicle length of 1 and 3 mm, 2 and 3 cm. The newly identified genes were named as OsHAP3D to OsHAP3K. OsHAP3K was identical to OsNF-YB1 . They encode a protein with a conserved domain in their middle, as do the OsHAP3A, OsHAP3B and OsHAP3C proteins. A phylogenetic analysis based on the amino acid sequences of the conserved domain showed that the OsHAP3 proteins were located in several different clades (Fig1astro). Among them OsHAP3D and OsHAP3E belonged to a clade which include LEC1 and L1L, both of which play critical roles in embryogenesis (Fig1astro).
We previously showed that OsHAP3A, OsHAP3B and OsHAP3C were expressed ubiquitously (Miyoshi et al. 2003), and Masiero et al. (2002) reported that OsHAP3K/OsNF-YB1 was expressed in seed. We examined expression patterns of the newly identified OsHAP3 genes. Because LEC1 and L1L were shown to be expressed in embryogenesis (Lotan et al. 1998; Kwong et al. 2003), the expression of the OsHAP3 genes at several different stages of embryogenesis and regeneration was examined in addition to various organs. Because it is extremely difficult to isolate early-stage embryo from ovary, RNAs isolated from ovary containing developing embryo were used for RT-PCR analysis and designated as embryo. The results showed that OsHAP3D to OsHAP3G showed expression at specific stages of embryogenesis . Expression of OsHAP3D in callus was first detected at 2 day-after-regeneration (DAR) and continued weakly at later stages. Expression of OsHAP3D was also detected in 2 day-after-pollination (DAP) embryo. Expression of OsHAP3E in callus was detected weakly at 0 and 2 DARs, and became stronger at 4 DAR and further at 6 DAR. Expression of OsHAP3E in embryo was detected at 6 DAP and weakly at 8 DAP. Expression of OsHAP3E was also detected in panicle with a length of 2–3 cm. Expression of OsHAP3F was detected at 8 DAR callus, 0 DAP and 8 DAP embryos and young leaf. Very weak signals were also detected in shoot apex and root. Expression of OsHAP3G was detected in 0 DAP embryo and 10 cm panicle. No expression of OsHAP3I was detected in the organs and at the developmental stages examined (data not shown), although the database searches identified several OsHAP3I cDNAs from etiolated seedling, in which we did not examine the expression, and one more cDNA from seedling and callus. This indicates that OsHAP3I was mainly expressed in etiolated seedling. No expression of OsHAP3H was also detected by the RT-PCR analysis in the examined tissues (data not shown) and a few cDNA was identified as Heading date clones by the database search. OsHAP3J showed ubiquitous expression in almost all tissues examined here like OsHAP3A, OsHAP3B and OsHAP3C but at a low level, although no cDNAs or EST sequences have been identified in any DNA databases.
The subunit contains an evolutionary conserved domain, which is responsible for DNA binding and protein–protein interaction, and non-conserved regions.
HAP genes in rice
Fig 1astro HAP genes in rice. Schematic presentation of OsHAP2 proteins (a), OsHAP3 proteins (b) and OsHAP5 proteins (c). Shaded boxes indicate conserved domains in each HAP subunit. Boxes with a one-letter code of amino acid residues indicate stretches of five or more corresponding amino acid residues. Phylogenetic trees of HAP2 (d), HAP3 (e) and HAP5 (f). The trees were drawn on the basis of the amino acid sequences of the conserved domains in each subunit. OsHAP5D and OsHAP5E, and AtNF-YC3 and AtNF-YC9 have identical amino acid sequences in the conserved domain, respectively. ScHAP2 (M15243), ScHAP3 (M59079) and ScHAP5 (U19932) are proteins from yeast Saccharomyces cerevisiae, and HsNF-YA (M59079), HsNF-YB (L06145) and HsNF-YC (U78774) are proteins from human Homo sapiens
OsMADS18 Interacts with OsNF-YB1
MADS-box proteins are transcription factors present in different eukaryotic kingdoms.A rice seed-specific NF-YB was identified as partner of OsMADS18 by two-hybrid screening. NF-YB contains a histone fold motif, HFM,1 and is part of the trimeric CCAAT-binding NF-Y complex. OsMADS18, alone or in combination with a natural partner, interacts with OsNF-YB1 through the MADS and I regions. Other rice MADS-box proteins do not interact in these assays, indicating specificity for the interaction. OsNF-YB1 is capable of heterodimerizing with NF-YC, but not trimerizing with NF-YA, thus precluding CCAAT binding. Mutation of the variant Asp at position 99 of the HFM α2-helix into a conserved serine recovers the capacity to interact with NF-YA, but not with DNA. This is the first indication that members of the NF-YB family work through mechanisms independent of the CCAAT box. MADS-box genes have been identified in a large number of different plant species . They have been shown to play key roles in plant development, regulating the transition from vegetative to reproductive growth , determining the identity of the floral meristem, floral organs , ovules , root development, shattering, and plant aging. The best studied plant MADS-box genes are those determining floral organ identity in Arabidopsis andAntirrhinum, where many flower homeotic mutants,i.e. ag, ap3, pi, andap1 in Arabidopsis and squa,def, glo, and ple inAntirrhinum, have been described and found to be caused by mutations in MADS-box genes.
Plant MADS-box proteins share a similar modular secondary structure. The MADS-box (M), at the N terminus, is by far the most conserved part of these proteins and is the DNA-binding domain. The MADS-box is followed by the I region (I), the K-box (K), and the C terminus (C), which is the most divergent part. The K and I are both involved in MADS-box protein dimer formation.
MADS-box proteins bind in vitro to a CArG box. This conservation in the binding sites does not reflect the specific functions that different MADS-box factors seem to havein vivo. The specific regulation of target genes will be dependent on the interactions MADS-box proteins make to form homo- or heterodimers and on the interactions these dimers make with ternary proteins. Using the two-hybrid system, it was shown that theAntirrhinum MADS-box proteins DEFICIENS (DEF) and GLOBOSA (GLO) exclusively interact with each other. On the contrary, theAntirrhinum PLENA (PLE) and Arabidopsis AGAMOUS (AG) proteins, both necessary for reproductive organ development, interact with several other MADS-box proteins . The specificity of the interactions between the different MADS-box transcription factors is undoubtedly one of the mechanisms to enhance the selectivity for target gene activation. In addition, ternary complex formation between the Antirrhinum MADS-box proteins SQUAMOSA (SQUA), DEF, and GLO has been described: the DEF-GLO heterodimer is able to form a ternary complex with the SQUA homodimer via the C termini. A putative target promoter of DEF and GLO, containing two adjacent CArG boxes, showed that the ternary complex binds much stronger than the separate hetero- or homodimers.
Despite this wealth of information, ternary complex formation between MADS-box proteins and unrelated polypeptides has not been described in plants. On the other hand the human MADS-box protein SRF, which activates numerous growth factor-inducible genes such as c-fos, binds as a homodimer to the serum response element and interacts with the ternary complex factors ELK-1 and SAP-1. The ternary complex factors make additional contacts with DNA at an ETS motif, which is two base pairs 5′ of the serum response element. Autonomous binding of ELK-1 and SAP-1 to the DNA is possible, however, with restricted sequence specificity compared with the ternary complex.
Interaction of OsHAP3A with OsHAP2 and OsHAP5 proteins
We examined interactions among three HAP subunits by a yeast two-hybrid system (Fig. 4). OsHAP3A was chosen to use as the bait, due to its ubiquitous expression in almost all tissues. It should be very useful for studying how a ubiquitous subunit can establish specific interactions with other subunits. An entire coding region of OsHAP3A was fused to a DNA-binding domain of GAL4 (BD-H3A), and BD-H3A was expressed in yeast cells together with a GAL4 activation domain fusion of entire OsHAP2 or OsHAP5 proteins (AD-H2A to AD-H2H and AD-H5A to AD-H5G). We could not examine the interaction of OsHAP3A with OsHAP2I and OsHAP2J, because only very low expression of these two genes was detected by RT-PCR, and no cDNA was obtained. Presence of introns in their genomic sequences also made it difficult to use their genomic DNAs to generate their activation domain fusion constructs. After all, we examined the interaction of OsHAP3A with eight OsHAP2 proteins out of ten and all seven OsHAP5 proteins. Interaction of OsHAP3A with HAP2 and HAP5 subunits. OsHAP3A was fused to a DNA-binding domain (BD) of GAL4 (BD-H3A), and HAP2, HAP5 and OsDrAp proteins were fused to a transcription activation domain (AD) of GAL4 (AD-H2A to AD-H2H, AD-H5A to AD-H5G and AD-DrAp1 and 2, respectively). Yeast cells expressing BD-H3A (right plates) or BD alone (vector, middle plates) and each of AD fusion proteins or AD alone were grown on a medium lacking histidine and supplemented with 20 mM 3-AT. Left pictures indicate positions of yeast cells expressing AD-H2A to AD-H2H or AD alone (upper), and AD-H5A to AD-H5G, AD-OsDrAp1 and 2 or AD alone (lower)
Labs working on this gene
(1)Genetic Strains Research Center, National Institute of Genetics, Shizuoka-ken, Japan
(2)School of Life Science, Graduate University for Advanced Studies, 1111 Yata, Mishima, Shizuoka-ken 411-8540, Japan
(3)Present address: Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
Nori Kurata Email: firstname.lastname@example.org
(4)Dipartimento di Genetica e Biologia dei Microrganismi and ‖Dipartimento di Biologia, Universitá di Milano, Italy and the §Dipartimento di Biologia Animale, Universitá di Modena e Reggio, Via Campi 213/d, 41100 Modena, Italy
Thirumurugan T, Ito Y, Kubo T, Serizawa A, Kurata N. Identification, characterization and interaction of HAP family genes in rice.Mol Genet Genomics. 2008 Mar;279(3):279-89. doi: 10.1007/s00438-007-0312-3. Epub 2008 Jan 9
Kazumaru Miyoshi, Yukihiro Ito, Akiko Serizawa1 andNori Kurata.(2003)OsHAP3 genes regulate chloroplast biogenesis in rice.Article first published online: 16 OCT 2003 DOI: 10.1046/j.1365-313X.2003.01897.x
Simona Masiero, Carol Imbrian, Federica Ravasio, Rebecca Favaro, Nilla Pelucchi, Mirella Sari Gorla, Roberto Mantovani, Lucia Colombo and Martin M. Kater,(2002)Ternary Complex Formation between MADS-box Transcription Factors and the Histone Fold Protein NF-YB.First Published on April 23, 2002, doi: 10.1074/jbc.M202546200 July 19, 2002 The Journal of Biological Chemistry, 277, 26429-26435.
Gusmaroli G, Tonelli C, Mantovani R (2001) Regulation of the CCAAT-binding NF-Y subunits in Arabidopsis thaliana. Gene 264:173–185 PubMedCrossRef
Gusmaroli G, Tonelli C, Mantovani R (2002) Regulation of novel members of the Arabidopsis thaliana CCAAT-binding nuclear factor Y subunits. Gene 283:41–48