HAP3 Subunit Research Articles

Natural Variation of OsHd8 Regulates Heading Date in Rice

Heading date, as one of the most important agronomic traits, is a fundamental factor determining crop yield. Although diverse genes related to heading date have already been reported in rice, the key gene that regulates heading date is still poorly understood. Here, we identified a heading date regulator, heading date 8 (OsHd8), which promoted the heading date under long-day conditions and encoded a putative HAP3 subunit of the CCAAT-box-binding transcription factor. It is localized in the nucleus and expressed in various tissues. Sequence analysis revealed that there were four SNPs and one InDel in the promoter region of OsHd8, which was involved in the regulation of some floral regulators including GHD7.1, SDG718, OsGI and HDT1. Further evolutionary analysis showed that OsHd8 presents divergence between indica and japonica, showing natural selection during the domestication of cultivated rice. These results indicate that OsHd8 plays an important role in the regulation of heading date, and may be an important target for rice breeding programs.

Cloning and functional characterization of seed-specific LEC1A promoter from peanut (Arachis hypogaea L.).

LEAFY COTYLEDON1 (LEC1) is a HAP3 subunit of CCAAT-binding transcription factor, which controls several aspects of embryo and postembryo development, including embryo morphogenesis, storage reserve accumulation and skotomorphogenesis. Herein, using the method of chromosomal walking, a 2707bp upstream sequence from the ATG initiation codon site of AhLEC1A which is a homolog of Arabidopsis LEC1 was isolated in peanut. Its transcriptional start site confirmed by 5' RACE was located at 82 nt from 5' upstream of ATG. The bioinformatics analysis revealed that there existed many tissue-specific elements and light responsive motifs in its promoter. To identify the functional region of the AhLEC1A promoter, seven plant expression vectors expressing the GUS (β-glucuronidase) gene, driven by 5' terminal series deleted fragments of AhLEC1A promoter, were constructed and transformed into Arabidopsis. Results of GUS histochemical staining showed that the regulatory region containing 82bp of 5' UTR and 2228bp promoter could facilitate GUS to express preferentially in the embryos at different development periods of Arabidopsis. Taken together, it was inferred that the expression of AhLEC1A during seed development of peanut might be controlled positively by several seed-specific regulatory elements, as well as negatively by some other regulatory elements inhibiting its expression in other organs. Moreover, the GUS expression pattern of transgenic seedlings in darkness and in light was relevant to the light-responsive elements scattered in AhLEC1A promoter segment, implying that these light-responsive elements harbored in the AhLEC1A promoter regulate skotomorphogenesis of peanut seeds, and AhLEC1A expression was inhibited after the germinated seedlings were transferred from darkness to light.

The HAP Complex Governs Fumonisin Biosynthesis and Maize Kernel Pathogenesis in Fusarium verticillioides

Contamination of maize ( Zea mays ) with fumonisins produced by the fungus Fusarium verticillioides is a global concern for food safety. Fumonisins are a group of polyketide-derived secondary metabolites linked to esophageal cancer and neural tube birth defects in humans and numerous toxicoses in livestock. Despite the importance of fumonisins in global maize production, the regulation of fumonisin biosynthesis during kernel pathogenesis is poorly understood. The HAP complex is a conserved, heterotrimeric transcriptional regulator that binds the consensus sequence CCAAT to modulate gene expression. Recently, functional characterization of the Hap3 subunit linked the HAP complex to the regulation of secondary metabolism and stalk rot pathogenesis in F. verticillioides . Here, we determine the involvement of HAP3 in fumonisin biosynthesis and kernel pathogenesis. Deletion of HAP3 suppressed fumonisin biosynthesis on both nonviable and live maize kernels and impaired pathogenesis in living kernels. Transcriptional profiling via RNA sequencing indicated that the HAP complex regulates at least 1,223 genes in F. verticillioides , representing nearly 10% of all predicted genes. Disruption of the HAP complex caused the misregulation of biosynthetic gene clusters underlying the production of secondary metabolites, including fusarins. Taken together, these results reveal that the HAP complex is a central regulator of fumonisin biosynthesis and kernel pathogenesis and works as both a positive and negative regulator of secondary metabolism in F. verticillioides .

Effects of altered expression of LEAFY COTYLEDON1 and FUSCA3 on microspore-derived embryogenesis of Brassica napus L.

Brassica napus (Bn) microspore-derived embryogenesis has become a model system to study basic aspects of plant development. Recognized transcription factors governing embryogenesis include: FUSCA3 (FUS3), a member of the plant-specific B3-domain family, and LEAFY COTYLEDON1 (LEC1), a member of the HAP3 subunit of the CCAAT binding factor family. The effects of altered expression of both genes were investigated during microspore-derived embryogenesis in established B. napus lines over-expressing or down-regulating BnLEC1, as well as in tilling lines where BnFUS3 was mutated. While over-expression of BnLEC1 decreases the yield of microspore-derived embryos (MDEs) without affecting their ability to regenerate plants, suppression of BnLEC1 or BnFUS3 reduced both embryo number and regeneration frequency. Embryos produced by these lines showed structural abnormalities accompanied by alterations in the expression of several embryogenesis-marker genes. Oil accumulation was also altered in the transgenic MDEs. Total oil content was increased in MDEs over-expressing BnLEC1 and decreased in those suppressing BnLEC1 or BnFUS3. Mutation of BnFUS3 also resulted in a small but significant increase in linoleic (C18:2) acid. Together this study demonstrates the crucial role of BnLEC1 and BnFUS3 during in vitro embryogenesis.

EF8 is involved in photoperiodic flowering pathway and chlorophyll biogenesis in rice.

A DTH8/Ghd8 allele suppresses flowering by altering the expression patterns of the 'florigen' genes, but also negatively regulates chlorophyll biogenesis. Flowering time is a critical agronomic trait determining the growing season, regional adaptation and yield potential in rice (Oryza sativa L.). We characterized a mutant named early flowering 8 (ef8) selected from an ethyl methanesulfonate (EMS)-treated population of indica cultivar 93-11. It showed earlier flowering, less grains per main panicle and slightly darker green leaves than the wild-type 93-11 under natural long-day conditions, but was not significantly different from 93-11 under natural short-day conditions. We isolated the Early Flowering 8 (EF8) gene by map-based cloning. EF8 encodes a putative HAP3 subunit of the CCAAT-box-binding transcription factor, which is localized to the nucleus. EF8 was expressed in various tissues, especially in leaves, with a rhythmic expression pattern. Our data showed that EF8 delayed flowering time under long-day conditions by altering the rhythmic expression patterns of 'florigen' genes Hd3a and RFT1. We also found that EF8 negatively regulates the expression of chlorophyll biosynthetic genes to reduce the chlorophyll content. Our data indicate that EF8 plays an important role in rice photoperiodic flowering pathway as well as yield potential and chlorophyll biogenesis and will be an important target for rice breeding programs.

The roles of two transcription factors, ABI4 and CBFA, in ABA and plastid signalling and stress responses

Genetic and physiological studies have revealed evidences for multiple signaling pathways by which the plastid exerts retrograde control over photosynthesis-associated-nuclear-genes. In this study we have examined the mechanisms of control of transcription by plastid signals, focusing on transcription factors. We have also further addressed the physical nature of plastid signals and the physiological role, in stress acclimation of this regulatory pathway. ABI4, a master Apetala 2 (AP2)-type transcription factor (TF), is targeted by multiple signalling pathways in plant cells, such as abscisic acid (ABA) signals, sugar signals and plastid signals derived from reactive oxygen species (ROS) and chlorophyll intermediates. ABI4 binds the promoter of target genes to prevent their transcription by competing with other competitive TFs. However, we found that once ABI4 bound the element (CCACGT), it may not be bound by other TFs, therefore making the signalling long-lasting. Downstream of ABI4, CBFA (CCAAT binding factor A) is a subunit of the HAP2/HAP3/HAP5 (Heme activator protein) trimeric transcription complex. CBFA however is a redundant HAP3 subunit. When emergency occurs (such as herbicide treatments or environmental stresses followed by ABA and ROS accumulation), the master transcription factor ABI4 down-regulates some TFs, like CBFA, and then some other TF subunits enter the transcription complex and transcriptional efficiency of stress-responsive genes (including the transcription co-factor CBP) is improved instantaneously. abi4, cbfA and cbp mutants showed weaker drought-tolerance after a herbicide norflurazon treatment, which indicated the physiological role of these key transcription factors.

Homologous HAP5 subunit from Picea wilsonii improved tolerance to salt and decreased sensitivity to ABA in transformed Arabidopsis

HAP is a ubiquitous transcription factor family which consists of three distinct subunits, namely HAP2, HAP3, and HAP5. Among them, HAP2 and HAP3 subunits have been reported to be involved in plant response to abiotic stress. Here, a HAP5 subunit was identified from Picea wilsonii Mast. and transformed to Arabidopsis to investigate its functions in plant stress response. We found that transformed Arabidopsis with over-expressing PwHAP5 exhibited higher seed germination under salinity, osmotic and abscisic acid (ABA) stress treatment compared to Col-0 plants. The seedlings of transformed Arabidopsis also showed improved tolerance to salinity and decreased sensitivity to ABA treatment. Over-expression of PwHAP5 in Arabidopsis athap5 mutant rescued partly tolerance to NaCl, mannitol and ABA treatment. Furthermore, we examined transcription levels of several stress-related genes in transformed seedlings. Among them, mRNA expression levels of COR15a, KIN1, DREB2A, and RD29A genes were substantially higher in transformed Arabidopsis than those in wild-type (Col-0) plants. Therefore, our data revealed that PwHAP5 plays positive roles in response to salinity, osmotic and ABA stress at different developmental stages in plants, respectively, via possibly regulating stress-related genes.

DTH8 Suppresses Flowering in Rice, Influencing Plant Height and Yield Potential Simultaneously

The three most important agronomic traits of rice (Oryza sativa), yield, plant height, and flowering time, are controlled by many quantitative trait loci (QTLs). In this study, a newly identified QTL, DTH8 (QTL for days to heading on chromosome 8), was found to regulate these three traits in rice. Map-based cloning reveals that DTH8 encodes a putative HAP3 subunit of the CCAAT-box-binding transcription factor and the complementary experiment increased significantly days to heading, plant height, and number of grains per panicle in CSSL61 (a chromosome segment substitution line that carries the nonfunctional DTH8 allele) with the Asominori functional DTH8 allele under long-day conditions. DTH8 is expressed in most tissues and its protein is localized to the nucleus exclusively. The quantitative real-time PCR assay revealed that DTH8 could down-regulate the transcriptions of Ehd1 (for Early heading date1) and Hd3a (for Heading date3a; a rice ortholog of FLOWERING LOCUS T) under long-day conditions. Ehd1 and Hd3a can also be down-regulated by the photoperiodic flowering genes Ghd7 and Hd1 (a rice ortholog of CONSTANS). Meanwhile, the transcription of DTH8 has been proved to be independent of Ghd7 and Hd1, and the natural mutation of this gene caused weak photoperiod sensitivity and shorter plant height. Taken together, these data indicate that DTH8 probably plays an important role in the signal network of photoperiodic flowering as a novel suppressor as well as in the regulation of plant height and yield potential.

Identification, characterization and interaction of HAP family genes in rice

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 HAP5 genes 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.

AtHAP3b Plays a Crucial Role in the Regulation of Flowering Time in Arabidopsis during Osmotic Stress

The HAP complex has been found in many eukaryotic organisms. HAP recognizes the CCAAT box present in the promoters of 30% of all eukaryotic genes. The HAP complex consists of three subunits--HAP2, HAP3 and HAP5. In this paper, we report the biological function of the AtHAP3b gene that encodes one of the HAP3 subunits in Arabidopsis. Compared with wild-type plants, hap3b-1 and hap3b-2 mutants exhibited a delayed flowering time under long-day photoperiod conditions. Moreover, the transcription levels of FT were substantially lower in the mutants than in the wild-type plants. These results imply that AtHAP3b may function in the control of flowering time by regulating the expression of FT in Arabidopsis. In a subsequent study, AtHAP3b was found to be induced by osmotic stress. Under osmotic stress conditions, the hap3b-1 and hap3b-2 mutants flowered considerably later than the wild-type plants. These results suggest that the AtHAP3b gene plays more important roles in the control of lowering under osmotic stress in Arabidopsis.

Identification and characterization of carrot HAP factors that form a complex with the embryo-specific transcription factor C-LEC1

C-LEC1, an orthologue of Arabidopsis LEC1, is thought to be an essential transcriptional activator required for normal development during the early and late phases of embryogenesis. C-LEC1 is similar in sequence to the HAP3 subunits of other organisms. To understand C-LEC1 function better, a cDNA library of carrot somatic embryos was screened for factors that form complexes with C-LEC1. Two carrot HAP5 homologues and two carrot HAP2 homologues were identified; these factors have significant sequence similarity to the conserved regions of HAP5 and HAP2, respectively. Some of these proteins form heterotrimeric complexes that bind specifically to DNA fragments containing a CCAAT sequence in vitro. The results suggest that C-LEC1 is a component of the CCAAT-box-binding factor and forms a complex with C-HAP2B and C-HAP5A or C-HAP5B that regulates gene expression during carrot embryo development.

Arabidopsis LEAFY COTYLEDON1 represents a functionally specialized subunit of the CCAAT binding transcription factor.

LEAFY COTYLEDON1 (LEC1) is a central regulator that is required for many aspects of Arabidopsis embryogenesis and sufficient to induce embryo development in vegetative cells when expressed ectopically. We previously showed that LEC1 encodes an HAP3 subunit of the CCAAT binding factor and that the 10 Arabidopsis HAP3 (AHAP3) subunits can be divided into two classes, the LEC1-type and the non-LEC1-type, based on sequence similarity within their B domains. By analyzing the ability of chimeric HAP3 subunits containing different combinations of domains from LEC1 and a non-LEC1-type AHAP3 subunit to suppress genetically the lec1 mutation, we show that the B domain of LEC1 is necessary and sufficient within the context of the protein for its activity in embryogenesis. Moreover, we identify one amino acid residue, Asp-55, specific to the LEC1-type B domain that is required for LEC1 activity in embryogenesis and sufficient to confer partial LEC1 activity to a non-LEC1-type B domain. Based on structural similarities between the HAP3 B domain and histone fold motif, we discuss how the Asp-55 residue may functionally differentiate LEC1 from the non-LEC1-type AHAP3 subunits.

LEAFY COTYLEDON1-LIKE defines a class of regulators essential for embryo development.

Arabidopsis LEAFY COTYLEDON1 (LEC1) is a critical regulator required for normal development during the early and late phases of embryogenesis that is sufficient to induce embryonic development in vegetative cells. LEC1 encodes a HAP3 subunit of the CCAAT binding transcription factor. We show that the 10 Arabidopsis HAP3 (AHAP3) subunits can be divided into two classes based on sequence identity in their central, conserved B domain. LEC1 and its most closely related subunit, LEC1-LIKE (L1L), constitute LEC1-type AHAP3 subunits, whereas the remaining AHAP3 subunits are designated non-LEC1-type. Similar to LEC1, L1L is expressed primarily during seed development. However, suppression of L1L gene expression induced defects in embryo development that differed from those of lec1 mutants, suggesting that LEC1 and L1L play unique roles in embryogenesis. We show that L1L expressed under the control of DNA sequences flanking the LEC1 gene suppressed genetically the lec1 mutation, suggesting that the LEC1-type B domains of L1L and LEC1 are critical for their function in embryogenesis. Our results also suggest that LEC1-type HAP3 subunits arose from a common origin uniquely in plants. Thus, L1L, an essential regulator of embryo development, defines a unique class of plant HAP3 subunits.

Arabidopsis LEAFY COTYLEDON1 Is Sufficient to Induce Embryo Development in Vegetative Cells

The Arabidopsis LEAFY COTYLEDON1 ( LEC1) gene is required for the specification of cotyledon identity and the completion of embryo maturation. We isolated the LEC1 gene and showed that it functions at an early developmental stage to maintain embryonic cell fate. The LEC1 gene encodes a transcription factor homolog, the CCAAT box–binding factor HAP3 subunit. LEC1 RNA accumulates only during seed development in embryo cell types and in endosperm tissue. Ectopic postembryonic expression of the LEC1 gene in vegetative cells induces the expression of embryo-specific genes and initiates formation of embryo-like structures. Our results suggest that LEC1 is an important regulator of embryo development that activates the transcription of genes required for both embryo morphogenesis and cellular differentiation.