JXB Advance Access originally published online on January 22, 2008
Journal of Experimental Botany 2008 59(2):225-234; doi:10.1093/jxb/erm300
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
HDA6 is required for jasmonate response, senescence and flowering in Arabidopsis
1Institute of Plant Biology, College of Life Science, National Taiwan University, Taipei 10617, Taiwan
2Department of Biology, West Virginia University, Morgantown, WV 26506-6057, USA
* To whom correspondence should be addressed. E-mail: kewu{at}ntu.edu.tw
Received 3 September 2007; Revised 3 November 2007 Accepted 5 November 2007
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Abstract |
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Post-translational modifications of histones, including acetylation, play a key role in modulating dynamic changes in chromatin structure and gene activity. Histone acetylation is modulated through the action of histone acetyltransferases and deacetylases. HDA6 is a RPD3-type histone deacetylase in Arabidopsis. The Arabidopsis HDA6 mutant, axe1-5, and HDA6 RNA-interfering (HDA6-RNAi) plants displayed higher levels of acetylated H3 compared with wild-type, suggesting that HDA6 affects histone acetylation levels globally. The expression of the jasmonate responsive genes, PDF1.2, VSP2, JIN1, and ERF1, was down-regulated in axe1-5 and HDA6-RNAi plants. Furthermore, axe1-5 and HDA6-RNAi plants displayed increased leaf longevity compared with the wild type. The expression of the senescence-associated genes, SAG12 and SEN4, was down-regulated in the axe1-5 and HDA6-RNAi plants. In addition, axe1-5 and HDA6-RNAi plants displayed late-flowering. The expression of FLC was up-regulated and hyperacetylated in axe1-5 and HDA6-RNAi plants, suggesting that HDA6 is required to deacetylate FLC chromatin and thereby repress its expression. Our results suggest that HDA6 is involved in jasmonate response, senescence, and flowering in Arabidopsis.
Key words: HDA6, flowering, histone deacetylases, jasmonic acid, senescence
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Introduction |
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In higher organisms, many different patterns of gene expression are required for proper development to occur and to allow for specific responses to environmental cues. Transcriptional regulation of gene expression plays a fundamental role in plant response to environmental stimuli. A fundamental mechanism controlling gene expression is the ability of many transcription factors to access the genome of eukaryotes. Post-translational modifications of histones, including acetylation, methylation, phosphorylation, and ubiquitinylation play a key role in modulating dynamic changes in chromatin structure and gene activity (Berger, 2002; Reyes et al., 2002). Histone acetylation levels are determined by the action of histone acetyltransferases and histone deacetylases (HDACs). Plant HDACs can be grouped into four different classes, namely, RPD3-class, HDA1-class, SIR2-class, and HD2-class (Pandey et al., 2002). RPD3-class HDACs are the homologues of the yeast protein, RPD3 (Taunton et al., 1996). Four RPD3-class HDACs, HDA19 (also called AtRPD3A or AtHD1), HDA6 (AtRPD3B), HDA7, and HDA9 (Wu et al., 2000; Murfett et al., 2001; Tian and Chen, 2001; Pandey et al., 2002) were identified in Arabidopsis. Mutations in HDA6 affected transgene expression, DNA methylation, and regulation of rRNA genes (Murfett et al., 2001; Aufsatz et al., 2002; Probst et al., 2004; Earley et al., 2006). Antisense suppression and T-DNA disruption of HDA19 expression resulted in a range of developmental abnormalities including apical defect, reduced fertility, delayed flowering and altered light response (Wu et al., 2000; Tian and Chen, 2001; Tian et al., 2003; Benhamed et al., 2006; Long et al., 2006). More recently, it was found that HDA18, a HDA1-class HDAC, is required for normal cellular patterning of the Arabidopsis root epidermis (Xu et al., 2005).
There is increasing evidence indicating that histone acetylation is involved in the plant response to abiotic and biotic stresses (Stockinger et al., 2001; Devoto et al., 2002; Jang et al., 2003; Kim et al., 2004; Song et al., 2005; Zhou et al., 2005). It was suggested that histone acetyltransferases may be recruited through transcription factors, such as CBF1, to cold-induced genes, through multiprotein complexes similar to those found in other eukaryotes (Stockinger et al., 2001). Recently, it was demonstrated that HDA6 can interact with COI1, an F-box protein, that is involved in jasmonate (JA)-mediated plant defence responses, suggesting a possible role for HDA6 in plant–pathogen interaction (Devoto et al., 2002). Our recent studies indicate that the expression of Arabidopsis HDA19 and HDA6 can be induced by ethylene and JA (Zhou et al., 2005). In addition, overexpression of HDA19 in Arabidopsis induced ethylene- and JA-regulated PR gene expression and resulted in increased resistance to the pathogen Alternaria brassicicola. These studies provide evidence that RPD3-type HDACs, HDA19, and HDA6, may play an important role in ethylene- and JA-signalling and pathogen responses. A recent study by Song et al. (2005) provides direct evidences that HDACs are involved in ABA and abiotic stress responses. It was found that an AP2/EREBP transcription repressor, AtERF7, interacts with the Arabidopsis thaliana homologue of a human global corepressor of transcription, AtSin3, which, in turn, may interact with a HDAC.
In the present study, it is reported that HDA6 is required for the JA response, senescence, and flowering in Arabidopsis. The expression of the JA-responsive genes, PDF1.2, VSP2, JIN1, and ERF1, was down-regulated in the Arabidopsis HDA6 mutant, axe1-5, and HDA6-RNA interfering (HDA6-RNAi) plants. Furthermore, axe1-5 and HDA6-RNAi plants displayed increased leaf longevity compared with the wild type. The expression of the senescence-associated genes, SAG12 and SEN4, was also down-regulated in the axe1-5 and HDA6-RNAi plants. In addition, axe1-5 and HDA6-RNAi plants displayed late-flowering. The expression of FLC was up-regulated and hyperacetylated in axe1-5 and HDA6-RNAi plants, suggesting that HDA6 is required to deacetylate FLC chromatin and thereby repress its expression.
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Materials and methods |
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Plant materials
Arabidopsis thaliana was grown in a growth chamber under long-day (LD, 16 h light and 8 h dark) or short-day (SD, 8 h light and 16 h dark) conditions after a 2–4 d stratification period. For growth under sterile conditions, seeds were surface-sterilized [15 min incubation in 5% (v/v) sodium hypochlorite, and rinsed three times in sterile distilled water] and sown on half-strength Murashige and Skoog (MS) salts (Sigma, St Louis, MO) supplemented with 1% sucrose, pH 5.7, and 0.8% (w/v) agar in Petri dishes.
Plasmid construction
To generate the HDA6:GUS construct, a 1.3 kb promoter for HDA6 was PCR amplified by using the primer pairs, 5'-TCCAGATCTGCAGTTGTAGG-3' and 5'-GCCTCCATCTCCGTCTCTCACTCAGAATC-3'. The resulting PCR product was then digested by PstI and NcoI and subcloned into the pCAMBIA1381 binary vector (Cambia, Canberra, Australia). To generate 35S:HDA6-GFP, the HDA6 coding region was PCR amplified using the primer pairs, 5'-AATTTCCCGGGCATGAGGCAGACGAAAGCGGCA-3' and 5'-AATTGAGCTCTTAAGACGATGGAGGATTCACG-3', to replace AtHD2A in the AtHD2A-GFP construct (Zhou et al., 2004). DNA and protein sequence analysis was carried out using BLAST searches (http://www.ncbi.nlm.nih.gov/BLAST/) and the Vector NTI Suite program (InforMax Inc., Bethesda, MD).
Plant transformation and selection
Plant transformation plasmids were electroporated into Agrobacterium tumefaciens GV3101 as described by Shaw (1995). The Agrobacterium-mediated transformation of Arabidopsis thaliana was performed as described by Clough and Bent (1998). T1 seeds were harvested, dried at 25 °C, and germinated on sterile medium containing 40 µg ml–1 hygromycin to select the transformants. Surviving T1 plantlets were transferred to soil to set seeds (T2).
β-glucuronidase assays and GFP localization
For histochemical GUS assay, Arabidopsis tissues were incubated in a 0.5 mg ml–1 solution of 5-bromo-4-chloro-indolyl β-D-glucuronide (X-Glu) in 100 mM sodium phosphate buffer, pH 7.0, and incubated at 37 °C overnight, followed by washing with 70% ethanol to remove the chlorophyll (Jefferson, 1988).
For GFP localization, transgenic seeds were germinated on a MS medium. Protoplasts were isolated from transgenic Arabidopsis seedlings as described by Weigel and Glazebrook (2002). The fluorescence photographs of protoplasts were taken using an Olympus florescent microscope (Tokyo, Japan) fitted with fluorescein isothiocyanate filters (excitation filter, 450–490 nm; emission filter, 520 nm; and dichroic mirror, 510 nm).
Semi-quantitative RT-PCR analysis
One microgram of total RNA was used for the first-strand cDNA synthesis after incubation at 65 °C for 10 min. cDNA was synthesized in a volume of 20 µl that contained MoMLV reverse transcriptase buffer (Promega, Madison, Wisconsin, USA), 10 mM dithiothreitol, 1.5 µM poly(dT) primer, 0.5 mM dNTPs, 2 U of MoMLV reverse transcriptase at 37 °C for 1 h. All PCR reactions were performed with 0.5 U of Taq polymerase (PGC Scientific, Gaithersburg, Maryland, USA), the buffer provided by the supplier, 0.2 mM dNTPs, and a pair of primers (0.1 µM each) in a final volume of 20 µl. PCR parameters differed for each gene: thermocycling conditions were 94 °C for 2 min followed by 25–35 cycles of 94 °C for 1 min, 55–68 °C for 1 min, and 72 °C for 2 min, with a final polymerization step at 72 °C for 10 min. The gene-specific primer pairs used for the RT-PCR are listed in the Table 1.
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Protein gel blot analysis
Nuclear proteins were isolated as described by Weigel and Glazebrook (2002). 500 mg of Arabidopsis seedling tissues were homogenized in 1 ml of Honda buffer (2.5% Ficoll 400, 5% dextran T40, 0.4 M sucrose, 25 mM TRIS–HCl [pH 7.4], 10 mM MgCl2, 10 mM β-mercaptoethenol, 100 µg ml–1 phenylmethylsulphonyl fluoride, 0.5 µg ml–1 antipain, 0.5 µg ml–1 leupeptin) and filtered through a 62 µm nylon mesh. 0.5% Triton X-100 was then added to the extract, which was incubated for 15 min on ice and centrifuged at 1500 g for 5 min. The pellet was washed with Honda buffer containing 0.1% Triton X-100, gently resuspended in 1 ml of Honda buffer, and centrifuged at 100 g for 5 min to pellet starch and cell debris. The supernatant was transferred to a microcentrifuge tube and centrifuged at 1800 g for 5 min to pellet the nuclear.
The nuclear extract was suspended in 200 µl of 5x SDS-PAGE loading buffer [0.2 M TRIS–HCl (pH 6.8), 25% SDS, 25% glycerol and 12.5% 2-mercaptoethanol]. The protein samples were loaded on 15% polyacrylamide gel and blotted onto a nitrocellulose membrane. The membrane was blocked in PBS containing 3% dry milk for 60 min and then incubated with 0.01–0.05 µg ml–1 of anti-acetyl-histone H3 K9 and K14 antibody (Catalogue no. 06–599, Upstate, Charlottesville, VA) for 2 h at room temperature. After washing, the primary antibody was detected with secondary anti-rabbit horseradish peroxidase coupled antibody (Amersham, Buckinghamshire, England) at room temperature for 45 min. Visualization was achieved using the ECL system (Amersham).
Chromatin immunoprecipitation analysis
Chromatin immunoprecipitation (ChIP) analysis was carried out as described previously (Johnson et al., 2002; Gendrel et al., 2005). Chromatin extracts were prepared from young leaves treated by formaldehyde. The chromatin was sheared to an average length of 500–1500 bp by sonication and immunoprecipitated with the anti-acetyl-histone H3 K9 and K14 antibody (Catalogue no. 06–599, Upstate, Charlottesville, VA). The immunocomplexes were harvested with Protein A agarose and heated at 65 °C for 5 h to release DNA cross-linked to the immunoprecipitated proteins. The DNA cross-linked to the immunoprecipitated proteins was analysed by PCR. To assess non-specific binding, the immunoprecipitation reaction was also performed in the absence of antibody. The primers used to amplify FLC were FLC-B (5'-TGTAGGCACGACTTTGGTAACACC-3' and 5'- GCAGAAAGAACCTCCACTCTACATC-3'), FLC-P (5'-CGACTTGAACCCAAACCTGAGGATCAAAT-3' and 5'-AGAAGATAAAAGGGGGAACAAATGAAAAC-3'), FLC-CH (5'-CTGCGACCATGATAGATACATGAGA-3' and 5'- TTCACTCAACAACATCGAGCACG-3'), FLC-V2 (5'- ATAGATTTGCCTCATATTTATGTGATTGT-3' and 5'-TTCATTATAGATCCGTACCAAAGAGGTTG-3'), and FLC-U (5'-GCTGATAAGGGCGAGCGTTTG-3' and 5'-AAGCCGTAGGCTTCTTCACTG-3'). The primer pair JP1595 (5'-CGTTTCGCTTTCCTTAGTGTTAGCT-3') and JP1596 (5'-AGCGAACGGATCTAGAGACTCACCTTG-3') was used to amplify ACTIN (Johnson et al., 2002). 30 cycles of PCR (94 °C for 25 s, 59 °C for 35 s, 72 °C for 30 s) were performed as separate reactions for FLC and ACTIN amplification.
Measurement of chlorophyll content and photochemical efficiency
From about 15 DAE (days after leaf emergence) onwards, the sixth rosette leaf, which was fully grown, was chosen for chlorophyll extraction and photochemical efficiency of photosystem II (PSII) measurement. Chlorophyll was extracted from equal volumes of leaf discs by grinding the leaves in liquid nitrogen and dissolving in 80% acetone. Chlorophyll concentration per g fresh weight of leaf was calculated as described by Porra et al. (1989). The photochemical efficiency of PSII was deduced from the characteristics of chlorophyll fluorescence (Oh et al., 1997) using a portable plant efficiency analyser (Hansatech Instruments, Morfolk, England). The ratio of maximum variable fluorescence to maximum yield of fluorescence (Fv/Fm), which corresponds to the potential quantum yield of the photochemical reactions of PSII, was used as the measurement of the photochemical efficiency of PSII (Oh et al., 1997).
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Results |
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Expression and localization of HDA6
The expression pattern of HDA6:GUS was analysed by fusing the 1.3 kb HDA6 promoter fragment to a GUS reporter gene in Arabidopsis plants. In 2-week-old transgenic seedlings, GUS was strongly expressed in cotyledons and hypocotyls (Fig. 1A). In adult plants, GUS expression was detected in the mature leaves and stems. GUS activity was also detected in all parts of flowers except the stamens. However, GUS activity was not detected in the seeds (Fig. 1A). The expression pattern of HDA6:GUS is therefore different from that of HDA19:GUS, which was expressed in all of the organs analysed (Zhou et al., 2005). Expression of HDA6 and other RPD3-type HDAs was analysed further by using RT-PCR. As shown in Fig. 1B, HDA6 and HDA19 transcripts were detected in roots, leaves, stems, and flowers of Arabidopsis. By comparison, HDA9 transcript could only be detected in flowers.
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To investigate the cellular distribution of HDA6 protein, an in vivo targeting experiment was performed using green fluorescent protein (GFP). HDA6-GFP gene fusion driven by the 35S promoter was created and introduced into Arabidopsis using the Agrobacterium-mediated floral dip method (Clough and Bent, 1998). To confirm that the fusion protein entered the nucleus, the fluorescence of GFP was monitored at the cellular level. Protoplasts were isolated from seedlings of transgenic Arabidopsis, and localization of the fusion protein was determined by a fluorescence microscope. As shown in Fig. 1C, bright green fluorescence was observed only in the nuclei of HDA6-GFP transgenic plants. This indicates that HDA6-GFP fusion protein was localized in the nucleus of the Arabidopsis cells. This result is consistent with the observation of Earley et al. (2006), who demonstrated the nuclear localization of HDA6-Flag by immunolocalization. These results support the idea that HDA6 is involved in transcription regulation.
HDA6 affected JA-responsive gene expression
To investigate the function of HDA6 further, two HDA6-RNAi lines, CS24038 and CS24039 (ecotype Wassilewskija [Ws]), generated by expressing a transgene that encodes double-stranded HDA6 RNA (Plant Chromatin Database: http://chromdb.org) were analysed. RT-PCR analysis indicated that HDA6 transcript levels in the HDA6-RNAi plants were significantly reduced relative to non-transgenic control plants (Fig. 2A). CS24039 plants had greater reduction in HDA6 expression compared with CS24038 plants. The expression of other RPD3-class HDACs, HDA19 and HDA9, was not affected in the HDA6-RNAi plants.
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The levels of acetylated histone H3 in HDA6-RNAi plants were analysed by protein gel blot analysis. As shown in Fig. 2B, there were obvious increased levels of acetylated H3 in two HDA6-RNAi lines compared with the Ws wild type, suggesting that the HDA6 transcript level affects histone acetylation levels globally. Similarly, increased level of acetylated H3 was also observed in the HDA6 mutant, axe1-5 [ecotype Columbia (Col)], when compared with the Col wild type (Fig. 2B). axe1-5 is a splice site mutant that has a base change at an intron splice site resulting in two HDA6 transcripts with altered lengths (Murfett et al., 2001). Changed levels of acetylated H3 in axe1-5 and HDA6-RNAi plants support the observation that HDA6 has a HDAC activity (Earley et al., 2006).
The interaction of HDA6 with COI1, an F-box protein that was required for JA-mediated plant defence responses, has been demonstrated (Devoto et al, 2002), suggesting a possible role of HDA6 in JA response. To investigate further the involvement of HDA6 in the JA-responsive pathway, semi-quantitative RT-PCR was conducted to examine transcript levels of JA-responsive genes, PDF1.2, VSP2, JIN1, and ERF1 (Lorenzo et al., 2003, 2004), in the axe1-5 and HDA6-RNAi plants. As shown in Fig. 3A, expression of PDF1.2, VSP2, JIN1, and ERF1 was down-regulated in axe1-5 and HDA6-RNAi plants. Expression of PDF1.2, VSP2, JIN1, and ERF1 can be induced by JA in Col wild-type plants (Fig. 3B). By comparison, expression of these JA-responsive genes can not be induced by JA in axe1-5 plants (Fig. 3B). Down-regulation of PDF1.2, VSP2, JIN1, and ERF1 in axe1-5 and HDA6-RNAi plants suggests that HDA6 is required for expression of the JA-responsive genes.
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axe1-5 and HDA6-RNAi plants displayed increased leaf longevity
JA was found to be involved in leaf senescence in Arabidopsis (He et al., 2002). To investigate whether HDA6 is required for leaf senescence, leaf longevity of the axe1-5 and HDA6-RNAi plants was examined. The phenotype of individual leaves grown under the long-day (LD, 16 h light and 8 h dark) condition was followed from the formation of a visually recognizable leaf primordium (1 mm in size) and the leaf was considered dead when the entire leaf turned yellow (Grbi and Bleecker, 1995). As shown in Fig. 4A, the leaves of axe1-5 and HDA6-RNAi plants turned yellow much slower and showed increased leaf longevity when compared with their wild-type counterparts.
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15).Leaf longevity was also assessed by measuring typical senescence-associated physiological markers, such as chlorophyll contents and photochemical efficiency of PSII (Fan et al., 1997; Oh et al., 1997). Chlorophyll contents decline at the onset of senescence and it is considered as an important indicator of the rate of senescence (Nam, 1997). Chlorophyll contents were measured from 15 DAE (days after leaf emergence), which was also the day of the 6th rosette leaf that was fully grown. At 45 DAE, the leaves of Ws and Col wild type lost 65–75% of their chlorophylls, whereas these of axe1-5 and HDA6-RNAi just lost 15–35% (Fig. 4B). Delayed senescence of the mutants was also defined as delay in the decrease in photosynthetic activity (Fig. 4C). The PSII efficiency has also been shown to be an effective indicator of leaf senescence in plants (Lu and Zhang, 1998a, b). It was demonstrated that, during senescence, the PSII efficiency declines rapidly, leading to a loss of photosynthetic capabilities of the leaves and leading to the eventual death of the leaves. This analysis indicated that axe1-5 and HDA6-RNAi leaves consistently showed later development of senescence-associated changes. These results suggest that decreased expression of HDA6 causes increased leaf longevity in Arabidopsis.
Leaf senescence is accompanied by the decreased expression of genes related to photosynthesis and protein synthesis genes (PAGs) (Bate et al., 1991) and increased expression of senescence-associated genes (SAGs) (Nam, 1997). The expression patterns were examined of two SAG genes, SAG12 and SEN4, which have been shown to be up-regulated during senescence (Gan and Amasino, 1997; Park et al, 1998), in the axe1-5 and HDA6-RNAi plants. As shown in Fig. 5, the expression of SAG12 and SEN4 was down-regulated in the axe1-5 and HDA6-RNAi plants when compared with the wild-type. By comparison, the expression of RPS17, a PAG gene that encodes the chloroplast ribosomal protein S17 (Woo et al., 2002), was up-regulated in axe1-5 and HDA6-RNAi plants (Fig. 5). These results support the idea that HDA6 is required for SAGs expression and therefore it is involved in senescence progression. In the absence of HDA6, PAG genes are up-regulated, which leads to a higher rate of photosynthesis, resulting in higher PSII efficiencies and higher chlorophyll contents in axe1-5 and HDA6-RNAi plants.
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axe1-5 and HDA6-RNAi plants displyed delayed flowering
axe1-5 and HDA6-RNAi mutants displayed later flowering phenotypes, as measured by the days to bolting and the rosette leaf numbers at flowering (Fig. 6). Col, Ws, and HDA6 mutants were grown in long-day (LD, 16 h light and 8 h dark) and short-day (SD, 8 h light and 16 h dark) conditions, and the flowering time was compared. The flowering of axe1-5 and HDA6-RNAi plants was greatly delayed in SD as well as in LD in terms of both the days to flowering and the rosette leaf numbers at flowering initiation (Fig. 6). axe1-5 plants did not flower even at 104 d after germination in SD, when some rosette leaves showed senescence. The delay in flowering time of axe1-5 and HDA6-RNAi plants was completely corrected by 45 d of vernalization at 4 °C (Fig. 6B). These observations suggest that HDA6 is involved in the autonomous pathway of flowering.
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The delayed flowering of the axe1-5 and HDA6-RNAi plants prompted us to analyse whether the expression of FLC, a transcription factor that controls the transition from vegetative to reproductive development, was affected. RT-PCR analysis indicated that expression of FLC was increased in axe1-5 and HDA6-RNAi plants compared with the wild type (Fig. 7). In addition to FLC, other MADS-box transcriptional factors, such as MAF1, MAF2, MAF4, and MAF5, are closely related to FLC and also function as repressors of flowering in Arabidopsis (Scortecci et al., 2001; Ratcliffe et al., 2003). It was found that expression of MAF4 and MAF5 was also increased in axe1-5 and HDA6-RNAi plants (Fig. 7).
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Chromatin immunoprecipitation (ChIP) analysis was carried out using the anti-acetyl-histone H3 antibody to determine if the acetylation level of FLC was affected in HDA6 mutants. A 295 base pair region (FLC-CH) of the first intron of FLC was shown to be hyperacetylated in the fld and fve mutants of the autonomous flowering pathway (He et al., 2003). Therefore the histone acetylation status of this region was analysed in FLC. As shown in Fig. 8B, FLC displayed hyperacetylation of histone H3 in the axe1-5 and HDA6-RNAi plants compared with the wild type, suggesting that HDA6 is required to deacetylate FLC chromatin and repress FLC expression. The histone acetylation status of other regions of FLC was also analysed in the axe1-5 mutant. There was increased acetylation of histone H3 in the 1st exon and 1st intron of FLC, but not in the promoter and the 3' UTR regions (Fig. 8C).
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The HDA6 mutant, axe1, was isolated based on deregulated expression of auxin-responsive transgenes (Murfett et al., 2001). Since then, other mutant alleles of HDA6, rts1 and sil1, were also isolated based on their effects on specific transgene expression (Aufsatz et al., 2002; Probst et al., 2004). It was shown that mutations in HDA6 result in loss of transcriptional silencing from several repetitive transgenic and endogenous templates. In the axe1-5 mutant, significant hyperacetylation is identified in the nucleolus organizer regions that contain the rDNA repeats (Probst et al., 2004). It was suggested that HDA6 might play a role in regulating activity of rRNA genes (Earley et al., 2006), and this control might be functionally linked to silencing of other repetitive templates and to its role in RNA-directed DNA methylation (Probst et al., 2004).
This study indicates that HDA6 is involved in JA response, senescence, and flowering. It has been observed that the JA-responsive genes, PDF1.2, JIN1, ERF1, and VSP2, were down-regulated in axe1-5 and HDA6-RNAi plants, suggesting that HDA6 is required for the JA-responsive pathway. The finding that HDA6 expression can be induced by JA also supports a role for HDA6 in the JA-response pathway (Zhou et al., 2005). Our previous studies indicate that HDA19 plays an important role in the JA-signalling pathway (Zhou et al., 2005). It would be interesting to know whether HDA6 and HDA19 have redundant function in the JA-response pathway. Further study by analysing HDA6 and HDA19 double mutants will be useful to address this question. HDA6 mutants displayed delayed leaf senescence compared with wild-type plants. The delayed leaf senescence in axe1-5 and HDA6-RNAi plants was demonstrated by measuring changes in typical senescence-associated physiological markers such as chlorophyll content and photochemical efficiency (Fan et al., 1997; Oh et al., 1997). Expression of two SAG genes, SAG12 and SEN4, was decreased in axe1-5 and HDA6-RNAi plants when compared with the wild type. By comparison, expression of a PAG gene, RPS17, was up-regulated in axe1-5 and HDA6-RNAi plants. These results suggest that HDA6 is required for SAGs expression and is involved in senescence progression.
Although histone deacetylases have often been associated with the repression of gene expression, recent studies in yeast and animal cells indicate that histone deacetylation can also be required as a transcriptional activation signal. Deletion of yeast histone deacetylases Rpd3, or Hda1 results in decreased transcription of a number of genes and an increase rather than a loss of silencing, pointing to an alternative role of HDACs as transcriptional activators (Rundlett et al., 1996; Bernstein et al., 2000). In addition, SIN3, a major component of the HDAC-containing transcriptional repressor complex, can function as both a transcriptional corepressor and a transcriptional coactivator (Nawaz et al., 1994). Furthermore, Mouse Histone Deacetylase 1 (HDAC1) can act as a negative regulator as well as a positive regulator of transcription (Zupkovitz et al., 2006). These studies indicate that gene regulation by acetylation is more dynamic and HDACs may also function as activators (Nusinzon and Horvath, 2005). An alternative explanation for the repression of gene activity in axe1-5 and HDA6 RNAi plants could be that HDA6 may target transcription repressors.
The interaction of HDA6 with COI1, an F-box protein that was required for JA-mediated plant defence responses, has been demonstrated (Devoto et al., 2002). However, the function and biological significance of this interaction is unknown. F-box proteins interact with SKP1 and cullin proteins to form E3 ubiquitin ligases known as the SCF complexes that selectively recruit regulatory proteins targeted for ubiquitination (Vierstra, 2003). Co-immunoprecipitation experiments confirmed the interaction of COI1 with SKP1-like proteins and HDA6 in planta. Regulation of HDAC activities by ubiquitination has been demonstrated in mammalian cells (Gaughan et al., 2005). It was proposed that COI1 may form a functional E3-type ubiquitin ligase in plants to regulate expression of JA-responsive genes, possibly by targeted ubiquitination of a histone deacetylase (Devoto and Turner, 2003). In addition to its role in targeting proteins for proteolytic degradation by the proteasome, ubiquitination can also regulate protein location, activity, and interaction with binding partners (Schnell and Hicke, 2003; Caron et al., 2005). Further analysis is required to reveal whether HDA6 is regulated by ubiquitination and the biological function of this modification.
Plant flowering is regulated by both environmental and endogenous cues. Molecular genetic studies on the mechanism of flowering in Arabidopsis, a quantitative long-day plant, have revealed four major flowering pathways: the photoperiod, autonomous, vernalization, and gibberellin pathways (Mouradov et al., 2002; Boss et al., 2004; Henderson and Dean, 2004). A central player in the autonomous and vernalization flowering pathways is FLC, which blocks flowering by inhibiting genes required to switch the meristem from vegetative to flower development. axe1-5 and HDA6-RNAi plants flowered later than the wild-type plants in both long-day and short-day photoperiods and flowered rapidly after exposure to a prolonged period of cold (vernalization), which is characteristic of autonomous-pathway mutants (He et al., 2003). The genes in the autonomous pathway, such as FVE and FLD, promote flowering by suppressing FLC. More recent studies indicated that histone acetylation is involved in plant flowering (He et al., 2003; Ausin et al., 2004; Kim et al., 2004). Mutations in FLD and FVE, two proteins that were found in histone deacetylase complexes, result in hyperacetylation of histones in FLC chromatin, up-regulation of FLC expression, and delayed flowering, indicating that the autonomous pathway regulates flowering in part by histone deacetylation (He et al., 2003; Ausin et al., 2004; Kim et al., 2004). It was found that FLC was up-regulated and hyperacetylated in the axe1-5 and HDA6-RNAi plants, suggesting that HDA6 is required to deacetylate FLC chromatin and repress its expression. FVE and FLD may therefore act with HDA6 to repress FLC expression.
In summary, our studies indicate that, in addition to its role in transgene expression, DNA methylation, and regulating activity of rRNA genes (Murfett et al., 2001; Aufsatz et al., 2002; Probst et al., 2004; Earley et al., 2006), HDA6 is involved in many aspects of plant development. In particular, HDA6 is required for JA response, senescence, and flowering in Arabidopsis. The role for JA in leaf senescence in Arabidopsis was demonstrated based on the fact that exogenous application of JA induces leaf senescence, and this induction requires an intact JA signalling pathway (He et al., 2002). In addition, it was shown that the endogenous JA level in senescing leaves increased to nearly 500% of that in non-senescent counterpart leaves. Thus HDA6 may be involved in leaf senescence via the JA-signalling pathway. Arabidopsis mutants that affect both senescence and flowering time were isolated previously (Barth et al., 2006). For example, the Arabidopsis ascorbic acid-deficient mutant vtc1 exhibits a delayed senescence and flowering phenotype (Pavet et al., 2005). By comparison, mutation of the matrix metalloproteinase At2-MMP that is important for the degradation and remodelling of the extracellular matrix causes late flowering but early senescence in Arabidopsis (Golldack et al., 2002). These results suggest that flowering and senescence could be regulated independently. It remains to be determined whether HDA6 is independently involved in these processes. Analysis of the genetic interaction between the HDA6 mutation and mutations affecting JA signalling, senescence or flowering is required to dissect further the role of HDA6 in these pathways.
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Acknowledgements |
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We thank Brian Miki for critical reading of the manuscript. We are grateful to Tom Guilfoyle for providing axe1-5 seeds, and Chromatin Functional Genomics Consortium and Arabidopsis Biological Resource Center for providing HDA6-RNAi seeds. This work was supported by grants from the National Science Council of Taiwan (NSC 95-2321-B-002-026 and 96-2311-B-002-013-MY2).
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