The Arabidopsis Histone Deacetylases HDA6 and HDA19 Contribute to the Repression of Embryonic Properties after Germination1[W] Motoki Tanaka, Akira Kikuchi, and Hiroshi Kamada* Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki 305–8572, Japan
Histone deacetylase (HDAC) is a chromatin-remodeling factor that contributes to transcriptional repression in eukaryotes. In Arabidopsis (Arabidopsis thaliana), the transcription factors LEAFY COTYLEDON1 (LEC1), FUSCA3 (FUS3), and ABSCISIC ACID INSENSITIVE3 (ABI3) play key roles in embryogenesis. Although the repression of embryogenesis-related genes during germination has been proposed to occur, the role of HDAC in this process has not been elucidated. To address this question, the effects of an HDAC inhibitor and suppression of the Arabidopsis HDAC genes on this process were investigated. Here, we show that treatment of an HDA6 repression line with the HDAC inhibitor trichostatin A resulted in growth arrest and elevated transcription of LEC1, FUS3, and ABI3 during germination. The growth-arrest phenotype of the repression line was suppressed by lec1, fus3, and abi3. An HDA6/HDA19 double-repression line displayed arrested growth after germination and the formation of embryo-like structures on the true leaves of 6-week-old plants even without trichostatin A. The growth-arrest phenotype of this line was rescued by lec1. These results suggest that during germination in Arabidopsis, HDA6 and HDA19 redundantly regulate the repression of embryonic properties directly or indirectly via repression of embryo-specific gene function.
The basic unit of chromatin is the nucleosome. A nucleosome is composed of 147 bp of DNA wrapped around a histone octamer, which consists of the histone H2A, H2B, H3, and H4 proteins (Luger et al., 1997). The N tails of the histones, which are exposed on the surface of the nucleosome, may be methylated, acetylated, ubiquitinated, or phosphorylated (Jenuwein and Allis, 2001). Chromatin remodeling induced by histone modification plays important roles in several biological phenomena in eukaryotes (Francis and Kingston, 2001; Narlikar et al., 2002), and genetic studies have suggested the importance of chromatin remodeling factors in the control of plant development (Habu et al., 2001; Meyer, 2001; Verbsky and Richards, 2001; Reyes et al., 2002; Reyes, 2006). Acetylation and deacetylation of the Lys residues in histones are involved in the reversible modulation of chromatin structure and mediate the positive-negative regulation of transcription (Berger, 2002). The acetylation and deacetylation of histones is thought to control gene expression by changing the accessibility of DNA to DNA-binding transcription factors. Histone acetyltransferase catalyzes histone acetylation. The recruitment 1
This work was supported by the Ministry of Education, Science, Culture, and Sports, Japan (grant-in-aid no. 16370017), and for Special Research on Priority Areas (grant-in-aid no. 19043007 to H.K.). * Corresponding author; e-mail
[email protected]. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Hiroshi Kamada (
[email protected]). [W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.107.111674
of histone acetyltransferases to a promoter results in histone hyperacetylation and transcriptional activation (Brownell and Allis, 1996; Kuo and Allis, 1998; Kuo et al., 2000). In contrast, histone deacetylase (HDAC) catalyzes histone deacetylation, a phenomenon associated with transcriptional repression (Kadosh and Struhl, 1998; Rundlett et al., 1998). The eukaryotic HDACs have been grouped into three families, largely based on their homology with the yeast (Saccharomyces cerevisiae) proteins RPD3 (reduced potassium deficiency 3)/HDA1 and SIR2 (silent information regulator 2; Pandey et al., 2002; Yang and Seto, 2003), as well as other proteins. Arabidopsis (Arabidopsis thaliana) has 18 putative HDAC family genes (Pandey et al., 2002), 12 RPD3/HDA1 family genes, two SIR2 family genes, and four plant-specific HDACs (Tian et al., 2003; Wu et al., 2003). There have been several reports on the putative functions of HDACs in plants (Chen and Tian, 2007). Antisense inhibition of the expression of HDA19 (or AtHD1), an RPD3/HDA1 family gene, leads to early senescence, serrated leaves, the formation of aerial rosettes, and delayed flowering accompanied by defects in floral organ identity (Wu et al., 2000a; Tian and Chen, 2001; Tian et al., 2003). HDA19 is also involved in jasmonic acid and ethylene signaling during the response to pathogens (Zhou et al., 2005). One RPD3/ HDA1 family protein, HDA6, is involved in plantpathogen interactions and has been shown to interact with an F-box protein involved in jasmonic acidmediated plant defense responses (Devoto et al., 2002). HDA6 has also been reported to be involved in transgene silencing and the regulation of ribosomal RNA transcription in Arabidopsis (Murfett et al., 2001;
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Probst et al., 2004; Earley et al., 2006). Additionally, HD2-type HDACs, which are plant-specific proteins, are thought to be involved in the regulation of embryogenesis. Antisense inhibition of the expression of AtHD2A, a plant-specific HDAC gene, leads to seed abortion (Wu et al., 2000b). AtHD2A is transcribed in flowers and embryonic tissues, including siliques and somatic embryos (Zhou et al., 2004). Furthermore, seed development-related gene expression is repressed by AtHD2A overexpression. Another plant-specific HDAC, AtHD2C, is said to be involved in the abscisic acid (ABA) response (Sridha and Wu, 2006). AtHD2C is repressed by ABA, and AtHD2C overexpression represses the ABA response, as monitored by germination and expression of the LATE EMBRYOGENESIS ABUNDANT PROTEIN (LEA)-class genes, which are activated by ABA. These findings suggest that deacetylation of core histones by HD2-type HDACs contributes to the control of LEA-class gene expression during germination. It is also thought that changes in HDAC activation occur during germination at 1 d after imbibition, at which point core histones at the promoter and coding regions of some LEA-class genes are deacetylated (Tai et al., 2005). Additionally, evidence suggests a role for HDACs in the regulation of embryonic properties during vegetative growth. PICKLE (PKL) was isolated as a repressor of the embryonic program during the vegetative growth phase (Ogas et al., 1997, 1999; Henderson et al., 2004; Li et al., 2005). pkl mutants form swollen primary roots, referred to as pickle roots, and show ectopic expression of LEAFY COTYLEDON1 (LEC1), LEC2, and FUSCA3 (FUS3) in their primary roots (Rider et al., 2003). PKL is thought to repress the transcription of embryo-specific genes and to establish repression of embryonic properties after germination. PKL encodes an SWI/SNF-class chromatin-remodeling factor that belongs to the CHD3 group (Ogas et al., 1999). In animals, CHD3-type chromatin remodeling factors have been found to be components of RPD3-containing HDAC complexes (Tong et al., 1998; Wade et al., 1998; Xue et al., 1998; Zhang et al., 1998). These results suggest that HDACs are involved in the suppression of embryonic properties after germination by repressing embryo-specific transcription factors. However, there are no reports of HDAC involvement in the repression of embryonic properties after germination. Here, we show that an HDAC is involved in the repression of embryonic properties upon germination, based on the inhibition of HDAC activity in germinated Arabidopsis seeds following treatment with trichostatin A (TSA), an HDAC inhibitor. Treatment with TSA during germination strongly inhibited growth and induced the expression of embryo-specific transcription factors, including LEC1, FUS3, and ABSCISIC ACID INSENSITIVE3 (ABI3). Among several mutants defective in putative HDAC genes, the HDA6 repression line showed growth arrest in the presence of low concentrations of TSA. Additionally, a double repression line of HDA6 and HDA19 displayed growth arrest 150
after germination and the formation of embryo-like structures on the true leaves of 6-week-old plants even without TSA exposure; however, lec1 mutation rescued the growth-arrest phenotype of this line. We conclude that HDA6 and HDA19 contribute to the repression of embryogenic properties via direct or indirect repression of embryo-specific transcription factors after germination. RESULTS Repression of Postgermination Growth by TSA Treatment
To examine the role of HDAC in seed germination, Arabidopsis seeds were sown in media containing various concentrations of TSA (Fig. 1). Under inhibitorfree conditions, most seeds showed radicle emergence with cotyledon expansion and greening within 7 d after sowing (Fig. 1A). In contrast, following treatment with TSA, most seeds showed radicle emergence but no cotyledon expansion or greening (Fig. 1B). At 14 d after sowing, most of the control seedlings exhibited expanded cotyledons and true leaves (Fig. 1C), whereas most of the TSA-treated seedlings were pale with unexpanded cotyledons (Fig. 1D). We examined the growth arrest during germination caused by TSA through observations of radicle emergence (germination, Fig. 1E) and cotyledon expansion and greening (postgermination growth, Fig. 1F). At every concentration of TSA, the germination rate 3 d after sowing was lower than that observed for the control seeds; however, at 7 d after sowing, more than 80% of the seeds treated with 50 mM TSA had germinated (Fig. 1E). In contrast, the extent of postgermination growth was dependent on the concentration of TSA; 85% of seeds treated with 50 mM TSA showed no postgermination growth even at 14 d after sowing (Fig. 1F). It is possible that TSA disrupted the biosynthesis of gibberellin (GA3), a key phytohormone that stimulates seed germination. To eliminate this possibility, GA3 was added along with the HDAC inhibitor. Simultaneous treatment of seeds with GA3 and TSA (both 50 mM) did not alter the effects of the inhibitor on postgermination growth (Fig. 1F). Expression of Embryogenesis-Related Genes in TSA-Treated Seeds
HDACs are thought to contribute to the repression of embryogenesis-related gene expression during germination (Rider et al., 2003; Zhou et al., 2004; Tai et al., 2005). Because treatment of seeds with TSA resulted in the arrest of postgermination growth, embryogenesisrelated gene expression was examined in TSA-treated seeds. Four embryo-specific transcription factor genes, LEC1, LEC2, FUS3, and ABI3, and five LEA-class genes, AtECP31, AtECP63, CRUCIFERIN A (CRA), CRB, and CRC were selected as representative embryogenesisrelated genes, and the expression of these genes in Plant Physiol. Vol. 146, 2008
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seeds treated with 50 mM TSA was monitored by reverse transcription (RT)-PCR (Fig. 2A). No transcripts of any of the genes were detected in germinated seedlings at 14 d after sowing under inhibitor-free conditions. In contrast, all of the genes were expressed in TSA-treated seeds. To examine whether the expression of embryo-specific transcription factors in HDAC inhibitor-treated seeds is maintained in dry seeds or whether they are induced during germination, the expression of LEC1 and FUS3 was analyzed in seeds treated with TSA at 12, 24, 48, and 72 h after sowing and in untreated controls. During embryogenesis, LEC1 expression begins 4 d after pollination and decreases in mature seeds (Baumbusch et al., 2004). LEC1 expression was not observed in dry seeds and was not induced in germinating control seeds (Fig. 2B). In contrast, elevated LEC1 expression was observed after sowing in seeds treated with 50 mM TSA. FUS3 was expressed in dry control seeds, but the level of expression was decreased in germinating control seeds (Fig. 2C). In seeds treated with TSA, FUS3 expression was decreased at 12 h after sowing but was elevated 12 h later. These results indicate that LEC1 and FUS3 were induced in inhibitor-treated seeds during germination. Induction of Embryo-Like Structures by TSA
To evaluate the viability of the growth-arrested seeds, seeds treated with TSA for 14 d were transferred to inhibitor-free medium. All of the growth-arrested seeds began to form true leaves with embryo-like structures on their surfaces (Fig. 3, A and B). Dissection of the leaves was followed by an analysis of embryogenesis-related gene expression (LEC1, LEC2, FUS3, ABI3, AtECP31, AtECP63, CRA, CRB, and CRC) in the dissected parts. Little or no expression was detected in the true leaves, whereas strong expression of all of the genes was detected in the embryo-like structures (Fig. 3C). Arrested Postgermination Growth in an HDA6 Repression Line Caused by Treatment with Low Concentrations of TSA Figure 1. Influence of the HDAC inhibitor TSA on the germination and postgermination growth of wild-type seeds. Wild-type seeds (Col) were incubated for 4 d at 4°C and then sown on B5 solid medium containing or lacking 50 mM TSA. A, Untreated seeds 7 d after sowing. B, 50 mM TSA-treated seeds at 7 d after sowing. C, Untreated seeds at 14 d after sowing. D, Seeds treated with 50 mM TSA at 14 d after sowing. Bar 5 1 mm. E, Effect of TSA on seed germination. Wild-type seeds (Col) were incubated for 4 d at 4°C and then sown on B5 solid medium containing various concentrations of TSA and containing or lacking GA3. Germination, defined as the protrusion of the radicle through the seed coat, was recorded 3, 7, and 14 d after sowing. Control, No inhibitor; 2, without GA3; 1, with 50 mM GA3. Each experiment was repeated three times and the average value is shown with the SD. F, Effect of TSA on postgermination growth. Postgermination growth was defined as cotyledon expansion and greening. The percentage of seeds showing postgermination growth was checked at 7 and 14 d after sowing. Each experiment was repeated three times and the average value is shown with the SD. Plant Physiol. Vol. 146, 2008
Arabidopsis contains 18 putative HDAC family genes grouped into three families (Pandey et al., 2002). There has been no report of the ectopic expression of embryospecific transcription factors during vegetative growth in a single HDAC mutant. These facts suggest that some HDACs act redundantly in the repression of embryonic properties and that a high concentration of TSA simultaneously inhibits the activity of related HDACs. Thus, we expected that a mutant deficient in an HDAC known to repress embryonic properties would exhibit growth arrest following treatment with low concentrations of TSA. To identify which HDAC contributes to seedling growth after germination and the repression of embryo-specific transcription factors, knockout and repressed HDAC lines (Supplemental 151
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Table S1) were treated with low concentrations of TSA. Wild-type and mutant seeds were sown in water containing 0.5 mM TSA. At this TSA concentration, the postgermination growth of the wild-type plants was unimpaired (Fig. 4). However, an HDA6 repression line (Arabidopsis Biological Resource Center [ABRC], Ohio State University, Columbus, stock no. CS24039, referred to as HDA6:RNAi) ceased postgermination growth after TSA treatment (Fig. 4). The germination rate of untreated HDA6:RNAi seeds was equal to that of wild-type seeds (Fig. 5A). Additionally, in medium containing 0.5 mM TSA, 90% of the HDA6:RNAi seeds germinated within 7 d after sowing; however, none showed cotyledon expansion or greening (Figs. 4 and 5B). Following treatment with even 0.1 mM TSA, the postgermination growth of the HDA6:RNAi seeds was strongly arrested (Fig. 5B). Arrested growth following treatment with a low concentration of TSA was also observed in another HDA6 RNAi line (ABRC stock no. CS24038), and the intensity of the arrest was related to the level of HDA6 expression (Supplemental Figs. S1 and S5). In contrast, the growth of the other HDAC mutants with or without TSA treatment was not statistically different (Supplemental Fig. S1). LEC1, FUS3, and ABI3 expression was analyzed in HDA6:RNAi seeds at 7 d after sowing. LEC1 expression was not induced in untreated HDA6:RNAi seeds but was induced by exposure to 0.5 mM TSA in HDA6:RNAi seeds (Fig. 5C; Supplemental Fig. S2). Likewise, FUS3 and ABI3 were not expressed in untreated HDA6:RNAi seeds, but were strongly expressed following TSA treatment (Fig. 5, D and E). Effects of TSA on Postgermination Growth in Embryo-Specific Transcription Factor Mutants
Figure 2. Expression of embryogenesis-related genes in seeds treated with the HDAC inhibitor TSA. A, Expression of embryogenesis-related genes in wild-type (Col) seedlings treated with TSA for 14 d. Control, No inhibitor; TSA, 50 mM TSA. The expression of LEC1, LEC2, FUS3, ABI3, AtECP31, AtECP63, CRA, CRB, and CRC was analyzed by RTPCR using specific primers. The UBQ10 gene was used as an internal standard. B, Expression of LEC1 in seeds treated with TSA at various times after sowing. Wild-type seeds (Col) were treated with (1) or without (2) 50 mM TSA. The LEC1 expression in seeds at 12, 24, 48, and 72 h after sowing, wild-type siliques at 3 d after flowering, and dry seeds was analyzed by quantitative RT-PCR. The horizontal axis indicates the time (h) after sowing. The vertical line indicates the level of LEC1 cDNA normalized to UBQ10. C, Expression of FUS3 in seeds treated with TSA at various times after sowing. The vertical line indicates the level of FUS3 cDNA normalized to UBQ10. Each experiment was repeated three times; the average value is shown with the SD.
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The seeds in which growth arrest was induced by treatment with TSA showed expression of the embryospecific transcription factor genes LEC1, FUS3, and ABI3 (Fig. 2). The transcription factors encoded by these genes have been reported to regulate embryo development and dormancy (Meinke et al., 1994; West et al., 1994) and are also involved in the maintenance of embryonic properties and the suppression of precocious germination (Meinke et al., 1994; Nambara et al., 2000). We examined whether the growth arrest of TSA-treated HDA6:RNAi seeds was caused by embryo-specific transcription factor expression by introgressing the lec1, fus3, and abi3 mutations into the HDA6:RNAi line (CS24039). Immature HDA6:RNAi lec1-1, HDA6: RNAi fus3-3, and HDA6:RNAi abi3-6 seeds were treated with 0.5 mM TSA during germination. Almost all immature HDA6:RNAi seeds showed arrested cotyledon expansion after treatment with 0.5 mM TSA. In contrast, immature HDA6:RNAi lec1-1, HDA6:RNAi fus3-3, and HDA6:RNAi abi3-6 seeds produced expanded green cotyledons on medium containing 0.5 mM TSA (Fig. 6). Plant Physiol. Vol. 146, 2008
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occasionally observed on the shoots (Fig. 7, F and G). No expression of LEC1, FUS3, or ABI3 was observed in HDA6:RNAi or HDA19:RNAi seedlings germinated on inhibitor-free medium, but all three genes were expressed in untreated HDA6/19:RNAi seedlings 7 d after sowing (Fig. 8, A–C; Supplemental Fig. S3). However, the expression of LEC1, FUS3, and ABI3 in HDA6/19:RNAi was 2 to 6 times lower than that in TSA-treated HDA6:RNAi seeds. In true leaves of the HDA6/19:RNAi line, no LEC1 or ABI3 expression was detected (data not shown), but FUS3 expression was observed (Fig. 8D). However, the level of FUS3 expression in true leaves was much lower than that in seedlings. Arrested Postgermination Growth in an HDA6:RNAi pkl Double Mutant Figure 3. Effects of TSA treatment on plant growth after transfer to inhibitor-free conditions. Wild-type seeds (Col) treated with 50 mM TSA for 14 d on phytohormone-free Gamborg’s B5 solid medium were transferred to inhibitor-free B5 liquid medium and cultured for an additional 4 weeks. A, Seedlings grown from seeds after the transfer to inhibitor-free medium. Note the formation of embryo-like structures on the true leaves (white arrows). B, Enlarged image of the embryo-like structures in A. Bar 5 1 mm. C, Expression of embryogenesis-related genes in the embryo-like structures. Embryogenesis-related gene expression, as indicated in Figure 2A, was analyzed by RT-PCR in the embryo-like structures (SE) and in the remaining true leaves after elimination of the embryo-like structures (Le). UBQ10 was used as an internal standard.
Arrested Postgermination Growth and Expression of Embryo-Specific Transcription Factor Genes in an HDA6-HDA19 Double Repression Line
HDA6:RNAi showed hypersensitivity to TSA, but in the absence of TSA, this line did not show arrested postgermination growth or embryo-specific transcription factor expression (Figs. 4 and 5). These results suggest that other HDACs act redundantly with HDA6 in the repression of embryonic properties. Thus, we crossed HDAC knockout and repression lines with the HDA6:RNAi line (CS24039). Among the resulting HDAC double mutants, one line, produced from a cross between HDA6:RNAi and an HDA19 repression line (ABRC stock no. CS30925, referred to as HDA19: RNAi), showed abnormal postgermination growth. Repression lines of either HDA6 or HDA19 showed wild-type-like cotyledon expansion and greening at 7 d after sowing (Fig. 7, A–C). However, approximately 70% of the seeds produced by the HDA6/HDA19 double-repression line (HDA6/19:RNAi) showed arrested cotyledon expansion and greening on inhibitorfree medium (Fig. 7D; Supplemental Fig. S4). In contrast, cotyledon expansion in HDA6/19:RNAi was restored by introgression of the lec1-1 mutation, as found in HDA6:RNAi (Fig. 7E). After cultivation for more than 1 month on Murashige and Skoog (MS) medium, the growth-arrested HDA6/19:RNAi seedlings began to develop true leaves, with embryo-like structures Plant Physiol. Vol. 146, 2008
The HDA6 repression line showed LEC1 and FUS3 expression after germination in the presence of low concentrations of TSA. Additionally, the doublerepression line HDA6/19:RNAi showed the same phenomenon without inhibitor treatment. Ectopic expression of LEC1 and FUS3 after germination in a PKLdeficient mutant has been reported (Ogas et al., 1997, 1999; Rider et al., 2003; Li et al., 2005). To examine the relationship between HDA6 and PKL, HDA6:RNAi was crossed with the PKL-deficient mutant pkl1-1. On TSA-free medium, the HDA6:RNAi and pkl1-1 lines germinated and produced expanded green cotyledons (Figs. 7B and 9A). In contrast, approximately 20% of the HDA6:RNAi pkl1-1 double-mutant seeds showed arrested cotyledon expansion and did not progress to vegetative growth (Fig. 9B). After cultivation for more than 1 month on MS medium, the growth-arrested seeds began to form true leaves that sometimes bore embryo-like structures (Fig. 9, C and D). Additionally,
Figure 4. Arrest of postgermination growth in HDA6:RNAi by treatment with low concentrations of TSA. Morphological features of growth-arrested HDA6:RNAi seedlings. Photographs were taken 7 d after the sowing of wild-type (Ws) and HDA6:RNAi (CS24039) seeds in distilled water containing or lacking 0.5 mM TSA. Bar 5 1 mm. 153
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Figure 5. Influence of the HDAC inhibitor TSA on the germination and postgermination growth of HDA6:RNAi. A, Germination rates of wild-type (Ws) and HDA6:RNAi seeds (CS24039). Seeds were incubated for 4 d at 4°C and then sown in distilled water containing or lacking 0.5 mM TSA. Germination, defined as protrusion of the radicle through the seed coat, was recorded at 1, 2, 3, 5, and 7 d after sowing. Untreated wild-type seeds (white squares), wild-type seeds exposed to 0.5 mM TSA (black squares), untreated HDA6:RNAi seeds (white triangles), and HDA6:RNAi seeds exposed to 0.5 mM TSA (black triangles) are shown. Each experiment was repeated three times, and the average value is shown with the SD. B, Effect of TSA on the postgermination growth of wild-type and HDA6:RNAi seeds. Wild-type (Ws) and HDA6:RNAi seeds (CS24039) were incubated for 4 d at 4°C and then sown in distilled water with or without TSA. Postgermination growth was defined as cotyledon expansion and greening by 7 d after sowing. Each experiment was repeated three times, and the average value is shown with the SD. *, None of the HDA6:RNAi seeds showed postgermination growth in the presence of 0.5 mM TSA. C, The expression of LEC1 in wild-type (Ws) and HDA6:RNAi seeds (CS24039) was analyzed by quantitative RT-PCR. Seeds were treated with (1) or without (2) 0.5 mM TSA for 7 d. D, FUS3 expression in wild-type (Ws) and HDA6:RNAi seeds. E, ABI3 expression in wild-type (Ws) and HDA6:RNAi seeds. The vertical line indicates the expression level of each gene normalized to UBQ10. Each experiment was repeated three times and the average value is shown with the SD.
embryo-like structures were occasionally observed on shoots of HDA6:RNAi pkl1-1 plants that did not form true leaves (Fig. 9E). In contrast, no embryo-like structures had formed on the pkl1-1 single mutant even after cultivation for 2 months on MS medium (Fig. 9F). LEC1, FUS3, and ABI3 expression was analyzed in HDA6:RNAi pkl1-1 double-mutant plants with embryolike structures on their shoots, and the level of expression of all three genes was higher than in the pkl1-1 single mutant (Fig. 9, G–I). DISCUSSION HDA6 and HDA19 Are Involved in the Repression of Embryonic Properties after Germination
The treatment of germinating seeds with TSA resulted in growth arrest (Fig. 1) accompanied by the expression of embryogenesis-related genes (Fig. 2A). 154
The corresponding embryo-specific transcription factors were highly expressed in seedlings whose growth was arrested due to HDAC inhibitor treatment. In wild-type plants, LEC1 was not expressed in dry seeds, but LEC1 expression was induced during germination by TSA treatment (Fig. 2B). Furthermore, in inhibitor-treated seeds, FUS3 expression temporarily decreased after sowing but could be increased again (Fig. 2C). The expression profiles of LEC1 and FUS3 in TSA-treated seeds exclude the possibility that the transcription of these embryogenesis-related genes was maintained by the growth arrest. Instead, both genes were re-induced during germination by TSA treatment. Of the HDAC mutants treated with low concentrations of TSA, only HDA6:RNAi failed to undergo postgermination growth and showed expression of embryo-specific transcription factor genes (Figs. 4 and 5). However, the germination rate of untreated HDA6:RNAi seeds was the same as that of wild-type Plant Physiol. Vol. 146, 2008
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Figure 6. Possible involvement of LEC1, FUS3, and ABI3 in the TSA-hypersensitive phenotype of HDA6: RNAi. Immature seeds of HDA6:RNAi (CS24039), HDA6:RNAi lec1-1 (CS24039/lec1-1), HDA6:RNAi fus3-3 (CS24039/fus3-3), and HDA6:RNAi abi3-6 (CS24039/abi3-6) were sown in B5 liquid medium containing (1) or lacking (2) 0.5 mM TSA for 7 d. In the presence of TSA, HDA6:RNAi seeds exhibited arrested cotyledon expansion, whereas HDA6:RNAi lec1-1, HDA6:RNAi fus3-3, and HDA6:RNAi abi3-6 showed cotyledon expansion and greening by 7 d after sowing. Bar 5 1 mm.
seeds (Fig. 5A), suggesting that the postgermination growth arrest in HDA6:RNAi was not caused by poor germination but by TSA. HDA6 belongs to the RPD3/ HDA1 family, and its deacetylase activity is directly inhibited by TSA (Earley et al., 2006). HDA6 has been reported to be involved in the silencing of transgenes and rDNA (Murfett et al., 2001; Probst et al., 2004; Earley et al., 2006). However, there has been no report of developmental abnormalities in an HDA6-deficient mutant. In this study, untreated seeds of the HDA6 repression line HDA6:RNAi did not show arrested postgermination growth or expression of embryospecific transcription factors after germination (Fig. 5). This suggests that other HDAC factors act redundantly with HDA6. HD2-type HDACs have been reported to be involved in embryo development, seed maturation, dormancy, and embryogenesis-related gene expression (Wu et al., 2000b; Zhou et al., 2004; Sridha and Wu, 2006). However, repression lines of these HDACs treated with a low concentration of TSA did not show inhibited growth after germination (Supplemental Fig. S1). Furthermore, we produced Plant Physiol. Vol. 146, 2008
double repression lines by crossing HDA6:RNAi with single repression lines of other HD2-type HDACs, but no growth arrest was observed in these lines after germination (data not shown). In contrast, in the Arabidopsis genome, 12 genes encode HDAC proteins belonging to the RPD3/HDA1 family (Pandey et al., 2002), and some RPD3/HDA1-type HDACs have been suggested to regulate development (Wu et al., 2000a; Tian and Chen, 2001; Tian et al., 2003; Zhou et al., 2005; Xu et al., 2005). These RPD3/HDA1-type HDACs were expected to have functional redundancy with HDA6 in the repression of embryonic properties after germination. In fact, the double repression of HDA6 and HDA19, which encodes an RPD3/HDA1-type HDAC, inhibited postgermination growth (Fig. 7D) with expression of LEC1, FUS3, and ABI3 after radicle emergence (Fig. 8, A–C) in the absence of TSA. Additionally, in the HDA6/19:RNAi line, FUS3 expression was observed in both seedlings and true leaves (Fig. 8D). These results suggest that HDA6 and HDA19 are involved in the repression of embryo-specific genes after germination. 155
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It has been reported that HDA19 repression and knockout lines show pleiotropic abnormalities in development, including seed development (Tian and Chen, 2001; Tian et al., 2003). Additionally, HDA19 has been suggested to be involved in the genome-wide regulation of developmental gene expression (Tian et al., 2005). However, there is no evidence to suggest the involvement of HDA19 in the repression of embryogenesis-related genes after germination. The HDA19 repression line did not show arrested postgermination growth even after treatment with low concentrations of TSA (Supplemental Fig. S1). This suggests that HDA6 is a key factor in the repression of embryonic properties after germination and that the
Figure 7. Morphological features of the HDA6/19:RNAi double repression line. A, Wild type (Ws). B, HDA6:RNAi (CS24039). C, HDA19:RNAi (CS30925). D, HDA6/19:RNAi (CS24039/CS30925). E, HDA6/19:RNAi lec1-1 (CS24039/CS30925/lec1-1) seedlings. A to E, Photographs were taken 7 d after sowing on inhibitor-free medium. F, HDA6/19:RNAi plants (CS24039/CS30925) at 6 weeks after sowing. The arrow indicates a somatic embryo-like structure that formed on the shoot. G, Enlarged image of the embryo-like structures in F. Bar 5 1 mm. 156
Figure 8. Expression of embryo-specific transcription factors in HDA6/ 19:RNAi seedlings. A, The LEC1 expression in wild-type (Ws), HDA6: RNAi (CS24039), HDA19:RNAi (CS30925), and HDA6/19:RNAi (CS24039/CS30925) seedlings at 7 d after sowing was analyzed by quantitative RT-PCR. B, FUS3 expression in Ws, HDA6:RNAi (CS24039), HDA19:RNAi (CS30925), and HDA6/19:RNAi (CS24039/ CS30925) seedlings at 7 d after sowing. C, ABI3 expression in Ws HDA6:RNAi (CS24039), HDA19:RNAi (CS30925), and HDA6/19:RNAi (CS24039/CS30925) seedlings at 7 d after sowing. D, FUS3 expression in the true leaves of Ws HDA6:RNAi (CS24039), HDA19:RNAi (CS30925), and HDA6/19:RNAi (CS24039/CS30925). True leaves were collected from each plant at 3 weeks after sowing. The vertical lines indicate the expression level of each gene normalized to UBQ10. Each experiment was repeated three times and the average value is shown with the SD.
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Figure 9. Morphological features of HDA6 pkl1-1 sown on inhibitor-free medium. A, pkl1-1 seedlings at 7 d after sowing. B, HDA6:RNAi pkl1-1 (CS24039/pkl1-1) seedlings at 7 d after sowing. C, HDA6:RNAi pkl1-1 (CS24039/pkl1-1) plants at 9 weeks after sowing. The arrow indicates a somatic embryo-like structure that formed on the true leaves. D, Enlarged image of the embryo-like structure in C. E, HDA6:RNAi pkl1-1 (CS24039/pkl1-1) plants at 6 weeks after sowing. The arrow indicates a somatic embryo-like structure formed on a shoot that lacked true leaf development. F, pkl1-1 plants at 6 weeks after sowing. Bar 5 1 mm. G, LEC1 expression in true leaves at 6 weeks after sowing of the pkl1-1 single mutant and HDA6:RNAi pkl1-1 (CS24039/pkl1-1). H, FUS3 expression in true leaves at 6 weeks after sowing of pkl1-1 and HDA6:RNAi pkl1-1 (CS24039/pkl1-1). I, ABI3 expression in true leaves at 6 weeks after sowing of pkl1-1 and HDA6:RNAi pkl1-1 (CS24039/pkl1-1). The vertical line indicates the expression level of each gene normalized to UBQ10. Each experiment was repeated three times and the average value is shown with the SD.
contribution of HDA19 is smaller than that of HDA6. In contrast, LEC1, FUS3, and ABI3 expression in HDA6/ 19:RNAi was lower than in TSA-treated HDA6:RNAi seeds (Figs. 5 and 8). Furthermore, LEC2 was expressed in wild-type seeds after TSA treatment (Fig. 2A) but not in HDA6:RNAi or HDA6/19:RNAi (data not shown). These results suggest that HDACs other than HDA6 and HDA19 act redundantly in the repression of embryonic properties after germination. Another possibility is that TSA acts via an unknown target to increase embryo-specific transcription factor expression. Ectopic expression of LEC1 and FUS3 after germination was reported in a PKL-deficient mutant (Ogas et al., 1997, 1999; Rider et al., 2003; Li et al., 2005). PKL encodes an SWI/SNF-class chromatin-remodeling factor that belongs to the CHD3 group (Ogas et al., 1999). CHD3 proteins are components of RPD3-containing HDAC complexes in animals (Tong et al., 1998; Wade et al., 1998; Xue et al., 1998; Zhang et al., 1998). Additionally, HDA6 and HDA19 are classified as RPD3/HDA1-type HDACs (Pandey et al., 2002). Therefore, HDA6 and/or HDA19 are thought to act with PKL in the repression of embryonic properties after germination. pkl plants form swollen primary roots, known as pickle roots, and exhibit ectopic exPlant Physiol. Vol. 146, 2008
pression of embryogenesis-related genes after germination (Rider et al., 2003). In this study, the pkl single mutant germinated and produced expanded green cotyledons on TSA-free medium (Fig. 9A), whereas the HDA6:RNAi pkl1-1 double mutant showed arrested cotyledon expansion (Fig. 9B). Embryo-like structures have been shown to form on hormone-free medium from the detached pickle roots, cotyledons, and hypocotyls of pkl seedlings (Ogas et al., 1997; Henderson et al., 2004). However, embryo-like structures were not observed even after a long period of cultivation on MS medium (more than 2 months) without detaching tissues (Fig. 9F). In contrast, after 1 month of cultivation, some HDA6:RNAi pkl1-1 double-mutant seedlings began to form embryo-like structures on their shoots (Fig. 9, C–E) that were visible in the absence of dissection. Additionally, the expression of LEC1, FUS3, and ABI3 in the double mutant was higher than that in either single mutant (Fig. 9, G–I). These results suggest that derepression of embryonic properties in the pkl mutant was enhanced by the repression of HDA6. In contrast, no obvious root abnormalities were observed in HDA6/19:RNAi seedlings (Fig. 7D). Additionally, ABI3 was expressed in HDA6/19:RNAi and HDA6 pkl1-1 (Figs. 8C and 9I), but ectopic ABI3 expression 157
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was not observed in pkl plants (Rider et al., 2003). Furthermore, the penetrance of the pkl phenotype was affected by GA3 and GA3 inhibitors, but the TSAinduced postgermination growth arrest was not eliminated by GA3 application (Fig. 1F). These facts suggest that HDA6 and/or HDA19 act independently of PKL to repress embryonic properties. It would be interesting to determine whether the phenotype of an HDA6/ HDA19/PKL triple mutant is more severe than that of either double mutant. Additionally, there is no evidence to suggest a molecular interaction between HDACs and PKL. It is therefore necessary to examine whether HDA6 and/or HDA19 form a complex with PKL that represses embryonic properties after germination. Embryo-Specific Transcription Factor Expression by Inhibition of HDAC Activity Induces Growth Arrest after Germination
The growth of wild-type seedlings was arrested by TSA treatment, but when the seedlings were transferred to TSA-free medium, they began to grow and formed embryo-like structures on their true leaves (Fig. 3, A and B). After a long period of cultivation on TSA-free MS medium, HDA6/19:RNAi and HDA6: RNAi pkl1-1 formed embryo-like structures (Figs. 7G and 8C). Embryo-like structures expressed not only LEA-class genes but also embryo-specific transcription factor genes (Fig. 3C). This observation suggests that the embryo-like structures are not formed by morphological conversion to a cotyledon-like organ but that they have embryonic properties at the molecular level, as do somatic embryos. The formation of embryo-like structures on vegetative tissues occurs when LEC1 or LEC2 is expressed ectopically in seedlings (Lotan et al., 1998; Stone et al., 2001). In addition, ectopic FUS3 expression results in the accumulation of seed storage proteins in true leaves (Gazzarrini et al., 2004). It has been reported that LEC1, LEC2, and FUS3 are important for the production of somatic embryos from immature seeds after 2,4-dichlorophenoxyacetic acid treatment (Gaj et al., 2005). This suggests that the formation of embryo-like structures by inhibition of HDAC activity is caused by the irregular expression of these embryo-specific transcription factors after germination. The arrest of growth after germination caused by TSA, as evidenced by the lack of cotyledon expansion or greening, was abrogated by the introgression of a lec1, fus3, or abi3 mutation (Fig. 6), each of which affects seed dormancy (Meinke et al., 1994; West et al., 1994). Seed dormancy and germination are controlled by phytohormones, particularly GA3 and ABA (Yamaguchi and Kamiya, 2000; Finkelstein and Gibson, 2002; Peng and Harberd, 2002). These observations suggest that the growth arrest caused by decreased HDAC activity is due to alterations in the endogenous levels of GA3, ABA, or both. However, the growth arrest following TSA treatment was not eliminated by the simultaneous application of GA3 (Fig. 1F). Further158
more, the arrest of postgermination growth induced by TSA was not eliminated by the application of fluridone, which inhibits ABA synthesis (Supplemental Fig. S6). This result excludes the possibility that the growth arrest triggered by the inhibition of HDAC activity was caused by the disruption of endogenous levels of GA3 and ABA. In contrast, several reports have shown that the HDACs in higher plants mediate transcriptional regulation during phytohormone signaling (Devoto et al., 2002; Gao et al., 2004; Song et al., 2005; Zhou et al., 2005; Sridha and Wu, 2006). Overexpression of AtHD2C, which encodes an HD2-type HDAC, enhances germination, even in ABA-containing medium (Sridha and Wu, 2006), indicating that the growth arrest caused by TSA is due to enhanced ABA sensitivity. In contrast, the TSA-triggered growth arrest was negated by introgression of the fus3 mutation (Fig. 6), but the fus3 mutant is still sensitive to germination arrest after ABA treatment (Parcy et al., 1997). Thus, it appears that the growth arrest triggered by TSA treatment is not caused by enhanced sensitivity to ABA. In contrast, it has been reported that the overexpression of LEC1 in seedlings causes growth arrest after germination (Lotan et al., 1998). Furthermore, ectopic LEC1 expression in seedlings alters the hypocotyl structure and produces ectopic accumulation of starch and lipids, phenomena that are enhanced in the presence of exogenous auxin and sugars but not in the presence of GA3 or ABA (Casson and Lindsey, 2006). Sugars are important regulators of germination and seedling development (Gibson, 2005). Furthermore, sugar signaling is thought to be involved in the repression of embryonic properties after germination (Tsukagoshi et al., 2007). The above result suggests that sugar signaling mediated by LEC1, FUS3, and/or ABI3 is involved in the growth arrest caused by the inhibition of HDAC activity. Together, these data suggest that the inhibition of HDAC activity produces growth arrest and the formation of embryo-like structures after germination through the expression of embryo-specific factors. To elucidate the molecular mechanisms of the phenomena induced by inhibiting HDAC activity, it will be necessary to compare HDA6/19:RNAi with HDA6/ 19:RNAi introgressed with lec1, fus3, or abi3 at the molecular level. HDAC Contributes to the Repression of Embryonic Properties via Repression of Embryo-Specific Transcription Factors after Germination
The embryo-specific transcription factors LEC1, FUS3, and ABI3 were highly expressed in TSA-treated seeds (Figs. 2 and 5) and in HDA6/19:RNAi (Fig. 8) after germination, suggesting that these genes are directly or indirectly repressed via the deacetylation of histones by HDAC. It has been reported that FUS3 is directly regulated by histone methylation by polycomb group proteins (Makarevich et al., 2006). Histone methylation is sometimes coupled with histone deacetPlant Physiol. Vol. 146, 2008
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ylation (Peterson and Laniel, 2004). Because FUS3 expression was observed in the true leaves of HDA6/19:RNAi (Fig. 8D), it is possible that the repression of FUS3 expression in vegetative tissues is regulated by histone deacetylation. However, FUS3 expression in true leaves was weaker than in seedlings (Fig. 8, B and D), indicating that the increase in FUS3 expression after germination was due to the ectopic expression of another factor(s) via inhibition of HDAC activity. It has been suggested that FUS3 expression is controlled by LEC1 during embryogenesis (To et al., 2006) and that ectopic LEC1 expression can induce the expression of FUS3 and ABI3 in vegetative tissues (Kagaya et al., 2005). LEC1 expression was observed in HDA6/19:RNAi seedlings but not in true leaves (data not shown). This suggests that the expression of FUS3 after germination is induced by ectopic LEC1 expression. In contrast, multiple VP1/ABI3-LIKE (VAL) mutants have been reported to form embryo-like structures on their vegetative tissues showing ectopic expression of LEC1, FUS3, and ABI3 (Suzuki et al., 2007). The VAL genes encode B3 proteins, which contain a domain associated with chromatin remodeling. The consensusbinding site of the B3 DNA-binding domain is in the promoter region and first intron of VAL-regulated genes. Additionally, this consensus sequence is present in the promoter and/or first intron of LEC1, FUS3, and ABI3, suggesting that HDAC simultaneously represses these embryo-specific transcription factors after germination via histone deacetylation. However, it is unclear which genes are directly regulated by HDAC after germination, repressing the embryonic properties of the plant during the vegetative phase. In summary, these results suggest that HDACs are involved in the repression of embryonic properties after germination via repression of the embryo-specific transcription factors LEC1, FUS3, and ABI3. Among the Arabidopsis HDACs, HDA6 and HDA19 act redundantly in this regulatory mechanism. Our findings suggest a role for HDAC following germination in the repression of embryonic properties in Arabidopsis. To elucidate the mechanism of repression, it will be necessary to identify the genes that are regulated by HDA6 and HDA19 via histone deacetylation.
Inhibitor Treatment TSA (Sigma Chemical) was dissolved in dimethylsulfoxide and stored at 220°C until use. GA3 (Sigma Chemical) was dissolved in water immediately before use. Surface-sterilized seeds were incubated at 4°C for 4 d and then sown on Gamborg’s B5 medium containing 5 to 50 mM TSA with or without 50 mM GA3 under a 16-h-light:8-h-dark cycle at 25°C. Germination was defined as protrusion of the radicle through the seed coat. Postgermination growth was defined as the appearance of expanded green cotyledons. To confirm that the growth-arrested seeds were still alive, seeds treated with 50 mM TSA for 14 d were transferred to TSA-free Gamborg’s B5 solid or liquid medium and cultured for 3 to 4 weeks. To examine the effect of low concentrations of TSA, wild-type and HDA6: RNAi seeds were sown in tissue-culture dishes (6-cm diameter) containing distilled water containing or lacking 0.5 mM TSA under a 16-h-light:8-h-dark cycle at 25°C. In comparative experiments using HDA6:RNAi lec1-1, HDA6: RNAi fus3-3, and HDA6:RNAi abi3-6, immature zygotic embryos were used in place of dry seeds. Immature seeds were grown for 7 d in B5 liquid medium containing or lacking 0.5 mM TSA.
RT-PCR Expression Analysis Total RNA was isolated using an RNAqueous kit (Ambion). cDNAs were synthesized using the SuperScript First-Strand Synthesis system for RT-PCR (Invitrogen). Each gene was amplified under the following conditions: 94°C for 10 min, followed by 25 to 40 cycles of 94°C for 15 s, 55°C for 15 s, and 72°C for 1 min. The primers (forward and reverse) used were as follows: for LEC1, 5#-AGACGGCAGAGAAACAATGG-3# and 5#-ATTCATCTTGACCCGACGAC-3#; for LEC2, 5#-TGAATCCTCAGCCGGTTTAC-3# and 5#-ACCCAACATCGCTGTTCTTC-3#; for FUS3, 5#-GTGGCAAGTGTTGATCATGG-3# and 5#-CGTGAAAACCGTCCAAATCT-3#; for ABI3, 5#-GCAGGACAAATGAGAGATCAG-3# and 5#-TCATTTAACAGTTTGAGAAG#; for AtECP31, 5#ATGAGCCAAGAGCAACCAAGGAG-3# and 5#-GGCATCTTCCCTCGTCACAGC-3#; for AtECP63, 5#-CGGTGGAAGCAAAGGATAAGACG-3# and 5#-CTTTCCGAGCATCATCATCCATC-3#; for CRA, 5#-CACTCTCAACAGTTACGATC-3# and 5#-GTGAGTCAAAGTGGTCTCGA-3#; for CRB, 5#-TGACCGCAACCTTAGACCAT-3# and 5#-TGCTATGGGTCAGTGTGGTC-3#; for CRC, 5#-ACAACATGAACGCTAACGAGA-3# and 5#-CTCCTCGATCAACTGTTGTT-3#; and for UBIQUITIN10 (UBQ10), 5#-GATCTTTGCCGGAAAACAATTGGAGGATGGT-3# and 5#-CGACTTGTCATTAGAAAGAAAGAGATAACAGG-3#. Quantitative RT-PCR was performed in a LightCycler (Roche Applied Science) using the LightCycler FastStart DNA Master SYBR Green I kit (Roche Applied Science). The LEC1 transcript was amplified using the primers 5#-AGACGGCAGAGAAACAATGG-3# and 5#-ACGATACCATTGTTCTTGTCACC-3#, the FUS3 transcript was amplified using the primers 5#-GGTACTGGCCAAACAACAATAGC-3# and 5#-CTAGCTGCAGACCATGAGCATT-3#, and the ABI3 transcript was amplified using the primers 5#-CAGGGATGGAAACCAGAAAAGA-3# and 5#-TTACCCACGTCGCTTTGCTT-3#. The amounts of cDNA were calculated using LightCycler 3.1 (Roche Applied Science). LEC1, FUS3, and ABI3 were normalized to UBQ10, which was amplified using the primers 5#-GTACTTTGGCGGATTACAACATC-3# and 5#-GAATACCTCCTTGTCCTGGATCT-3#.
Supplemental Data MATERIALS AND METHODS Plant Materials The Arabidopsis (Arabidopsis thaliana) ecotypes Columbia (Col) and Wassilewskija (Ws) were used in this study. The lec1-1, fus3-3, abi3-6, pkl1-1, and HDAC mutants (see Supplemental Table S1; Supplemental Fig. S7) were obtained from the ABRC (Ohio State University). The HDA6 repression line HDA6:RNAi (ABRC stock no. CS24039) was crossed with lec1-1, fus3-3, abi3-6, the HDA19 repression line HDA19:RNAi (CS30925), or pkl1-1 to introgress the respective mutations. For germination, seeds were surface sterilized with a sodium hypochlorite solution (available chlorite concentration of 1%) for 5 min and then rinsed five times with sterile distilled water. The surfacesterilized seeds were incubated at 4°C for 4 d, sown in plastic petri dishes (9-cm diameter) containing 20 mL of phytohormone-free Gamborg’s B5 solid medium (0.8% agar; w/v), and incubated in a 16-h-light:8-h-dark cycle at 25°C.
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The following materials are available in the online version of this article. Supplemental Figure S1. Effects of the HDAC inhibitor TSA on postgermination growth in the HDAC mutant lines. Supplemental Figure S2. Expression of embryo-specific transcription factors in HDA6:RNAi seeds after HDAC inhibitor treatment. Supplemental Figure S3. Expression of embryo-specific transcription factors in single and double repression lines of HDA6 and HDA19. Supplemental Figure S4. Morphological features of another HDA6/ HDA19:RNAi line. Supplemental Figure S5. HDA6, HDA19, and UBQ10 expression in single and double repression lines of HDA6 and HDA19. Supplemental Figure S6. Effects of fluridone on the arrest of postgermination growth by TSA treatment.
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Supplemental Figure S7. Target sequences for HDA6 and HDA19 RNAi. Supplemental Table S1. HDAC mutant lines used to screen for TSAhypersensitive mutants. Received October 25, 2007; accepted November 11, 2007; published November 16, 2007.
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