The Arabidopsis Histone Deacetylases HDA6 and HDA19 ... · Motoki Tanaka, Akira Kikuchi, and Hiroshi Kamada* Graduate School of Life and Environmental Sciences, University of Tsukuba,

The Arabidopsis Histone Deacetylases HDA6 and HDA19Contribute to the Repression of Embryonic Propertiesafter 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. InArabidopsis (Arabidopsis thaliana), the transcription factors LEAFY COTYLEDON1 (LEC1), FUSCA3 (FUS3), and ABSCISICACID INSENSITIVE3 (ABI3) play key roles in embryogenesis. Although the repression of embryogenesis-related genes duringgermination has been proposed to occur, the role of HDAC in this process has not been elucidated. To address this question, theeffects of an HDAC inhibitor and suppression of the Arabidopsis HDAC genes on this process were investigated. Here, weshow that treatment of an HDA6 repression line with the HDAC inhibitor trichostatin A resulted in growth arrest and elevatedtranscription of LEC1, FUS3, and ABI3 during germination. The growth-arrest phenotype of the repression line was suppressedby lec1, fus3, and abi3. An HDA6/HDA19 double-repression line displayed arrested growth after germination and the formationof embryo-like structures on the true leaves of 6-week-old plants even without trichostatin A. The growth-arrest phenotype of thisline was rescued by lec1. These results suggest that during germination in Arabidopsis, HDA6 and HDA19 redundantly regulatethe repression of embryonic properties directly or indirectly via repression of embryo-specific gene function.

The basic unit of chromatin is the nucleosome. Anucleosome is composed of 147 bp of DNA wrappedaround a histone octamer, which consists of the his-tone H2A, H2B, H3, and H4 proteins (Luger et al.,1997). The N tails of the histones, which are exposedon the surface of the nucleosome, may be methylated,acetylated, ubiquitinated, or phosphorylated (Jenuweinand Allis, 2001). Chromatin remodeling induced byhistone modification plays important roles in severalbiological phenomena in eukaryotes (Francis andKingston, 2001; Narlikar et al., 2002), and genetic stud-ies have suggested the importance of chromatin re-modeling 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 inhistones are involved in the reversible modulation ofchromatin structure and mediate the positive-negativeregulation of transcription (Berger, 2002). The acetyla-tion and deacetylation of histones is thought to controlgene expression by changing the accessibility of DNAto DNA-binding transcription factors. Histone acetyl-transferase catalyzes histone acetylation. The recruitment

of histone acetyltransferases to a promoter results inhistone hyperacetylation and transcriptional activa-tion (Brownell and Allis, 1996; Kuo and Allis, 1998;Kuo et al., 2000). In contrast, histone deacetylase (HDAC)catalyzes histone deacetylation, a phenomenon asso-ciated with transcriptional repression (Kadosh andStruhl, 1998; Rundlett et al., 1998).

The eukaryotic HDACs have been grouped intothree families, largely based on their homology withthe yeast (Saccharomyces cerevisiae) proteins RPD3 (re-duced potassium deficiency 3)/HDA1 and SIR2 (silentinformation regulator 2; Pandey et al., 2002; Yang andSeto, 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 putativefunctions of HDACs in plants (Chen and Tian, 2007).Antisense inhibition of the expression of HDA19 (orAtHD1), an RPD3/HDA1 family gene, leads to earlysenescence, serrated leaves, the formation of aerialrosettes, and delayed flowering accompanied by de-fects in floral organ identity (Wu et al., 2000a; Tian andChen, 2001; Tian et al., 2003). HDA19 is also involvedin jasmonic acid and ethylene signaling during theresponse to pathogens (Zhou et al., 2005). One RPD3/HDA1 family protein, HDA6, is involved in plant-pathogen interactions and has been shown to interactwith an F-box protein involved in jasmonic acid-mediated plant defense responses (Devoto et al., 2002).HDA6 has also been reported to be involved in trans-gene silencing and the regulation of ribosomal RNAtranscription in Arabidopsis (Murfett et al., 2001;

1 This work was supported by the Ministry of Education, Science,Culture, and Sports, Japan (grant-in-aid no. 16370017), and forSpecial Research on Priority Areas (grant-in-aid no. 19043007 to H.K.).

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed 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

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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 embryo-genesis. Antisense inhibition of the expression ofAtHD2A, a plant-specific HDAC gene, leads to seedabortion (Wu et al., 2000b). AtHD2A is transcribed inflowers and embryonic tissues, including siliques andsomatic embryos (Zhou et al., 2004). Furthermore, seeddevelopment-related gene expression is repressed byAtHD2A overexpression. Another plant-specific HDAC,AtHD2C, is said to be involved in the abscisic acid(ABA) response (Sridha and Wu, 2006). AtHD2C isrepressed by ABA, and AtHD2C overexpression re-presses the ABA response, as monitored by germina-tion and expression of the LATE EMBRYOGENESISABUNDANT PROTEIN (LEA)-class genes, which areactivated by ABA. These findings suggest that deacet-ylation of core histones by HD2-type HDACs contrib-utes to the control of LEA-class gene expression duringgermination. It is also thought that changes in HDACactivation occur during germination at 1 d after imbi-bition, at which point core histones at the promoterand coding regions of some LEA-class genes aredeacetylated (Tai et al., 2005).

Additionally, evidence suggests a role for HDACs inthe regulation of embryonic properties during vege-tative growth. PICKLE (PKL) was isolated as a repres-sor of the embryonic program during the vegetativegrowth phase (Ogas et al., 1997, 1999; Henderson et al.,2004; Li et al., 2005). pkl mutants form swollen primaryroots, referred to as pickle roots, and show ectopicexpression of LEAFY COTYLEDON1 (LEC1), LEC2,and FUSCA3 (FUS3) in their primary roots (Rideret al., 2003). PKL is thought to repress the transcriptionof embryo-specific genes and to establish repression ofembryonic properties after germination. PKL encodesan SWI/SNF-class chromatin-remodeling factor that be-longs to the CHD3 group (Ogas et al., 1999). In animals,CHD3-type chromatin remodeling factors have beenfound to be components of RPD3-containing HDACcomplexes (Tong et al., 1998; Wade et al., 1998; Xueet al., 1998; Zhang et al., 1998). These results suggestthat HDACs are involved in the suppression of em-bryonic properties after germination by repressingembryo-specific transcription factors. However, thereare no reports of HDAC involvement in the repressionof embryonic properties after germination.

Here, we show that an HDAC is involved in therepression of embryonic properties upon germination,based on the inhibition of HDAC activity in germinatedArabidopsis seeds following treatment with tricho-statin A (TSA), an HDAC inhibitor. Treatment withTSA during germination strongly inhibited growth andinduced the expression of embryo-specific transcrip-tion factors, including LEC1, FUS3, and ABSCISICACID INSENSITIVE3 (ABI3). Among several mutantsdefective in putative HDAC genes, the HDA6 repres-sion line showed growth arrest in the presence of lowconcentrations of TSA. Additionally, a double repres-sion line of HDA6 and HDA19 displayed growth arrest

after germination and the formation of embryo-likestructures on the true leaves of 6-week-old plants evenwithout TSA exposure; however, lec1 mutation rescuedthe growth-arrest phenotype of this line. We concludethat HDA6 and HDA19 contribute to the repressionof embryogenic properties via direct or indirect re-pression of embryo-specific transcription factors aftergermination.

RESULTS

Repression of Postgermination Growth by

TSA Treatment

To examine the role of HDAC in seed germination,Arabidopsis seeds were sown in media containing var-ious concentrations of TSA (Fig. 1). Under inhibitor-free conditions, most seeds showed radicle emergencewith cotyledon expansion and greening within 7 dafter sowing (Fig. 1A). In contrast, following treatmentwith TSA, most seeds showed radicle emergence butno cotyledon expansion or greening (Fig. 1B). At 14 dafter sowing, most of the control seedlings exhibitedexpanded cotyledons and true leaves (Fig. 1C), whereasmost of the TSA-treated seedlings were pale withunexpanded cotyledons (Fig. 1D). We examined thegrowth arrest during germination caused by TSAthrough observations of radicle emergence (germina-tion, Fig. 1E) and cotyledon expansion and greening(postgermination growth, Fig. 1F). At every concentra-tion of TSA, the germination rate 3 d after sowing waslower than that observed for the control seeds; how-ever, at 7 d after sowing, more than 80% of the seedstreated with 50 mM TSA had germinated (Fig. 1E). Incontrast, the extent of postgermination growth wasdependent on the concentration of TSA; 85% of seedstreated with 50 mM TSA showed no postgerminationgrowth even at 14 d after sowing (Fig. 1F). It is possiblethat TSA disrupted the biosynthesis of gibberellin(GA3), a key phytohormone that stimulates seed ger-mination. To eliminate this possibility, GA3 was addedalong with the HDAC inhibitor. Simultaneous treat-ment of seeds with GA3 and TSA (both 50 mM) did notalter the effects of the inhibitor on postgerminationgrowth (Fig. 1F).

Expression of Embryogenesis-Related Genes in

TSA-Treated Seeds

HDACs are thought to contribute to the repressionof embryogenesis-related gene expression during ger-mination (Rider et al., 2003; Zhou et al., 2004; Tai et al.,2005). Because treatment of seeds with TSA resulted inthe arrest of postgermination growth, embryogenesis-related gene expression was examined in TSA-treatedseeds. Four embryo-specific transcription factor genes,LEC1, LEC2, FUS3, and ABI3, and five LEA-class genes,AtECP31, AtECP63, CRUCIFERIN A (CRA), CRB, andCRC were selected as representative embryogenesis-related genes, and the expression of these genes in

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seeds treated with 50 mM TSA was monitored byreverse transcription (RT)-PCR (Fig. 2A). No tran-scripts of any of the genes were detected in germinatedseedlings at 14 d after sowing under inhibitor-freeconditions. In contrast, all of the genes were expressedin TSA-treated seeds. To examine whether the expres-sion of embryo-specific transcription factors in HDACinhibitor-treated seeds is maintained in dry seeds orwhether they are induced during germination, theexpression of LEC1 and FUS3 was analyzed in seedstreated with TSA at 12, 24, 48, and 72 h after sowingand in untreated controls. During embryogenesis, LEC1expression begins 4 d after pollination and decreasesin mature seeds (Baumbusch et al., 2004). LEC1 ex-pression was not observed in dry seeds and was notinduced in germinating control seeds (Fig. 2B). Incontrast, elevated LEC1 expression was observed aftersowing in seeds treated with 50 mM TSA. FUS3 wasexpressed in dry control seeds, but the level of ex-pression was decreased in germinating control seeds(Fig. 2C). In seeds treated with TSA, FUS3 expres-sion was decreased at 12 h after sowing but waselevated 12 h later. These results indicate that LEC1and FUS3 were induced in inhibitor-treated seedsduring germination.

Induction of Embryo-Like Structures by TSA

To evaluate the viability of the growth-arrestedseeds, seeds treated with TSA for 14 d were transferredto inhibitor-free medium. All of the growth-arrestedseeds began to form true leaves with embryo-likestructures on their surfaces (Fig. 3, A and B). Dissec-tion of the leaves was followed by an analysis ofembryogenesis-related gene expression (LEC1, LEC2,FUS3, ABI3, AtECP31, AtECP63, CRA, CRB, and CRC)in the dissected parts. Little or no expression wasdetected in the true leaves, whereas strong expressionof all of the genes was detected in the embryo-likestructures (Fig. 3C).

Arrested Postgermination Growth in an HDA6Repression Line Caused by Treatment with LowConcentrations of TSA

Arabidopsis contains 18 putative HDAC family genesgrouped into three families (Pandey et al., 2002). Therehas been no report of the ectopic expression of embryo-specific transcription factors during vegetative growthin a single HDAC mutant. These facts suggest thatsome HDACs act redundantly in the repression of em-bryonic properties and that a high concentration ofTSA simultaneously inhibits the activity of relatedHDACs. Thus, we expected that a mutant deficient inan HDAC known to repress embryonic propertieswould exhibit growth arrest following treatment withlow concentrations of TSA. To identify which HDACcontributes to seedling growth after germination andthe repression of embryo-specific transcription factors,knockout and repressed HDAC lines (Supplemental

Figure 1. Influence of the HDAC inhibitor TSA on the germination andpostgermination growth of wild-type seeds. Wild-type seeds (Col) wereincubated for 4 d at 4�C and then sown on B5 solid medium containing orlacking50mM TSA.A,Untreatedseeds7dafter sowing.B,50mM TSA-treatedseeds at 7 d after sowing. C, Untreated seeds at 14 d after sowing. D, Seedstreated with 50 mM TSA at 14 d after sowing. Bar 5 1 mm. E, Effect of TSA onseed germination. Wild-type seeds (Col) were incubated for 4 d at 4�C andthensownonB5solidmediumcontainingvariousconcentrationsofTSAandcontaining or lacking GA3. Germination, defined as the protrusion of theradicle through the seed coat, was recorded 3, 7, and 14 d after sowing.Control,No inhibitor;2,withoutGA3;1,with 50 mM GA3. Eachexperimentwas repeated three times and the average value is shown with the SD. F, Effectof TSA on postgermination growth. Postgermination growth was defined ascotyledon expansion and greening. The percentage of seeds showing post-germination growth was checked at 7 and 14 d after sowing. Each experi-ment was repeated three times and the average value is shown with the SD.

Effects of HDAC Inhibition on Postgermination Growth

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Table S1) were treated with low concentrations of TSA.Wild-type and mutant seeds were sown in watercontaining 0.5 mM TSA. At this TSA concentration,the postgermination growth of the wild-type plantswas unimpaired (Fig. 4). However, an HDA6 repres-sion line (Arabidopsis Biological Resource Center[ABRC], Ohio State University, Columbus, stock no.CS24039, referred to as HDA6:RNAi) ceased postger-mination growth after TSA treatment (Fig. 4). Thegermination rate of untreated HDA6:RNAi seeds wasequal to that of wild-type seeds (Fig. 5A). Additionally,in medium containing 0.5 mM TSA, 90% of theHDA6:RNAi seeds germinated within 7 d after sow-ing; however, none showed cotyledon expansion orgreening (Figs. 4 and 5B). Following treatment witheven 0.1 mM TSA, the postgermination growth of theHDA6:RNAi seeds was strongly arrested (Fig. 5B).Arrested growth following treatment with a low con-centration of TSA was also observed in another HDA6RNAi line (ABRC stock no. CS24038), and the intensityof the arrest was related to the level of HDA6 expres-sion (Supplemental Figs. S1 and S5). In contrast, thegrowth of the other HDAC mutants with or withoutTSA treatment was not statistically different (Supple-mental Fig. S1).

LEC1, FUS3, and ABI3 expression was analyzed inHDA6:RNAi seeds at 7 d after sowing. LEC1 expres-sion was not induced in untreated HDA6:RNAi seedsbut was induced by exposure to 0.5 mM TSA inHDA6:RNAi seeds (Fig. 5C; Supplemental Fig. S2).Likewise, FUS3 and ABI3 were not expressed in un-treated HDA6:RNAi seeds, but were strongly ex-pressed following TSA treatment (Fig. 5, D and E).

Effects of TSA on Postgermination Growth inEmbryo-Specific Transcription Factor Mutants

The seeds in which growth arrest was induced bytreatment with TSA showed expression of the embryo-specific transcription factor genes LEC1, FUS3, andABI3 (Fig. 2). The transcription factors encoded bythese genes have been reported to regulate embryodevelopment and dormancy (Meinke et al., 1994; Westet al., 1994) and are also involved in the maintenanceof embryonic properties and the suppression of pre-cocious germination (Meinke et al., 1994; Nambaraet al., 2000).

We examined whether the growth arrest of TSA-treatedHDA6:RNAi seeds was caused by embryo-specifictranscription factor expression by introgressing thelec1, fus3, and abi3 mutations into the HDA6:RNAiline (CS24039). Immature HDA6:RNAi lec1-1, HDA6:RNAi fus3-3, and HDA6:RNAi abi3-6 seeds weretreated with 0.5 mM TSA during germination. Almostall immature HDA6:RNAi seeds showed arrested cot-yledon expansion after treatment with 0.5 mM TSA. Incontrast, immature HDA6:RNAi lec1-1, HDA6:RNAifus3-3, and HDA6:RNAi abi3-6 seeds produced ex-panded green cotyledons on medium containing0.5 mM TSA (Fig. 6).

Figure 2. Expression of embryogenesis-related genes in seeds treatedwith the HDAC inhibitor TSA. A, Expression of embryogenesis-relatedgenes 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 RT-PCR using specific primers. The UBQ10 gene was used as an internalstandard. B, Expression of LEC1 in seeds treated with TSA at varioustimes after sowing. Wild-type seeds (Col) were treated with (1) orwithout (2) 50 mM TSA. The LEC1 expression in seeds at 12, 24, 48, and72 h after sowing, wild-type siliques at 3 d after flowering, and dryseeds was analyzed by quantitative RT-PCR. The horizontal axisindicates the time (h) after sowing. The vertical line indicates the levelof LEC1 cDNA normalized to UBQ10. C, Expression of FUS3 in seedstreated with TSA at various times after sowing. The vertical line indicatesthe level of FUS3 cDNA normalized to UBQ10. Each experiment wasrepeated three times; the average value is shown with the SD.

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Arrested Postgermination Growth and Expression of

Embryo-Specific Transcription Factor Genes in anHDA6-HDA19 Double Repression Line

HDA6:RNAi showed hypersensitivity to TSA, but inthe absence of TSA, this line did not show arrestedpostgermination growth or embryo-specific transcrip-tion factor expression (Figs. 4 and 5). These results sug-gest that other HDACs act redundantly with HDA6in the repression of embryonic properties. Thus, wecrossed HDAC knockout and repression lines withthe HDA6:RNAi line (CS24039). Among the resultingHDAC double mutants, one line, produced from across between HDA6:RNAi and an HDA19 repressionline (ABRC stock no. CS30925, referred to as HDA19:RNAi), showed abnormal postgermination growth.Repression lines of either HDA6 or HDA19 showedwild-type-like cotyledon expansion and greening at 7d after sowing (Fig. 7, A–C). However, approximately70% of the seeds produced by the HDA6/HDA19double-repression line (HDA6/19:RNAi) showed ar-rested cotyledon expansion and greening on inhibitor-free medium (Fig. 7D; Supplemental Fig. S4). In contrast,cotyledon expansion in HDA6/19:RNAi was restoredby introgression of the lec1-1 mutation, as found inHDA6:RNAi (Fig. 7E). After cultivation for more than1 month on Murashige and Skoog (MS) medium,the growth-arrested HDA6/19:RNAi seedlings beganto develop true leaves, with embryo-like structures

occasionally observed on the shoots (Fig. 7, F and G).No expression of LEC1, FUS3, or ABI3 was observed inHDA6:RNAi or HDA19:RNAi seedlings germinatedon inhibitor-free medium, but all three genes wereexpressed in untreated HDA6/19:RNAi seedlings 7 dafter sowing (Fig. 8, A–C; Supplemental Fig. S3). How-ever, the expression of LEC1, FUS3, and ABI3 inHDA6/19:RNAi was 2 to 6 times lower than that inTSA-treated HDA6:RNAi seeds. In true leaves of theHDA6/19:RNAi line, no LEC1 or ABI3 expressionwas detected (data not shown), but FUS3 expressionwas observed (Fig. 8D). However, the level of FUS3expression in true leaves was much lower than thatin seedlings.

Arrested Postgermination Growth in an HDA6:RNAi pklDouble Mutant

The HDA6 repression line showed LEC1 and FUS3expression after germination in the presence of lowconcentrations of TSA. Additionally, the double-repression line HDA6/19:RNAi showed the samephenomenon without inhibitor treatment. Ectopic ex-pression of LEC1 and FUS3 after germination in a PKL-deficient mutant has been reported (Ogas et al., 1997,1999; Rider et al., 2003; Li et al., 2005). To examine therelationship between HDA6 and PKL, HDA6:RNAiwas crossed with the PKL-deficient mutant pkl1-1. OnTSA-free medium, the HDA6:RNAi and pkl1-1 linesgerminated and produced expanded green cotyledons(Figs. 7B and 9A). In contrast, approximately 20% ofthe HDA6:RNAi pkl1-1 double-mutant seeds showedarrested cotyledon expansion and did not progress tovegetative growth (Fig. 9B). After cultivation for morethan 1 month on MS medium, the growth-arrestedseeds began to form true leaves that sometimes boreembryo-like structures (Fig. 9, C and D). Additionally,

Figure 3. Effects of TSA treatment on plant growth after transfer toinhibitor-free conditions. Wild-type seeds (Col) treated with 50 mM TSAfor 14 d on phytohormone-free Gamborg’s B5 solid medium weretransferred to inhibitor-free B5 liquid medium and cultured for anadditional 4 weeks. A, Seedlings grown from seeds after the transfer toinhibitor-free medium. Note the formation of embryo-like structures onthe true leaves (white arrows). B, Enlarged image of the embryo-likestructures in A. Bar 5 1 mm. C, Expression of embryogenesis-relatedgenes in the embryo-like structures. Embryogenesis-related gene ex-pression, as indicated in Figure 2A, was analyzed by RT-PCR in theembryo-like structures (SE) and in the remaining true leaves afterelimination of the embryo-like structures (Le). UBQ10 was used asan internal standard.

Figure 4. Arrest of postgermination growth in HDA6:RNAi by treat-ment with low concentrations of TSA. Morphological features ofgrowth-arrested HDA6:RNAi seedlings. Photographs were taken 7 dafter the sowing of wild-type (Ws) and HDA6:RNAi (CS24039) seeds indistilled water containing or lacking 0.5 mM TSA. Bar 5 1 mm.





embryo-like structures were occasionally observed onshoots of HDA6:RNAi pkl1-1 plants that did not formtrue leaves (Fig. 9E). In contrast, no embryo-like struc-tures had formed on the pkl1-1 single mutant evenafter cultivation for 2 months on MS medium (Fig. 9F).LEC1, FUS3, and ABI3 expression was analyzed inHDA6:RNAi pkl1-1 double-mutant plants with embryo-like structures on their shoots, and the level of ex-pression of all three genes was higher than in the pkl1-1single mutant (Fig. 9, G–I).

DISCUSSION

HDA6 and HDA19 Are Involved in the Repression ofEmbryonic Properties after Germination

The treatment of germinating seeds with TSA re-sulted in growth arrest (Fig. 1) accompanied by theexpression of embryogenesis-related genes (Fig. 2A).

The corresponding embryo-specific transcription fac-tors were highly expressed in seedlings whose growthwas arrested due to HDAC inhibitor treatment. Inwild-type plants, LEC1 was not expressed in dryseeds, but LEC1 expression was induced during ger-mination by TSA treatment (Fig. 2B). Furthermore, ininhibitor-treated seeds, FUS3 expression temporarilydecreased after sowing but could be increased again(Fig. 2C). The expression profiles of LEC1 and FUS3 inTSA-treated seeds exclude the possibility that the tran-scription of these embryogenesis-related genes wasmaintained by the growth arrest. Instead, both geneswere re-induced during germination by TSA treatment.

Of the HDAC mutants treated with low concentra-tions of TSA, only HDA6:RNAi failed to undergopostgermination growth and showed expression ofembryo-specific transcription factor genes (Figs. 4 and5). However, the germination rate of untreatedHDA6:RNAi seeds was the same as that of wild-type

Figure 5. Influence of the HDAC inhibitor TSA on the germination and postgermination growth of HDA6:RNAi. A, Germinationrates of wild-type (Ws) and HDA6:RNAi seeds (CS24039). Seeds were incubated for 4 d at 4�C and then sown in distilled watercontaining 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. Eachexperiment was repeated three times, and the average value is shown with the SD. B, Effect of TSA on the postgermination growthof wild-type and HDA6:RNAi seeds. Wild-type (Ws) and HDA6:RNAi seeds (CS24039) were incubated for 4 d at 4�C and thensown in distilled water with or without TSA. Postgermination growth was defined as cotyledon expansion and greening by 7 dafter sowing. Each experiment was repeated three times, and the average value is shown with the SD. *, None of the HDA6:RNAiseeds showed postgermination growth in the presence of 0.5 mM TSA. C, The expression of LEC1 in wild-type (Ws) andHDA6:RNAi seeds (CS24039) was analyzed by quantitative RT-PCR. Seeds were treated with (1) or without (2) 0.5 mM TSA for7 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 timesand the average value is shown with the SD.

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seeds (Fig. 5A), suggesting that the postgerminationgrowth arrest in HDA6:RNAi was not caused by poorgermination but by TSA. HDA6 belongs to the RPD3/HDA1 family, and its deacetylase activity is directlyinhibited by TSA (Earley et al., 2006). HDA6 has beenreported to be involved in the silencing of transgenesand rDNA (Murfett et al., 2001; Probst et al., 2004;Earley et al., 2006). However, there has been no reportof developmental abnormalities in an HDA6-deficientmutant. In this study, untreated seeds of the HDA6repression line HDA6:RNAi did not show arrestedpostgermination growth or expression of embryo-specific transcription factors after germination (Fig. 5).This suggests that other HDAC factors act redun-dantly with HDA6. HD2-type HDACs have beenreported to be involved in embryo development, seedmaturation, dormancy, and embryogenesis-relatedgene expression (Wu et al., 2000b; Zhou et al., 2004;Sridha and Wu, 2006). However, repression lines ofthese HDACs treated with a low concentration ofTSA did not show inhibited growth after germination(Supplemental Fig. S1). Furthermore, we produced

double repression lines by crossing HDA6:RNAi withsingle repression lines of other HD2-type HDACs, butno growth arrest was observed in these lines aftergermination (data not shown). In contrast, in theArabidopsis genome, 12 genes encode HDAC proteinsbelonging to the RPD3/HDA1 family (Pandey et al.,2002), and some RPD3/HDA1-type HDACs have beensuggested 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 wereexpected to have functional redundancy with HDA6in the repression of embryonic properties after germi-nation. In fact, the double repression of HDA6 andHDA19, which encodes an RPD3/HDA1-type HDAC,inhibited postgermination growth (Fig. 7D) with ex-pression of LEC1, FUS3, and ABI3 after radicle emer-gence (Fig. 8, A–C) in the absence of TSA. Additionally,in the HDA6/19:RNAi line, FUS3 expression was ob-served in both seedlings and true leaves (Fig. 8D).These results suggest that HDA6 and HDA19 are in-volved in the repression of embryo-specific genes aftergermination.

Figure 6. Possible involvement of LEC1, FUS3, andABI3 in the TSA-hypersensitive phenotype of HDA6:RNAi. Immature seeds of HDA6:RNAi (CS24039),HDA6:RNAi lec1-1 (CS24039/lec1-1), HDA6:RNAifus3-3 (CS24039/fus3-3), and HDA6:RNAi abi3-6(CS24039/abi3-6) were sown in B5 liquid mediumcontaining (1) or lacking (2) 0.5 mM TSA for 7 d. Inthe presence of TSA, HDA6:RNAi seeds exhibitedarrested cotyledon expansion, whereas HDA6:RNAilec1-1, HDA6:RNAi fus3-3, and HDA6:RNAi abi3-6showed cotyledon expansion and greening by 7 dafter sowing. Bar 5 1 mm.





It has been reported that HDA19 repression andknockout lines show pleiotropic abnormalities in de-velopment, including seed development (Tian andChen, 2001; Tian et al., 2003). Additionally, HDA19has been suggested to be involved in the genome-wideregulation of developmental gene expression (Tianet al., 2005). However, there is no evidence to sug-gest the involvement of HDA19 in the repression ofembryogenesis-related genes after germination. TheHDA19 repression line did not show arrested post-germination growth even after treatment with lowconcentrations of TSA (Supplemental Fig. S1). Thissuggests that HDA6 is a key factor in the repression ofembryonic properties after germination and that the

Figure 7. Morphological features of the HDA6/19:RNAi double re-pression 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 onthe shoot. G, Enlarged image of the embryo-like structures in F.Bar 5 1 mm.

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 byquantitative 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 WsHDA6:RNAi (CS24039), HDA19:RNAi (CS30925), and HDA6/19:RNAi(CS24039/CS30925) seedlings at 7 d after sowing. D, FUS3 expressionin the true leaves of Ws HDA6:RNAi (CS24039), HDA19:RNAi (CS30925),and HDA6/19:RNAi (CS24039/CS30925). True leaves were collectedfrom each plant at 3 weeks after sowing. The vertical lines indicatethe expression level of each gene normalized to UBQ10. Each ex-periment was repeated three times and the average value is shownwith the SD.

Tanaka et al.




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:RNAiseeds (Figs. 5 and 8). Furthermore, LEC2 was expressedin wild-type seeds after TSA treatment (Fig. 2A) butnot in HDA6:RNAi or HDA6/19:RNAi (data not shown).These results suggest that HDACs other than HDA6and HDA19 act redundantly in the repression ofembryonic properties after germination. Another pos-sibility is that TSA acts via an unknown target to in-crease embryo-specific transcription factor expression.

Ectopic expression of LEC1 and FUS3 after germi-nation was reported in a PKL-deficient mutant (Ogaset al., 1997, 1999; Rider et al., 2003; Li et al., 2005). PKLencodes an SWI/SNF-class chromatin-remodeling fac-tor that belongs to the CHD3 group (Ogas et al., 1999).CHD3 proteins are components of RPD3-containingHDAC complexes in animals (Tong et al., 1998; Wadeet al., 1998; Xue et al., 1998; Zhang et al., 1998).Additionally, HDA6 and HDA19 are classified asRPD3/HDA1-type HDACs (Pandey et al., 2002).Therefore, HDA6 and/or HDA19 are thought to actwith PKL in the repression of embryonic propertiesafter germination. pkl plants form swollen primaryroots, known as pickle roots, and exhibit ectopic ex-

pression of embryogenesis-related genes after germi-nation (Rider et al., 2003). In this study, the pkl singlemutant germinated and produced expanded greencotyledons on TSA-free medium (Fig. 9A), whereas theHDA6:RNAi pkl1-1 double mutant showed arrestedcotyledon expansion (Fig. 9B). Embryo-like structureshave been shown to form on hormone-free mediumfrom the detached pickle roots, cotyledons, and hypo-cotyls of pkl seedlings (Ogas et al., 1997; Hendersonet al., 2004). However, embryo-like structures were notobserved even after a long period of cultivation on MSmedium (more than 2 months) without detachingtissues (Fig. 9F). In contrast, after 1 month of cultiva-tion, some HDA6:RNAi pkl1-1 double-mutant seed-lings began to form embryo-like structures on theirshoots (Fig. 9, C–E) that were visible in the absence ofdissection. Additionally, the expression of LEC1, FUS3,and ABI3 in the double mutant was higher than that ineither single mutant (Fig. 9, G–I). These results suggestthat derepression of embryonic properties in the pklmutant was enhanced by the repression of HDA6. Incontrast, no obvious root abnormalities were observedin HDA6/19:RNAi seedlings (Fig. 7D). Additionally,ABI3 was expressed in HDA6/19:RNAi and HDA6pkl1-1 (Figs. 8C and 9I), but ectopic ABI3 expression

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 weeksafter sowing. The arrow indicates a somatic embryo-like structure that formed on the true leaves. D, Enlarged image of theembryo-like structure in C. E, HDA6:RNAi pkl1-1 (CS24039/pkl1-1) plants at 6 weeks after sowing. The arrow indicates asomatic 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 lineindicates the expression level of each gene normalized to UBQ10. Each experiment was repeated three times and the averagevalue is shown with the SD.





was not observed in pkl plants (Rider et al., 2003).Furthermore, the penetrance of the pkl phenotype wasaffected by GA3 and GA3 inhibitors, but the TSA-induced postgermination growth arrest was not elim-inated by GA3 application (Fig. 1F). These facts suggestthat HDA6 and/or HDA19 act independently of PKLto repress embryonic properties. It would be interest-ing to determine whether the phenotype of an HDA6/HDA19/PKL triple mutant is more severe than that ofeither double mutant. Additionally, there is no evidenceto suggest a molecular interaction between HDACs andPKL. It is therefore necessary to examine whetherHDA6 and/or HDA19 form a complex with PKL thatrepresses embryonic properties after germination.

Embryo-Specific Transcription Factor Expression byInhibition of HDAC Activity Induces Growth Arrestafter Germination

The growth of wild-type seedlings was arrested byTSA treatment, but when the seedlings were trans-ferred to TSA-free medium, they began to grow andformed embryo-like structures on their true leaves(Fig. 3, A and B). After a long period of cultivation onTSA-free MS medium, HDA6/19:RNAi and HDA6:RNAi pkl1-1 formed embryo-like structures (Figs. 7Gand 8C). Embryo-like structures expressed not onlyLEA-class genes but also embryo-specific transcriptionfactor genes (Fig. 3C). This observation suggests thatthe embryo-like structures are not formed by morpho-logical conversion to a cotyledon-like organ but thatthey have embryonic properties at the molecular level,as do somatic embryos. The formation of embryo-likestructures on vegetative tissues occurs when LEC1 orLEC2 is expressed ectopically in seedlings (Lotan et al.,1998; Stone et al., 2001). In addition, ectopic FUS3expression results in the accumulation of seed storageproteins in true leaves (Gazzarrini et al., 2004). It hasbeen reported that LEC1, LEC2, and FUS3 are impor-tant for the production of somatic embryos fromimmature seeds after 2,4-dichlorophenoxyacetic acidtreatment (Gaj et al., 2005). This suggests that theformation of embryo-like structures by inhibition ofHDAC activity is caused by the irregular expression ofthese embryo-specific transcription factors after ger-mination.

The arrest of growth after germination caused byTSA, as evidenced by the lack of cotyledon expansionor greening, was abrogated by the introgression of alec1, fus3, or abi3 mutation (Fig. 6), each of whichaffects seed dormancy (Meinke et al., 1994; Westet al., 1994). Seed dormancy and germination are con-trolled by phytohormones, particularly GA3 and ABA(Yamaguchi and Kamiya, 2000; Finkelstein and Gibson,2002; Peng and Harberd, 2002). These observations sug-gest that the growth arrest caused by decreased HDACactivity is due to alterations in the endogenous levelsof GA3, ABA, or both. However, the growth arrestfollowing TSA treatment was not eliminated by thesimultaneous application of GA3 (Fig. 1F). Further-

more, the arrest of postgermination growth inducedby TSA was not eliminated by the application offluridone, which inhibits ABA synthesis (Supplemen-tal Fig. S6). This result excludes the possibility that thegrowth arrest triggered by the inhibition of HDACactivity was caused by the disruption of endogenouslevels of GA3 and ABA. In contrast, several reportshave shown that the HDACs in higher plants mediatetranscriptional regulation during phytohormone sig-naling (Devoto et al., 2002; Gao et al., 2004; Song et al.,2005; Zhou et al., 2005; Sridha and Wu, 2006). Over-expression of AtHD2C, which encodes an HD2-typeHDAC, enhances germination, even in ABA-containingmedium (Sridha and Wu, 2006), indicating that thegrowth arrest caused by TSA is due to enhanced ABAsensitivity. In contrast, the TSA-triggered growth ar-rest was negated by introgression of the fus3 mutation(Fig. 6), but the fus3 mutant is still sensitive to germi-nation arrest after ABA treatment (Parcy et al., 1997).Thus, it appears that the growth arrest triggered byTSA treatment is not caused by enhanced sensitivity toABA. In contrast, it has been reported that the over-expression of LEC1 in seedlings causes growth arrestafter germination (Lotan et al., 1998). Furthermore,ectopic LEC1 expression in seedlings alters the hypo-cotyl structure and produces ectopic accumulation ofstarch and lipids, phenomena that are enhanced in thepresence of exogenous auxin and sugars but not in thepresence of GA3 or ABA (Casson and Lindsey, 2006).Sugars are important regulators of germination andseedling development (Gibson, 2005). Furthermore,sugar signaling is thought to be involved in the re-pression of embryonic properties after germination(Tsukagoshi et al., 2007). The above result suggeststhat sugar signaling mediated by LEC1, FUS3, and/orABI3 is involved in the growth arrest caused by theinhibition of HDAC activity.

Together, these data suggest that the inhibition ofHDAC activity produces growth arrest and the for-mation of embryo-like structures after germinationthrough the expression of embryo-specific factors. Toelucidate the molecular mechanisms of the phenom-ena induced by inhibiting HDAC activity, it will benecessary to compare HDA6/19:RNAi with HDA6/19:RNAi introgressed with lec1, fus3, or abi3 at themolecular level.

HDAC Contributes to the Repression of EmbryonicProperties via Repression of Embryo-SpecificTranscription Factors after Germination

The embryo-specific transcription factors LEC1,FUS3, and ABI3 were highly expressed in TSA-treatedseeds (Figs. 2 and 5) and in HDA6/19:RNAi (Fig. 8)after germination, suggesting that these genes aredirectly or indirectly repressed via the deacetylationof histones by HDAC. It has been reported that FUS3 isdirectly regulated by histone methylation by poly-comb group proteins (Makarevich et al., 2006). Histonemethylation is sometimes coupled with histone deacet-

Tanaka et al.




ylation (Peterson and Laniel, 2004). Because FUS3expression was observed in the true leaves ofHDA6/19:RNAi (Fig. 8D), it is possible that the re-pression of FUS3 expression in vegetative tissues isregulated by histone deacetylation. However, FUS3expression in true leaves was weaker than in seedlings(Fig. 8, B and D), indicating that the increase in FUS3expression after germination was due to the ectopicexpression of another factor(s) via inhibition of HDACactivity. It has been suggested that FUS3 expression iscontrolled by LEC1 during embryogenesis (To et al.,2006) and that ectopic LEC1 expression can induce theexpression of FUS3 and ABI3 in vegetative tissues(Kagaya et al., 2005). LEC1 expression was observed inHDA6/19:RNAi seedlings but not in true leaves (datanot shown). This suggests that the expression of FUS3after germination is induced by ectopic LEC1 expres-sion. In contrast, multiple VP1/ABI3-LIKE (VAL) mu-tants have been reported to form embryo-like structureson their vegetative tissues showing ectopic expressionof LEC1, FUS3, and ABI3 (Suzuki et al., 2007). The VALgenes encode B3 proteins, which contain a domainassociated with chromatin remodeling. The consensus-binding site of the B3 DNA-binding domain is in thepromoter region and first intron of VAL-regulatedgenes. Additionally, this consensus sequence is pres-ent in the promoter and/or first intron of LEC1, FUS3,and ABI3, suggesting that HDAC simultaneouslyrepresses these embryo-specific transcription factorsafter germination via histone deacetylation. However,it is unclear which genes are directly regulated byHDAC after germination, repressing the embryonicproperties of the plant during the vegetative phase.

In summary, these results suggest that HDACs areinvolved in the repression of embryonic propertiesafter germination via repression of the embryo-specifictranscription factors LEC1, FUS3, and ABI3. Amongthe Arabidopsis HDACs, HDA6 and HDA19 act re-dundantly in this regulatory mechanism. Our findingssuggest a role for HDAC following germination in therepression of embryonic properties in Arabidopsis. Toelucidate the mechanism of repression, it will benecessary to identify the genes that are regulated byHDA6 and HDA19 via histone deacetylation.

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 surface-

sterilized 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.

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#-ATTCATCTTGACCCGAC-

GAC-3#; for LEC2, 5#-TGAATCCTCAGCCGGTTTAC-3# and 5#-ACCCAAC-

ATCGCTGTTCTTC-3#; for FUS3, 5#-GTGGCAAGTGTTGATCATGG-3# and

5#-CGTGAAAACCGTCCAAATCT-3#; for ABI3, 5#-GCAGGACAAATGA-

GAGATCAG-3# and 5#-TCATTTAACAGTTTGAGAAG#; for AtECP31, 5#-

ATGAGCCAAGAGCAACCAAGGAG-3# and 5#-GGCATCTTCCCTCGTCA-

CAGC-3#; for AtECP63, 5#-CGGTGGAAGCAAAGGATAAGACG-3# and

5#-CTTTCCGAGCATCATCATCCATC-3#; for CRA, 5#-CACTCTCAACAGT-

TACGATC-3# and 5#-GTGAGTCAAAGTGGTCTCGA-3#; for CRB, 5#-TGA-

CCGCAACCTTAGACCAT-3# and 5#-TGCTATGGGTCAGTGTGGTC-3#; for

CRC, 5#-ACAACATGAACGCTAACGAGA-3# and 5#-CTCCTCGATCAAC-

TGTTGTT-3#; and for UBIQUITIN10 (UBQ10), 5#-GATCTTTGCCGGAAAA-

CAATTGGAGGATGGT-3# and 5#-CGACTTGTCATTAGAAAGAAAGAGA-

TAACAGG-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#-ACGATACCATTGTTCTTGT-

CACC-3#, the FUS3 transcript was amplified using the primers 5#-GGT-

ACTGGCCAAACAACAATAGC-3# and 5#-CTAGCTGCAGACCATGAGC-

ATT-3#, and the ABI3 transcript was amplified using the primers 5#-CAG-

GGATGGAAACCAGAAAAGA-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

The following materials are available in the online version of this article.

Supplemental Figure S1. Effects of the HDAC inhibitor TSA on post-

germination 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 postgermi-

nation growth by TSA treatment.





Supplemental Figure S7. Target sequences for HDA6 and HDA19 RNAi.

Supplemental Table S1. HDAC mutant lines used to screen for TSA-

hypersensitive mutants.

Received October 25, 2007; accepted November 11, 2007; published November

16, 2007.

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