Genetic Control of Developmental Changes Induced by Disruption of
Arabidopsis Histone Deacetylase 1 (AtHD1) Expression
Lu Tian,*
,†Jianlin Wang,* M. Paulus Fong,*
,‡Meng Chen,* Hongbin Cao,
§Stanton B. Gelvin
§and Z. Jeffrey Chen*
,†,‡,1*Department of Soil and Crop Sciences and Intercollegiate Programs in†Genetics and‡Molecular and Environmental Plant Sciences,
Texas A&M University, College Station, Texas 77843-2474 and§Department of Biological Sciences,
Purdue University, West Lafayette, Indiana 47907-1392
Manuscript received April 6, 2003 Accepted for publication May 23, 2003
ABSTRACT
Little is known about the role of genetic and epigenetic control in the spatial and temporal regulation of plant development. Overexpressing antisenseArabidopsis thaliana HD1(AtHD1) encoding a putative ma-jor histone deacetylase induces pleiotropic effects on plant growth and development. It is unclear whether the developmental abnormalities are caused by a defectiveAtHD1or related homologs and are heritable in selfing progeny. We isolated a stable antisenseAtHD1(CASH) transgenic line and a T-DNA insertion line in exon 2 ofAtHD1, resulting in a null allele (athd1-t1). Bothathd1-t1and CASH lines display increased levels of histone acetylation and similar developmental abnormalities, which are heritable in the presence of antisenseAtHD1or in the progeny of homozygous (athd1-t1/athd1-t1) plants. Furthermore, when the
athd1-t1/athd1-t1plants are crossed to wild-type plants, the pleiotropic developmental abnormalities are immediately restored in the F1hybrids, which correlates withAtHD1expression and reduction of histone H4 Lys12 acetylation. Unlike the situation with the stable code of DNA and histone methylation, developmental changes induced by histone deacetylase defects are immediately reversible, probably through the resto-ration of a reversible histone acetylation code needed for the normal control of gene regulation and development.
P
LANT development is plastic and affected by ge- in environmental signals. Disruption of histone deacety-lases results in growth and developmental abnormalities netic, epigenetic, and environmental factors.Vege-tative and reproductive (inflorescence) development is and aging in yeast cells (Imaiet al. 2000). These develop-mental changes are associated with down- or upregula-initiated at shoot apical meristems and/or axillary
meri-tion of several hundred genes (Bernsteinet al. 2000; stems that can be induced by internal and external
Robyret al. 2002), suggesting that histone deacetylases signals (Bernier 1986; Walbot 1996; Bleecker and
are key regulators of eukaryotic development. Histone Patterson 1997; Meyerowitz 1997). The molecular
acetylation and deacetylation may also play a role in mechanisms underlying the plastic nature of plant
de-gene expression and development in animals. For exam-velopment are largely unknown. Both genetic and
epi-ple, mouse histone deacetylase 1 (HD1) is a growth fac-genetic changes may contribute to the developmental
tor-inducible gene (Bartlet al. 1997). Histone hyper-plasticity of plants (Walbot1996;Meyerowitz 1997,
acetylation plays a role in establishing stable states of 2002; Srinivas 2000; Habu et al. 2001; Martienssen
differential gene activity during gastrulation in Xenopus andColot2001).
(Almouzniet al. 1994). Epigenetic regulation is a major aspect of gene
con-The Arabidopsis genome contains 18 putative HDs
trol by which heritable changes in gene expression
oc-(HDAsorHDACs) and 12 putative histone acetyltransfer-cur without an alteration in DNA sequence. Changes in
ases (HATs) distributed among all five chromosomes chromatin structure may affect accessibility of promoter
(Arabidopsis Genome Initiative2000;Pandey et al. elements to the transcriptional machinery and thus
af-2002). There are four classes of histone deacetylases in fect transcription. Modifications on core histones and
plants (Lusseret al. 2001). First, the RPD3-like protein their associated covalent bonds are known as the
“his-is the major h“his-istone deacetylase in yeast and mammals. tone code” (JenuweinandAllis2001). Changes in the
Mutations in RPD3 affect ⵑ500 genes in yeast ( Bern-histone code may facilitate fine tuning of gene
expres-steinet al. 2000;Robyret al. 2002).Arabidopsis thaliana
sion in response to developmental programs or changes
HD1 (AtHD1; GenBank accession no. AAB66486), also known as AtHDA19 (Pandey et al. 2002), is a putative homolog of yeast RPD3 and the major histone deacety-1Corresponding author:Molecular Genetics/MS 2474, Department of
lase (HD1) in humans and mice (Rundlettet al. 1996;
Soil and Crop Sciences, Texas A&M University, College Station, TX
77843-2474. E-mail: [email protected] Taunton et al. 1996; Bartl et al. 1997; Lusser et al.
1997). This class consists of at least two isoforms (HD1B-I ever, it is not known whether the abnormal phenotypes observed in the antisenseAtHD1plants result from the and -II) in maize embryos (Rossiet al. 1998;Lechner
et al. 2000) and four genes (HDA6, -7, -9, and -19) in disruption of single gene or related homologs in the
AtHDmulti-gene family. Moreover, it is unclear whether Arabidopsis. One of the maize genes,ZmRpd3or
HD1B-II(Lechneret al. 2000), complements a yeastrpd3null disruption of histone deacetylation induces other epi-genetic lesions and whether the induced phenotypic mutant (Rossiet al. 1998). Second, on the basis of DNA
sequences, HDA- and HOS-like HDs are related to RPD3 changes are heritable in the absence of the original
AtHD1 defect. Finally, the acetylation code is revers-but have different specific activities in deacetylating
his-tones (VidalandGaber1991;De Rubertiset al. 1996; ible and dynamic because core histones can be ace-tylated or deaceace-tylated through the activities of histone Rundlett et al. 1996, 1998). Eight Arabidopsis genes
in this category are predicted and some of them may acetyltransferases (HATs) or histone deacetylases (HDs, HADs, HDACs) during growth and development ( All-be distinct from other memAll-bers in this group (Pandey
et al. 2002). Third, HD2 is a plant-specific histone deace- freyet al. 1964), whereas the code of DNA and histone methylation is stable because no active demethylation tylase (Lusser et al. 1997) localized in the nucleolus.
At least two isoforms exist in maize and four genes in pathway has been identified (Jenuwein and Allis 2001). However, both the stable and reversible codes Arabidopsis (Lusser et al. 1997; Dangl et al. 2001).
Fourth, a NAD-dependent HD (SIR2-like protein) forms can be stored in chromatin and propagated through meiosis. Thus, compared to the stable code, the revers-a newly discovered clrevers-ass of HDs (Imaiet al. 2000;Landry
et al. 2000). In yeast, the deacetylation by SIR2 is NAD ible histone acetylation code may contribute differently to gene regulation and development. In this study, we dependent and possibly coupled to ADP ribosylation
(Tanner et al. 2000). Arabidopsis has two SIR2-like show that defects induced by expressing antisense
AtHD1, or by T-DNA insertion mutagenesis ofAtHD1, genes (Pandeyet al. 2002).
The role of histone acetylation and deacetylation in result in similar developmental pleiotropy. Unlike the situation withddm1(decrease in DNA methylation) mu-plant gene regulation is poorly understood (Verbsky
andRichards 2001;Liet al. 2002). Transgenic plants tants, defects in AtHD1do not induce cumulative epi-genetic lesions after four to five generations of selfing. treated with propionic or butyric acid, chemical
inhi-bitors of histone deacetylases, display increased levels Furthermore, genetic analyses indicate that disruption of developmental programs,AtHD1expression, and his-of DNA methylation and epigenetic variegation (ten
Lohuis et al. 1995). HC toxin, the host-selective toxin tone acetylation is immediately corrected in AtHD1/ athd1-t1heterozygous plants probably through restora-of the maize fungal pathogenCochliobolus carbonum,
in-hibits histone deacetylases in host plants (Broschet al. tion of the reversible histone acetylation code. 1995). Blocking deacetylation by sodium butyrate or
trichostatin A derepresses silent rRNA genes subject to
MATERIALS AND METHODS
nucleolar dominance (ChenandPikaard1997). In a
genetic screen for auxin-insensitive mutants,Murfett Plant materials:Constitutiveantisense histone deacetylase
et al. (2001) identified mutagenized plants with en- (CASH) transgenic plants were described previously (Tian
andChen2001). All plants were grown in vermiculite mixed
hanced expression ofgusAandhptIItransgenes. Further
with 10% soil in a growth chamber with growth conditions of
analysis indicated that several of these mutations were
22⬚/18⬚(day/night) and 16 hr of illumination per day, except
inAtHDA6, a presumed histone deacetylase, suggesting
as noted otherwise. Seeds from CASH plants were germinated
that this gene is important for transcriptional regulation in Murashige/Skoog medium (Sigma, St. Louis) in the pres-of the promoters controlling these transgenes. Indeed, ence of 15–50g/ml of kanamycin. After 2 weeks the plants
were transferred to soil and grown in a growth chamber. The
AtHDA6is required to maintain DNA methylation
pat-T-DNA insertion line was grown without kanamycin selection
terns induced by double-stranded RNA (Aufsatzet al.
except as noted otherwise. All photographs, except those
2002). Overexpression ofOsHDAC1, a putative histone
taken with the imaging system in a scanning electron
micro-deacetylase 1 gene in rice, is correlated with the induc- scope, were taken using a Nikon N-900 digital camera or the tion of OsHDAC1, increased growth rate, and altered CCD system of a Nikon SMZ-100 fluorescence microscope.
Scanning electron microscopy:Scanning electron
micros-plant morphology (Janget al. 2003).
Antisense-medi-copy was performed using a modified protocol (Muraiet al.
ated downregulation of the Arabidopsis genes AtHD1
2002) with a Hitachi s570 scanning electron microscope (Tokyo)
orAtHD2, which putatively encode histone deacetylases,
at an accelerating voltage of 5–15 kV. Tissue samples were fixed in
results in a variety of abnormal phenotypes in early a solution containing 3% (v/v) formaldehyde/glutaraldehyde and late stages of Arabidopsis development (Wuet al. and 0.1msodium cacodylate buffer (pH 7.4) at room
tempera-ture for 13 hr, washed three times with 0.2msodium cacodylate
2000a,b;Tian andChen2001).
buffer (pH 7.4), dehydrated through an ethanol series, critical
Downregulation of histone deacetylation induces
point dried with CO2, and sputter coated with gold before
pleiotropic effects on Arabidopsis development (Tian
viewing by scanning electron microscopy.
andChen2001), suggesting that histone deacetylation Nucleic acid preparation and analysis:RNA and DNA were directly or indirectly affects the expression of many isolated from at least five leaves of each plant at the same
How-1997). For DNA and RNA blot analyses, hybridization was into the plasmid pGEM T-easy (Promega) and sequenced us-ing an ABI Prism Big Dye terminator cycle sequencus-ing reaction performed following the method ofChurch and Gilbert
(1984). The DNA and RNA blots were washed in 2⫻SSC and kit (PE Applied Biosystems). The small fragment that ap-peared in one-step reverse transcriptase (RT)-PCR was cloned 0.2⫻SDS at 65⬚for 30–60 min and hybridization signals were
detected using a digital imaging system or exposure to X-ray into pGEM T-easy (Promega). The plasmid DNA was isolated using a QIAprep spin miniprep kit (QIAGEN) and sequenced. film or to a PhosphorImager.
PCR-based reverse genetic approach to identify T-DNA in- RT-PCR:RT-PCR was carried out using 500 ng of total RNA
prepared from leaf tissues. SuperScript one-step RT-PCR was
sertions in theAtHD1gene:We used a PCR-based approach
similar to that described byKrysan et al. (1996) to identify performed using the PlatinumTaq system (Invitrogen, San Diego) according to the manufacturer’s instructions. The Arabidopsis (ecotype Ws) mutants containing a T-DNA
inser-tion in or near theAtHD1gene. Pooled samples of DNA from primers used for detectingAtHD1transcripts were AtHD1-R, 5⬘-GCT TAC AAC AAC AAC AAC TCC AGA AAC TT-3⬘and 1000, 100, and 20 plants from the Feldmann T-DNA insertion
library (FeldmannandMarks1987;Forsthoefelet al. 1992) AtHD1-F, 5⬘-AGA AAG CCA GAG AGA GAG AGA GAG ATC AT-3⬘. For detectingCYC2btranscripts, we used the following were successively assayed for insertions, followed by assay of
individual plants from the pool of 20 mutant plants. The primers: Cyc2b-F, 5⬘-TCG GTG TAG AGA TGA AGA GAC AGA-3⬘and Cyc2b-R, 5⬘-GCA ACT AAA CCA ACA AGC TGA zygosity of a mutant allele was determined using forward and
reverse primers specific to theAtHD1gene and one primer AGC-3⬘.
Strand-specific first-strand cDNAs were synthesized using specific to theAtHD1gene in combination with either a T-DNA
left- or right-border primer.AtHD1 primer sequences were 500 ng of total RNA and Omniscript reverse transcriptase (QIAGEN). AtHD1-R and AtHD1-F primers were used to syn-5⬘-GCACTAGTGGCGCGCCATGGATACTGGCGGCAA-3⬘
and 5⬘-GCAGATCTATTTAAATCGCCTGCTCCGCCCCACC-3⬘. thesize sense and antisense strands ofAtHD1, respectively. RT was performed at 37⬚ for 60 min and the enzyme was then T-DNA primer sequences were left border, 5⬘-GATGCACTC
GAAATCAGCCAATTTTAGAC-3⬘; right border, 5⬘-TCCTTC inactivated by incubation at 93⬚for 5 min. For antisenseAtHD1
detection, following an initial denaturation step, 35 cycles of AATCGTTGCGGTTCTGTCAGTTC-3⬘. PCR was carried out
using 0.5 unit ExTaq (Takara, Berkeley, CA) DNA polymerase PCR were performed using the program of 94⬚for 30 sec, 57⬚ for 30 sec, and 72⬚for 1.5 min with a final extension of 10 with a robocycler (Stratagene, La Jolla, CA) using one cycle
of 95⬚for 5 min, 30–36 cycles of 94⬚for 40–60 sec, 56⬚for 1 min at 72⬚. A 5-l aliquot of PCR products was resolved by electrophoresis through a 1% agarose gel and subjected to min, and 72⬚for 3 min, plus an extension cycle of 72⬚for 10
DNA blot analysis. For sense AtHD1analysis, PCR was per-min followed by 4⬚on hold. We used 0.24mof each primer
formed using the same conditions as described above, except and 0.2 mmof each dNTP and either 100 ng DNA (for
screen-that 20 cycles were used. Hybridization was performed using ing superpools of 1000 plants) or 20 ng DNA (for screening
a full-lengthAtHD1cDNA as a probe. The actin geneAct2(An
pools of 100, 20, or individual plants) in a 50-l final
reac-et al. 1996) was used as an internal control for quantification. tion volume.
Relative intensities of individual DNA fragments were
mea-T-DNA junction sequence identification in antisense
trans-sured using a PhosphorImager (Molecular Dynamics,
Sunny-genic lines: T-DNA junction sequences in the CASH lines
vale, CA). were identified using a modification of a procedure previously
Western blot analysis:Western blot analysis was carried out described (Zhouet al. 1997). Briefly, 1g genomic DNA and
according toTianandChen(2001). In brief, crude protein 1g pBluescript plasmid DNA were separately digested at 37⬚
and histone extracts were prepared and subjected to electro-overnight in a 40-l reaction containing 10 units ofPstI. The
phoresis through 8 and 15% SDS polyacrylamide gels. Immu-digested genomic DNA solution was extracted with phenol/
noblots were prepared and probed with antisera against the chloroform/isoamylalcohol (25:24:1; Fisher) and precipitated
N-terminal portion of AtHD1 (TianandChen2001) or with using 2 volumes of ethanol and 1/10 vol of 3msodium acetate
a site-specific antibody against histone H4 Lys12 (Upstate Bio-(pH 5.2). The linear form of pBluescript plasmid was purified
technology, Lake Placid, NY) and developed by enhanced using a Gelpure kit (GeneMate; ISC Bioexpress, Kaysville,
chemiluminescence (ECL; Amersham, Buckinghamshire, UK). UT). For DNA ligation, 250 ng of digested genomic DNA and
180 ng of digested pBluescript plasmid DNA were mixed in a 20-l solution containing 3 units of T4 DNA ligase (Promega,
Madison, WI) and incubated at 16⬚overnight. RESULTS Two consecutive PCR reactions were performed using three
different primers. T3 primer (5⬘-AAT TAA CCC TCA CTA Isolation of a stable antisenseAtHD1transgenic plant: AAG GG-3⬘) was derived from an endogenous sequence of AtHD1 is constitutively expressed throughout plant de-the pBluescript plasmid. TR2 (5⬘-GAT GGG GAT CAG ATT velopment, although a slightly high level of expression GTC GTT TC-3⬘) and TR3 (5⬘-GTC GTT TCC CGC CTT CAG
is detected in reproductive tissues. Expressing CASH in
TTT A-3⬘) were two nested primers derived from the T-DNA
transgenic Arabidopsis plants results in the reduction
sequence close to the right border. The first PCR reaction
was performed using TR2 and T3 primers and 5l of ligation of tetra-acetylated histone H4 and a variety of
develop-solution as template. After purification using a QIAquick PCR mental abnormalities (Tian and Chen 2001). Many purification kit (QIAGEN, Chatsworth, CA), an aliquot of 2 lines showed variable phenotypes and deceased in early l PCR product from the first reaction was added to the second
developmental stages. To understand better theAtHD1
PCR reaction containing TR3 and T3 primers. Both PCR
reac-effects, we isolated a CASH126 line that displayed
consis-tions were performed for 35 cycles with a program of 30 sec
at 94⬚for denaturation, 30 sec at 52⬚for annealing, and 3 min tent phenotypes in selfing progeny. CASH126 plants
at 72⬚for extension, followed by a final extension at 72⬚for displayed phenotypes in the vegetative and flowering 8 min. The products of the second PCR amplification were stages in the fourth generation (Figure 1) similar to subjected to electrophoresis through a 1.0% agarose gel. The
the “pinhead” phenotype observed inArgonautemutants
band containing the DNA fragment of interest was excised
(Moussianet al. 1998;Lynnet al. 1999;Morelet al. 2002).
from the gel and the DNA was purified using a DNA
Figure1.—Development and inheritance of phenotypes in a stable CASH126 plant. Only the plants in the fourth genera-tion are shown. (A) A 7-day-old seedling displayed a defective SAM. (B) The defective SAM in the 10-day-old seedling devel-oped a pair of lobed true leaves. (C) A lateral SAM was initiated after 3 weeks. (D) The transition from vegetative to flowering development was delayed. (E) In the flowering stage, the plants display low fertility. About 75% of siliques were small and contained few viable seeds.
grew slowlyin vitro. After the plants were transferred to soil, cotyledonary leaves became bleached and yellow. The first pair of true leaves was very small, narrowly elongated, and lacked chlorophyll (Figure 1, A and B). Starting with the second pair of true leaves, leaf develop-ment appeared normal except for a delay of the transi-tion from adult vegetative to inflorescence development
(Figure 1, C and D). CASH126 plants developed ad- Figure2.—Detection ofAtHD1transgenes in the genome and expression of the transgene and endogenousAtHD1in
ditional true leaves, probably resulting from a
develop-CASH126 plants. (A) Map of the transgene construction
indi-mental transition initiated from axillary meristems.
Veg-cating thenptIIand antisenseAtHD1(asAtHD1) genes located
etative development could continue up to 4–5 weeks between the left and right T-DNA borders (LB and RB, respec-under long-day (16/8 hr of day/night) conditions. tively). (B) DNA blot containing genomic DNA digested with Eventually inflorescences developed from lateral meri- BamHI andEcoRI was hybridized with thenptIIprobe. Two copies of the transgene containingnptIIandAtHD1were
de-stems similar to those of wild-type plants. Although some
tected in CASH126 plants (lanes 2 and 3), whereas no
trans-inflorescence branches were sterile (Figure 1E), seeds
gene was present in the control plant (lane 1). (C) One copy
that could be harvested and germinated and the re- of theasAtHD1transgene is inserted into the 3⬘untranslated sulting plants showed developmental abnormalities sim- region (UTR) of theCYC2bgene, as shown in the top diagram. ilar to those observed in plants of previous generations. RT-PCR analysis indicated thatCYC2bexpression was similar in control (lane 2) and CASH126 (lane 3) plants. DNA size
One explanation for the abnormal phenotypes in
markers are shown in lane 1. PCR product amplified from a
CASH126 plants would be that insertion of the
trans-genomic DNA is shown in lane 4. (D) Strand-specific
RT-genes into the genome disrupted a locus important for PCR analysis in CASH126 plants. (Top)asAtHD1was highly plant development, such as a homeotic gene control- expressed in CASH 126 plants (lanes 3–5) and undetectable ling flowering. The endogenousAtHD1is a single-copy in control plants (ecotype Columbia, lane 1, and ecotype Ws, lane 2). (Middle) RT-PCR and DNA blot analyses were
per-gene located on chromosome 4 (Arabidopsis Genome
formed to detect the expression levels of sense AtHD1 Initiative 2000; Tian and Chen2001). We analyzed
(sAtHD1). The strand-specific RT-PCR product was resolved
the copy number of transgenes in CASH126 plants using by electrophoresis through a 1.0% agarose gel and transferred DNA blot analysis and detected two fragments of the onto a Hybond-N⫹membrane (Amersham Pharmacia). The transgenes (Figure 2, A and B), indicating that these blot was hybridized with anAtHD1full-length cDNA probe. The relative expression levels (%) of senseAtHD1(sAtHD1)
transgenic plants contain two copies of theAtHD1
trans-transcripts were estimated usingAct2as an internal control.
gene. We further used a PCR method (Zhouet al. 1997) to identify the transgene insertion sites in the genome in CASH126. As expected, two fragments were amplified
from DNA of transgenic plants. DNA sequencing results mately equal amount of CYC2btranscripts in the con-indicated that one of the transgene insertion sites was trol and CASH126 plants (Figure 2C). The other inser-located ⵑ150 bp downstream of the CYC2b stop co- tion site remains unknown, as the sequenced fragment don of chromosome 4. The insertion did not affect did not match Arabidopsis sequence in the database.
We used strand-specific RT-PCR to determine the
approxi-pression levels of antisense and endogenous AtHD1
genes in transgenic and control plants. AntisenseAtHD1
(asAtHD1) transcripts were abundant in each of three CASH126 plants that were derived from single-seed de-scent, but absent in control plants (Figure 2D, lanes 1 and 2). To determine the expression levels of the endogenous senseAtHD1(sAtHD1) transcript, we per-formed a semiquantitative RT-PCR analysis usingAct2
(Anet al. 1996) as an internal control.sAtHD1andAct2
Figure 3.—A null mutation (athd1-t1) is generated by
transcripts were amplified and subjected to DNA blot T-DNA insertion into theAtHD1gene. (A)AtHD1 contains analysis, and the relative expression ratios ofsAtHD1and seven exons. The T-DNA is inserted into exon 2. Primers used
Act2transcripts were calculated in control and CASH126 in RT-PCR for detectingAtHD1expression are shown above the first exon and below the last exon. Primers used to amplify
plants. The results indicated that endogenous sense
thenptIIgene in RT-PCR reactions (C) and thenptIIfragment
AtHD1transcripts were greatly reduced in the CASH126
used for DNA blot analysis (B) are shown below and above
plants, ranging from 5 to 60% of the level detected in
the T-DNA diagram, respectively. Striped box indicates the
the control plants. It is notable thatsAtHD1RNA levels location of theAtHD1fragment used for DNA blot analysis were inversely correlated with the amount ofasAtHD1 in Figure 7. (B) DNA blot analysis shows a single T-DNA insertion in theathd1-t1line. (C) RT-PCR analysis indicates
transcripts (Figure 2D: compare lanes 3–5).
Overexpress-thatnptIIgene expression is detected in theathd1-t1 plants
ing the antisenseAtHD1gene reduced the accumulation
(lanes 1 and 2), but absent in wild-type plants (ecotype
Colum-of endogenousAtHD1 transcripts, suggesting that the bia, lane 3; Ws, lane 4). T-DNA insertion produces null alleles penetrance of developmental abnormalities is corre- in theAtHD1 locus. AtHD1 expression was detected in Ws lated with the level ofsAtHD1transcript. (lane 6) but absent in theathd1-t1line (lane 7).Act2was am-plified as a positive internal control. (D) AtHD1 production A T-DNA insertion inAtHD1results in anAtHD1null
is reduced in theathd1-t1plants. Crude protein extracts (25g) mutation:Phenotypic abnormalities in transgenic plants
were subjected to electrophoresis though an
SDS-polyacryl-may fluctuate because of variable levels of transgene amide gene and immunoblotted onto Immobilon-P (Milli-expression (Figure 2D). Moreover, although downregu- pore, Bedford, MA) or Hybond-ECL (Amersham) membranes. lation ofAtHD1in antisense transgenic plants results in The membrane was probed with antibodies against the N terminus of AtHD1. A band cross-reacting with anti-AtHD1
a variety of developmental abnormalities, it is unclear
antibodies in athd1-t1 plants was reduced to only a trace
whether these phenotypic changes result from disrupted
amount (lane 4) compared to the band detected in the control
expression ofAtHD1. The expression of antisenseAtHD1 plant (lane 3). A protein loading control is shown in an 8%
may also affect the expression of other genes, because SDS-PAGE stained with Coomassie Blue (lanes 1 and 2). there are severalAtHD1homologs (Pandeyet al. 2002).
We therefore identified an Arabidopsis (ecotype Ws) mutant line that contains a T-DNA insertion in the
AtHD1 gene (athd1-t1; Figure 3A). DNA blot analysis CASH126 andathd1-t1plants display similar develop-mental abnormalities:To determine whether CASH126 indicated only one T-DNA insertion in the genome
(Fig-ure 3B). Using a PCR-based reverse genetic approach andathd1-t1plants show similar changes in early devel-opmental stages, we grew them side by side in a growth (Krysan et al. 1996) and DNA sequence analysis, we
determined that the T-DNA was inserted into exon 2 chamber under short-day conditions (8/16 hr of day/ night). The leaves of both CASH126 andathd1-t1plants ofAtHD1(Figure 3A). This insertion caused a null
muta-tion of AtHD1; noAtHD1 expression was detected in were slightly chlorotic and showed disrupted radial sym-metry (Figure 4, A–D), probably resulting from de-homozygous insertion lines (lane 7, Figure 3C).
Further-more, Western blot analysis using antibodies against the fective shoot apical meristems as previously described (Figure 1). A prominent phenotype was the somewhat N terminus of AtHD1 (TianandChen2001) indicated
that, in the homozygous mutant line, the level of AtHD1 left-handed “twist” of the longitudinal axis of rosette leaves inathd1-t1plants (Figure 4, B and F). This twist protein was greatly reduced (Figure 3D, lane 4). Only
a small amount of protein (ⵑ5% that of the wild type) did not occur in other parts of the plants and was differ-ent from the previously described “lefty” mutants ( Thi-was detected in homozygous plants, most likely resulting
from cross-reaction of the antibodies to other AtHD1 tamadee et al. 2002). This left-handed twist was not obvious in the leaves of CASH126 plants (Figure 4C). homologs. For example, AtHD1 and AtHD6 share 69
and 84% of amino-acid sequence identity and similarity However, under close examination, the leaves of CASH126 plants were also twisted (Figure 4G), although in the N termini, respectively. Alternatively, the low level
of AtHD1 detected could be due to residual expression the orientation of the distortion was not consistent. Both wild-type andAtHD1disruption lines flowered at ofathd1-t1; however, a different-sized protein would be
observed because the T-DNA was inserted in the coding approximately the same time, suggesting that vegetative development initiated from axillary meristems had little sequence (Figure 3A). Taken together, the data suggest
had low seed set. To investigate the cause of sterility, we examined the flower structures of athd1-t1 plants using light and scanning electron microscopy. Flower morphology ofathd1-t1plants was irregular. Many flow-ers had missing petals and sepals (Figure 5, B, C, and E) compared to the typical “crucifer-shaped” flowers of wild-type plants (Figure 5, A and D). Some flowers had fewer than six stamens, and some of these were fused (Figure 5F). The stamens were short (Figure 5E). As a result, pollen would have difficulty reaching the “tall” stigma. The incompatibility between “impotent” male stamens and tall female stigmas is likely associated with sterility because, compared with wild-type stigmas that were completely covered with pollen grains (Figure 5G), only a few pollen grains could reach the stigmas of homozygous athd1-t1plants (arrows in Figure 5H). As a result, siliques of homozygous athd1-t1 plants were short and contained few seeds (Figure 5, M and O), whereas siliques of wild-type plants were long and con-tained many fully developed seeds (Figure 5, M and N). Pollen grains ofathd1-t1plants were apparently normal (Figure 5, K and L). Although structural incompatibility between stigma and stamen is likely related to sterility, Figure4.—CASH126 andathd1-t1plants show similar ab- we do not rule out other possibilities, such as biological normal developmental phenotypes. CASH126 and athd1-t1
incompatibility between pollen and stigma interactions
plants were grown side by side in a growth chamber under
during pollination in athd1-t1 plants, which may also
short-day (A–H) or long-day (I–L) conditions. (A) Wild-type
cause sterility.
plant (Ws) 3 weeks after germination. (B) Homozygous
athd1-t1/athd1-t1plants 3 weeks after germination. (C) CASH126 The pleiotropic phenotype resulting fromAtHD1 de-plants 3 weeks after germination. (D) Wild-type plant (Col, fects is immediately restored in heterozygous plants and Columbia) 3 weeks after germination. (E) Mature
inflores-displays Mendelian segregation:The developmental
de-cence branches inathd1-t1and Ws. (F) Rosette leaves of Ws
fects of CASH and athd1-t1/athd1-t1 plants are
consis-andathd1-t1plants. (G) Rosette leaves of CASH126 and
con-tently observed in selfing generations. However, it is
trol plants. (H) Florescence branches of CASH126 and
Colum-bia plants. (I) Enlarged picture of mildly defective flowers in unknown whether the phenotypic abnormalities were
athd1-t1plants. ( J) Plant morphology ofathd1-t1and CASH126 dependent on the continued deficiency inAtHD1 ex-plants. (K and L) Enlarged pictures of inflorescence branches
pression and core histone acetylation profiles. To
ad-showing severely defective flower organs (K) and mildly
abnor-dress this, we made F1hybrids between wild-type (Ws) mal flowers (L) of CASH126 plants.
and homozygous athd1-t1/athd1-t1 plants (Figure 6A) and examined the phenotypes in the resulting F1hybrids
and F2 siblings. In all the F1plants examined, most of
the developmental abnormalities, including rosette leaf conditions (16/8 hr of day/night). As with the CASH
lines,athd1-t1lines floweredⵑ2–5 days late (31.9⫾1.1 morphology and fertility, were reversed to those of wild-type plants, except that F1plants still developed slightly
days,n⫽43) compared to the wild-type plants (28.1⫾
2.3 days, n ⫽ 42). Although severe phenotypes in the shorter siliques. These results indicate that most of the developmental defects induced by the homozygous mu-vegetative stage were often observed under short-day
conditions (Figure 4, A–D, F, G), similar abnormalities tants are immediately corrected inAtHD1/athd1-t1 het-erozygotes. Indeed, the expression level of AtHD1 in in the reproductive stage were observed under both
long- and short-day conditions (Figure 4, E, H, I–L). the heterozygous plants was equal to that of wild-type plants (Figure 6B). Furthermore, the acetylation level CASH126 and athd1-t1 plants developed abnormal
flower structures, including reduced numbers of petals of histone H4 Lys12 was increased approximately three-fold in theathd1-t1/athd1-t1homozygous mutants com-(Figure 4, I, K, and L), split flowers com-(Figure 4K), and
sterility (Figure 4, E and H). The abnormal flower phe- pared to the wild-type plants (Figure 6C). H4 Lys12 is one of the primary deacetylation sites targeted by RPD3 notypes inathd1-t1plants (Figure 4I) were not as severe
as those of CASH126 plants (Figure 4J), but were more in yeast (VidalandGaber1991;Rundlettet al. 1996). The H4 Lys12 level was reversed to that of the wild-uniform. CASH126 lines, however, displayed a range
of penetrance in developmental abnormalities, ranging type plants in the heterozygous plants, implying that acetylation at some specific sites is responsible for the from severe (Figure 4K) to mild (Figure 4L).
ex-pression in these plants. In the F2 progeny, the wild- selfing we did not observe abnormal phenotypes in these
plants in addition to developmental abnormalities ob-type and abnormal phenoob-types segregated 3:1,
coseg-regating with the level of AtHD1 expression in these served in the original homozygous plants (Figure 7). Phenotypic abnormalities were less severe under long-plants. These data indicate that the developmental
ab-normalities are dependent on disruption ofAtHD1ex- day conditions than under short-day conditions, sug-gesting that the penetrance of phenotypes is dependent pression and of some specific acetylation sites (e.g., H4
Lys12) in theathd1-t1lines. on day length.AtHD1 expression was not detected in
the plants after four generations of selfing. These data,
Does athd1-t1induce additional epigenetic changes?
Theddm1mutant induces epigenetic lesions in addition together with the immediate restoration of normal de-velopment inAtHD1/athd1-t1heterozygous plants, sug-to those observed in the originalddm1mutant (Stokes
et al. 2002). To determine whetherathd1-t1 acts as an gest that developmental changes induced by disruption of AtHD1expression are reversible and dependent on epigenetic modulator, we examined phenotypic changes
in the plants derived from single-seed descent of one AtHD1expression and histone deacetylation.
athd1-t1/athd1-t1homozygous plant. Consistent with the results in CASH plants, within four generations of
DISCUSSION
Heritable changes of developmental abnormalities in-duced byAtHD1disruption:Blocking histone deacetyla-tion induces a wide range of developmental changes. The abnormal development includes defective shoot apical meristems (SAM), irregular trichomes and cellu-lar patterns, late flowering, abnormal inflorescences and flowers, and aborted seeds. These developmental abnormalities are stable in the presence ofAtHD1 dis-ruption after four to five generations of selfing. More-over, a T-DNA insertion into theAtHD1gene (athd1-t1) results in a mutant line (Ws ecotype) that shows devel-opmental abnormalities similar to those of CASH126 plants (Columbia ecotype), confirming that AtHD1 plays an important role in reprogramming developmental processes. The affected plants develop through initiat-ing axillary meristems and additional changes that en-sure the completion of a life cycle, although they have to overcome structural and developmental incompati-bilities resulting from irregularly orchestrated patterns and tempos of organogenesis. Histone deacetylation and the resulting effects on gene regulation may con-tribute to the fundamental and dynamic process of de-velopmental plasticity in plants (Walbot1996;
Meyer-Figure5.—Abnormal flower development and sterility in
athd1-t1plants. (A) Wild-type (Ws) plants have a typical “cruci-fer” flower with four petals, six stamens, and a regular stigma. (B and C) Transformation of the “crucifer” shape into an irregular form inathd1-t1plants resulting from fused petals (B) and missing flower parts (C). (D) Normal flower morphol-ogy of wild-type plants revealed by scanning electron micros-copy. (E and F) Short stamens and missing petals (E) and fused stamens (F) inathd1-t1plants. (G) The stigma of the wild-type flower is completely covered with pollen grains. (H) Only a few pollen grains (arrows) are found on the stigma of
Figure7.—athd1-t1/athd1-t1plants showed constant devel-opmental abnormalities during four generations (G1–G4) of
Figure 6.—(A) Developmental abnormalities in rosette
selfing. The plants were grown side by side in the same growth leaves and siliques of athd1-t1/athd1-t1 homozygous plants
chamber. No AtHD1expression was detected by RT-PCR in were restored in F1(AtHD1/athd1-t1) progeny. The F1hybrid,
the homozygousathd1-t1/athd1-t1plants obtained in four gen-heterozygous for theathd1-t1locus, was generated by crossing
erations of selfing. a wild-type Ws plant with a homozygousathd1-t1plant. (Right)
Genomic DNA was digested withEcoRI and SacII, subjected to electrophoresis through a 1% agarose gel, and blotted onto a Hybond⫹membrane. The blot was hybridized with a DNA
fragment containing exons 1 and 2 ofAtHD1(see Figure 3A). ond, no additional visual abnormalities accumulate in The F1plant contained a normalAtHD1allele and anathd1- the selfing progeny of
athd1-t1/athd1-t1 homozygous t1allele. (B) Equal quantities ofAtHD1transcripts were
de-plants. The data suggest that the dynamic and reversible
tected by RT-PCR in the F1 and Ws plants, but no AtHD1
process of acetylation and deacetylation provides a
spa-transcript was detected in theathd1-t1/athd1-t1homozygous
plants. RT-PCR amplification ofAct2was used as an internal tial and temporal code for gene activation and silencing.
control. (C) Western blot indicates that histone H4 Lys12 Consistent with this hypothesis, histone deacetylation acetylation is increased in the homozygous athd1-t1 plants.
may affect the expression of genes in response to
envi-Hybridization intensities of a nonspecific (n.s.) protein
de-ronmental (e.g., light, day length) and developmental
tected by the anti-H4 Lys12 antibody were used as internal
(e.g., homeotic genes) changes. Indeed, both antisense
controls to calculate the relative ratio of H4 Lys12 acetylation
accumulation, which is shown in a histogram below the West- AtHD1 transgenic and athd-t1 homozygous lines show
ern blot. variable flowering time and severity of developmental
abnormalities under short- and long-day conditions.
AtHD1is a member of a multi-gene family that may diverge in functions. For example, AtHD6, a RPD3-like owitz 1997). Disruption of AtHDs may directly affect
homolog, is involved in the release of transgene silenc-expression of genes such assuperman(TianandChen
ing (Murfett et al. 2001) and is associated with the 2001) in specific developmental stages. Alternatively,
maintenance of DNA methylation patterns of trans-the disruption may induce a series of changes in
regula-genes (Aufsatz et al. 2002). However, recessive muta-tory networks. Complex phenotypes such as defective
tions inAtHDA6do not induce visible abnormal pheno-apical shoot meristems and abnormal flowers could be
types (Murfettet al. 2001). AntisenseAtHD2lines (Wu some of these pleiotropic effects. It will be interesting
et al. 2000a,b) show less severe developmental abnormal-to identify target genes whose expression is affected in
ities than antisense AtHD1 lines do (Tian and Chen theathd1-t1and antisenseAtHD1transgenic lines.
2001). Significantly, although histone deacetylases are Developmental abnormalities induced by disruption
encoded by a multi-gene family and share sequence of histone deacetylation are different from those
in-homology (Pandeyet al.2002), other AtHD homologs duced by DNA methylation defects (Vongset al. 1993;
cannot compensate for the loss of AtHD1 activity in the Finneganet al. 1996;Ronemuset al. 1996;Gendrelet al.
athd1-t1lines, suggesting that AtHD1is a key member 2002;StokesandRichards2002). First, the abnormal
of the histone deacetylase gene family. Unlike the tetra-development in theathd1-t1homozygous plants can be
anti-senseAtHD1(TianandChen2001), hyper-acetylation and developmental programs return to normal, inde-pendent of reprogramming through meiosis. The con-of core histones in theathd1-t1 line is restricted to a
specific set of lysine residues including H4 Lys12. These cept of a stable or reversible code is also supported by the results obtained from a recent study indicating that data suggest that specific patterns of histone acetylation
may be established by AtHD1, consequently controlling the loss of histone H4 Lys16 acetylation inddm1 homozy-gous plants is compensated in DDM1/ddm1 heterozy-the expression of a subset of genes during development.
The role of a histone code in genetic and epigenetic gous plants, whereas DNA and histone H3 Lys9 methyla-tion remain unchanged (Soppeet al. 2002).
regulation:Chromatin-based gene regulation in
eukary-otes is controlled by a chromatin code (Jenuweinand The relationship between reversible and stable codes is largely unknown. Acetylation and deacetylation may Allis 2001) that can be further classified as stable or
reversible. The stable code includes DNA and histone act on active or inactive chromatin as a competitor for histone methylation sites (JenuweinandAllis2001). methylation (RichardsandElgin2002). Once the
sta-ble code is established, it is difficult to alter unless For example, histone acetyltransferases and methyl-transferases may compete for histone H3 Lys9 to estab-through a reset of developmental programming during
meiosis, because no active demethylation pathway has lish an active or inactive form of chromatin (Jenuwein andAllis2001;Littet al. 2001). Histone deacetylation been identified (Jenuwein and Allis 2001; Li 2002;
Richards and Elgin 2002). As a result, changing a and DNA methylation may also be interdependent (Selker1998;Soppeet al. 2002). Moreover, a putative stable code results in epigenetic inheritance or variation
(Stokeset al. 2002;StokesandRichards2002). More- histone deactylase (AtHDA6) is needed to enhance DNA methylation induced by double-stranded RNA over, alteration in the stable code may serve as an
epi-genetic “modulator” that induces changes at other loci, (Aufsatzet al. 2002). Finally, some DNA methyltransfer-ases (e.g., CMT) or histone deacetylases (e.g., HD2) were independent of the original chromatin conformation.
Indeed, in theddm1genetic background, epigenetic le- identified only in plants (Lusseret al. 1997, 2001; Heni-koffandComai1998;Lindrothet al. 2001), suggesting sions are induced in a genomic region containing
multi-ple repeats associated with disease resistance genes, pre- that plants may control gene regulation via both general and unique chromatin pathways. It will be interesting sumably because they are vulnerable in response to
changes in DNA methylation (Stokes and Richards to test how the reversible code of histone acetylation controlled by AtHD1 interacts with the biochemically 2002). Moreover, these epialleles may be affected not
only by demethylation, but also by allelic interactions stable code of DNA and histone methylation and affects the plastic nature of plant development.
among loci or within a locus (Stokeset al. 2002), a
phe-nomenon similar to meiotic transvection (Aramayo We thank Eric J. Richards for insightful discussions and Gary E. andMetzenberg1996) or paramutation (Hollicket al. Hart and Karel Riha for critical suggestions on improving the manu-script, Joe Wang for assistance in photography, Hyeon-Se Lee and
1997). Methylation-associated epialleles have also been
Jenny Lee for helpful discussions, and Mike Pendleton in the
Micros-reported in mutants with defective flower structure (e.g.,
copy and Imaging Center at Texas A&M University for providing
SUPERMAN) or a delay in flowering time (e.g., FWA),
excellent technical services. Work in S.B.G.’s laboratory was supported
which may result from aberrant DNA methylation pat- by grants (99-75715 and 99-75930) from the National Science Founda-terns (Jacobsen and Meyerowitz 1997; Kakutani tion Plant Genome Research Program. Research in the Chen labora-tory was supported by a grant (00-77774) from the National Science
1997;Soppe et al. 2000).
Foundation Plant Genome Research program and the Texas
Agricul-The reversible code (e.g., histone acetylation and
tural Experiment Station.
deacetylation) is heritable (JenuweinandAllis2001) but the action of the code is dependent on the respective biochemical activities that set the code. The code is
LITERATURE CITED
dynamic and reversible during any developmental stage
and is independent of meiosis. Although the connection Allfrey, V. G., R. FaulknerandA. E. Mirsky, 1964 Acetylation and methylation of histones and their possible role in regulation
between a specific acetylation code and gene regulation
of RNA synthesis. Proc. Natl. Acad. Sci. USA51:786–794.
remains to be elucidated, the heritable and reversible Almouzni, G., S. Khochbin, S. DimitrovandA. P. Wolffe, 1994 nature of developmental abnormalities observed in Histone acetylation influences both gene expression and
develop-ment ofXenopus laevis.Dev. Biol.165:654–669.
AtHD1disruption lines is likely associated with changes
An, Y. Q., J. M. McDowell, S. Huang, E. C. McKinney, S. Chambliss in histone acetylation (e.g., H4 Lys12 acetylation) and
et al., 1996 Strong, constitutive expression of the Arabidopsis methylation. It is conceivable that under normal condi- Act2/Act8 actin subclass in vegetative tissues. Plant J.10:107–121.
Arabidopsis Genome Initiative, 2000 Analysis of the genome
se-tions, genes, including homeotic genes (e.g.,superman;
quence of the flowering plantArabidopsis thaliana.Nature408:
Tian and Chen 2001) responsible for plant
develop-796–815.
ment, may be controlled by the reversible code of acetyl- Aramayo, R., and R. L.Metzenberg, 1996 Meiotic transvection in
fungi. Cell86:103–113.
ation and deacetylation. However, the acetylation code
Aufsatz, W., M. F.Mette, J.Van Der Winden, M.Matzkeand A. J.
is reversible and dependent on histone deacetylases or
Matzke, 2002 HDA6, a putative histone deacetylase needed to
other factors in the chromatin control. As soon as the enhance DNA methylation induced by double-stranded RNA.
EMBO J.21:6832–6841.
Bartl, S., J. Taplick, G. Lagger, H. Khier, K. Kuchleret al., 1997 RPD3-type histone deacetylases in maize embryos. Biochemistry
39:1683–1692. Identification of mouse histone deacetylase 1 as a growth
factor-Li, E., 2002 Chromatin modification and epigenetic reprogramming inducible gene. Mol. Cell. Biol.17:5033–5043.
in mammalian development. Nat. Rev. Genet.3:662–673.
Bernier, G., 1986 The flowering process as an example of plastic
Li, G., T. C. Hall andR. Holmes-Davis, 2002 Plant chromatin: development. Symp. Soc. Exp. Biol.40:257–286.
development and gene control. Bioessays24:234–243.
Bernstein, B. E., J. K. TongandS. L. Schreiber, 2000 Genomewide
Lindroth, A. M., X. Cao, J. P. Jackson, D. Zilberman, C. M.
McCal-studies of histone deacetylase function in yeast. Proc. Natl. Acad.
lumet al., 2001 Requirement of CHROMOMETHYLASE3 for Sci. USA97:13708–13713.
maintenance of CpXpG methylation. Science292:2077–2080.
Bleecker, A. B., andS. E. Patterson, 1997 Last exit: senescence,
Litt, M. D., M. Simpson, M. Gaszner, C. D. AllisandG. Felsenfeld, abscission, and meristem arrest in Arabidopsis. Plant Cell 9:
2001 Correlation between histone lysine methylation and devel-1169–1179.
opmental changes at the chicken beta-globin locus. Science293:
Brosch, G., R. Ransom, T. Lechner, J. D. WaltonandP. Loidl,
2453–2455. 1995 Inhibition of maize histone deacetylases by HC toxin,
Lusser, A., G. Brosch, A. Loidl, H. HaasandP. Loidl, 1997 Identi-the host-selective toxin of Cochliobolus carbonum. Plant Cell 7:
fication of maize histone deacetylase HD2 as an acidic nucleolar 1941–1950.
phosphoprotein. Science277:88–91.
Chen, Z. J., andC. S. Pikaard, 1997 Epigenetic silencing of RNA
Lusser, A., D. KolleandP. Loidl, 2001 Histone acetylation: lessons polymerase I transcription: a role for DNA methylation and
from the plant kingdom. Trends Plant Sci.6:59–65. histone modification in nucleolar dominance. Genes Dev. 11:
Lynn, K., A. Fernandez, M. Aida, J. Sedbrook, M. Tasakaet al., 1999 2124–2136.
The PINHEAD/ZWILLE gene acts pleiotropically in
Arabidop-Church, G. M., andW. Gilbert, 1984 Genomic sequencing. Proc.
sis development and has overlapping functions with the ARGO-Natl. Acad. Sci. USA81:1991–1995.
NAUTE1 gene. Development126:469–481.
Dangl, M., G. Brosch, H. Haas, P. LoidlandA. Lusser, 2001
Com-Martienssen, R., andV. Colot, 2001 DNA methylation and epi-parative analysis of HD2 type histone deacetylases in higher
genetic inheritance in plants and filamentous fungi. Science293:
plants. Planta213:280–285.
1070–1074.
De Rubertis, F., D. Kadosh, S. Henchoz, D. Pauli, G. Reuter
Meyerowitz, E. M., 1997 Genetic control of cell division patterns et al., 1996 The histone deacetylase RPD3 counteracts genomic
in developing plants. Cell88:299–308. silencing inDrosophilaand yeast. Nature384:589–591.
Meyerowitz, E. M., 2002 Plants compared to animals: the broadest
Feldmann, K. A., andM. D. Marks, 1987 Agrobacterium-mediated
comparative study of development. Science295:1482–1485. transformation of germinating seeds ofArabidopsis thaliana: a
Morel, J. B., C. Godon, P. Mourrain, C. Beclin, S. Boutetet al., non-tissue culture approach. Mol. Gen. Genet.208:1–9.
2002 Fertile hypomorphic ARGONAUTE (ago1) mutants
im-Finnegan, E. J., 2001 Is plant gene expression regulated globally?
paired in post-transcriptional gene silencing and virus resistance. Trends Genet.17:361–365.
Plant Cell14:629–639.
Finnegan, E. J., W. J. PeacockandE. S. Dennis, 1996 Reduced
Moussian, B., H. Schoof, A. Haecker, G. Jurgens andT. Laux, DNA methylation inArabidopsis thalianaresults in abnormal plant
1998 Role of the ZWILLE gene in the regulation of central shoot development. Proc. Natl. Acad. Sci. USA93:8449–8454.
meristem cell fate during Arabidopsis embryogenesis. EMBO J.
Forsthoefel, N. R., Y. Wu, B. Schulz, M. J. Bennett andK. A.
17:1799–1809.
Feldmann, 1992 T-DNA insertion mutagenesis inArabidopsis:
Murai, K., S. Takumi, H. KogaandY. Ogihara, 2002 Pistillody, prospects and perspectives. Aust J. Plant Physiol.19:353–366.
homoeotic transformation of stamens into pistil-like structures,
Gendrel, A. V., Z. Lippman, C. Yordan, V. ColotandR. A.
Mar-caused by nuclear-cytoplasm interaction in wheat. Plant J. 29:
tienssen, 2002 Dependence of heterochromatic histone H3
169–181. methylation patterns on the Arabidopsis gene DDM1. Science
Murfett, J., X. J. Wang, G. HagenandT. J. Guilfoyle, 2001
Identi-297:1871–1873.
fication ofArabidopsishistone deacetylase hda6 mutants that affect
Habu, Y., T. KakutaniandJ. Paszkowski, 2001 Epigenetic
develop-transgene expression. Plant Cell13:1047–1061. mental mechanisms in plants: molecules and targets of plant
Pandey, R., A. Muller, C. A. Napoli, D. A. Selinger, C. S. Pikaard
epigenetic regulation. Curr. Opin. Genet. Dev.11:215–220.
et al., 2002 Analysis of histone acetyltransferase and histone
Henikoff, S., andL. Comai, 1998 A DNA methyltransferase
homo-deacetylase families ofArabidopsis thaliana suggests functional log with a chromodomain exists in multiple polymorphic forms
diversification of chromatin modification among multicellular in Arabidopsis. Genetics149:307–318. eukaryotes. Nucleic Acids Res.
30:5036–5055.
Hollick, J. B., J. E. DorweilerandV. L. Chandler, 1997 Paramuta- Richards, E. J., andS. C. Elgin, 2002 Epigenetic codes for hetero-tion and related allelic interachetero-tions. Trends Genet.13:302–308. chromatin formation and silencing: rounding up the usual
sus-Imai, S., C. M. Armstrong, M. KaeberleinandL. Guarente, 2000 pects. Cell108:489–500.
Transcriptional silencing and longevity protein Sir2 is an NAD- Robyr, D., Y. Suka, I. Xenarios, S. K. Kurdistani, A. Wanget al., dependent histone deacetylase. Nature403:795–800. 2002 Microarray deacetylation maps determine genome-wide
Jacobsen, S. E., and E. M. Meyerowitz, 1997 Hypermethyl- functions for yeast histone deacetylases. Cell109:437–446. atedSUPERMAN epigenetic alleles inArabidopsis.Science277: Ronemus, M. J., M. Galbiati, C. Ticknor, J. ChenandS. L.
Della-1100–1103. porta, 1996 Demethylation-induced developmental pleiotropy
Jang, I. C., Y. M. Pahk, S. I. Song, H. J. Kwon, B. H. Nahmet al., inArabidopsis.Science273:654–657.
2003 Structure and expression of the rice class-I type histone Rossi, V., H. Hartingsand M. Motto, 1998 Identification and deacetylase genes OsHDAC1–3: OsHDAC1 overexpression in characterisation of anRPD3homologue from maize (Zea mays transgenic plants leads to increased growth rate and altered archi- L.) that is able to complement anrpd3null mutant ofSaccharomyces tecture. Plant J.33:531–541. cerevisiae.Mol. Gen. Genet.258:288–296.
Jenuwein, T., andC. D. Allis, 2001 Translating the histone code. Rundlett, S. E., A. A. Carmen, R. Kobayashi, S. Bavykin, B. M. Science293:1074–1080. Turneret al., 1996 HDA1 and RPD3 are members of distinct
Kakutani, T., 1997 Genetic characterization of late-flowering traits yeast histone deacetylase complexes that regulate silencing and induced by DNA hypomethylation mutation inArabidopsis thali- transcription. Proc. Natl. Acad. Sci. USA93:14503–14508. ana.Plant J.12:1447–1451. Rundlett, S. E., A. A. Carmen, N. Suka, B. M. Turner andM.
Krysan, P. J., J. C. Young, F. TaxandM. R. Sussman, 1996 Identifi- Grunstein, 1998 Transcriptional repression by UME6 involves cation of transferred DNA insertions within Arabidopsis genes deacetylation of lysine 5 of histone H4 by RPD3. Nature392: involved in signal transduction and ion transport. Proc. Natl. 831–835.
Acad. Sci. USA93:8145–8150. Selker, E. U., 1998 Trichostatin A causes selective loss of DNA
methyla-Landry, J., A. Sutton, S. T. Tafrov, R. C. Heller, J. Stebbinset tion in Neurospora. Proc. Natl. Acad. Sci. USA95:9430–9435. al., 2000 The silencing protein SIR2 and its homologs are NAD- Soppe, W. J., S. E. Jacobsen, C. Alonso-Blanco, J. P. Jackson, T.
dependent protein deacetylases. Proc. Natl. Acad. Sci. USA97: Kakutaniet al., 2000 The late flowering phenotype offwa mu-5807–5811. tants is caused by gain-of-function epigenetic alleles of a
homeo-domain gene. Mol. Cell6:791–802.
Soppe, W. J. J., Z. Jasencakova, A. Houben, T. Kakutani, A. Mei- Tian, L., andZ. J. Chen, 2001 Blocking histone deacetylation in Arabidopsisinduces pleiotropic effects on plant gene regulation
steret al., 2002 DNA methylation controls histone H3 lysine
9 methylation and heterochromatin assembly in Arabidopsis. and development. Proc. Natl. Acad. Sci. USA98:200–205.
Verbsky, M. L., andE. J. Richards, 2001 Chromatin remodeling EMBO J.21:6549–6559.
Srinivas, S., 2000 Development: nature and nurture. J. Cell Sci. in plants. Curr. Opin. Plant Biol.4:494–500.
Vidal, M., andR. F. Gaber, 1991 RPD3 encodes a second factor
re-113:3549–3550.
Stokes, T. L., andE. J. Richards, 2002 Induced instability of two quired to achieve maximum positive and negative transcriptional states inSaccharomyces cerevisiae.Mol. Cell. Biol.11:6317–6327. Arabidopsis constitutive pathogen-response alleles. Proc. Natl.
Acad. Sci. USA99:7792–7796. Vongs, A., T. Kakutani, R. A. Martienssen andE. J. Richards, 1993 Arabidopsis thaliana DNA methylation mutants. Science
Stokes, T. L., B. N. KunkelandE. J. Richards, 2002 Epigenetic
variation inArabidopsisdisease resistance. Genes Dev.16:171–182. 260:1926–1928.
Walbot, V., 1996 Sources and consequences of phenotypic and
Tanner, K. G., J. Landry, R. SternglanzandJ. M. Denu, 2000
Silent information regulator 2 family of NAD-dependent histone/ genotypic plasticity in flowering plants. Trends Plant Sci.1:27–32.
Wu, K., K. Malik, L. Tian, D. BrownandB. Miki, 2000a Functional protein deacetylases generates a unique product,
1-O-acetyl-ADP-ribose. Proc. Natl. Acad. Sci. USA97:14178–14182. analysis of a RPD3 histone deacetylase homologue inArabidopsis
Taunton, J., C. A. HassigandS. L. Schreiber, 1996 A mammalian thaliana.Plant Mol. Biol.44:167–176.
histone deacetylase related to the yeast transcriptional regulator Wu, K., L. Tian, K. Malik, D. BrownandB. Miki, 2000b Functional Rpd3p. Science272:408–411. analysis of HD2 histone deacetylase homologues inArabidopsis
ten Lohuis, M., H. Galliano, I. HeidmannandP. Meyer, 1995 thaliana.Plant J.22:19–27.
Treatment with propionic and butyric acid enhances expression Zhou, Y. X., R. J. NewtonandJ. H. Gould, 1997 A simple method variegation and promoter methylation in plant transgenes. Biol. for identifying plant/T-DNA junction sequences resulting from Chem. Hoppe Seyler376:311–320. Agrobacterium-mediated DNA transformation. Plant Mol. Biol.
Thitamadee, S., K. TuchiharaandT. Hashimoto, 2002 Microtu- Rep.15:246–254. bule basis for left-handed helical growth in Arabidopsis.Nature
