constitutive expression exposes functional redundancy between … · constitutive expression...
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Constitutive Expression Exposes Functional Redundancybetween the Arabidopsis Histone H2A Gene HTA1and Other H2A Gene Family Members OA
HoChul Yi,1 Nagesh Sardesai, Toshinori Fujinuma,2 Chien-Wei Chan,3 Veena,4 and Stanton B. Gelvin5
Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907-1392
The Arabidopsis thaliana histone H2A gene HTA1 is essential for efficient transformation of Arabidopsis roots by Agro-
bacterium tumefaciens. Disruption of this gene in the rat5mutant results in decreased transformation. InArabidopsis, histone
H2A proteins are encoded by a 13-member gene family. RNA encoded by these genes accumulates to differing levels in roots
and whole plants; HTA1 transcripts accumulate to levels up to 1000-fold lower than do transcripts of other HTA genes. We
examined the extent to which other HTA genes or cDNAs could compensate for loss of HTA1 activity when overexpressed in
rat5mutant plants.Overexpressionof all testedHTAcDNAs restored transformation competence to the rat5mutant.However,
only theHTA1 gene, but not otherHTA genes, could phenotypically complement rat5mutant plantswhen expressed from their
nativepromoters. Expressionanalysis ofHTApromoters indicated that theyhaddistinct but somewhatoverlappingpatternsof
expression inmatureplants.However, only theHTA1promoterwas inducedbywoundingorbyAgrobacterium infectionof root
segments. Our data suggest that, with respect toAgrobacterium-mediated transformation, all tested histoneH2Aproteins are
functionally redundant. However, this functional redundancy is not normally evidenced because of the different expression
patterns of the HTA genes.
INTRODUCTION
Histones are highly conserved components of eukaryotic chro-
matin. They are classified into four subcategories of core his-
tones (H2A, H2B, H3, and H4) and linker histones (H1). Core
histones are among the most abundant proteins in eukaryotic
organisms. The expression of many histone genes is coupled to
the S phase of the cell cycle and correlates with DNA replication
and cell proliferation (Sundas et al., 1993; Brandstadter et al.,
1994; Kouchi et al., 1995; Kanazin et al., 1996; Reichheld et al.,
1998; Zhao et al., 2000; Nelson et al., 2002). However, some
histones are expressed in tissues with low proliferation and cell
division activity (Waterborg and Robertson, 1996; van den
Heuvel et al., 1999; Xu et al., 1999). This latter expression pattern
is characteristic of replacement histones andmay result from the
natural turnover of histones in nondividing mature cells or in cells
undergoing substantial chromatin reorganization.
For many years, histones were considered as relatively pas-
sive structural units of eukaryotic chromatin. During the past
decade, however, an increasing number of reports has empha-
sized the roles of histone modification and histone variants in the
regulation of gene expression (Paull et al., 2000; Rice and Allis,
2001; Verbsky and Richards, 2001; Ausio and Abbott, 2002;
Fransz and de Jong, 2002; Redon et al., 2002). Histone acety-
lation and methylation in particular have been implicated in gene
expression (Vongs et al., 1993;Hirochika et al., 2000; Beisel et al.,
2002; Volpe et al., 2002). Minor histone variants may contribute
to diverse nuclear functions by directing distinct or unique chro-
matin architectures (Brown, 2001; Ahmad and Henikoff, 2002;
Smith, 2002; Talbert et al., 2002). These more recent findings
have brought histones once again into the limelight as important
mediators of cellular events.
For most species, histone genes are members of multigene
families. In addition to histone modification, the nature of these
histone multigene families has attracted considerable attention
relating to the specific roles of individual histones in mediating
divergent cellular responses. Several publications have focused
on H2A variants. One such variant, H2AX, becomes phosphor-
ylated following induction of double-strand DNA breaks and is
associated with the recruitment of repair factors to damaged
DNA (Andegeko et al., 2001; Celeste et al., 2002; Tsukuda et al.,
2005). Another histone H2A variant, H2A.Z, was first identified by
West and Bonner (1980) and is highly conserved throughout
evolution. H2A.Z proteins are nonuniformly spread among chro-
matin (Leach et al., 2000), and theymay confer properties distinct
from other H2A variants upon the organism (Iouzalen et al., 1996;
1Current address: Vector Research, 710 West Main Street, Durham, NC27701.2 Current address: DNA Chip Research, Yokohama 230-0045, Japan.3 Current address: Well Home Health Products, Wurih Township,Taichung 414, Taiwan.4 Current address: Donald Danforth Plant Science Center, St. Louis, MO63132-2918.5 To whom correspondence should be addressed. E-mail [email protected]; fax 765-496-1496.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with policy described inthe Instructions for Authors (www.plantcell.org) is: Stanton B. Gelvin([email protected]).OAOpen Access articles can be viewed online without a subscription.Article, publication date, and citation information can be found atwww.plantcell.org/cgi/doi/10.1105/tpc.105.039719.
The Plant Cell, Vol. 18, 1575–1589, July 2006, www.plantcell.orgª 2006 American Society of Plant Biologists
Jackson andGorovsky, 2000; Santisteban et al., 2000; Fan et al.,
2002). Recently, this histone variant has been implicated in
preventing the spread of silent heterochromatin into euchromatic
regions of the genome (Mizuguchi et al., 2004). Histone mac-
roH2A1may be involved in inactivation of X chromosomes and in
transcriptional silencing (Costanzi et al., 2000).
Although several plant histone H2A genes have been iden-
tified and their expression patterns analyzed (Koning et al.,
1991; Callard and Mazzolini, 1997; Huh et al., 1997; van den
Heuvel et al., 1999; Xu et al., 1999), their functions have not yet
been well defined. We have identified an Arabidopsis thaliana
mutant, rat5, in which the histone H2A gene HTA1 is disrupted.
This mutant is resistant to Agrobacterium tumefaciens–mediated
root transformation caused by a deficiency in T-DNA integration
into the plant genome (Mysore et al., 2000b). More recently, we
showed that this Arabidopsis HTA1 gene is expressed in nondi-
viding tissues and that its expression is highly correlated to root
cells that are most susceptible to Agrobacterium-mediated
transformation (Yi et al., 2002). The expression of HTA1 in cells
that are not undergoing mitotic division suggests that histone
H2A-1 is a replacement histone variant. As with the situation in
most species, the Arabidopsis HTA genes constitute amultigene
family. The fact that disruption of the HTA1 gene causes a rat
phenotype indicates that expression of other histone H2A family
members at normal levels does not compensate for loss ofHTA1
function in roots. In this article, we investigated the expression
levels and patterns of several Arabidopsis HTA genes and
directly tested the degree of functional redundancy afforded by
HTA family members with regard to complementation of the rat
phenotype in rat5. Our results indicate that multiple histone HTA
genes can, when overexpressed from a strong promoter, com-
pensate for loss ofHTA1 gene activity. However,whenexpressed
from their native promoters, only HTA1 can complement rat5.
These results suggest that the tested histone H2A proteins are
functionally redundant with respect to Agrobacterium-mediated
transformation but that the levels and/or patterns of expression
from their native promoters are insufficient to compensate for loss
of HTA1 function.
RESULTS
Histone H2A Genes in Arabidopsis
The Arabidopsis genome contains 13 histone H2A (HTA) genes,
each of which encodes a protein of a different sequence (http://
www.chromdb.org/). Individual histoneH2Agenes are dispersed
among the five Arabidopsis chromosomes (Table 1) with sub-
stantial distance not only between H2A genes but also between
H2A genes and other histone genes H2B, H3, H4, and H1. Com-
parison of the 13Arabidopsis histoneH2A amino acid sequences
indicates that they can be categorized into four major groups,
plus one unique member (Figures 1 and 2). These histones share
very high sequence similarity among themselves and to histone
H2Aproteins in other species. Four histone H2As (H2A-1, -2, -10,
and -13) display >92% amino acid sequence identity. We set
these histones into group I. H2A-3 and H2A-5 are classified as
group II; they contain a SQEF motif in their C-terminal regions.
This sequence is characteristic of H2AX, a histone H2A variant
involved in double-strand DNA break repair induced by g radiation
(Rogakou et al., 1998). H2A-3 and H2A-5 show;76%amino acid
sequence identitywithH2A-1. Three histoneH2As, H2A-6, -7, and
-12, show ;57% amino acid sequence identity with H2A-1 and
;70% among themselves. These members have a SPKKmotif in
their C-terminal regions. This motif is found in plant histone H2As
that are cell cycle regulated (Huh et al., 1995). The fourth group
includes H2A-8, -9, and -11. These histones share 80 to 90%
intragroup amino acid sequence identity and ;52% sequence
identity with H2A-1. Group IV members show strong (;80%)
identity with histone H2A F/Z variants in other organisms.
Histone H2A-4 is quite different from the other Arabidopsis
histone H2As. H2A-4 has only 35%amino acid sequence identity
with the other histone H2A family members and contains only
119 amino acids (compared with other family members that are
130 to 150 amino acids in length). A full-length cDNA clone is not
available, and we have been unable to generate a cDNA using
gene-specific primers. It is possible that the gene encoding
histone H2A-4 (HTA4) is a pseudogene.
Table 1. Arabidopsis Histone H2As
Gene Gene ID
Protein Length
(Amino Acids)
Number
of cDNAsaNumber of
EST ClonesaFunction or Subtype
(Consensus Sequences)
HTA1 At5g54640 132 2 11 Agrobacterium transformation
HTA2 At4g27230 131 2 22
HTA3 At1g54690 142 2 5 H2AX (SQEF)
HTA4 At4g13570 119 0 0
HTA5 At1g08880 143 2 33 H2AX (SQEF)
HTA6 At5g59870 151 1 17 DNA binding motif (SPKK)
HTA7 At5g27670 151 2 17 DNA binding motif (SPKK)
HTA8 At2g38810 137 0 (1)b 5 H2A.F/Z
HTA9 At1g52740 135 3 70 H2A.F/Z
HTA10 At1g51060 133 2 30
HTA11 At3g54560 137 1 12 H2A.F/Z
HTA12 At5g02560 153 0 (1)b 7 DNA binding motif (SPKK)
HTA13 At3g20670 133 7 22
a Data from The Arabidopsis Information Resource (www.arabidopsis.org).b Numbers inside parenthesis were amplified by RT-PCR based on longest EST information for this research.
1576 The Plant Cell
Arabidopsis Histone H2A Genes Are Not Functionally
Redundant with Regards to Agrobacterium-Mediated
Root Transformation
Histone H2A-1 plays an important role in Agrobacterium-medi-
ated transformation ofArabidopsis roots (Mysore et al., 2000b; Yi
et al., 2002). The rat5 mutant contains two tandem copies of
T-DNA inserted into the 39 untranslated region of theHTA1 gene.
This mutation results in a deficiency in T-DNA integration upon
subsequent attempts to retransform the roots (Mysore et al.,
2000b). In order to determine the extent of functional redundancy
among the histone H2A genes, we individually introduced var-
iousHTA genes (HTA1, -2, -3, -4, -5, -6, -8, and -10), representing
the four histone H2A clades, into the rat5 mutant using a flower
dip procedure (Clough and Bent, 1998). Mysore et al. (2000a)
previously showed that many rat mutants, including rat5, are
transformable by this method of Agrobacterium-mediated trans-
formation. For each introduced gene, we analyzed 11 to 29 lines
for restoration of transformation proficiency to the rat5 mutant.
Figure 3 shows that compared with wild-type plants, roots of
rat5mutant plants transformedonaverage at only 18% frequency,
and even those tumors that did form were very small compared
with those incited onwild-type roots (Figure 5). Despite high amino
acid sequence similarity among the introduced Arabidopsis his-
tone H2As, none of the heterologous H2A gene family members
could compensate for disruption of the HTA1 (RAT5) gene. As
previously shown (Mysore et al., 2000b), introduction of a genomic
copy of HTA1 into rat5 plants substantially increased their trans-
formation competence; among the transgenic lines examined,
transformationwas restored, on average, to 79% that of wild-type
plants. Thus, noneof the other testedHTAgeneswere functionally
redundant with HTA1. This makes sense in light of our ability to
discern the rat phenotype in rat5 (HTA1) mutant plants despite the
presence of intact copies of all other histone H2A genes.
Heterologous Histone H2A cDNAs, under the Control of a
Constitutive Cauliflower Mosaic Virus 35S Promoter, Can
Restore Transformation Proficiency to the rat5Mutant
It is possible that additional copies of HTA genes did not com-
plement the rat5 mutant because these genes could not ex-
press to a sufficient extent in the root cells most susceptible to
transformation by Agrobacterium (Yi et al., 2002). We therefore
Figure 1. Multiple Sequence Alignment of Arabidopsis H2A Proteins.
The 13 Arabidopsis histone H2A amino acid sequences were compared and analyzed by ClustalX (version 1.81) multiple alignment. Darkly shaded
amino acid residues indicate identity; lightly shaded residues indicate synonymous amino acids.
H2A Proteins and Agrobacterium Infection 1577
introduced into rat5 mutant plants cDNA copies of each of the
genes investigated above (except HTA4, for which a cDNA was
unavailable) and investigated the transformation efficiency of
the derived transgenic lines. In these plants, the cDNAs were
under the control of a strong, constitutive cauliflower mosaic
virus (CaMV) 35S promoter. Figure 4 shows that, unlike the
situationwith genomic copies of theHTA genes, transgenic rat5
plants containing any of the testedHTA cDNAswere restored to
transformation proficiency. In these experiments, root seg-
ments from rat5 mutant plants displayed a transformation
efficiency only 7% that of wild-type plants. Introduction of the
HTA1 cDNA into these plants restored transformation, on
average, to 81% of the wild-type controls. Other HTA cDNAs
restored transformation to lesser extents. Transgenic rat5
plants individually containing HTA groups I and II cDNA trans-
genes (HTA2, -3, -5, and -10) displayed a 50 to 70% restoration
of transformation efficiency. Histones encoded by these cDNAs
are closest in amino acid sequence to histone H2A-1 (Figures
1 and 2). Representatives of histone H2A groups III and IV,
HTA6 and HTA8, restored transformation to rat5 mutant plants
less efficiently (32 and 24%, respectively). Histones encoded
by familymembers of groups III and IV aremore distant in amino
acid sequence to histone H2A-1 than are members of groups I
and II.
Wild-type Arabidopsis ecotype Ws roots usually produce large
green teratomata when infected by A. tumefaciens A208. The
extent of tumorigenesis can be determined not only by the per-
centage of root segments that develop tumors but also by the
morphology and color of the resulting tumors; greater suscepti-
bilitygenerally correlateswith largerandgreener tumors (Zhuetal.,
2003). Figure 5 shows that rat5 lines individually transgenic for
HTA1, -2, -3, -5, and -10 cDNAs produced large green teratomata
when infected by A. tumefaciens A208, whereas rat5 lines trans-
genic for HTA6 or HTA8 produced less pigmented and more
amorphous tumors. These tumor phenotypes additionally indicate
that these transgenic lines are less susceptible to Agrobacterium-
mediated transformation than are lines containing the other
cDNAs. Taken together, our data indicate that, with regard to
Agrobacterium-mediated transformation, all tested histone H2A
proteins are (at least partially) functionally redundant. Our data
further suggest that the lackof functional redundancydisplayedby
the tested HTA genes may result from insufficient levels of
expression or from inappropriate patterns of expression in root
cells most susceptible to transformation by Agrobacterium.
Histone H2AmRNAs Accumulate to Different Extents
in Arabidopsis Plants
The ability of multiple HTA cDNAs, but not genomic DNAs, to
complement the rat phenotype of the rat5 mutant suggests that
the level of expression of the differentHTA genesmay also play a
role in Agrobacterium-mediated transformation. We therefore
measured the steady state levels of transcripts from eight HTA
genes (HTA1, -2, -3, -4, -5, -6, -8, and -10) that were previously
tested in the complementation assays described above. Be-
cause of the high level of sequence similarity among the histone
H2A genes, we performed quantitative real-time RT-PCR anal-
yses using primer sets specific for the individual HTA genes. In
our initial analyses, we were able to amplify RT-PCR products of
all tested HTA mRNAs except HTA4 and HTA8. As mentioned
above, HTA4 may be a pseudogene. The amplification of HTA8
mRNA was weak and variable; we presume that the steady state
level of HTA8 transcripts is very low. We analyzed the levels of
otherHTA transcripts from three independent RNA extractions of
roots or whole plants grown in vitro. Their individual transcript
levels were normalized to those of a-tubulin and glyceralde-
hydes-3-phosphate-dehydrogenase (GAPDH) and displayed as
the number of molecules per microgram of total RNA.
Figure 6 shows that HTA1 and HTA2 transcripts accumulated
to only a low level in preparations of RNA from whole plants and
root tissue.HTA3 andHTA5 transcripts were detected at a level 3
to 5 times higher than those of HTA1 and HTA2. HTA5, -6, and
-10 showed a root preferential expression pattern. Considering
that we use root tissues for transformation assays, it is interesting
to note that HTA1 transcripts do not accumulate to high levels in
roots. Despite this low level of HTA1 expression, disruption of
HTA1 in the rat5 mutant resulted in recalcitrance of Arabidopsis
Figure 2. Phylogenetic Reconstruction of Arabidopsis Histone H2A
Proteins Based on Amino Acid Sequence Similarity.
The Arabidopsis histone H2A proteins were classified into four groups
plus one unique member. The members marked with an asterisk were
used for further analysis. The topology and branch length of the phylo-
gram was produced by distance/neighbor-joining analyses performed
with PAUP* software (v4.10b) and reviewed using TreeView software
(version 1.6.6). The percentage of bootstrap replications supporting each
branch is shown at the nodes of the tree.
1578 The Plant Cell
toAgrobacterium-mediated transformation (Mysore et al., 2000b).
These results indicate that insufficient levels of HTA gene ex-
pression in the cells undergoing transformation may be a reason
for the nonredundant function of other HTA genes with regard to
Agrobacterium-mediated transformation.
Arabidopsis Histone H2A Promoters Display Different
Patterns of Expression
Because our data suggested that the lack of functional redun-
dancy among the various HTA genes may result from differential
patterns of expression, we examined the expression of nineHTA
promoters, plus the first encoded 10 amino acids of each H2A
protein fused to a gusA gene, in transgenic Arabidopsis plants.
Previous data indicated that b-glucuronidase (GUS) expression
directed by a similarHTA1-gusA translational fusion construction
paralleled expression of the HTA1 gene as determined by in situ
RNA hybridization (Yi et al., 2002). For these experiments, we
generated at least 10 lines of transgenic plants for each HTA
promoter-gusA fusion construction and examined the resulting
plants at weekly intervals after germination. Figures 7 and 8
indicate that in mature plants (i.e., plants that had just started to
flower), the HTA promoters showed distinct but, in some cases,
overlapping patterns of expression. These overlapping patterns
of expression could be further delimited in some cases to dis-
tinguish between two promoters on the basis of tissue specificity
(Figure 8).
The HTA2, -3, -6, -8, and -13 promoters directed GUS ex-
pression predominantly in the meristems and actively dividing
cells, including the root meristem and lateral root primordia
(Figures 7B and 8) as well as the floral and leaf meristematic
regions (Figure 7A). The HTA8 promoter could be distinguished
from the other promoters by the specificity of GUS expression in
the root tissues (Figure 8); expression was restricted to the
vasculature in the maturation zone for the HTA8 promoter.
HTA10 promoter-directed GUS expression was predominantly
restricted to the root cap cells in the apical zone (Figure 8). This
promoter expressed in nondividing regions of the plant, including
the root elongation and maturation zones (Figures 7B and 8) and
the leaf veins (Figure 7A). The HTA6 promoter displayed another
patternof expression. Thispromoter expressedstrongly through-
out the roots and leaves (Figures 7A and 7B). TheHTA4 promoter
expressed in a spotty manner at the margins of cauline and ro-
sette leaves (Figure 7A). The promoter also expressed weakly in
the root apical and elongation zones (Figures 7B and 8).
Although the expression patterns of the HTA1, -2, -3, -5, -6,
-10, and -13 promoters looked similar, there were clear differ-
ences in several aspects. As shown previously, HTA1 expressed
mainly in the root tip area, the elongation zone, and in lateral root
primodia (Figure 7B; Yi et al., 2002). HTA1 showed weak ex-
pression in root cap and epidermal cells of the apical region,
whereas HTA2, -3, -5, -6, -10, and -13 expression patterns
were somewhat broader than that of HTA1. The HTA1 and -5
Figure 3. Restoration of rat5 Transformation Proficiency by Histone H2A Genomic DNA Clones.
Roots segments from rat5 transgenic plants containing one of the histone H2A genomic DNA clones were inoculated with A. tumefaciens A208. After 2
d, the root segments were transferred to hormone-free Murashige and Skoog (MS) medium containing timentin. After 1 month, the root segments were
scored for tumor production. Note that only the HTA1 (RAT5) genomic clone complemented the rat5 mutant plant. n, number of root segments
evaluated; Ws, Wassilewskija. Error bars represent standard deviation.
H2A Proteins and Agrobacterium Infection 1579
promoters did not express well in the floral bud, whereas the
HTA2, -3, -10, and -13 promoters did (Figure 7A). The HTA5 and
HTA10 promoter did not express as well in newly emerging
leaves as did the HTA1 promoter.
Thus, although some of the HTA promoters showed over-
lapping patterns of expression, none of the promoters showed
identical expression patterns (Table 2, Figures 7 and 8). In addi-
tion, we did not find any correlation between the various pro-
moter activities and the extent of amino acid sequence similarity
among the histone H2A proteins. Based upon these data, the
tested Arabidopsis histone H2A genes are differentially regu-
lated. It is likely that this differential regulation is responsible for the
lack of functional redundancy among the histone H2A genes with
regards to Agrobacterium-mediated genetic transformation.
The Expression of Only HTA1 Is Increased in Response to
Root Wounding
We previously demonstrated that the expression of HTA1 is
induced by cutting Arabidopsis roots into small segments (Yi
et al., 2002). Because of the importance of wounding in the trans-
formation process (Baker et al., 1997), we examined the induc-
tion of different HTA promoter-gusA fusion constructions in
wounded root segments. For this experiment, we cut the roots
of transgenic plants containing one of seven different HTA-gusA
fusion genes into 3- to 5-mm segments and assayed them for
GUS activity immediately after cutting and 2 d later. We scored
the percentage of cut ends that stained blue with 5-bromo-
4-chloro-3-indolylglucuronide (X-gluc). Table 3 shows that, ac-
cording to this assay, onlyHTA1 showed an increase in activity in
response to wounding. All other tested HTA genes showed a
decrease in expression. These data indicate that HTA genes re-
spond differently to wounding and suggest that increased HTA1
expression in the region near thewound sitemay be important for
Agrobacterium-mediated transformation.
Response of HTA Genes to Infection of Root Segments
by Agrobacterium
We had previously shown that high levels of GUS activity are
directed by the HTA1 promoter after infection of root segments
by Agrobacterium (Yi et al., 2002). We therefore investigated
whether otherHTA genes respond similarly to bacterial infection.
We initially attempted to quantify various HTA mRNAs, using
quantitative real-time RT-PCR, in cut root segments that were
not inoculated or inoculated with either A. tumefaciens At793
(lacking a Ti-plasmid) or At804 (containing the disarmed Ti-
plasmid pEHA105) plus the T-DNA binary vector pBISN1
(Narasimhulu et al., 1996). However, over a period of 36 h after
inoculation, we could not detect any major changes in transcript
abundance for any HTA gene, including HTA1 (data not shown).
This lack of change in mRNA quantity is most likely explained by
the fact that only a very few cells near the cut end of the root
segment were infected by bacteria.
We therefore investigated expression of GUS activity directed
by various HTA promoter-gusA fusions in cut root segments
inoculated with A. tumefaciens A208 (a tumorigenic strain con-
taining pTiT37). GUS activity, measured as the percentage of
root segments staining blue with X-gluc, indicated the activity of
the various HTA promoters. As a control, we examined root
Figure 4. Restoration of rat5 Transformation Proficiency by Histone H2A cDNA Clones.
Roots segments from rat5 transgenic plants containing one of the histone H2A cDNA clones were inoculated with A. tumefaciens A208. After 2 d, the
root segments were transferred to hormone-free MS medium containing timentin. After 1 month, the root segments were scored for tumor production.
Note that all cDNA clones tested complemented the rat5 mutant plant. However, the HTA6 and HTA8 cDNAs did not complement as well as the other
cDNAs tested. n, number of root segments evaluated.
1580 The Plant Cell
segments inoculated with the transfer-deficient strain A. tume-
faciens A136 (which lacks a Ti-plasmid). Table 4 shows that the
HTA1 promoter specifically responded toA. tumefaciensA208, a
tumorigenic strain. More than five times as many root segments
showed GUS activity when cocultivated with this strain than
with A. tumefaciens A136, a nontumorigenic strain lacking a
Ti-plasmid. No otherHTA-gusA promoter fusion showed such an
increase in activity after infection by A. tumefaciens A208. These
experiments indicate that the various tested HTA promoters re-
spond differently to infection by Agrobacterium and that the
HTA1 promoter in particular is induced.
DISCUSSION
Arabidopsis Histone H2A Genes
Similar to the situation with other organisms,Arabidopsis histone
H2A genes constitute a multigene family. However, unlike the
arrangement of mammalian histone H2A genes (Albig and
Doenecke, 1997), the 13members of this family are not clustered
in the genome but rather are dispersed among the five Arabi-
dopsis chromosomes.
Mammalian H2A proteins are classified into four different
variant families: core H2A, macroH2A, H2A.F/Z, and H2AX.
Numerous groups have investigated the functions of each variant
type (Iouzalen et al., 1996; Liu et al., 1996; Reichheld et al., 1998;
Rogakou et al., 1998; Costanzi et al., 2000; Santisteban et al.,
2000). However, little is currently known about the function of
Arabidopsis histone H2A proteins. We compared Arabidopsis
and mammalian histone H2A amino acid sequences with regard
to classification into variant families. Arabidopsis H2A-8, -9, and
-11 are similar to H2A.F/Z variants, whereas H2A-3 and H2A-5
show strong similarity to H2AX variants. Callard and Mazzolini
(1997) reported that the expression of Arabidopsis H2AvAt
(HTA11), a H2A.F/Z variant, is tightly related to cell proliferation.
They suggested that expression of HTA11 might be associated
with a switch from a quiescent to an actively replicating state.
Arabidopsis H2A-3 and H2A-5 contain a conserved SQEF motif
that is found in most H2AX variants in mammals,Drosophila, and
yeast. The Ser residue of this motif becomes phosphorylated in
response to double strand DNA breaks, meiotic recombination
preceding synaptic cross-over, immune cell development, and
apoptotic DNA fragmentation (Rogakou et al., 1998, 2000;
Chen et al., 2000; Mahadevaiah et al., 2001; Tsukuda et al.,
2005).
H2A-6, -7, and -12 contain a SPKKmotif at their C termini. Huh
et al. (1995) suggested that wheat (Triticum aestivum) H2A
proteins can be divided into two groups based upon the exis-
tence of this SPKK motif, which is conserved in angiosperm
H2As. The SPKK motif can bind to the DNAminor groove in A/T-
rich regions (Suzuki, 1991). In sea urchins, histone H1 contains
N-terminal SPKK repeats that are targets for the cdc2 kinase,
and phosphorylation of these repeats is important for chromatin
condensation during sperm development (Roth and Allis, 1992).
The SPKKmotif may be a unique feature of plant H2As because it
is lacking in mammalian, insect, and yeast H2As.
Figure 5. Root Tumorigenesis Assays of rat5 Mutant Plants Containing Various Histone H2A cDNAs Controlled by a CaMV 35S Promoter.
Root segments from plants containing the indicated cDNAs, rat5 mutant plants, and Ws wild-type plants were inoculated with A. tumefaciens A208.
After 2 d, the root segments were transferred to solidified MSmedium containing timentin (to kill the bacteria) but lacking phytohormones. After 1 month,
representative plates from each infection were photographed.
H2A Proteins and Agrobacterium Infection 1581
Differential Expression of Histone H2AMembers
in Arabidopsis
In most organisms, core histone genes are expressed primarily
during the S phase of the cell cycle (Tanimoto et al., 1993;
Oswald et al., 1996; Downs et al., 2000). In addition, there are
histones, called replacement histones, that are expressed con-
stitutively in nondividing cells (Chaubet-Gigot et al., 2001). Re-
placement histones can be functionally redundant with core
histones and can exchange with preexisting histones during de-
velopment. We analyzed Arabidopsis histone H2A gene expres-
sion patterns using promoter-GUS fusions (Figures 7 and 8).
Previous work validated that the expression pattern of a HTA1-
gusA fusion gene using this technique was identical to that
revealed by RNA in situ hybridization (Yi et al., 2002).
HTA2, -3, -6, -8, and -13 expression was predominantly in
meristematic regions in a pattern similar to that of a cyclin gene,
whose expression is directly related to the cell cycle (Ferreira
et al., 1994; Yi et al., 2002). The other tested Arabidopsis his-
tone H2A genes (HTA1, -4, -5, and -10) showed a broad range
of expression patterns. Because they were expressed in cells
not undergoing mitotic divisions, these HTA genes display
some characteristics of replacement histones. It is notable that
HTA13 is expressed mainly in nondividing tissues of the plant
(Figure 7).
Based on amino acid sequence similarity, HTA8 is most
closely related to known H2A.F/Z variants. Vertebrate H2A.F/Z
variants are regarded as replacement histones that are used to
replace core H2A proteins during portions of the cell cycle other
than S phase (Wu et al., 1982; Dalton et al., 1989; Hatch and
Bonner, 1990). However, Drosophila melanogaster H2A.F/Z var-
iants are essential for viability and are expressed maximally
during DNA replication (van Daal and Elgin, 1992). Arabidopsis
HTA8 shows an expression pattern expected of a core histone in
that its expression is restricted to meristematic cells. The ex-
pressionof anotherArabidopsisH2A.F/Z variant, cH2AvAt (HTA11),
is tightly correlated with cell proliferation in cell suspension cul-
tures (Callard and Mazzolini, 1997).
Although we grouped Arabidopsis histone H2A proteins on the
basis of their primary protein structure, we did not find any
relationship between histone H2A gene expression patterns and
protein structural similarity. For example, H2A-3 and H2A-5 are
classified as H2A.X variants, yet their expression patterns were
quite disparate. Histone H2A-10 and H2A-13 also display strong
similarity in amino acid sequence but differ substantially in their
expression patterns (Figures 7 and 8, Table 2).
Functional Redundancy of Histone Genes
Although the rat5 mutant, which contains a disruption of the
HTA1 gene, appears phenotypically normal, it is deficient in
Figure 6. Quantification of HTA Transcripts in Roots and Whole Arabidopsis Plants.
RNAwas isolated from 3-week-oldArabidopsis roots or whole plants and used for reverse transcription. Equal amounts of the reverse transcription products
were used as templates for quantitative real-time PCR using primers directed against each histone HTA,GAPDH, or a-tubulin gene. The level of expression
of each HTA gene was determined after 30 cycles of reaction using Corbett Rotor gene software (version 1.4) and normalized to internal standards (GAPDH
and a-tubulin). Three different RNA samples isolated from different batches of plant material were used for reverse transcription, and at least two PCR
analyses were performed for each RNA sample. Closed bars, RNA from whole plants; open bars, RNA from roots. Error bars denote standard deviation.
1582 The Plant Cell
Figure 7. Expression of HTA Promoter-gusA Fusion Genes in Transgenic Arabidopsis Plants.
Transgenic plants containing each of the indicated HTA-gusA fusion genes were stained with X-gluc and photographed. Representative plants from
representative transgenic lines are shown.
(A) Aerial parts of plant; insets show flower bolts.
(B) Plant roots; bottom section of each panel shows root tips.
T-DNA integration (Mysore et al., 2000b) and thus is highly recal-
citrant to Agrobacterium-mediated root transformation (Nam
et al., 1999). Expression of the other HTA genes in this mutant is
similar to that of wild-type plants (H.C. Yi, unpublished data).
These findings suggest that the tested HTA genes are not
functionally redundant with HTA1, at least with respect to
Agrobacterium-mediated transformation. Indeed, none of the
heterologousHTA genes tested could compensate for the loss of
HTA1 when transformed into rat5 mutant plants (Figure 3).
However, Arabidopsis histone H2A proteins display a high de-
gree of structural similarity, and all tested Arabidopsis H2A
cDNAs were functionally redundant with HTA1 when they were
expressed constitutively (Figure 4). Taken together, these results
suggest that histone H2A proteins are functionally redundant
withhistoneH2A-1with respect toAgrobacterium-mediated trans-
formation. However, the expression patterns of these genes do
not permit this redundancy to be displayed. Placement of the
various HTA cDNAs under the regulation of the HTA1 promoter
may shed more light on the role of cell-specific regulation of
histone H2A expression in Agrobacterium-mediated transforma-
tion. These experiments are currently being conducted.
Functional redundancy of histone H2A genes differs among
species. Takami et al. (1997) removed approximately half of the
histone genes from a cultured chicken cell line. The results of
these experiments indicated that some of the histone family
members or variants are not completely redundant. In Tetrahy-
mena thermophila, disruption of either of the two major histone
H2A genes did not substantially affect viability of the organism.
However, mutation of theHTA3 gene encoding aH2A.F/Z variant
was not completely compensated by expression of the other
major H2A genes (Liu et al., 1996).
Individual members of a multigene family could support dispa-
rate cellular metabolic activities and developmental responses.
Histone H2A family members are abundant proteins that play
important and sometimes different roles in constituting chromo-
somal structure (Redon et al., 2002). For example, histone H2AX
may be involved in signaling double strand breaks that require
repair (Paull et al., 2000), whereas histone H2A.Z is involved in
marking telomeric heterochromatin and in preventing its spread
into euchromatic regions of the chromosome (Meneghini et al.,
2003; Mizuguchi et al., 2004). Particular histone variants may
associate with various chromosomes or chromosomal regions.
Figure 8. Transverse Sections of Roots of Transgenic Arabidopsis Plants Showing Expression of HTA Promoter-gusA Fusion Genes.
Approximately 20 transgenic lines for each promoter-gusA fusion were screened, and the roots were sectioned. Representative photomicrographs of
the root apical, elongation, and maturation zones from representative transgenic lines are shown. Schematic diagrams of the root apical, elongation,
and maturation zones are shown in the last panel. RC, root cap cells; Ep, epidermis; C, cortex; En, endodermis; P, pericycle; V, vasculature.
1584 The Plant Cell
Histone macroH2A1 is found on inactive X chromosomes of fe-
male mice (Costanzi et al., 2000), whereas certain histone H3
variants are specifically associatedwith centromeric heterochro-
matin (Talbert et al., 2002).
As a first step in elucidating control of histone H2A gene ex-
pression, we used quantitative real-time RT-PCR to monitor the
steady state level of transcripts encoded by six HTA genes
(HTA1, -2, -3, -5, -6, and -10). Real-time PCR has been used by
several research groups to estimate gene copy number and for
quantification of transcript levels (Bustin, 2000; Kandasamyet al.,
2002; Milligan et al., 2002; Toyooka et al., 2002). Because we
usedArabidopsis roots to test forAgrobacterium-mediated trans-
formation competence, we used this tissue for our RNA analysis
and subsequently compared these results with those using RNA
extracted fromwhole plants. The expression ofmany testedHTA
genes in roots paralleled that of whole plants (Figure 6). However,
HTA5, -6, and -10 showed enriched expression in root tissues.
The quantitative real-time RT-PCR results correlated well with
our HTA promoter-gusA fusion reporter gene expression data
(Figures 7 and 8, Table 2). HTA1 is expressed at low levels and
constitutes only 2 to 3% of total histone H2A gene expression. It
is interesting to note that, despite the low level of expression,
Arabidopsis HTA1 is essential for integration of Agrobacterium
T-DNA and cannot be compensated for by normal levels of ex-
pression of the other HTA genes.
HTA1 also responds differently to wounding and to Agro-
bacterium infection than do the other investigated HTA genes
(Tables 3 and 4). Thus, HTA1 shows a unique association with
Agrobacterium-mediated plant transformation that is not shared
by other Arabidopsis histone genes. We have searched for DNA
sequence motifs in the HTA1 promoter that may confer wound-
inducible expression using the database PLACE (http://www.
dna.affrc.go.jp/PLACE/; Higo et al., 1999). A W-box motif, com-
monly found in the promoters ofwound-inducible genes (Nishiuchi
et al., 2004), exists in theHTA1 promoter at position�354. How-
ever, this motif also appears (sometimes inmultiple places) in the
promoters of HTA3, HTA4, HTA6, HTA8, and HTA9, promoters
that are not wound inducible. Other DNA sequence motifs
commonly found in wound-inducible promoters, such as a GT1
box, also appear in the HTA1 promoter, although a MYC box
sometimes associatedwithwound-inducible promoters is absent
from the HTA1 promoter. Thus, specific motifs causing wound
inducibility of the HTA1 promoter remain to be established.
METHODS
Plant and Agrobacterium Growth
Arabidopsis thaliana seedswere surface sterilized and germinated in Petri
dishes containing B5 medium (Gibco BRL) solidified with 7.5 g/L bacto-
agar (Difco). When appropriate, antibiotics were added either to kill Agro-
bacterium tumefaciens (100 mg/mL timentin) or for transgene selection
(50 mg/mL kanamycin or 50 mg/mL hygromycin). The plates were incu-
bated under a 16-h-light/8-h-dark photoperiod at 258C for 1 week. Seed-
lings were individually transferred into baby food jars containing B5
Table 2. Expression Patterns of Nine HTA Promoter-gusA Reporter Gene Fusions in Transgenic Arabidopsis Plants
Gene
Root Apical
Zone
Lateral Root
Primordia
Root Elongation
Zone
Root Maturation
Zone Leaves
Center of
Rosette
Floral
Bud
HTA1 þ/� þ þ þ Expanded þ þHTA2 þ _ þ þ Veins þ þHTA3 þ þ þ þ Young þ þHTA4 þ/� � � � Margin � þ/�HTA5 þ þ þ þ Young þ �HTA6 þ þ þ þ Expanded þ þ/�HTA8 þ þ þ/� þ Young þ þHTA10 þ þ þ þ Veins � þHTA13 þ þ þ þ Expanded þ þ
The þ, �, and þ/� indicate expression, no expression, and weak expression of the gusA reporter gene, respectively.
Table 3. Changes of Arabidopsis H2A Promoter Activity Near the Cut Site of Root Segments
Freshly Cut Incubated for 2 d on MS Medium
Gene
GUS Expression
(%)aNumber of Root
Segments Assayed
GUS Expression
(%)aNumber of Root
Segments Assayed
HTA1 16.4 354 34.6 399
HTA2 51.0 776 17.3 1064
HTA3 9.2 981 4.1 398
HTA4 0.5 364 0.3 325
HTA5 50.5 871 24.5 1191
HTA6 49.1 793 29.7 1169
HTA13 38.7 860 26.8 1086
a Calculated as the percentage of root segments showing GUS activity near the cut site.
H2A Proteins and Agrobacterium Infection 1585
medium and grown for 2 to 3 weeks for tumorigenesis assays. Alterna-
tively, plants were grown in pots containing soil for harvesting seeds.
Agrobacterium was grown in YEP-rich or AB minimal medium
(Lichtenstein and Draper, 1986) at 308C. When appropriate, antibiotics
were usedat the following concentrations: 10mg/mL rifampicin, 100mg/mL
kanamycin (on solidified medium), or 25 mg/mL (in liquid medium).
Cloning of Histone H2A Genomic DNAs and cDNAs
Arabidopsis histone H2A genomic DNAs were cloned by PCR amplifica-
tion. PCR primers were designed to bind ;2 kb upstream and 1 kb
downstreamof the coding sequence of individual histone H2A genes. The
sequence of the primers used are: HTA1 forward, 59-TGGGTCGTGGA-
CATCAGATGGTTCGGA-39; HTA1 reverse, 59-GAGGCATAGACACTGT-
CACTCACTTGT-39; HTA2 forward, 59-GGCTATGGATCTTGAACAACC-
GGACCT-39; HTA2 reverse, 59-ACGAGAGCTCTAAACCGAATCGTC-
CCT-39; HTA3 forward, 59-GAGCAGTTTTTCGAGGAGGTGGTCTCC-39;
HTA3 reverse, 59-TTGAACGATGTTCTGCGGTGGCTCGGC-39; HTA4
forward, 59-TGGGACTGACCTTAAAATTGTCACA-39; HTA4 reverse,
59-GTCTCGCTTCGTGAAATACAGTTGT-39; HTA5 forward, 59-ACCTG-
TCGACGACCAATAACTGCAGCA-39; HTA5 reverse, 59-AGCGAGCTAA-
TTCAGGTGCGAACCAAC-39; HTA6 forward, 59-AGTAACAACCTGCCA-
AACCCGTCCCGC-39; HTA6 reverse, 59-GTCCATGGAAGCTAAGGCA-
CTTGCAGC-39; HTA8 forward, 59-TCCGCAATCTTGGTCACATCAG-
TCA-39; HTA8 reverse, 59-ACCCTGCAAACAAGAAGAGTCAGGC-39;
HTA10 forward, 59-TCGTCCCAAGCGTCACTCGAACCAA-39; HTA10
reverse, 59-ACAGAACCTTCCTTTCGCAATTGGT-39; HTA13 forward,
59-AGACAAGGCCAGAGGGGCAAGCCAT-39; HTA13 reverse, 59-GCC-
ATGGCTTCTTCGACTCTCTTCA-39.
For amplification accuracy and fidelity, AccuTaq LA DNA polymerase
(Sigma-Aldrich) and Takara EX Taq DNA polymerase were used. PCR
reactions were performed using the following cycling conditions: one
cycle of denaturation at 988C for 30 s; 35 cycles of denaturation at 948C for
30 s, annealing at 658C for 45 s, extension at 688C for 5 min; one cycle of
elongation at 688C for 30min. The annealing temperatures and timeswere
occasionally modified depending on primer specificity. The AccuTaq
reaction mixture contained 200 ng of Arabidopsis genomic DNA, 50 mM
Tris-HCl, 15 mM ammonium sulfate, pH 9.3, 2.5 mMMgCl2, 0.1% Tween
20, 500mMdeoxynucleotide triphosphate (dNTP)mix, 400 nMof primers,
and 5 units of Taq polymerase. The ExTaq reaction mixture contained
200 ng of Arabidopsis genomic DNA, 25 mM TAPS, pH 9.3, 50 mM KCl,
2 mMMgCl2, 1 mM 2-mercapoethanol, 200 mMdNTPmix, 1 mMprimers,
and 5 units of Takara ExTaq polymerase. Amplified PCR products were
cloned into pBluescript II SKþ (Stratagene), pGEM-T (Promega), or pCR
2.1 (Invitrogen) vectors. Five histone H2A cDNAs that had not been
isolated from an Arabiodopsis cDNA library in our laboratory were cloned
by RT-PCR. Total RNAwas isolated fromArabidopsis roots, and 1 to 2mg
of total RNAwas used for reverse transcription using oligo(dT) as a primer
(Promega). The reaction mixture was diluted 10 to 20 times, and 10 to
20 mL of the reverse transcription mixture was used for the PCR reaction.
Five PCR primer sets for HTA1, -4, -6, -8, and -13 were designed based
on Arabidopsis EST information. The sequences of the primers were as
follows:HTA1 forward, 59-CTCTTTGTGGGTTGTTGTTGTTGAA-39;HTA1
reverse, 59-GAAACAACATGTCGAAACAGAAACGG-39; HTA4 forward,
59-ACGGTCTCATTTTATGTTTCATGGT-39; HTA4 reverse, 59-GGTTGT-
TTTGTTGATGAGACTAGTG-39; HTA6 forward, 59-TCAGTAATCGATA-
ACCGTAGCAATG-39; HTA6 reverse, 59-GACCAAAAGATTAGACGA-
AGCGTAT-39; HTA8 forward, 59-CTTTGATTTCGACGACGGATCTTGA-39;
HTA8 reverse, 59-TAATAGAGACTTAGGACTCAGAGAG-39; HTA13
forward, 59-TTTCTATCTCTCTTCCCAAATCACA-39; HTA13 reverse,
59-CAACCACAACACAAATCCCTAATCT-39.
RT reactionmixtures (10 or 20mL)were added toPCR reactionmixtures
containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 100 mM
dNTP, and 3 units of high-fidelity Taq DNA polymerase (Boehringer
Mannheim). PCR was performed using the following conditions: one cy-
cle of denaturation at 958C for 2 min; 35 cycles of denaturation at 948C for
20 s, annealing at 588C for 40 s, and extension at 728C for 2.5 min; one
cycle of elongation at 728C for 15min. The PCRproductswere cloned into
pBSþ (Stratagene). All the cloned genomic and cDNA sequences were
confirmed by analysis at the Purdue University Genomics Center.
T-DNA Binary Vector Construction and Plant Transformation for
Complementation of the rat5Mutant
For cDNA complementation analysis, histone H2A cDNAs (HTA1, -2, -3,
-5, -6, -8, -10, and -13) were excised from plasmids using either SalI or
SalI plus XbaI and ligated into the multicloning site of the T-DNA binary
vector pS35-hpt behind the CaMV 35S promoter in the sense orientation.
For genomic DNA complementation, histone H2A genomic DNA frag-
ments (HTA1, -2, -3, -4, -5, -6, -8, -10, and -13) containing;2 kb of their
native promoter sequence and 1 kb of downstream sequence were
excised from plasmids using appropriate restriction sites: HTA1, -2, -5,
Table 4. Changes of Arabidopsis HTA Promoter Activity Near the Cut Site of Root Segments 2 d after Cocultivation with Various
Agrobacterium Strains
A. tumefaciers A208a A. tumefaciens A136b
Gene
GUS Expression
(%)c 6 SE
Number of Root
Segments Assayed
GUS Expression
(%)c 6 SE
Number of Root
Segments Assayed
HTA1 15.8 6.5 1040 2.8 0.1 1096
HTA2 20.8 3.2 1179 16.4 2.4 873
HTA3 4.8 0.7 1073 8.9 1.3 910
HTA4 0.8 0.3 615 1.3 0.4 455
HTA5 8.1 1.8 1378 5.9 0.3 1173
HTA6 19.0 2.5 1615 17.9 1.5 799
HTA8 6.1 0.7 655 8.3 1.4 495
HTA10 3.4 0.9 520 10.1 0.0 355
HTA13 20.7 2.4 1017 19.1 2.0 619
a T-DNA and Vir protein transfer-competent strain.b T-DNA and Vir protein transfer-deficient strain.c Calculated as the percentage of root segments showing GUS activity near the cut site.
1586 The Plant Cell
and -6, XbaI andSalI;HTA3 and -4,HindIII and SpeI;HTA8 and -10,SmaI;
HTA13, SalI and XhoI. The excised DNA fragments were cloned into the
T-DNA binary vector pGPTV-hpt. The various constructions carrying
histone H2A cDNAs or genes were introduced into A. tumefaciens
GV3101 (Koncz and Schell, 1986) by electroporation and used for floral
dip transformation (Clough and Bent, 1998) of rat5mutant plants (Mysore
et al., 2000a). Harvested seeds were spread on B5 medium containing
kanamycin and hygromycin for selection of transgenic plants.
Tumorigenesis Assays and Phenotypic Analysis
Arabidopsisplantswere grown axenically in baby food jars for 3 to 4weeks
at 258C. Root segments were cut and infected with Agrobacterium as
described previously (Nam et al., 1999). Root segments were separated
onto solidifiedMSmediumcontaining timentin but lacking phytohormones
and scored 1 month later for the number and phenotype of the tumors.
Construction of Histone H2A Promoter-gusA Fusion Genes
Approximately 2 kb upstream of each histone H2A genomic DNA, plus
sequences encoding the first 10 to 22 amino acids, were PCR amplified
with SmaI linker sequences. The primer sequences were as follows:
HTA1, 59-CCCGGGGGATCCAAGAGTTTTTCCACGACCA-39; HTA2,
59-CCCGGGCTTGCTACTACGAGAAGTAGACTTC-39;HTA3, 59-CCCGG-
GACCTTTAGTTGTTCCACTGCCGGCG-39; HTA4, 59-CCCGGGATCT-
TTTAGTATATTCGTGTTGCAC-39; HTA5, 59-CCCGGGGGTTGTTCCGC-
TTCCTGCGCCTGTA-39; HTA6, 59-CCCGGGAGCTTTCTTCACTTTTC-
CGGTGGAT-39, HTA8, 59-CCCGGGTAGAAGCCCTTTCCCACCTTTA-
CCA-39, HTA10, 59-CCCGGGAGATCCGAGTGTTTTACCACGACCC-39;
HTA13, 59-CCCGGGGATCCGAGAGTTTTGCCGCGACCCG-39. PCR re-
actions were performed using the same conditions as for genomic DNA
PCR. Amplified products were cloned in theSmaI site of pBluescript II SK.
Cloned DNA fragments were excised from the plasmid using appropriate
restriction enzymes (HTA1, -2, and -6, XbaI and SmaI; HTA3, -4, -5, -10,
and -13, SalI and SmaI; HTA8, SmaI) and cloned into the T-DNA binary
vector pBI101.3 as a translational fusion with the gusA gene. The con-
structionswere transferred intoA. tumefaciensGV3101 and used to trans-
form Arabidopsis ecotype Ws by a flower dip protocol (Clough and Bent,
1998). Transgenic plants were selected onmedium containing kanamycin
and used for analysis of promoter activity of each of the HTA genes.
Sequence Alignment and Phylogenetic Analyses
Protein sequences encoded by 13 histone H2A (HTA) genes were down-
loaded from the ChromDB database (http://www.chromdb.org/) and
aligned using ClustalX 1.81 (Thompson et al., 1997). Nomanual adjustment
was required for the alignment. The alignment was used for distance/
neighbor-joining phylogenetic analyses performed in PAUP* v4.10b
(Swofford, 2003) based on the total number of pairwise character differ-
ences. Confidence values for groupings in the treewere assessed by boot-
strap resampling (Felsenstein, 1985) with 10,000 repetitions for distance/
neighbor joining, and the tree was reviewed in TreeView 1.6.6 (Page, 1996).
Histochemical Analysis of GUS Activity
Detection of GUS expression in plant tissues was performed using X-gluc
according to Jefferson et al. (1987) with modifications. Briefly, plant
samples were harvested in 90% acetone incubated at room temperature
for 20 min. The samples were rinsed once with staining solution (0.2%
Triton X-100, 50 mM sodium phosphate buffer, pH 7.2, 2 mM potassium
ferrocyanide, and 2 mM potassium ferricyanide). The staining solution
was replaced by staining solution containing 2 mM X-gluc, and the plant
samples were vacuum infiltrated on ice for 20 min. Following overnight
incubation of the samples at 378C, the plants were processed for sec-
tioning by incubating them successively in 20, 35, and 50% ethanol at
room temperature for 30 min each. Ethanol was replaced by FAA (5%
formaldehyde, 10% glacial acetic acid, and 50% ethanol) for 30 min, and
the ethanol series (80, 90, and 95%) was continued until the plants were
finally in 100% ethanol. Plant tissuewas infiltrated and embedded in JB-4
resin (Electron Microscopy Sciences) following the manufacturer’s in-
structions. Tissues were sectioned at a thickness of 10mmusing a Sorvall
JB-4 microtome and examined under a compound light microscope.
Quantitative Real-Time RT-PCR of HTA Transcripts
Total RNA isolated from roots or whole plants was used for reverse tran-
scription and subsequent PCR amplification. Thirteen primer sets were
designed, eachwith amelting temperature of 55 to 658C. Each primerwas
>25 nucleotides and recognized gene-specific regions in the 59 or 39
untranslated regions of individual histone H2A genes. The sequences of
the primers were as follows: HTA1 forward, 59-CTCTTTGTGGGTTGTT-
GTTGTTGAAA-39; HTA1 reverse, 59-GAGAGACGTGTTCTATATCATT-
GTG-39; HTA2 forward, 59-CTATCTTGGGTAGTAGAGAGAAATG-39; HTA2
reverse, 59-AGCTTTCCCTATCTTCGTAATGAAC-39; HTA3 forward,
59-GCCTCTTCAAATTTCCCGATAAACA-39;HTA3 reverse, 59-GGAACAGA-
GAGCCATGTCTATGTTA-39; HTA4 forward, 59-ACGGTCTCATTTTATGT-
TTCATGGT-39; HTA4 reverse, 59-GGTTGTTTTGTTGATGAGACTAGTG-
39; HTA5 forward, 59-TCTGTTCTAAATTTCGAAGAAGACG-39; HTA5 re-
verse, 59-CTGAACTAAGAAGTCTAAGAACCTC-39; HTA6 forward,
59-TCAGTAATCGATAACCGTAGCAATG-39; HTA6 reverse, 59-CTAGC-
AACGAAAACTCTAGCAGATT-39;HTA7 forward, 59-CTTCATCTAAGATCC-
GAATATAACC-39; HTA7 reverse, 59-CTTTTGGAGCTTTTGAACAATG-
GAG-39; HTA8 forward, 59-CTTTGATTTCGACGACGGATCTTGA-39;
HTA8 reverse, 59-TAATAGAGACTTAGGACTCAGAGAG-39; HTA9 for-
ward, 59-CAATCTCCAAGGATTTTACTGTGA-39; HTA9 reverse, 59-CAA-
AGCGGGTAACTAAAAAAGTCCT-39; HTA10 forward, 59-CTGTTTCCC-
TCTCATCTTTACACAA-39; HTA10 reverse, 59-CACAAGAGAGTGGAT-
TTGGTTGATTA-39; HTA11 forward, 59-GTCTCAAGATCTAGAAGAAG-
GAAAC-39; HTA11 reverse, 59-GGAAAATGAGTCCAAGACGACAGAA-39;
HTA12 forward, 59-TCAGAATCAAATTTCTCGTCGTGTC-39; HTA12
reverse, 59-CAGTAGGTTATACAGAGGAATGAAG-39; HTA13 forward,
59-TTTCTATCTCTCTTCCCAAATCACA-39; HTA13 reverse, 59-CAACCA-
CAACACAAATCCCTAATCT-39. The PCR reactionwas performed using a
Rotor-Gene real-time thermocycler (Corbett Research). The PCR reac-
tionmixture contained 200mMeach dNTP, 2mMMgCl2, 10mMTris-HCl,
pH 9.0, 50 mM KCl, 0.1% Triton X-100, 0.24 mM of the gene-specific
primer combination, 13 dilution of the fluorescent dye Sybr-Green I
(Molecular Probes), and 2.5 units of Taq2000 DNA polymerase (Strata-
gene). As internal standards, GAPDH and a-tubulin genes were used.
Real-time PCR was performed using the following cycle conditions: 958C
for 2min; 30 to 32 cycles of 948C for 20 s, 578C for 45 s, and 728C for 2min;
a final 5-min elongation at 728C. After PCR amplification, the data were
analyzed using Corbett Rotor gene software (version 4.4).
Accession Numbers
Designator numbers for the histone H2A genes are listed in Table 1.
ACKNOWLEDGMENTS
This work was supported by grants from the National Science Foundation
Plant Genome program (9975715 and 9975930), the Biotechnology Re-
search and Development Corporation, the Corporation for Plant Biotech-
nologyResearch, and aP30grant to thePurdueUniversityCancerCenter.
We thank theexpertise ofDebraShermanof theLifeSciencesMicroscopy
Facility, Purdue University for help with the microscopy.
H2A Proteins and Agrobacterium Infection 1587
Received November 23, 2005; revised April 18, 2006; accepted May 9,
2006; published June 2, 2006.
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H2A Proteins and Agrobacterium Infection 1589
DOI 10.1105/tpc.105.039719; originally published online June 2, 2006; 2006;18;1575-1589Plant Cell
HoChul Yi, Nagesh Sardesai, Toshinori Fujinuma, Chien-Wei Chan, Veena and Stanton B. Gelvin and Other H2A Gene Family MembersHTA1Gene
Histone H2AArabidopsisConstitutive Expression Exposes Functional Redundancy between the
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