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The ATL Gene Family from Arabidopsis thaliana and Oryza sativa Comprises a
Large Number of Putative Ubiquitin Ligases of the RING-H2 Type
Mario Serrano,* Socorro Parra,
Luis D. Alcaraz, Plinio Guzma
´
n
Departamento de Ingenierı
´
a Gene
´
tica de Plantas, Centro de Investigacio
´
n y de Estudios Avanzados del IPN Unidad Irapuato,
Apartado Postal 629, Irapuato, Gto., 36500 Me
´
xico
Received: 19 February 2005 / Accepted: 30 September 2005 [Reviewing Editor: Dr. Martin Kreitman]
Abstract. Ubiquitin ligases play an important reg-
ulatory role in the control of protein degradation
processes via the ubiquitin/26S proteasome pathway
in eukaryotes. These enzymes participate in substrate
specification and mediate the transfer of ubiquitin to
target proteins. A large number of ubiquitin ligases
are predicted in the eukaryotes whose genomes have
been sequenced; in Arabidopsis thaliana more than
1300 genes are thought to encode ubiquitin ligases.
At least three classes of ubiquitin ligases are present
in Arabidopsis, one of which comprises about 470
RING zinc-finger domain proteins. Within this class
we have characterized the ATL family that encodes a
RING-H2 finger. We identified 80 members of this
family in A. thaliana and 121 in Oryza sativa. About
60% of the rice ATLs are clustered with A. thaliana
ATLs, and in many cases the gene products showed
sequence similarities beyond the ATLÕs conserved
features, suggesting that they could be orthologous
genes. Ninety percent of the ATLs are intronless
genes, suggesting that the structure of the basic ATL
protein may have evolved as a functional module. We
carried out a survey of T-DNA insertions in 30% of
the Arabidopsis ATL genes and screened for possible
phenotypes. Four of these genes are likely to be
essential for viability, since homozygous plants for
the T-DNA insertion were not recovered. One of
them, ATL8, is mainly expressed in young siliques,
suggesting a role during embryogenesis. We also
recovered a line carrying a T-DNA insertion in
ATL43 that showed an ABA-insensitive phenotype,
suggesting a role of this gene in the ABA response.
The organization of ATLs in Arabidopsis and rice in
this study will be a valuable comprehensive guide for
this multigene family.
Key words: Gene families — Ubiquitin ligases —
Insertional mutagenesis — ABA — Embryo lethal —
Intronless genes — Defense response — Gene struc-
ture
Introduction
The ubiquitin/26S proteasome is a primary pathway
for degradation of cellular proteins that is conserved
among eukaryotes (Glickman and Ciechanover
2002). This system exerts regulated degradation in
multiple cellular events such as the cell cycle, signal
transduction cascades, and stress responses and par-
ticipates in the elimination of aberrant and truncated
polypeptides. When a proteinÕs fate is to be degraded
by this system, the protein gets covalently linked to
ubiquitin molecules, converting it into a substrate for
degradation by the 26 proteasome. Ubiquitin conju-
gation occurs through a cascade of enzymatic reac-
tions involving three classes of enzymes: E1, the
ubiquitin-activating enzyme; E2, the ubiquitin-con-
jugating enzyme, which prepares the ubiquitin chains
*Present address: Max Planck Institute for Plant Breeding
Research, Ko
¨
ln, Germany
Present address: Atlas Operation Inc., FL, USA
Correspondence to: Plinio Guzma
´
n; email: pguzman@ira.cinvestav.
mx
J Mol Evol (2006) 62:434–445
DOI: 10.1007/s00239-005-0038-y
to be transferred; and E3, the ubiquitin ligase that
mediates the transfer of the ubiquitin to proteins and
targets substrates to the proteasome—E3s are thus
implicated in substrate specificity (Glickman and
Ciechanover 2002).
The Arabidopsis thaliana genome encodes more
than 1300 genes that may be involved in the ubiqu-
itin/26 proteasome pathway. Two genes correspond
to E1 enzymes, about 45 genes to E2 or E2-like
proteins, and a major and diverse group of almost
1200 genes corresponds to E3 enzymes. Other ele-
ments participating in this pathway are deubiquiti-
nation enzymes and components of the 26S
proteasome (Smalle and Vierstra 2004). This large
number of genes involved in the ubiquitin/26S pro-
teasome pathway indicates that the control of protein
degradation has a major role in controlling cellular
processes in plants. In fact, analysis of mutations in a
few particular genes of this pathway has revealed that
indeed it affects most cellular processes in plants
(Sullivan et al. 2003; Moon et al. 2004; Smalle and
Vierstra 2004).
Genes coding E3s can be classified in various
groups, depending on their having the HECT do-
main, the RING-finger domain, or the U-box (Moon
et al. 2004; Smalle and Vierstra 2004). The RING
(Really Interesting New Gene) finger is a particular
class of zinc-finger domain that uses a particular
disposition of cysteine and histidine residues to bind
two zinc ions (Freemont 1993; Joazeiro and Weiss-
man 2000). This domain is overrepresented in Ara-
bidopsis, as this superfamily contains almost 400
predicted members: 3 to 4 times more than those
predicted in Caenorhabditis elegans or Drosophila
melanogaster (Moon et al. 2004; Smalle and Vierstra
2004).
As in other eukaryotes, Arabidopsis E3s that con-
tain a RING-finger domain function as a single
subunit or as part of a multisubunit complex. As a
single subunit, the RING-finger domain binds to the
E2-conjugating enzyme and to the substrate; both the
RING-finger and the domain mediating the recog-
nition of the substrate are contained in the same
polypeptide.
We have previously shown that some RING zinc
finger genes are rapidly induced in response to elici-
tors. These RING fingers are members of a gene
family named ATL (Salinas-Mondragon et al. 1999).
The canonical RING motif comprises seven cysteine
residues and one histidine residue, which coordinate
the binding of two zinc ions. ATL genes contain the
variant RING-H2 in which there is a substitution of a
histidine residue for the fifth cysteine residue on the
zinc-binding residues (Salinas-Mondragon et al.
1999). Mutants that deregulate the expression of
various ATLs were isolated and their analysis re-
vealed a link between the induction of this putative
class of ubiquitin ligases and the plant defense sig-
naling pathways (Serrano and Guzman 2004). To
obtain a comprehensive guide for the ATL class of
plant RING-finger genes, we have classified the ATL
proteins from Arabidopsis and rice proteomes. The
comparative analysis revealed important information
about family organization and gene structure and
provided hints about gene function. We found that
the Arabidopsis family comprised 80 members and the
rice family 121. The 201 classified ATL proteins form
tentatively 14 groups. We analyzed potential pheno-
types associated with T-DNA insertion in 24 mem-
bers of the ATL family in Arabidopsis. The analysis
of these insertional mutants showed that four ATL
genes were essential while one more was affected in its
response to ABA/glucose.
Materials and Methods
Bioinformatics.
Arabidopsis sequences were retrieved from
MIPs (http://mips.gsf.de/proj/thal/db/search/search_frame.html)
and SIGnAL (http://signal.salk.edu/) databases. T-DNA insertion
lines were obtained from SIGnAL http://signal.salk.edu/cgi-bin/
tdnaexpress) (Alonso et al. 2003). Occurrence of cDNA clones and
expression of ATL genes were inspected using the Arabidopsis Tiling
Array Transcriptome Express Tool (http://signal.salk.edu/cgi-bin/
atta) (Yamada et al. 2003). The pattern matching tool at TAIR
(http://www.arabidopsis.org/cgi-bin/patmatch/nph-patmatch.pl)
was used to search for the signatures sequences PxCxHxxHxxC
and PxCxHxxHxxCxxxW in Arabidopsis. Rice sequences were ob-
tained from The Institute for Genomic Research database (http://
tigrblast.tigr.org/euk-blast/index.cgi?project=osa1) and from the
SIGnAL (http://signal.salk.edu/cgi-bin/RiceGE) databases; occur-
rence of cDNA clones in rice was assessed in SIGnAL. Phylogeny
reconstruction was generated using the neighbor-joining method
with a bootstrap of 1000 replicates (seed = 92,738), assuming a p-
distance model with homogeneous pattern of substitutions on all
tested genes (n = 201) of Arabidopsis and rice; analyses were con-
ducted using MEGA version 3.0 (Kumar et al. 2004). The multiple
sequence alignment of ATL proteins was performed using Clustal X
(v 1.81) (Thompson et al. 1997).
PCR Analysis of T-DNA Insertions in Arabidopsis
ATL Genes.
Genomic DNA was isolated from rosette leaves
of plants grown in soil as previously described (Murray and
Thompson 1980). PCRs were performed as follows: 35 cycles
(melting, 30 s at 94C; annealing, 1 min at 60C; extension, 5 min at
72C). We used a primer directed to the left border of the T-DNA:
LB, 5¢-GGCAATCAGCTGTTGCCCGTCTCACTGGTG-3¢.
Gene specific primers for Arabidopsis ATL genes are listed in
Supplementary Table 2. PCR amplification products were resolved
in 1.5% (w/v) agarose gels.
Expression Analysis by RT-PCR. RT-PCR screen
was used to determine tissue specific expression of ATL4, ATL6,
ATL8, and ATL10 genes. Samples of total RNA from 7-day-old
seedlings or selected tissues were isolated using Concert Plant RNA
Reagent (Invitrogene) treated with DNAse I. Reactions were per-
formed using Super Script One-Step RT-PCR with Platinum Taq
(Invitrogene) using 100 ng RNA (DNA-free) from each sample.
The thermal cycling conditions were 30 min at 50C, 2 min at 94C,
30–40 cycles of 30 s at 94C, 45 s at 54C, and 1 min at 72C, and a
435
final extension of 10 min at 72C. Amplification products were
fractionated into a 1.0% agarose gel. The gene specific primers used
are listed in Supplementary Table 3.
Seedling Treatments. Plants at the seedling stage
were obtained after germination of surface sterilized seeds in petri
dishes containing Murashige and Skoog (MS) medium (GIBCO
BRL, Gaithersburg, MD) supplemented with 0.6% agar and 2%
sucrose. Plates were incubated at 4C for 4 days and then at 24C
during 7 days in a growth chamber under continuous light.
Search for effects of various growth regulators was carried out at
the seedling stage at the following concentrations: 10 lM gib-
berellic acid, 0.5 mM abscisic acid, 0.5 mM salicylic acid, 50 lM
jasmonic acid, 100 lM kinetin, 50 lM 2,4-D, 0.1 and 10 lM
ACC (etiolated seedlings for 72 h), and 0.1 to 3.0 lM abscisic
acid; 1.0 to 7.0% glucose and mannitol were assessed in half-
strengh MS medium.
GUS Expression Assays of ATL6, ATL29, and
ATL36.
Promoter regions of ATL6, ATL29, and ATL36
were cloned into the pBI101.2 vector (Jefferson et al. 1987). Se-
quence of the primers used to amplify the promoter regions are
listed in Supplementary Table 4. Transformations were performed
by the dip floral method (Clough and Bent 1998). Five indepen-
dent homozygous T3 generation plants were propagated and the
pattern of GUS expression was analyzed; the pattern was very
similar in all five lines (data not shown). Histochemical and
fluorometric assays were performed as previously described (Jef-
ferson 1987).
Results
Genomewide Inspection for Arabidopsis and Rice ATL
Proteins
We initially based our survey of Arabidopsis ATL
proteins on a signature inferred for many ATL pro-
teins. This signature consisted of residues located at
the central region of the RING-H2 domain, two cy-
steines corresponding to the third and sixth zinc li-
gands, two histidines corresponding to the fourth and
fifth zinc ligands, a highly conserved proline spaced
out a residue upstream from the third zinc ligand, and
a highly conserved tryptophan spaced out three res-
idues downstream from the sixth zinc ligand (see
Fig. 1). The key residue for specifically detecting
ATL proteins is the proline residue. The tryptophan
residue within the domain is present in many RING-
finger domains as previously observed (Zheng et al.
2000). We found that almost the same number of
domains were predicted in the query whether or not
this tryptophan was included (see Fig. 1).
The sequences were then examined for the pres-
ence of the other modules predicted in ATL proteins
(see Fig. 1). One such module is a region rich in
hydrophobic amino acid residues that is located at
the amino-terminal end. This module is commonly a
single hydrophobic region that in most of the ATLs is
at least 18 residues. However, amino-terminal ends
containing two or three hydrophobic modules can
also be found. A conserved motif located between the
hydrophobic and the RING-H2 modules has been
also predicted in ATLs. This region, named GLD
(GLD denotes the first three conserved residues of the
sequence), comprises about 16 residues where a gly-
cine and a proline residues are highly conserved and
the distance between them is almost invariable. Al-
though a region rich in basic residues has also been
predicted in various ATLs, these basic modules were
not noticeable in most of them, and thus it was not
considered a requirement for classification in our
search for ATL members.
We confirmed and strengthened our results by
comparing our survey to a previous computational
analysis of RING fingers in Arabidopsis (Kosarev et
al. 2002). In that study, 387 RING domains were
predicted and grouped. Three main classes, corre-
Fig. 1. Features of Arabidopsis ATL proteins. Schematic repre-
sentation of the conserved domains in ATL proteins. The consen-
sus amino acid sequences are based on nine ATLs. The
hydrophobic region represents either single or multiple regions.
The basic region is present in several ATLs but not in most of them
and was not used to screen for ATLs. The pattern-matching tool at
TAIR was used to search for the signatures shown below the
RING-H2 sequence; the values indicate the resulting number of
domains and proteins. G, glycine; L, leucine; D aspartate; S, serine;
F, phenylalanine; C, cysteine; H, histidine; P, proline; W, trypto-
phan.
436
sponding to the canonical RING, to the RING-H2,
and to RING variants, were described. The RING-
H2 class was found to consist of 215 domains in 214
proteins. One of the clusters (cluster 2.1) groups 75
proteins that include most of the initially described
ATLs (Kosarev et al. 2002). The ATL proteins
identified in this work correspond to this cluster plus
two members from another cluster (Supplementary
Table 1). A few other differences were detected, such
as new genes or changes in gene names (see Supple-
mentary Table 1). At the end of our analysis, 80
Arabidopsis ATL proteins were predicted. This
number is also consistent with the results from an-
other analysis of Arabidopsis RING zinc fingers that
predicted 79 RING-H2 proteins containing a
hydrophobic domain (Greve et al. 2003).
A detailed draft of 95% of the rice genome is
currently available for analysis. To identify ATLs in
the rice proteome, reiterative searches for the RING-
H2 motif of ATL2 and ATL6 on the rice protein
sequences databases were performed using BLAST.
The sequences were then inspected for the presence of
both the hydrophobic and the GLD modules; 121 rice
proteins that contained these features were predicted.
Availability of cDNA clones and transcript verifica-
tion by whole-genome array showed that 70 of the
Arabidopsis ATL genes are expressed; likewise cDNA
or EST clones have been isolated from 62 of the rice
ATLs (see Supplementary Table 1).
Relationships Between ATL Proteins
Analysis of the phylogenetic tree containing 201 ATL
protein sequences revealed three distinct clades; these
clades were assembled in 14 groups based on a pro-
tein distance matrix (Fig. 2 and Supplementary Fig. 1
and Table 1). Clade 1 (Fig. 2; shadowed in yellow)
contained 75% of the protein sequences, of which a
great number belonged to rice. The other two clades
contained a similar number of Arabidopsis and rice
protein sequences (Fig. 2; shadowed in blue and red).
Defined groups of presumable paralogues are readily
identified, Rice paralogues were contained in groups
a, b, d, and f, and Arabidopsis paralogues in groups g,
k, l, and m. Likewise, several of the groups of the
Arabidopsis and rice predicted proteins had a wide
distribution across the dendrogram with comparable
numbers between clusters, suggesting that similar
selective pressure has driven the growth of the ATL
family by means of internal duplications. For in-
stance, clade 3 (Fig. 2; shadowed in red) included
mostly proteins encoded by genes containing introns,
a feature not shared by most ATLs (see below and
Fig. 5).
Fig. 2. Phylogeny of Arabidopsis
and rice ATL proteins. The
neighbor-joining method was used
to construct the tree (see Materials
and Methods and Supplementary
Information). Each one of the three
major clades is shown in a different
color. Green and red squares
highlight Arabidopsis and rice
ATLs, respectively; the
corresponding genes containing T-
DNA insertions analyzed are
highlighted in yellow. The branches
were assembled in 14 groups, a to
n, based on a protein distance
matrix. Pink squares point out
proteins with more than one exon;
blue squares, proteins with more
than one hydrophobic region
toward the amino end; gray
squares represent genes induced in
response to flagelin; black squares
indicate genes induced in eca
mutants; and light green shows
promoter fusions that are induced
by elicitor.
437
Features of ATL Genes
A distinct feature of the ATL family is the presence
of a hydrophobic domain located at the amino-ter-
minal end that typically spans over 15 amino acid
residues (Salinas-Mondragon et al. 1999). Most of
the ATLs include a single hydrophobic region
(Fig. 3; upper panel), but we found that two and up
to three hydrophobic regions can also be predicted
in some members (see Supplementary Table 1). Two
hydrophobic regions are common in proteins from
group g (Fig. 3; middle panel), while three hydro-
phobic regions are evident in group n (Fig. 3; lower
panel).
The position of the 80 Arabidopsis ATL genes on
the five Arabidopsis chromosomes is shown in Fig. 4.
Six tandem duplications of genes were detected; four
corresponded to sets of two genes, and two to sets of
four genes. Except for the ATL29/ATL53 set from
chromosome 4, proteins from each set were grouped
within the same cluster, indicating that they may have
arisen from recent gene duplication events. Similarly,
about seven tandem duplications of OsATLs were
also detecting (data not shown), supporting the fact
that various ATL genes are formed by means of lin-
ear-specific expansion.
Gene structure analysis showed that almost 90% of
the ATLs were intronless genes (70 of 80 Arabidopsis
ATLs lack introns and 102 of 121 rice ATLs lack
introns; see Supplementary Table 1). Eleven of the
intron-containing ATLs were clustered in a separate
clade (clade 3 or group n in Fig. 2). With the excep-
tion of OsATL98, the members of this group contain
four introns that separate distinct domains. The first
three exons encode regions rich in hydrophobic resi-
dues, the fourth exon includes the conserved region
and the first pair of cysteine residues from the RING-
H2 domain, and the fifth exon includes the rest of the
RING-H2 domain. OsATL98 lacks the second and
third introns and instead includes the GLD region
and the RING domain in a single exon (see Fig. 5;
group n).
Additionally, five sets of two to four genes con-
taining introns were randomly distributed among the
groups; three of them are depicted in Fig. 5. Ara-
bidopsis genes clustered within group a contained two
introns. In this case, each of the three modules, the
hydrophobic region, GLD region, and RING-H2
finger, is encoded in a different exon. Groups c and k
each comprise two rice genes in which two or three
modules are also separated by introns (Fig. 5).
Survey of T-DNA Insertions in ATL Genes
The function of ATL genes in plants has not yet been
established, although several of them catalyze poly-
ubiquitination in vitro (Takai et al. 2002; Stone et al.
2005). To gain insight into the role of members of this
Fig. 3. Prediction of the
hydrophobic domain in ATL
proteins. Diagrams were created by
the MacDNASIS Pro v3.5 program
using the method of Kyte and
Doolittle with a window size of 15.
In each diagram, the ordinate
displays hydrophobic and
hydrophilic values, plotted above
and below, respectively. The
abscissa shows the amino acid
number; / represents a
hydrophobic segment of the
protein.
438
family and to have an initial clue about the degree of
functional redundancy in this family, we looked for
phenotypes of Arabidopsis lines carrying mutations in
ATL genes. Since loss-of-function mutations in
RING-finger genes of the ATL family have not arisen
from genetic screens described in the literature, we
analyzed the phenotypic consequences of T-DNA
insertional mutations in ATL genes. T-DNA inser-
tion mutant lines for 24 ATL genes were obtained
from the SALK sequence-indexed insertion mutant
database (Alonso et al. 2003) (see Supplementary
Fig. 3); lines were genotyped to identify homozygous
lines for the corresponding T-DNA insertion.
Homozygous plants carrying T-DNA insertions
were obtained for 20 of the ATL genes, and in 4 cases
only hemizygous lines were recovered. The homozy-
gous lines were examined under standard growth
conditions; after visual inspection of the plants no
morphological, growth, or developmental alterations
were detected in any of these mutant lines (data not
shown). An assortment of conditions was then as-
sayed to try to find phenotypes in such mutant lines.
We tested for the effect of regulators of plant growth
and of various environmental conditions on the
growth of young seedlings (see Materials and Meth-
ods). In this survey, one mutant line exhibited an
altered response; a T-DNA insertion in ATL43 shows
insensitivity to abscisic acid (see below).
We reasoned that a cause for the lack of detect-
able phenotypes might be the occurrence of gene
redundancy. With the support of the phylogenetic
tree we investigated possible structural similarities
between ATLs. We mainly based our analysis on
comparing the protein sequences downstream from
the RING-H2 sequence. In most cases, one or a few
proteins similar in sequence were observed (see
alignments for ATL7, ATL3, ATL71, ATL16,
ATL12, ATL5, ATL13, ATL49, ATL67, ATL80,
ATL76, and ATL45 in Supplementary Fig. 2).
Likewise, from a branch of 13 Arabidopsis proteins,
4 mutant lines did not show a readily detectable
phenotype (ATL11, ATL9, ATL38, and ATL29,
from a branch in group g) (see Supplementary
Fig. 2e). These observations suggest that structurally
related genes exist for most ATLs. When similarities
were not detected toward the carboxy end, regions
flanking the hydrophobic domain were analyzed (see
ATL14 in Supplementary Fig. 2c). ATL43 got sep-
Fig. 4. Distribution of ATL genes on the five Arabidopsis chromosomes. The chromosomes of Arabidopsis are indicated and schematically
represented by lines. The relative position of the 80 Arabidopsis ATL genes is indicated. Regions containing possible gene duplications are
enclosed in dashed squares. Genes from clade 1 are indicated in black, those from clade 2 in gray, and genes from clade 3 in light-gray.
439
arated with a rice ATL in a unique branch. A T-
DNA insertion in this gene results in insensitivity to
abscisic acid; in this case functional redundancy may
be discarded.
Four Lines Carrying T-DNA Insertions in Essential
Genes
In four cases, only hemizygous lines were recovered,
suggesting that the homozygous lines for the T-DNA
insertion were not viable. This observation was fur-
ther confirmed by analyzing the segregating progeny
of a hemizygous line. T-DNA insertions analysis in
ATL4, ATL6, ATL8,andATL10 yielded lines lack-
ing the corresponding T-DNA and lines harboring
hemizygous T-DNA insertions but not lines homo-
zygous for the T-DNA insertions; the observed seg-
regation ratio is compatible with the expected 1:2
ratio between wild-type and hemizygous plants (see
Table 1). A seedling-lethal phenotype was not de-
tected in either of the mutant lines, suggesting that
the mutation may be affecting embryogenesis or
gamete formation (data not shown).
ATL4 is in the same branch with ATL12 and
ATL42, but it showed fewer similarities to these two
proteins, suggesting that there are no proteins clo-
sely related to ATL4 (see Fig. 2 and Supplementary
Fig. 2i). A structural difference was detected in
ATL10. The predicted sequence for ATL10 showed
three copies of a 13-amino acid residue sequence
that is present only once in the other three proteins
of the same branch (Supplementary Fig. 2n). The
other two ATLs had structurally related proteins.
ATL6 showed similarities to ATL31 which were
included in a branch on group g and ATL8 showed
similarities to ATL80 (Supplementary Figs. 2f and
m, respectively).
We evaluated the expression of these four ATL
genes in different tissues. RNA was extracted from
seedlings, roots, rosette leaves, stems, cauline leaves,
inflorescences, and immature siliques and expression
was assessed by RT-PCR (Fig. 6). Transcripts cor-
responding to ATL4 and ATL6 were detected in all
tissues tested, except for roots. It was shown previ-
ously that ATL6 is induced by elicitors, whereas
ATL4 is not (Salinas-Mondragon et al. 1999). For
ATL8, expression was mainly observed in inflores-
cences and immature siliques, suggesting that it may
be specific for reproductive tissue. Expression of
ATL10 was not detected in any tissue, indicating that
its expression may be restricted to certain conditions
not used by us, since there are cDNAs reported for
this gene (see Supplementary Table 1).
A Line Carrying a T-DNA Insertion in ATL43 Shows
Insensitivity to Abscisic Acid (ABA)
A line carrying a T-DNA insertion in ATL43 showed
a distinct phenotype: when germinated in ABA-con-
taining media, it displayed an ABA-insensitive phe-
notype. The progeny of 10 individual plants was
tested again for the phenotype and in all cases seed-
lings displayed the ABA-insensitive phenotype. A
further analysis of this phenotype indicated that it
was intermediate compared to the ABA-insensitive
mutant abi4 (Fig. 7a). The abi4 mutant germinates in
high levels of ABA as well as in high levels of glucose
(Leon and Sheen 2003). Since crosstalk between the
ABA and the glucose pathways occurs in plants, we
Fig. 5. Intron-exon organization in three groups of ATL genes.
Exons (boxes) and introns (dashed lines) are shown. The length of
introns in base pairs is indicated below the intron. Light-gray boxes
represent hydrophobic regions, dark-gray boxes the GLD region,
and black boxes the RING-H2 domain. The letter code to the left
indicates the group to which it belongs (see Fig. 2).
440
tested for the effect of glucose in the atl43 mutant
(Leon and Sheen 2003). atl43 showed an intermediate
glucose-insensitivity phenotype compared to abi4
(Fig. 7b); no effect was observed when mannose
medium was used as a control (Fig. 7c).
ATL Genes Participating in the Elicitor Response
We previously showed that ATL2 is induced by chitin
or cellulysin and that it exhibits a distinct expression
pattern after incubation with these elicitors of the
defense response. Likewise, by Northern blot analysis
we detected induction of ATL6 in response to the
same stimuli. ATL6 belongs to group g, most of
whose members encode two hydrophobic regions at
the amino terminus instead of one and a distinct
carboxy-terminal region. We therefore tested ATL6
and two other members of this group for chitin or
cellulysin iduction. Expression of GUS fusions to
promoters of ATL6, ATL29, and ATL36 was exam-
ined in light-grown young seedlings (Fig. 8). ATL29
and ATL36 displayed localized GUS staining to the
shoot apical meristem region, a pattern similar to that
previously reported for ATL2. ATL6 showed a higher
basal expression throughout the cotyledonary leaves.
An increase in GUS activity was detected in the three
plants carrying the GUS constructs after incubation
in a solution containing elicitor. Histochemical
Fig. 7. Inhibition of seed germination in Arabidopsis atl43 by
ABA and glucose. Vernalized seeds from wt, atl43, and abi4 were
germinated for 7 days in MS media containing increasing concen-
trations of ABA (A), glucose (B), and mannitol (C). Germination
percentage is indicated as the number of seeds showing cotyledon
emergence. Data are means of a single duplicated experiment.
Fig. 6. Expression pattern of essential ATL genes in Arabidopsis.
RT-PCR (reverse transcription with PCR) assays were performed
from total RNA isolated from seedlings (S), root (R), rosette leaves
(RL), stems (S), cauline leaves (CL), primary inflorescences (I), and
immature siliques (IS). One hundred nanograms of DNAse I-
treated RNA was used for each reaction and the resulting products
were fractionated in a 1.0% agarose gel. Sizes of the RT-PCR
products are as follows: ATL4, 1005 bp; ATL6, 1197 bp; ATL8,
398 bp; and ATL10, 310 bp. CF150 (At1g72150) was used as a
constitutive expression control. Under the conditions used no
amplification was detected for ATL10 (data not shown).
Table 1. Segregation analysis of insertion lines in essential ATL genes
Wild type (no T-DNA) Hemizygous (one copy) Homozygous (two copies) v
2
ATL4 12 34 None 1.0894; p < 0.05
ATL6 15 33 None 0.0937; p < 0.05
ATL8 12 35 None 1.2812; p < 0.05
ATL10 13 36 None 1.0226; p < 0.05
441
analysis showed that GUS activity after elicitor
treatment disseminates throughout the seedling as
previously seen for ATL2. These observations indi-
cate that structurally related ATL genes may be in-
volved in the defense response, as previously inferred
(Salinas-Mondragon et al. 1999; Serrano and
Guzman 2004).
Discussion
To develop a comprehensive guide for the ATL
family, a multigenic family of putative ubiquitin lig-
ases, we identified the predicted ATL genes from
Arabidopsis and rice. The ATL family is an important
class of RING-H2-finger genes since it represents
about 40% of the RING-H2-finger genes in Arabid-
opsis. Our study detected 201 ATL proteins, 80 from
Arabidopsis and 121 from rice. Inspection of data-
bases showed evidence of expression for about 90%
of Arabidopsis ATLs and 50% of rice ATLs (the re-
duced number for rice may only be a sign of the less
extensive analysis of the rice transcriptome). This
observation suggests that most ATLs are active gene
forms rather than pseudogenes. Although the func-
tion of this family of ubiquitin ligases is virtually
unknown, we show initial evidence of the involve-
ment of some of its members in the growth-regulator
response, response to biotic stress, and plant devel-
opment. These results support the suggestion that a
large number of members of this multigene family
participate in the regulation of plant-specific pro-
cesses. Selective constraints and the need for diver-
gence within this gene family to respond to a wider
number and variable stimuli may have driven its
expansion.
About 60% of the rice ATLs are clustered with
Arabidopsis ATLs, and for many of them their pre-
dicted gene products showed sequence similarities,
indicating that they may be orthologous (Supple-
mentary Figs. 2d, f–h, and k–m). Likewise, adapta-
tive radiation of ATLs within rice may be observed:
groups a, d, and f harbor large clades with only rice
proteins (see Fig. 2). We have also found tandem
duplications in ATL genes in Arabidopsis and rice,
suggesting that lineage-specific expansion may have
led to the generation of some members of this family
(Fig. 4 and data not shown). Two tandem genes that
may have arisen from duplication events are ATL10
and ATL76. For these two genes specialization seems
to have occurred since a T-DNA insertion in ATL10
renders a lethal phenotype (Table 1), while viable
plants are obtained from a T-DNA line in ATL76.
This suggests that there are functional differences
between the two genes and supports the fact that new
gene functions often evolve from gene duplication
(Copley et al. 2003).
Another feature of the gene structure analysis is
that about 90% of the ATL genes lack introns. This is
confirmed by the analysis of cDNA clones for almost
all of the Arabidopsis ATLs that are publicly available
(see Supplementary Table 1). Noteworthily, most of
the ATLs containing introns are clustered within
group n, cDNA clones are also available for all of
them, confirming the predicted gene structure (see
Fig. 2 and Supplementary Table 1). Group n, which
corresponds to a separate clade, is the only group
that includes members from both Arabidopsis and
rice. This fact suggests that these are genes of an
ancient origin, which probably arose before the sep-
aration of mono- and dicotyledonous plants. A re-
verse-transcribed cDNA could be the origin of
intronless genes (Maestre et al. 1995). If this is the
case, the origin of intronless ATL genes may also
have occurred before the separation between mono-
cot and dicot plants since both species contain a high
Fig. 8. Induction of ATL2, ATL6, ATL29, and ATL36 promoter
activity by incubation with cellulysin in young Arabidopsis seed-
lings carrying promoter fusions to GUS. A Histochemical locali-
zation of GUS activity in 6-day-old light-grown Arabidopsis
seedlings incubated in MS medium containing 100 mg/ml cellulysin
for 120 min; representative seedlings are shown. B Fluorometric
analysis of GUS activity was performed as described under
Materials and Methods; the result is the mean of the measurements
of two different samples.
442
number of putative orthologous intronless ATL
genes. It is tempting to speculate that the general
structure of the ATLs has then evolved as a basic
functional module.
We have also carried out a survey of T-DNA
insertions in 30% of the Arabidopsis ATL genes.
Analysis of loss-of-function mutations is an impor-
tant tool in the search for gene function. Loss-of-
function mutations in ATL genes have not been
previously described. Indeed, in comparisons of the
wild-type and the different plants carrying an inser-
tion in ATL genes, no obvious morphological varia-
tions were detected in any of the mutant lines
growing under standard conditions. One of the rea-
sons for the lack of detectable phenotypes is gene
redundancy since structurally related proteins that
clustered together were detected for almost every one
of the corresponding mutated genes. Supporting this
fact are the results for ATL43 and ATL4 insertional
mutants. Neither of these genes seems to have a
structurally related protein and their insertional
mutations displayed a phenotype. The absence of
evident phenotypes also suggests that atl mutants
may exhibit subtle alterations that are traceable only
under very specific conditions.
The ATL proteins are likely to be single-subunit
E3 ubiquitin ligases since EL5, a member of the
ATL family from rice, mediates in vitro autoubiq-
uitination in a reaction depending on E1, E2, and
ubiquitin. Additionally, nine of the RING-H2 pro-
teins inferred in our survey to be ATLs possess in
vitro ubiquitin ligase activity (Takai et al. 2002;
Stone et al. 2005). Other components of ubiquitin
ligases in Arabidopsis such as F-box, U-box, and
RING proteins encode additional distinct domains;
for instance, WD-40, LRR, or ARM domains are
commonly encoded in F-box, U-box, and RING-
finger proteins (Azevedo et al. 2001; Gagne et al.
2002; Mudgil et al. 2004; Stone et al. 2005). Apart
from the distinct modules described for ATLs, no
other well-conserved recognition or protein interac-
tion domains were detected, suggesting that the
structure of the ATL protein is sufficient to operate
as a functional module and that specific function
may arise from the regions of similarity that occur
within putative carboxy-terminal domains of un-
known function found in some of the ATLs (see
Supplementary Fig. 2).
Four members of the family, ATL4, ATL6, ATL8,
and ATL10, are likely to be essential for viability
since homozygous plants for T-DNA insertion were
not recovered. Analysis of expression revealed that
ATL8 is mainly expressed in young siliques, sug-
gesting a role during embryogenesis. Mutant analysis
on RING-H2 genes has been scarce. MsRH2-1, an
alfalfa gene highly related to ATL4, when expressed
ectopically in alfalfa or in Arabidopsis, renders dra-
matic and pleiotropic alterations in plant morphol-
ogy and development; alterations in hormone
response have been inferred for such a response
(Karlowski and Hirsch 2003). RIE1, a non-ATL
RING-H2-finger protein, shows an embryo-lethal
phenotype. It encodes the RING-H2-finger domain
at the carboxyl end, RIE1 is expressed in all tissues
tested, and embryo development is arrested from the
globular to the torpedo stages (Xu and Li 2003).
These observations indicate that ATL genes as well as
other RING-H2 zinc-finger genes may play pivotal
roles during early stages of plant development, link-
ing these putative ubiquitin ligases to such processes.
Compelling evidence indicates that several ATLs
may participate in defense response in plants. ATL2
mRNA is rapidly and transiently induced after
incubation with elicitors of pathogen response such
as chitin and cellulases (Salinas-Mondragon et al.
1999). In eca mutants, which show constitutive
expression of ATL2 and other ATL genes, expression
of defense-related genes is also impaired (see black
squares in Fig. 2) (Serrano and Guzman 2004). EL5,
a rice ATL (OsATL24), is also an elicitor-induced
gene (Takai et al. 2001). Recently, it has been shown
that induction of ATLs is not restricted to fungal-
associated elicitors; significant induction of seven
ATLs by bacterial flagellin, ranging between 1.9- and
30.2-fold, was reported (see gray squares in Fig. 2)
(Navarro et al. 2004; Zipfel et al. 2004). Together
these observations indicate that several ATLs may be
part of the innate immune response mediated by
pathogen molecules. This response, also known as
PAMPs (pathogen-associated molecular patterns), is
conserved among plants and animals (Nurnberger
et al. 2004). A previously unidentified role for ATLs
is also revealed in this work. A member of the ATL
family may be in involved in the ABA response since
the insertion line in ATL43 shows an ABA-insensitive
phenotype. This observation is supported by the fact
that the expression of ATL43 as well as other ATLs
have been reported to be induced after ABA treat-
ment in genomewide expression analysis (Hoth et al.
2002; Sanchez et al. 2004).
The function in plants for the enormous number of
ubiquitin ligases within the RING-H2 class awaits
unraveling. A two-hybrid screening detected interac-
tion between a NAC (NAM/ATAF1/2/CUC2) tran-
scription factor and the RING-H2 protein RHA2a
(Greve et al. 2003). It was inferred that RHA2a reg-
ulates the activity of the NAC transcription factor
since expression of b-glucuronidase fusions of both
proteins colocalized to the nucleus. RHA2a is highly
related to the ATL family but it lacks the highly
conserved proline residue located at the central re-
gion of the RING-H2 domain (see P residue in
Fig. 1). Indeed, RHA2a containing a mutation that
adds the proline residue partially abolishes the
443
interaction with the NAC transcription factor, indi-
cating that this residue is important for recognition
(Greve et al. 2003). Further analysis of plant lines
carrying a mutation in more than one ATL, analysis
of the ectopic expression of ATL genes, and the
search for possible targets of the putative ubiquitin
ligases will help to unravel the role of this multigene
family during the plant life cycle.
Acknowledgments. We thank Gabriela Olmedo for critical
comments on the manuscript and Laura Aguilar for technical
support. We thank the Salk Institute Genomic Analysis Labora-
tory for providing the sequence-indexed Arabidopsis T-DNA
insertion mutants and the Arabidopsis Biological Resource Center
Stock Center (Ohio State University, Columbus) for distributing
seeds. This work was supported by a grant from CONACYT,
Me
´
xico (to P.G.), and by fellowships from CONACYT to M.S.,
S.P., and L.D.A.
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