Expression profiling of the Arabidopsis annexin gene family during germination, de-etiolation and abiotic stress

Abstract

Annexins are a multigene family in most plant species and are suggested to play a role in a wide variety of essential cellular processes. In Arabidopsis thaliana there are eight different annexins (AnnAt1-8), which range from 29% to 83% in deduced amino acid sequence identity. As a first step toward clarifying the individual functions of these annexins, in this study we have used quantitative real time reverse transcription PCR to assess their differential expression in different tissues or after different stimuli. We determined which annexins are expressed during germination and early seedling growth by assaying annexin expression levels in dry and germinating seeds and in 7-day-old light-grown seedlings. Our results indicate that transcripts for all eight annexins are present in germinating seeds and that transcript levels for all the annexins increase by 7 days of normal growth. We assayed transcript levels in dark grown roots, cotyledons, and hypocotyls and found that the relative abundance of each annexin varied in these dark-grown tissues. We also examined the effects of red and far red light treatments on annexin expression in 5.5-day-old etiolated seedlings. Light treatments significantly altered transcript levels in hypocotyls and cotyledons for only two members of the gene family. Finally, we monitored annexin expression changes in response to a variety of abiotic stresses. We found that the expression of most of the Arabidopsis annexin genes is differentially regulated by exposure to salt, drought, and high- and low-temperature conditions, indicating a likely role for members of this gene family in stress responses.

Full text

Research paper
Expression profiling of the Arabidopsis annexin gene family
during germination, de-etiolation and abiotic stress
A. Cantero
a,1
, S. Barthakur
b,1
, T.J. Bushart
a
, S. Chou
c
, R.O. Morgan
d
, M.P. Fernandez
d
,
G.B. Clark
a
, S.J. Roux
a,*
a
Department of Molecular Cell and Developmental Biology, University of Texas, Austin, Texas 78713 USA
b
National Research Center on Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 110 012 India
c
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720-3200 USA
d
Department of Biochemistry and Molecular Biology, Edificio Santiago Gascon, Faculty of Medicine, University of Oviedo, 33006 Oviedo, Spain
Received 25 August 2005
Available online 28 February 2006
Abstract
Annexins are a multigene family in most plant species and are suggested to play a role in a wide variety of essential cellular processes. In
Arabidopsis thaliana there are eight different annexins (AnnAt1-8), which range from 29% to 83% in deduced amino acid sequence identity. As a
first step toward clarifying the individual functions of these annexins, in this study we have used quantitative real time reverse transcription PCR
to assess their differential expression in different tissues or after different stimuli. We determined which annexins are expressed during germina-
tion and early seedling growth by assaying annexin expression levels in dry and germinating seeds and in 7-day-old light-grown seedlings. Our
results indicate that transcripts for all eight annexins are present in germinating seeds and that transcript levels for all the annexins increase by
7 days of normal growth. We assayed transcript levels in dark grown roots, cotyledons, and hypocotyls and found that the relative abundance of
each annexin varied in these dark-grown tissues. We also examined the effects of red and far red light treatments on annexin expression in 5.5-
day-old etiolated seedlings. Light treatments significantly altered transcript levels in hypocotyls and cotyledons for only two members of the gene
family. Finally, we monitored annexin expression changes in response to a variety of abiotic stresses. We found that the expression of most of the
Arabidopsis annexin genes is differentially regulated by exposure to salt, drought, and high- and low-temperature conditions, indicating a likely
role for members of this gene family in stress responses.
© 2006 Elsevier SAS. All rights reserved.
Keywords: Annexin; Arabidopsis thaliana; Real time PCR; Expression; Abiotic stress
1. Introduction
Calcium is a universal signal in eukaryotic cells, and diverse
signaling pathways in plants include an increase in the concen-
tration of cytosolic Ca
2+
([Ca
2+
]
cyt
) in at least one of the steps.
Included among the calcium-binding proteins that transduce
these calcium signals into adaptive responses is the annexin
gene family [8,11]. Plant annexins have been localized at the
cell periphery of highly secretory cell types where they are
hypothesized to play a role in the Golgi-mediated secretion of
new wall materials and plasma membrane that occurs during
growth and development. Additionally, plant annexins have
been found in many other cellular locales including the cyto-
plasm, the vacuole and nucleus. They appear to be a multifunc-
tional gene family whose members play roles in a number of
diverse physiological processes. For example, the cellular dis-
tribution of certain annexins is changed in response to touch
[33], gravity [7] and diurnal cycles [19], and in Medicago an
annexin is involved in Nod signaling [4]. Plant annexins have
also been implicated in imparting tolerance to various abiotic
stresses [15,22]. Recent analyses of two Arabidopsis annexin
T-DNA insertional mutants provided evidence that these an-
nexins play an important role in germination during salt and
osmotic stress [26].
www.elsevier.com/locate/plaphy
Plant Physiology and Biochemistry 44 (2006) 13–24
Abbreviations: RT, reverse transcription; MD, monocot–dicot; APT1,
adenosine phosphoribosyl transferase.
*
Corresponding author. Tel.: +1 512 471 4238; fax: +1 512 232 3402.
E-mail address: sroux@uts.cc.utexas.edu (S.J. Roux).
1
These authors contributed equally to this work.
0981-9428/$ - see front matter © 2006 Elsevier SAS. All rights reserved.
doi:10.1016/j.plaphy.2006.02.002

There are eight different annexins in Arabidopsis, some of
which are likely to have unique individual functions [9]. Data
from individual annexin studies as well as transcriptome stu-
dies have established that plant annexin expression is con-
trolled by a number of environmental and developmental sig-
nals. Previously, we have shown that two of these annexins,
AnnAt1 and AnnAt2, have differential in situ RNA localization
patterns that are temporally and developmentally regulated [9].
We also noted differences in predicted functional motifs be-
tween the different Arabidopsis annexins.
Expression profiling has become an important tool for gain-
ing insights into the partitioning of functions and redundancy
within a multigene family [30]. Global surveys by microarray
analyses have provided a rough glimpse of how transcript
abundance of most Arabidopsis genes, including members of
the annexin gene family, changes under different circumstances
of development or environmental stimuli. However, recent re-
sults point to an especially important role for annexins in
growth control and in certain stress responses. Since none of
the global surveys carried out thus far were specifically fo-
cused on the role of annexins in these processes, they do not
provide adequate detail to resolve whether specific members of
the annexin family are differentially regulated in rapidly grow-
ing tissues or in tissues under abiotic stress. To address this
question we decided to use the quantitative real time RT-PCR
technique.
Quantitative real time RT-PCR allows quantification of the
transcript levels of different members of a conserved gene fa-
mily, all sharing a high percentage of identity, with both high
sensitivity and specificity [14]. Use of this technique facilitates
a determination of the absolute and relative level of any tran-
script of any type. Even transcripts with very high or extremely
low levels of expression can be quantified accurately. The ac-
curacy and the reliability of the real time PCR have previously
been demonstrated in studies on human genotyping and patho-
gen detection [17,12].
In plants the quantitative real time PCR technique has been
used to determine the number of T-DNA insertions in trans-
genic plants [20,37], to detect the presence of genetically mod-
ified organisms in food [18], and to quantify the transcript le-
vels in plant organs [25,28]. Particularly relevant to the present
studies, this technique has been successfully used to analyze
closely related genes in many different families of plant en-
zymes [31,38,39] and for expression profiling of signal trans-
duction protein families such as the entire Arabidopsis Shaggy-
like kinase multigene family [5], the CTR1 gene family in-
volved in ethylene signaling [1], and over 1400 Arabidopsis
transcription factor transcripts [10]. Quantitative real time RT-
PCR is also well suited to performing expression profiling in
response to environmental stimuli. For example, it was used to
analyze the expression of an Arabidopsis gene family encoding
plasma membrane aquaporins in response to abiotic stresses,
which allowed the authors to determine the stress-related bio-
logical function of certain gene members of this family [21].
In the experiments reported here we use quantitative real
time RT-PCR to profile how the transcript abundance of all
eight annexin genes change during seed germination, seedling
growth and development, and stress responses. Our results pro-
vide a clearer picture of which annexin genes respond most
strongly in each of these scenarios, and provide an important
database for directing future mutant and localization studies.
2. Results
2.1. Genomic, structural and phylogenetic analysis
of annexins in Arabidopsis
The schematic in Fig. 1 depicts the gene structures for all
eight annexins, and highlights functionally important features.
Notably, AnnAt3 and AnnAt4 are located on chromosome 2 in
tandem and AnnAt6 and AnnAt7 are located on chromosome 5
in tandem. AnnAt3 and AnnAt4 may share a 5′promoter region
and this could result in dual regulation of their transcripts. An-
other interesting observation arising from the gene structure
analyses is that four of the Arabidopsis annexins, AnnAt3,An-
nAt4,AnnAt5, and AnnAt8, have congruent gene structures,
each having six exons. In contrast, analysis of the gene struc-
ture for the other four annexins shows the common feature of
intron losses. AnnAt1 has lost the final three introns, AnnAt2
lacks a single final intron, and both the members of the tandem
pair, AnnAt6 and AnnAt7, have lost their final two introns.
Phylogenetic analysis of the eight Arabidopsis (dicot) an-
nexins (identical to diploid cotton, not shown), 10 Oryza
(monocot) annexins, and two Ceratopteris annexins as the out-
group resulted in a fully resolved tree (Fig. 2). All bifurcations
were supported with at least 70% maximum likelihood or boot-
strap values. Branch lengths are proportional to the amount of
evolution (i.e. the number of amino acid replacements per site).
The presence of direct orthologs in Arabidopsis and rice sug-
gests that several annexins are ancient, predating the monocot-
dicot (MD) separation 200 million years ago (Fig. 2). For ex-
ample, Arabidopsis and rice share an orthologous tandem pair,
with AnnAt3 and AnnAt4 being direct orthologs of ANXD14
and ANXD16 on chromosomes 2 and 5 in the respective spe-
cies. Several more recent lineage-specific gene duplications
have occurred in Arabidopsis and rice following the MD se-
paration. One such lineage-specific amplification in Arabidop-
sis resulted in paralogs AnnAt1,AnnAt2,AnnAt6, and AnnAt7
and at least four recent gene duplications within the rice line-
age created additional paralogs with distinct subfamily num-
bering.
In terms of protein evolution within the Arabidopsis annex-
in clade, AnnAt1,AnnAt2,AnnAt6, and AnnAt7 represent rela-
tively recent lineage-specific duplications and as such are ex-
pected to show the least divergence in protein structure and
function. The other tandem pair of AnnAt3 and AnnAt4 has a
longer history of divergence occurring before MD separation,
but possible concerted evolution due to their physical proxi-
mity may have limited the extent of their divergence.
In general, the primary sequences for plant annexin genes
are fairly divergent from each other. AnnAt2,AnnAt6, and An-
nAt7 are the most similar to each other, showing 80–86% iden-
A. Cantero et al. / Plant Physiology and Biochemistry 44 (2006) 13–2414

tity at the nucleotide level and 76–83% identity at the deduced
amino acid level. AnnAt3 and AnnAt4 appear to be the most
divergent at the amino acid level. The predicted protein se-
quences for the gene family range from 316–322 amino acids
with molecular masses between 34 and 39 kDa. When the core
repeat regions for all eight of the Arabidopsis annexins are
compared to the annexin consensus sequences taken from
many annexin core repeat regions as analyzed by Barton et
al. [2], it is evident that the structural repeats that help define
this gene family are present in Arabidopsis annexins. Overall it
is clear that the first and fourth repeats show the highest level
of conservation while the second and third repeats are the most
divergent.
In annexins the type II calcium-binding site is determined
by the conserved glycine-X-glycine–threonine loop followed
42 amino acids downstream of the first glycine residue by a
glutamic acid or an aspartic acid residue. Regarding predicted
type II calcium-binding sites, many plant annexins have a
Fig. 1. Schematic diagram of the genomic structures of the annexin gene family in Arabidopsis. Exons are depicted as white boxes and introns as lines. Untranslated
regions (UTRs) are represented as black boxes and non-coding regions between tandem genes as gray boxes.
Fig. 2. Molecular evolution of annexins in the completed genomes of Arabidopsis thaliana (thale cress) and Oryza sativa japonica (rice). The phylogenetic tree was
determined by maximum likelihood analysis with release 3.0 of the IQPNNI program [34]. Known fern annexins were used as outgroup for the comparison of
aligned, full-length protein sequences using eight gamma rates to account for site-specific variation. The tree was fully resolved, with confidence values for the
branching topology all above 90 and congruency with the neighbor-joining, bootstrap tree analysis by MEGA 3.1 [23] (not shown). Bifurcations containing solid
circles represent common gene duplications, while open circles signify intra-lineage gene duplications. Nomenclature for members of the plant annexin family
(ANXD) includes taxon names based on phylogenetic analysis of all known plant annexins (unpublished) together with the systematic names given by the
corresponding genome sequencing projects. Finished sequence data are available from The Arabidopsis Information Resource (http://www.arabidopsis.org/), the
International Rice Genome Sequencing Project (http://rgp.dna.affrc.go.jp/IRGSP/) and NCBI-GenBank for the fern sequences (AF308588, AF308589).
A. Cantero et al. / Plant Physiology and Biochemistry 44 (2006) 13–24 15

fairly well conserved site in either the first or fourth repeat.
AnnAt3 also appears to have a binding loop in the first and
fourth repeats, but it’s more divergent, tandem duplicate An-
nAt4 is essentially devoid of type II calcium binding sites
(Fig. 3).
2.2. Relative quantitation of annexin transcripts under normal
growth conditions
Raw C
T
values for the constitutively expressed housekeep-
ing gene, adenosine phosphoribosyl transferase (APT1), were
compared to each of the eight annexins in 7-day-old seedlings
(data not shown). Each of the eight genes shows significant
expression, indicating that all members of the family are tran-
scriptionally active under normal growth conditions.
In Fig. 4 we determined the relative quantitation of annexin
transcripts under normal growth conditions in dry seeds, ger-
minating seeds, and 7-day-old seedlings by using quantitative
real time reverse transcription polymerase chain reaction. For
this comparison, annexin levels were normalized to a grand
calibrator of AnnAt1 in dry seeds in order to allow for propor-
tional comparisons of annexins within a single time point or of
an annexin across time points. We were able to clearly distin-
guish quantitative differences in the relative transcript level of
the eight annexin family members. Every annexin was detect-
able in whole seeds and seedlings regardless of the time point.
With the exception of AnnAt4 transcript, all annexin transcript
levels decrease 26 hours after sowing, and this is followed by
an increase in transcript levels for all eight annexins after 7 days
of growth.
2.3. Relative quantitation of annexin transcripts in dark grown
tissues
Fig. 5 shows the RNA levels of all eight annexins as ana-
lyzed in the steady-state condition of 5.5-day-old dark-grown
seedlings. Generally, highest expression levels were seen in
roots and hypocotyls, while cotyledons had lower expression
levels. RNA levels for AnnAt4 are fairly even throughout the
plant while AnnAt5, 6, and 7 show the largest disparities be-
tween root tissue as compared to cotyledon (27.0-, 45.7-, 11.2-
fold decreases, respectively).
2.4. Red and Far-red light effects on annexin expression
Most of the light effects on annexin expression in germinat-
ing seedlings are not significant. However, the general trends
of expression changes are consistent, showing an increase in
Fig. 3. Sequence alignment of Arabidopsis annexin repeats. The amino acids highlighted at the 21 numbered positions are in agreement with the residues that have
conserved amino acid properties in each of the four annexin repeats of 22 annexin sequences as analyzed by Barton et al. [2]. The two asterisks (in repeat 1 of
AnnAt4 and in repeat 2 of AnnAt3) indicate locations of short amino acid insertions in these two annexin repeats that are not shown in this alignment. The amino
acids in the insertion for repeat 1 of AnnAt4 are EERAFEKCHDH and for repeat 2 of AnnAt3 are KKSLE. According to Barton et al. [2] in the 88 animal repeats
analyzed, the following amino acids occurred in the following sequence positions (P#) of repeats: P#11: A V; P#14: I L; P#17: A S; P#23: G; P#24: T V S; P#29:L I
W F; P#30: V I L T N; P#32: I L V N; P#33: M L I V F; P#34: T A C G V; P#36:R; P#37: S A T N; P#44: I V L T; P#48: Y F; P#56: M I L; P#60: I L V M; P#65: V
L I F T; P#67: G F; P#69: L F M Y I; P#72: V L A I M C G T; P#73: F L M I V.
A. Cantero et al. / Plant Physiology and Biochemistry 44 (2006) 13–2416

annexin expression after light exposure in cotyledons, and a
decrease in annexin levels in hypocotyls (Fig. 6). Within hypo-
cotyls, AnnAt5 is the only annexin that shows a change in RNA
levels significant to the 95% confidence level (Fig. 6A). An-
nAt5 shows a significant up-regulation in hypocotyls (sixfold)
that is reversed by far-red light treatment (sevenfold) (Fig. 6A).
AnnAt2 and AnnAt6 show a trend of decreased RNA levels
under red light with possible photoreversibility by far-red light
treatment (Fig. 6A). Within cotyledon tissues, AnnAt6 is the
only annexin that has statistically significant light-induced
changes (P< 0.05) (Fig. 6B). It shows significant up-regulation
after red light exposure as compared to dark grown seedlings,
and this up-regulation is reversed by far-red light treatment. In
contrast, AnnAt7 is up-regulated (10-fold) but this change is
not statistically significant and is not reversed by far-red light
treatment. Although it is true that physiological significance is
not based on magnitude of expression, AnnAt6 does show the
largest up-regulation (35-fold) compared to all other annexins,
which tend towards smaller (< 10-fold) expression changes.
2.5. Differential expression of annexin genes in whole
seedlings under various abiotic stresses
Comprehensive expression profiling of the eight members
of the annexin family in 7-day-old seedlings was carried out
under a series of abiotic treatments. Quantitative measurements
were done comparing expression of each gene under normal
growth conditions. The series of stresses included modifica-
tions of temperature down to 4 °C and up to 37 °C, dehydrat-
ing the plants for 2 hours, and modifying the salt concentration
Fig. 4. Changes in annexin RNA levels during germination. Real time RT-PCR
results are shown comparing RNA levels for all eight annexins within and
across three time points. Seeds were treated with cold stratification at 4 °C for
3 days before sowing on plates. Dry seed corresponds to unimbibed seeds,
germinated to whole seedlings collected at 26 hours post-sowing on nutrient
media plates, and 7-day-old seedlings to whole seedlings collected at 7 days
post-sowing on nutrient media plates. Total RNA was isolated from whole
seedlings collected at 26 hours post-sowing or after 7 days of growth. Results
are normalized in two directions, within treatment condition to AnnAt1 and
across time points to dry seed, giving a grand calibrator of AnnAt1 in dry seeds
(value of 1). With AnnAt1-dry seed as the grand calibrator, comparisons are
proportional both within any single time point, as well as with a single annexin
across time points. In order to keep both increases and decreases in relative fold
differences in RNA levels proportional the Y axis is presented in log scale.
Error bars represent standard error of the mean.
Fig. 5. Relative levels of annexins in dark grown tissues. Real time RT-PCR
results are shown comparing RNA levels for each annexin across three tissue
types. Roots, hypocotyls, and cotyledons were harvested for RNA isolation
from 5.5-day-old etiolated seedlings. Results are normalized to annexin levels
in roots (value of 1). In order to keep both increases and decreases in relative
fold differences in RNA levels proportional the Y axis is presented in log scale.
Error bars represent standard error of the mean.
Fig. 6. Effects of Red and far-red light on annexin RNA levels in cotyledons
and hypocotyls. A. Real time RT-PCR results are shown comparing RNA
levels of each annexin in hypocotyls 30 min after three different light
treatments, which were: Dark, plants grown for 5.5 days in complete darkness;
Red, dark grown plants exposed to red light for 2 min; Red + far-red, dark
grown plants exposed to red light for 2 min followed by far-red light for 3 min.
Results are normalized within each annexin to levels in dark treated plants
(value of 1). In order to keep both increases and decreases in relative fold
differences in RNA levels proportional the Y axis is presented in log scale.
Error bars represent standard error of the mean. B. Real time RT-PCR results as
in panel A, but showing changes in annexin RNA levels from cotyledon tissue.
A. Cantero et al. / Plant Physiology and Biochemistry 44 (2006) 13–24 17

by adding 250 mM NaCl for 2 hours. Fig. 7 shows the relative
expression changes for each annexin gene family member and
the range of the transcript levels observed in the conditions
described above. The error bars represent the 95% confidence
intervals for each gene under different treatments. All the
changes noted below are statistically significant. Table 1 shows
a summary of the relative fold differences in the transcript le-
vels for the eight annexin genes after various stress treatments.
AnnAt1 and AnnAt2 responded quite differently to these
four abiotic stresses. AnnAt1 message levels were increased
by all four treatments with the highest induction observed in
response to NaCl treatment (42-fold) and dehydration treat-
ment (39-fold). In contrast, AnnAt2 message levels were
down-regulated in response to salt and drought treatments as
well as cold treatment. Under high temperature for 2 h at
37 °C, AnnAt2 message level was significantly up-regulated
(43-fold) while AnnAt1 message level increased only margin-
ally (less than twofold).
AnnAt3 was up-regulated by cold temperature (sevenfold)
and dehydration (13-fold) while remaining almost unchanged
in its transcript level under NaCl stress. In contrast it was down
regulated 19-fold by heat exposure. AnnAt4 responded strongly
at the transcriptional level to NaCl treatment showing a very
high rate (383-fold) compared to control expression under dis-
tilled water treatment controls, while changing little in response
to cold treatment and decreasing in response to both heat and
dehydration treatments (eightfold and fivefold, respectively).
AnnAt5 showed higher transcript levels after all four abiotic
stress treatments with highest transcript abundance in response
to saline treatment (64-fold).
Qualitatively, AnnAt6 and AnnAt8 showed similar response
patterns, being positively affected by high temperature, dehy-
dration and NaCl, and relatively unaffected by low tempera-
ture. Quantitatively, AnnAt6 showed a moderate increase in ex-
pression, and AnnAt8 showed high increases in transcript
abundance: 434 times more under NaCl stress and 175 times
under dehydration.
There was not a significant change in expression of AnnAt7
under dehydration stress, but 42 times more expression under
NaCl stress. Like all the other annexins except AnnAt2 and
AnnAt3,AnnAt7 was positively modulated under NaCl stress
when exposed to 250 mM NaCl solution for 2 hours. AnnAt7
and AnnAt5 were the only two genes that were strongly in-
duced by both high temperature and low temperature.
In this study we also used reference genes known to be spe-
cifically involved in abiotic stress tolerance and these genes
were quantified under similar experimental conditions in order
to validate our stress treatment induced annexin gene expres-
sion changes. Results indicate that Rd29 was up-regulated un-
der all the four different stress treatments, being positively
modulated by 630-fold by dehydration and 223-fold by NaCl
treatment (data not shown). Proline synthesizing enzyme P5CS
was induced by dehydration (512-fold) and by NaCl (79-fold),
whereas proline catabolizing enzyme PDH showed marginal
induction under the stress treatments as expected (data not
shown).
3. Discussion
The temporal and spatial expression of genes and the sub-
cellular localization of the proteins encoded by those genes
provide important clues to their functions. As expected for a
multifunctional multigene family, annexins showed differential
expression during growth and development and in response to
specific environmental stimuli. Previously, we have noted that
for AnnAt1 and AnnAt2, mRNA and protein localization pat-
terns are almost identical in seedlings, possibly indicating little
or no post-translational regulation of expression for these two
annexins during normal growth and development [5]. Post-
Fig. 7. Stress effects on annexin levels. Real time RT-PCR results are shown
comparing annexin RNA levels under stress conditions as compared to
untreated controls. All plants were 7-day-old with various treatments ending at
the time of RNA collection. Treatments and durations are as follows: 4 °C,
plants held at an ambient temperature of 4 °C for 2 hours; 37 °C, plants held at
an ambient temperature of 37 °C for 2 hours; Dehyd, dehydration on bench for
2 hours; NaCl, plants transferred to nutrient plates containing 250 mM NaCl for
2 hours. Error bars represent 95% confidence intervals calculated as described
in Section 4. Data is presented as the fold change in RNA levels as normalized
to Control untreated 7-day-old plants (value of 1). In order to keep both
increases and decreases in relative fold differences in RNA levels proportional
the Y axis is presented in log scale. All changes due to treatment condition are
statistically significant at the 95% confidence level and as such control data is
omitted for visual simplicity.
Table 1
Relative fold differences for each annexin in response to various stress
treatments
Gene 4 °C 37 °C Dehydration 250 mM NaCl
AnnAt1 13.3 2 39.2 44.2
AnnAt2 –7.2 43.5 –11.4 –11
AnnAt3 7–18.9 13.1 –1.3
AnnAt4 2–8.1 –5.5 383.2
AnnAt5 5.8 8.4 4.4 63.7
AnnAt6 –2.4 11.1 25.8 13.7
AnnAt7 5.3 20.8 1.5 42.2
AnnAt8 2.3 2.3 175.4 433.5
A. Cantero et al. / Plant Physiology and Biochemistry 44 (2006) 13–2418

translational regulation of active protein levels through signal-
mediated protein degradation via proteasomes has recently
been shown to be an important and commonly used level of
gene control in plant cells. In fact, it has been suggested that
altered protein levels of AnnAt1 in seeds in response to stress
may be due to proteasomal protein degradation [26]. In this
study we focus only on mRNA expression levels for the Ara-
bidopsis annexin gene family.
Although the results of many plant microarray studies that
include information on annexin expression patterns are avail-
able, the high throughput methodology of microarray analysis
often does not distinguish between related members of a gene
family.
Quantitative real time RT-PCR is more accurate at detecting
relative expression over a larger linear range because the scan-
ning and processing involved in microarray analysis often
eliminates the lowest and highest ends of its detectable range
[10]. For these reasons we used quantitative real time RT-PCR
as a precise, sensitive and reproducible technique to measure
the expression profiles of the annexin multigene family.
Gene structure and phylogenetic analyses of the Arabidopsis
annexins reveal genome-based differences within this gene fa-
mily. For example, these analyses show that the feature of in-
tron loss in the family is a shared characteristic of the more
recent, evolutionary clade. Sequence analysis of the core re-
peats indicate that second and third repeats may be sources of
functional differences between annexins as these repeat regions
have diverged more than the first and fourth repeats.
All eight annexins are detectable in dry seeds. This may be
indicative that germinating seeds use their RNA stores for in-
itial translation needs. A germinating seed has to start growth
rapidly despite its limited resources, so, as with other messages
and proteins, annexins may be part of a pool of pre-synthesized
material allowing for a quicker initiation of growth.
Increase in annexin transcript levels between a dry seed and
a germinated plant could be expected for a gene encoding a
growth-related protein, because transitioning from a dormant
mature embryo to a metabolically active seedling requires sig-
nificant growth. Surprisingly, seven of the eight annexins show
a decrease in RNA levels 26 hours after sowing as compared to
a dry seed. The decreased levels of annexin messages 26 hours
after sowing seems counterintuitive, since growth is not slow-
ing down during this period. Perhaps levels of annexin protein
optimal for growth have been reached at this time, reducing the
need to maintain high message levels. An alternative explana-
tion for this trend of down-regulation of annexin message le-
vels during germination would be that increases in annexin
message levels do occur in certain cells or tissues but we are
missing these changes by averaging message levels over all the
tissues in the germinating seed. In situ RNA localization of
each annexin, an extension of our previous report on cell-type
specific expression patterns in germinating seedlings for An-
nAt1 and AnnAt2 [9], would provide a rigorous test of this al-
ternative explanation.
It appears that all eight annexin transcripts are fairly abun-
dant after 7 days of growth in the light. This may indicate that
the whole annexin gene family contributes importantly to the
diverse cellular functions needed for seedling growth. These
results also show that the quantitative real time RT-PCR meth-
od is suitable for the identification and quantitative comparison
of the relative transcript levels of different annexin encoding
genes.
Localization of RNA across a plant can give clues as to the
roles and importance of various members of a gene family. In
order to survey the distribution of annexin transcripts, RNA
levels were examined with real time RT-PCR on tissues taken
from dark grown seedlings. All of the annexins exhibit the
trend of having higher levels in roots than in cotyledons. This
fits nicely with previous histological investigations indicating
that AnnAt1, for example, is found primarily in the roots of
young germinating seedlings [6,9]. The observed trend also
meshes with the general notion that annexins are involved in
growth, since in an etiolated plant, the majority of cell expan-
sion is occurring in the roots and hypocotyls, while the cotyle-
dons exhibit much less growth. We have previously used stan-
dard RT-PCR to assay annexin transcript levels in various light
grown tissues of older plants, but not many comparisons be-
tween the previous data and the present data can be made [9].
AnnAt4 transcript levels are similar in all three tissues ex-
amined and show the weakest changes during the germination
process with only a slow increase over time. In contrast to An-
nAt4,AnnAt5,AnnAt6, and AnnAt7 are not expressed evenly in
all three tissues within young etiolated seedlings. Each of these
genes shows strongest expression in roots and hypocotyls with
markedly decreased levels in cotyledons. Potential roles for
these annexins are discussed in relation to light effects on their
expression.
Light triggers global changes in gene expression in young
seedlings, and recent microarray studies have begun to elucidate
some of the specific transcript changes regulated by phyto-
chrome during seedling de-etiolation [32]. Because of potential
involvement of annexins in mediating growth and development
we examined phytochrome-mediated changes in annexin ex-
pression in cotyledons and hypocotyls, two tissues with differ-
ent growth responses to light. The growth changes induced by
red light are very rapid, so we examined the early gene expres-
sion changes that could be detected after only 30 min, on the
assumption that these changes would be the most influential in
mediating the light effects. At this early time point the RNA
levels of AnnAt5 in hypocotyls changed in a significant manner,
increasing sevenfold after red light, and this change was re-
versed by far-red light, indicating that AnnAt5 is among the
“early-response”genes rapidly regulated by phytochrome in this
tissue. This result suggests that AnnAt5 could be participating in
cellular changes leading to growth inhibition.
In cotyledons, where red light induces cell division and cell
expansion, red light induces a statistically significant 35-fold
increase in transcript abundance for AnnAt6, and far-red light
reverses this effect, making AnnAt6 a second annexin gene ra-
pidly regulated by phytochrome in seedlings. However, be-
cause this light-induced expression change happens in cotyle-
dons, AnnAt6, unlike AnnAt5 is associated with growth
A. Cantero et al. / Plant Physiology and Biochemistry 44 (2006) 13–24 19

promotion rather than growth inhibition. Interestingly, AnnAt6
expression is down-regulated eightfold by red light in hypoco-
tyls. This change is not statistically significant because of the
large standard error, but if additional tests show this trend to be
significant they would support the conclusion that rapid up-
and down-regulation of growth in different seedling tissues
by red light closely parallels its rapid up- and down-regulation
of AnnAt6 transcript levels, suggesting an important role for
AnnAt6 in light-regulated growth.
Both red light and far-red light treatments of seedlings have
only minor effects on the transcript abundance of all other an-
nexin genes in cotyledons and hypocotyls, suggesting that
most annexins are not among the genes rapidly regulated by
phytochrome. Whether any of these other annexins are among
the “late-response”genes that are regulated by phytochrome
remains to be tested.
Prior studies have demonstrated that AnnAt1 and AnnAt2
expression and localization are positively correlated with secre-
tion and growth activities [6], and the results discussed above
could indicate that AnnAt6 expression may also be positively
correlated with growth. Testing this hypothesis would require
developing specific antibody tools, as has been done for An-
nAt1 and AnnAt2 [6]. However, the negative correlation of An-
nAt5 transcript expression with hypocotyl growth seems to re-
fute the conclusion that all annexins are engaged in activities
that favor growth, and lends further evidence that annexin
functions are not redundant.
One emerging theme from recent plant annexin studies is
that annexins participate in abiotic stress responses, therefore
we wanted to examine expression patterns for the entire Arabi-
dopsis annexin gene family in response to a variety of abiotic
stresses. After 2 hours exposure to different abiotic stress con-
ditions most of the annexin genes were differentially regulated.
The rapidity of these responses makes it less likely that they
arose from secondary effects. There is evidence supporting a
role for calcium signaling in the responses to the different abio-
tic stresses we used [35], thus the differential regulation of ex-
pression for the annexin family of calcium-binding proteins is
probably a meaningful component of these responses.
As discussed earlier Lee et al. have described evidence sup-
porting a role for two Arabidopsis annexins, AnnAt1 and An-
nAt4, in germination under stress conditions of high osmoti-
cum and salt [26]. In their study, treatment with NaCl
appeared to decrease levels of immunodetectable AnnAt1 and
induced cytoplasmic AnnAt1 to move to the membrane frac-
tion. Their RNA gel blot analysis of AnnAt1 transcript levels
in roots of 2-week-old seedlings that were responding to
250 mM NaCl indicated no change in AnnAt1 transcript levels
at 2 hours or later time points. Based on these results, the
authors suggest that salt treatment may cause AnnAt1 to be
targeted to the proteasome for degradation.
In contrast to the Lee et al. study [26], our results show
more than a 40-fold increase in AnnAt1 transcript levels in
whole 7-day-old seedlings 2 hours after treatment with
250 mM NaCl. The difference between our results may be
due to the use of different developmental stages, different tis-
sue sources, or different techniques. However, in order to un-
derstand the potential role of AnnAt1 in response to salt stress,
this discrepancy in reports on its expression will need to be
resolved.
AnnAt1 has previously been implicated in stress responses
based on its ability to complement the oxy5 bacterial strain.
This ability was apparently due to inherent peroxidase activity,
and, indeed, AnnAt1 does have a catalase motif in the first re-
peat of its protein structure [15]. A second report described
additional experiments supporting the ability of this protein to
protect against oxidative cellular damage [24]. More recently,
the property of inherent peroxidase activity for AnnAt1 has
been confirmed [16], and since H
2
O
2
is involved in many
stress and defense responses, this feature of AnnAt1 could al-
low it to play a key role in stress signaling.
Although we separately tested the effects of each abiotic
stress on annexin expression, often these individual stresses
can occur together and there is cross-talk between the various
stress pathways. Drought and salinity can both result in osmo-
tic stress, and both involve calcium and gene expression
changes in their signal transduction pathways. Drought and salt
treatments appear to affect the level of certain annexin mes-
sages similarly. For example, drought and salt treatments in-
crease the message level for AnnAt1 (39-fold and 44-fold, re-
spectively) and decrease the message levels for AnnAt2 (11.4-
fold and 11-fold, respectively). AnnAt5,AnnAt6, and AnnAt8
are all up-regulated by both salt and drought treatments,
although for these annexins there are differences in the level
of induction by the two treatments.
Transcript levels for all the annexins except AnnAt2 and
AnnAt3 increased following NaCl treatment. Two of them, An-
nAt4 and AnnAt8, showed a particularly high positive response
that was comparable to that of Rd29 and P5Cs (two marker
genes for salt stress in Arabidopsis [36]) under similar condi-
tions (data not shown). This result suggests an important role
for annexin gene family members during salt stress. Presum-
ably their function would be to help transduce the signaling
effects of Ca
2+
, which has at least two documented roles in salt
tolerance: to mediate adaptation to higher salt levels in the
growth medium or soil, and to regulate Na
+
transport systems
[13,40,41]. Transcriptional induction of annexin genes in re-
sponse to salt and other abiotic stresses open a new area of
investigation on the roles played by these proteins in the trans-
duction of stress signals.
Interestingly, the message levels of AnnAt3 and AnnAt4,
which share a common 2.5 kb 5′region in their gene structure,
do not appear to be regulated in the same fashion by the dif-
ferent abiotic stress treatments. Analysis of the regulatory ele-
ments found in the shared 5′region reveals some differences
within the 300 bp region immediately upstream of the respec-
tive start sites for these two genes. For example, the proximal
promoter region of AnnAt4 has a salt responsive sequence mo-
tif which is not found in this region for AnnAt3. This motif
difference might explain our results showing salt-induced in-
crease in AnnAt4 message levels and lack of salt regulation
for AnnAt3 message levels.
A. Cantero et al. / Plant Physiology and Biochemistry 44 (2006) 13–2420

Previous results obtained in wheat provide evidence for a
potential role of annexins in cold responses. Breton et al. [3]
found that two annexins redistributed from the cytoplasm to
the plasma membrane within 30 min after cold treatment. Ad-
ditionally, the protein levels for these two wheat annexins gra-
dually increased in response to cold treatment reaching a peak
1 day after cold treatment. In this study, we observed that cold
treatment leads to a fairly substantial increase in the transcript
levels for four of the annexins, AnnAt1,AnnAt3,AnnAt5, and
AnnAt7, possibly indicating a role for these annexins in cold
responses in Arabidopsis.
The results presented and discussed here provide clues to-
wards elucidating the function of Arabidopsis annexin gene
family during growth and development and under adverse en-
vironmental conditions. Differential expression pattern collec-
tively suggest annexin genes do not serve completely redun-
dant function and have both distinct and overlapping function
in certain signal transduction pathway. In general the expres-
sion patterns also suggest a role for this gene family in seedling
growth and development and response to abiotic stress. This
information will be a valuable starting point for future gene
knockout and other genetic studies that will be needed to clar-
ify the precise function of each individual annexin and to re-
solve which annexins have overlapping or distinct functions.
These genetic studies plus the expression results discussed here
will help reveal just how important role these proteins play in
plant Ca
2+
signaling.
4. Methods
4.1. Plant material and growth conditions
Arabidopsis thaliana ecotype Columbia (Col) and Wassi-
lewskija (Ws) seeds were sown on Murashige and Skoog med-
ium [27], supplemented with 2% sucrose and solidified with
0.7% agar. Ws seeds were used for all experiments except
stress studies which used Col seeds. All seeds were treated
with cold stratification at 4 °C for 3 days prior to sowing. Nor-
mal growth was at 22 °C under continuous light (140 μmol/m
2
per s). Seven-day-old seedlings were harvested after germina-
tion and frozen in liquid nitrogen before storage at –80 °C.
4.2. Stress treatments
To test the affect of various abiotic stresses 7-day-old seed-
lings were exposed to the following stress treatments for
2 hours each: 250 mM NaCl, dehydration on the bench,
37 °C, and 4 °C. Wild-type plants without any stress treatments
were used as controls, substituting normal growth condition for
temperature stress, distilled water for NaCl treatment, and nor-
mal growth temperature in closed media as control for dehy-
dration stress.
4.3. Light treatments
To test the effects of light on annexin gene expression, 5.5-
day-old etiolated seedlings were either exposed to 2 min red
light (76 μmol/m
2
per s) followed by 30 min dark, 2 min red
light followed immediately by 3 min far-red light
(112 μmol/m
2
per s) and then 30 min dark, or no light treat-
ment before harvesting hypocotyls and cotyledons.
4.4. RNA isolation and cDNA synthesis
Total RNA was isolated from whole seedlings by using the
Plant RNeasy extraction kit (Qiagen USA, Valencia, CA,
USA). Total RNA was isolated from dry seeds by using the
method described by Salmi et al. [29] for RNA isolation from
dry spores with slight modifications. Briefly, the modifications
include a precipitation of carbohydrates using 20% isopropanol
followed by 100% isopropanol at room temperature instead of
95% ethanol at –80 °C. To eliminate residual genomic DNA,
the RNA was treated with RNase free DNase (Invitrogen,
Carlsbad, CA, USA) and quantified in a spectrophotometer
(Nanodrop). cDNA was synthesized using Superscript II re-
verse transcriptase (Invitrogen) according to the manufacturer’s
instructions. The efficiency of cDNA synthesized was assessed
by end point RT-PCR using constitutive gene specific primers
encoding APT1 (Adenosine phosphoribosyl transferase).
4.5. Oligonucleotide probes and primers for real time RT-PCR
To facilitate real time RT-PCR measurement of transcripts
of the eight annexins, oligonucleotide primers were required to
meet a stringent set of criteria to ensure maximum specificity
and efficiency. For designing the individual annexin specific
primers sequences were selected within the divergent structural
repeats of individual annexins or, if distinct sequence was not
available within a particular gene, the UTRs were used.
LUX™fluorogenic labeled primers were designed using the
web-based LUX™designer software of Invitrogen according
to the specifications of 18–24 nucleotides in length, product
size ranging between 80 and 150 bp, and a melting temperature
range of 60–80 °C. Table 2 shows primer sequences for annex-
in and APT1 genes. The forward experimental primers were
labeled by the fluorophore FAM and forward control house-
keeping gene primers by the fluorophore JOE. Global align-
ments of the selected primer sequences for individual annexins
with genomic sequences and transcript sequences of all the an-
nexin genes were performed to retain and maintain specificity
for individual annexins.
We also used several positive control genes known to be
involved in abiotic stress tolerance. Specifically, gene specific
primers were designed and synthesized for Rd29 (Accession
no. D13044) (shown to respond to a variety of environmental
stresses including dehydration, low temperature, saline treat-
ments and osmotic changes) and two components of proline
biosynthetic pathway P5CS (Accession no. NM123232) (delta
A. Cantero et al. / Plant Physiology and Biochemistry 44 (2006) 13–24 21

1-pyrroline-5-carboxylate synthetase) and PDH (Accession no.
NM113981) (proline dehydrogenase).
Gene specific primers were designed and synthesized for the
constitutive housekeeping gene APT1 (Accession no.
AF325045) which was used as an internal control. The internal
control gene was chosen on the basis of its constant expression
levels over experimental condition (time/treatment), which is
ideally a constitutive housekeeping gene. For use with the
comparative C
T
method, the control gene should have similar
amplification efficiency to experimental genes, which was con-
firmed after running a validation experiment. Efficiencies be-
tween the control gene APT1 and each of the eight annexins
were compared through analysis of serial template dilutions.
The slope of the log template versus ΔC
T
was found to be less
than 0.1 for each comparison, validating the use of the com-
parative C
T
method. Melting curves on products from each
primer set gave discrete peaks, indicating single target amplifi-
cation. Conventional agarose gel electrophoresis also showed
single bands of expected size for each primer set.
4.6. Quantitative real time RT-PCR conditions
Reverse transcription polymerase chain reactions were per-
formed using an ABI Prism 7700 or 7900HT Sequence Detec-
tion System (Applied Biosystems, Foster City, CA, USA). The
thermal cycling profile was performed with either a two-step or
three-step cycle. The two-step program was: 50 °C for 2 min,
95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and
60 °C for 1 min. The three-step program was: 50 °C for 2 min,
95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s,
55 °C for 30 s and 72 °C for 30 s. A dissociation curve was
also run to check for presence of artifacts. On the 7700 this
was done by running the samples at 95 °C for 20 s, 60 °C
for 20 s and 95 °C for 15 s with a Ramp of 19 min and 59 s
from 60 °C to 95 °C, on the 7900HT the automated dissocia-
tion program was used.
PCR reactions were performed in 96- or 384-well polypro-
pylene plates sealed with transparent adhesive covers. Plati-
num® Quantitative PCR SuperMix-UDG (Invitrogen) was
used for 20 μl final volume reactions with 1 μl of ROX refer-
ence dye. Primers were used at 100 nM each with the equiva-
lent of 75–200 ng of reverse-transcribed RNA template per re-
action. In all experiments, appropriate negative controls
containing no template RNA were subjected to the same pro-
cedure to exclude or detect any possible contamination or carry
over. Before proceeding with the actual experiments, a series
of primer dilutions and template dilution were carried out to
determine optimum primer and template concentrations to be
used in the experiments for optimum amplification of target.
Each sample was analyzed at least three times and three repli-
cates were used in each case. Reactions were repeated as ne-
cessary to give a minimum of six useable data points based on
a minimum requirement of two technical replicates for each of
three biological replicates. Actual data averaged an nof 9.6
across all experiments, with a range of 7–21.
4.7. Data analysis
C
T
values were obtained using the manufacturer supplied
SDS 1.91 or SDS 2.2.1 software. Baselines and thresholds
were set manually based on amplification plots, with typical
baselines running from cycles 3 through 15 and threshold va-
lues selected within the early exponential portion of the plot.
C
T
values were exported for expression calculations with Mi-
crosoft Excel. Data points with irregular amplification or melt-
ing curves were excluded from further computations.
Relative fold expression changes were calculated using the
comparative C
T
method, where fold change is calculated as
2
–ΔΔ CT
. Validation experiments for the comparative C
T
meth-
od were performed as described above.
Calculations were performed in the following manner. ΔC
T
values were calculated as the difference between the mean C
T
Table 2
Real time RT-PCR primers
Gene Sequence Strand
AnnAt1 5′-CATCTTTCGCACTTCTCGGTGAAGA[FAM]G-3′Forward
5′-GAGCAGCTTATGTTTCTCTGTGGA-3′Reverse
AnnAt2 5′-GACTAATGACGCCATTGTTGGGATTAG[FAM]C-3′Forward
5′-ACGTGCGTCTGCTTCGTCTC-3′Reverse
AnnAt3 5′-CACCAGGCATGGACAATGCTATTACTGG[FAM]G-3′Forward
5′-CAAGTCACAAGCAAAGCCAAGAAT-3′Reverse
AnnAt4 5′-CAACTCGGGATGGGAATGGGAG[FAM]TG-3′Forward
5′-CGCAGTATTGAAGCGGGAGAA-3′Reverse
AnnAt5 5′-GTATCGTTCAAAGGAAGAGGCTGCGA[FAM]AC-3′Forward
5′-TTGTGTTGCATTGCGATGAG-3′Reverse
AnnAt6 5′-GACGAGAGACAGTGTGCATGTTCCTCG[FAM]C-3′Forward
5′-GCCATTCTCCGACCACTGAA-3′Reverse
AnnAt7 5′-CACCATCCATTGCTAAAGACACTCATGG[FAM]G-3′Forward
5′-TGCAAGTCCAACCACAACTG-3′Reverse
AnnAt8 5′-CATCAACTTGACAAGCCGCCTTGA[FAM]G-3′Forward
5′-CCACCATTGTTTCTCCTCCACA-3′Reverse
APT1 5′-CAACGTGGCCCTCCTATTGCG[JOE]TG-3′Forward
5′-CCGAAATAACCTTCCCAGGTAGC-3′Reverse
A. Cantero et al. / Plant Physiology and Biochemistry 44 (2006) 13–2422

value of APT over three replicates and each individual C
T
va-
lue of the corresponding annexins. For each treatment condi-
tion and annexin, ΔC
T
values were then averaged and standard
deviations calculated across all biological and technical repli-
cates.
Standard error of the means was estimated using:
S:E:¼s
ffiffiffi
n
p
sis the standard deviation of nnumber of data points.
Where indicated, 95% confidence intervals were determined
using the two-tailed distribution formula:
ΔΔCT±tα½n1
s
ffiffiffi
n
p
ΔΔC
T
values are naturally normalized to a value of 1 based
on the treatment used as the calibrator. AnnAt1 or specific
treatments or conditions were arbitrarily used as the calibrator
in order to give reasonable standardization across graphs.
Acknowledgements
We would like to thank Dr. Stuart Reichler and Andy Al-
verson with help in preparation of the manuscript. Sharmistha
Barthakur was funded by Department of Science and Technol-
ogy (DST), Government of India under BOYSCAST fellow-
ship. This work was supported by a NASA grant (NAGW
1519) to G.B. Clark and S.J. Roux.
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