1
Running title: AtECA3 plays a crucial role in Mn nutrition.
ECA3, a Golgi-localised P2A-type-ATPase, plays a crucial role in manganese
nutrition in Arabidopsis.
Rebecca F. Mills1, Melissa Louise Doherty1, Rosa L. López-Marqués2, Thilo Weimar3,
Paul Dupree3, Michael G. Palmgren2 Jon K. Pittman4 and Lorraine E. Williams1
1School of Biological Sciences, University of Southampton, Bassett Crescent East,
Southampton, SO16 7PX, UK 2 Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National
Research Foundation, Department of Plant Biology, University of Copenhagen,
Thorvaldsensvej 40, DK-1871 Frederiksberg, Denmark. 3 Department of Biochemistry, Building O, Downing Site, Cambridge CB2 1QW, UK 4 Faculty of Life Sciences, University of Manchester, 3.614 Stopford Building, Oxford
Road, Manchester M13 9PT, UK
Corresponding author:
Dr. Lorraine E. Williams,
Phone: +44 (0)23 8059 4278
Fax: +44 (0)23 8059 4319
Email: [email protected]
Journal Research Area: Signal Transduction and Hormone Action
Keywords: calcium, heavy metal transport, manganese, plant membrane transport,
PMR1, secretory-pathway Ca-ATPases, T-DNA insertion mutants, zinc
Plant Physiology Preview. Published on November 16, 2007, as DOI:10.1104/pp.107.110817
Copyright 2007 by the American Society of Plant Biologists
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Abstract
Calcium and manganese are essential nutrients required for normal plant
growth and development, and transport processes play a key role in regulating their
cellular levels. Arabidopsis contains four P2A-type ATPase genes, AtECA1-4, which
are expressed in all major organs of Arabidopsis. To elucidate the physiological role
of AtECA2 and AtECA3 in Arabidopsis, several independent T-DNA insertion mutant
alleles were isolated. When grown on medium lacking Mn, eca3 mutants, but not
eca2 mutants, displayed a striking difference from wild-type plants. After
approximately 8-9 d on this medium, the eca3 mutants became chlorotic and root
and shoot growth were strongly inhibited compared to wild-type plants. These severe
deficiency symptoms were suppressed by low levels of Mn indicating a crucial role
for ECA3 in Mn nutrition in Arabidopsis. eca3 mutants were also more sensitive than
wild-type plants and eca2 mutants on media lacking Ca, however the differences
were not so striking as in this case all plants were severely affected. ECA3 partially
restored the growth defect on high Mn of the S. cerevisiae pmr1 mutant, which is
defective in a Golgi Ca/Mn pump (PMR1), and the S. cerevisiae K616 mutant (pmc1
pmr1 cnb1), defective in Golgi and vacuolar Ca/Mn pumps. ECA3 also rescued the
growth defect of K616 on low Ca. Promoter:GUS studies show that ECA3 is
expressed in a range of tissues and cells including primary root tips, root vascular
tissue, hydathodes, and guard cells. When transiently expressed in tobacco, an
ECA3-YFP fusion protein showed overlapping expression with the Golgi protein,
GONST1. We propose that ECA3 is important for Mn and Ca homeostasis, possibly
functioning in the transport of these ions into the Golgi. ECA3 is the first P-type
ATPase to be identified in plants that is required under Mn-deficient conditions.
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Introduction
Calcium and manganese are essential nutrients required for normal plant
growth and development. Both Mn deficiency and excess Mn accumulation can result
in severely decreased crop yield. Mn deficiency is one of the most widespread trace
element deficiencies for arable crops, often seen in cereals such as wheat and barley
(Jiang et al. 2006). Plants deficient in Mn appear chlorotic and growth is reduced
(Shenker et al. 2004). Mn2+ is essential throughout all stages of plant development; it
activates a number of different enzymes such as decarboxylases and
dehydrogenases in the Krebs cycle (Marschner 1995), and several
glycosyltransferases in the Golgi (White et al. 1993; Nunan and Scheller 2003). Mn2+
is associated with photosystem II where it is required for light-induced water oxidation
through which oxygen is produced. Mn is also required for mitochondrial superoxide
dismutase (MnSOD) which is involved in scavenging of superoxides (Bowler et al.
1991) and is important in the biosynthesis of fatty acids and carotenoids (Marschner
1995). As well as being an essential element, Mn2+ is also toxic at higher
concentrations causing chlorosis, brown speckles on mature leaves, and necrosis
(Marschner 1995).
Transport proteins play a crucial role in maintaining the correct concentrations
of Mn in the cytoplasm and also in supplying Mn to particular sub-cellular
compartments where it may be required to act as an enzyme cofactor. In the yeast,
Saccharomyces cerevisiae, and also in many bacterial systems, several pathways for
Mn2+ transport have been identified (Kehres and Maguire, 2003; Culotta et al. 2005).
In plants the situation is less clear but several proteins which may transport Mn have
been identified in Arabidopsis including: AtECA1, an ER P2A-type ATPase; AtCAX2, a
vacuolar cation/H+antiporter; AtIRT1, a plasma membrane ZIP transporter, and
AtNramp3, a vacuolar Nramp (reviewed in Williams et al. 2000; Hall and Williams,
2003; Pittman 2005). Certain members of the cation diffusion facilitator (CDF) family
also appear to transport Mn including AtMTP11 (Delhaize et al. 2007; Peiter et al.
2007).
Ca is required at much higher concentrations than Mn and plays many
fundamental roles in plants, including stabilization of cell walls and membranes,
facilitating root extension and protection against potentially damaging factors in the
soil such as salinity (Sanders et al. 1999). Ca deficiency is rare in nature but
disorders that are observed in horticulture tend to be seen in fast-growing tissues and
include blossom-end rot in tomato, tip-burn in leafy vegetables and fruit cracking
(Marschner 1995). Ca is also important in signalling, coupling a wide range of
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extracellular stimuli to a vast array of intracellular responses (Sanders et al. 1999).
As such the cytosolic Ca concentration must be carefully regulated and resting levels
are usually around 0.1–0.2 µM. Ca2+ enters the cytoplasm through calcium channels
present at both the plasma membrane and intracellular membranes (White 2002).
Ca2+ efflux is mediated by high affinity P-type Ca-ATPases and lower affinity Ca/H
antiporters (Evans and Williams 1998; Geisler et al. 2000, Cheng et al. 2005). The
complexity of Ca signalling and homeostasis is reflected in the diversity of Ca2+-
transporters. Molecular analyses of Ca-ATPases in plants indicates that they divide
into two types: P2A-type Ca-ATPases (also known as ECAs), which show homology
to the sarcoplasmic/endoplasmic reticulum Ca-ATPases (SERCAs), and P2B-type Ca
-ATPases (also known as ACAs) which are generally stimulated by calmodulin and
show homology to the calmodulin-binding Ca-ATPases found at the PM of animal
cells (PMCAs) (Axelsen and Palmgren, 1998; Geisler et al., 2000). A subset of P2A-
type Ca ATPases, the secretory-pathway Ca-transport ATPases (SPCAs) are found
in other organisms (PMR1 is an example in S.cerevisiae) but these do not appear to
exist in plants.
Ca-pumps may have a variety of functions: loading of Ca into intracellular
stores such as the ER and vacuole for them to act as sources of Ca for signalling;
shaping and terminating a Ca signal; providing Ca for vesicle trafficking and fusion;
supplying Ca and possibly other divalent cations such as Mn and Zn to organelles to
support their biochemical reactions (Sze et al. 2000). Genetic studies using mutants
are now starting to reveal specific functions for individual Ca-pumps (Wu et al. 2002;
Schiott et al. 2004).
There are four P2A-type ATPases in Arabidopsis (AtECA1-4). Of these,
AtECA1 was the first to be studied (Liang et al., 1997) and was shown to function at
the ER in the transport of Ca and Mn (Liang et al. 1997, Liang and Sze 1998). The
growth of the eca1-1 mutant is sensitive to low Ca and high Mn suggesting that
pumping of these cations into the ER is required to support plant growth under
conditions of Ca deficiency and Mn toxicity (Wu et al. 2002). Having shown that
additional P2A-type ATPases, ECA2 and ECA3, are expressed in Arabidopsis
(Pittman et al. 1999), the main objective of the present study was to investigate their
physiological role. This was achieved by isolating T-DNA insertion mutants in which
the genes encoding these proteins had been disrupted and comparing the phenotype
of the mutants to wild-type Arabidopsis. We present evidence showing that eca3
mutants have a unique and distinct phenotype from eca1 and eca2 mutants and
demonstrate that ECA3 has a crucial role in Mn nutrition.
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Results
Primary structure of Arabidopsis ECA3
AtECA3 from Col-0 contains an open reading frame of 998 amino acids with a
predicted molecular mass of 109.062 kDa. It is identical to the sequence predicted
from genome sequencing (The Arabidopsis Information Resource,
http://arabdopsis.org) but differs from the ECA3 sequence (AJ132388) that we cloned
previously from Landsberg (Pittman et al. 1999) in four bases: A336G, T453A,
T1647G, C2934T. In the ECA3 sequence from Landsberg, the second and fourth
base changes are silent but the first and third give changes in the amino acid
sequence: R46G and S549R. Conpred II, a consensus prediction method for
obtaining transmembrane topology models (http://bioinfo.si.hirosaki-
u.ac.jp/~ConPred2), indicates that AtECA3 contains ten transmembrane domains, a
small cytoplasmic loop between TM 2 and 3 and a large cytoplasmic loop between
TM 4 and 5. The latter contains the highly conserved phosphorylation domain
CSDKTGTLT which includes the phosphorylated aspartate residue (Asp347 for
AtECA3) that is phosphorylated during the reaction-cycle in all P-type ATPases. This
model is consistent with structural models that have been presented for the
sarcoplasmic reticulum Ca-ATPase from animal cells (Toyoshima et al. 2000).
Isolation of T-DNA insertion mutant alleles of ECA2 and ECA3
To determine the physiological function of ECA2 and ECA3, we obtained mutant
lines in which ECA2 and ECA3 were likely to be functionally disrupted. We isolated
two independent mutant alleles for each (eca2-1, eca2-2, eca3-1 and eca3-2). These
T-DNA insertion alleles were identified using the reverse genetics approach outlined
in Materials and Methods. Figure 1A illustrates the genotyping of plants from the
Salk line 045567; Figure 1B shows that for each particular mutant, no product with
the appropriate gene-specific primers could be detected in reverse-transcribed cDNA
from plants homozygous for the insert. Figure 1C and Table I summarises the T-DNA
insertion positions in the resulting mutant alleles and indicate where in the protein the
insertion would occur. The position of the inserts in each of the mutants means that
these are predicted loss-of-function mutants.
The effect of low Ca on eca mutants
To elucidate the biological function of ECA2 and ECA3 in Arabidopsis, we monitored
insertion mutants under a wide range of conditions comparing their growth and
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development to wild-type plants. When grown on soil, no major differences from wild-
type plants were observed (Supplementary Figure 1).
Because eca1-1 mutants were previously shown to be extremely retarded
when grown on low Ca medium (0.2 mM) (Wu et al. 2002), eca2 and eca3 mutants
were tested in this range. At basal levels of Ca (1.5 mM), eca3-1, eca3-2, eca2-1,
and eca2-2 mutants were similar in their fresh weight and chlorophyll content to wild-
type plants; results are shown only for fresh weight for eca2-1 and eca3-1 (Figure
2A). In contrast to results reported previously for eca1-1 mutants (Wu et al. 2002),
eca2 and eca3 mutants were no more retarded in growth than wild-type plants at low
Ca (in this case 0.1 mM). We next tested whether there was a difference in the
growth of the mutants when Ca was omitted from the medium. Under these
conditions both wild-type and mutant seedlings were adversely affected (Figure 2B).
They became chlorotic and stopped growing at an early stage. In general, both eca3
mutants were slightly more sensitive than wild-type and eca2 mutants, but in this
experiment only the eca3-1 were significantly lower in fresh wt (P<0.05) (Figure 2B).
eca3 mutants are susceptible to growth in Mn-deficient conditions
eca1-1 mutants were previously shown to be more adversely affected in growth on
high (0.5 mM) Mn medium (Wu et al. 2002). We found no difference in the response
of eca2 and eca3 mutants to this level of Mn compared to wild-type plants (results
not shown). Therefore we investigated whether the mutants showed any responses
under Mn-deficient conditions. A striking difference was observed in the eca3
mutants compared to eca2 and wild-type when grown on medium with Mn omitted
(Figure 3). Under these conditions, eca3 mutants showed a marked reduction in
growth, which first became apparent after approximately 8-9 days. Both root and
shoot growth were reduced (Figure 3A, B). In addition, the cotyledons became yellow
and the marked reduction in chlorophyll is indicated in Figure 3B. There were some
interesting features observed when individual seedlings were examined closely
(Figure 3A, Supplementary Figure 2). In some seedlings dark areas were seen at the
tips of developing leaves (Supplementary Figure 2). On leaves where the lamina was
expanding, a brown band was often observed across the petiole or lamina with
bleaching occurring distal to the band. In many cases the leaf lamina failed to expand
further and senescing areas were observed. When leaves did develop, these and the
expanded cotyledons were seen to bleach. Addition of Mn to the medium at a
concentration of around 1 µM was sufficient to suppress these phenotypes (Figure
4).
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The response of the Arabidopsis mutants to other ion deficiencies was also
investigated. When Zn was omitted from the growth medium there was no significant
difference in the performance of the eca3 (Figure 5A) or eca2 mutants (results not
shown) compared to wild-type. eca3 mutants were inhibited in their growth to the
same extent as wild-type and there were no significant differences in terms of fresh
weight of shoot or chlorophyll content. Omission of Fe from the medium had a
marked effect on growth and chlorophyll content of all plants but there was no
specific detrimental effect on the mutants compared to wild-type plants (Figure 5B).
Omitting Cu from the media also had no specific effect on the mutants compared to
wild-type (Figure 5B).
Elemental analysis of eca3 mutants
Since the eca3 mutants showed a clear phenotype under Mn-deficient conditions, the
Mn content was determined under nutrient sufficient (basal, 50 µM Mn) and Mn-
deficient conditions (0µM Mn). The levels observed in each genotype are shown in
Figure 6. To make this clearer, the level relative to the wild-type (expressed as a
percentage) is shown in Spplementary Figure 3, with the level in the wild-type (100%)
indicated as a dashed line. Despite the marked differences observed in the growth
phenotype of eca3 mutants compared to wild-type plants when grown under Mn-
deficient conditions, there were no major differences in metal-content changes when
eca3 mutants and wild-type plants were compared. Generally the Mn content of the
eca3 mutants was slightly lower than wild-type plants under both conditions (i.e.
basal Mn supplied and Mn omitted). Ca content was slightly higher under both
conditions in the eca3 mutants compared to wild-type plants and Zn was similar or
slightly lower (Figure 6). Again, for both Ca and Zn, there was little difference in the
response to reducing the Mn concentration in the media of eca3 mutants compared
to wild-type plants. We cannot rule out that extended growth under Mn-deficient
conditions could lead to metal content changes, but we chose a point at which the
symptoms although apparent were not too severe.
Expression analysis of Type 2A Ca-ATPases
Semi-quantitative RT-PCR was used to determine whether ECAs were present in
various organs of hydroponically grown Arabidopsis. Primers designed to regions of
AtECA1-4 amplified products of 127 bp, 325 bp, 279 bp and 127 bp respectively (Figure
7A). Amplification of a 122 bp product with primers to the constitutively expressed
ribosomal protein S16, a component of the 40S subunit (Kim et al. 2006) was used as a
control. Transcripts for all ECAs are present in all major organs (Figure 7A). Results for
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ECA2 and ECA3 are consistent with data from publicly-available microarray studies
(https://www.genevestigator.ethz.ch/) (Zimmermann et al 2004), which also show that
these genes are expressed in all major organs (Figure 7B). Expression of ECA1 and
ECA4 cannot be determined from microarray analysis as the probe sets are not distinct.
To confirm these results and obtain further information about the tissue-specific
expression of ECA3 within the different plant organs, a β-glucuronidase (GUS) reporter
gene was cloned under the control of the promoter region for ECA3. GUS staining of
seedlings showed high expression levels in guard cells, hydathodes and the vascular
tissue in leaves (Figure 8). In primary roots, high levels of expression were seen at the
root tip and vascular system. Transverse sections of roots showed high GUS activity in
the stele particularly in pericycle and xylem parenchyma cells. Staining was also
observed in developing lateral roots, and tip and vascular staining was seen in more
mature lateral roots. A strong staining was also found in different flower tissues
including stamens, petals and sepals, as well as in siliques (Figure 8).
Some metal transporters are regulated at the transcriptional level by the
metals they transport. For example, AtHMA4, a Zn-transporting ATPase is up-
regulated by elevated levels of Zn (Mills et al. 2003; Williams and Mills 2005) and the
Fe-transporter, IRT1, is up-regulated under Fe-deficient conditions (Vert et al. 2001).
Others, such as MPT1/ZAT, show no obvious regulation by metals at the level of
transcription (van der Zaal et al. 1999). Real-time PCR was performed to determine
whether expression of ECA2 and ECA3 changed under conditions of metal deficiency.
Root and shoot expression levels of ECA2 and ECA3 relative to the S16 component of
ribosomal 40S are shown for seedlings grown on basal medium (control) or in the
absence of either Mn or Zn (Figure 9). There were no significant differences in the
expression of either gene when grown under these conditions. For a comparison, we
measured expression levels of the transporter ZIP4 and in this case there was a
marked up-regulation under Zn-deficient conditions as reported previously (Wintz et
al 2003; Van de Mortel et al 2006).
ECA3 is localised to the Golgi
To determine the sub-cellular localisation of ECA3, fusions with yellow fluorescent
protein (YFP) were generated. Transient expression in tobacco epidermal cells
followed by confocal laser scanning microscopy revealed small motile organelles
characteristic of Golgi (Figure 10). There was a high level of colocalisation with the
Golgi protein GONST1 (Handford et al. 2004) visualised using GONST1-GFP
fusions.
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ECA3 restores the growth defects of yeast K616 mutant on Ca-depleted medium and
of K616 and pmr1 mutants on high Mn medium.
Heterologous expression in yeast has been used in a number of studies to
functionally characterize animal and plant transporters. The K616 (∆pmr1, ∆pmc1,
∆cnb1) yeast mutant is defective in endogenous Ca/Mn-pumps and has been very
useful in functional analysis of plant Ca-pumps (Liang and Sze 1998; Harper et al.
1998). Growth of K616 yeast is inhibited in the presence of elevated Mn or high Zn in
the media (Wu et al. 2002). ECA3 rescues K616 on high Zn, restoring growth almost
to the level of control K601 cells (Figure 11A). ECA3 also partially restored growth of
K616 on elevated Mn, but not to the extent of the K601 cells. Increased sensitivity to
elevated Mn concentrations is also observed in the pmr1 mutant (Lapinskas et al.
1995) and ECA3 reduced this growth defect compared to vector controls (Figure
11C). K616 vector transformants are also unable to grow on media containing
reduced levels of Ca (i.e. in the presence of EGTA). ECA3 suppresses the EGTA-
sensitive phenotype, allowing growth in the presence of EGTA to levels similar to
those seen in the control K601 strain (Figure 11B).
Discussion
Arabidopsis contains four P2A-type ATPases (ECA1-4) which are related to
the well-characterized SERCA Ca-ATPases in mammals, and ten P2B-type ATPases
related to the mammalian plasma membrane calmodulin-stimulated Ca2+-ATPases.
These are all members of the P-type superfamily of ATPases. Understanding the
functional properties, expression patterns and localisation of each of these pumps is
important if we are to understand how they contribute to ion homeostasis and
signalling in plants. The aim of this study was to gain insight into the physiological
roles of ECA2 and ECA3 by studying Arabidopsis T-DNA insertion mutants in which
the genes encoding these ATPases have been disrupted. In addition, we have used
heterologous expression in yeast to study functional properties of ECA3.
The studies conducted here with eca3 mutants have revealed a crucial role
for ECA3 in Mn nutrition. eca3 mutants showed poor growth on Mn-deficient medium
compare to wild-type plants (Figure 3 and Supplementary Figure 2). The response to
Mn-deficiency is first seen after about 8-9 days on this medium; prior to this, growth
is similar to wild-type. Omitting Zn, Fe or Cu from the medium had little specific effect
on the mutant compared to wild-type (Figure 5). In the absence of Mn, eca3 mutants
showed a reduction in both root and shoot fresh weight and in chlorophyll content;
this phenotype could be rescued by adding 1 µM Mn to the medium (Figure 4). The
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fact that eca3 mutants show little difference to wild-type plants when Mn was present
in the medium at normal levels indicates that other genes can compensate for the
loss of ECA3 under these conditions. Notably on soil there were no marked
differences in the development of the eca3 mutants, which grew similarly to wild-type;
in this case there was probably sufficient Mn for normal growth and development.
There appear to be distinct differences in the roles the ECAs play in Mn and
Ca tolerance. Wu et al. (2002) found that eca1 mutants were sensitive to high Mn
and suggested that ECA1 may confer tolerance to this metal by removing it from the
cytosol. Physical symptoms of Ca deficiency have also been reported in eca1-1
mutants when the external Ca was reduced to 0.2-0.4 mM, suggesting that ECA1
may be the main pump capable of loading Ca into the ER lumen under these
conditions (Wu et al. 2002). Both eca2 and eca3 mutants grew normally in this range
but when Ca was omitted from the medium altogether, eca3 mutants seemed slightly
more affected than eca2 mutants or wild-type, suggesting a role for ECA3 in Ca
homeostasis (Figure 2). However, it should be stressed that under these conditions,
all plants were severely affected and the differences in the mutants and wild-type
were not as marked as seen for the situation when Mn was omitted from the medium.
Results from semi-quantitative RT-PCR and microarray analysis showed that
ECA3 is expressed in all major organs of Arabidopsis (Figure 7). Promoter:GUS
studies confirmed this and showed that ECA3 is highly expressed in a range of
tissues and cells including root tips, hydathodes, guard cells and vascular tissue
(Figure 8). The markedly different metal-dependent phenotypes observed in eca1
mutants (Wu et al. 2002) and eca3 mutants (this study) indicates that these ATPases
have different physiological roles in Arabidopsis. This could be related to differences
in their membrane location. ECA1 is associated primarily with the endoplasmic
reticulum functioning in the transport of Ca and Mn into this compartment (Liang et al.
1997). Recent proteomic analysis (Dunkley et al. 2006) confirms an ER localisation
for ECA1 and a Golgi localisation for ECA3 (Paul Dupree, paper in preparation).
Using transient expression of YFP–ECA3 fusion proteins in tobacco, we show here
that ECA3 colocalises with GONST1 (Figure 10), a protein previously shown to be
located in the Golgi (Handford et al. 2004).
From the data we have presented in this paper we propose that ECA3 could
serve a similar function to Golgi-associated secretory-pathway Ca-transport ATPases
(SPCAs) found in other organisms. In mammalian cells there are three distinct
classes of P2-type Ca-ATPases: SERCA-type, PMCA-type and SPCAs (also referred
to as PMR1-type) and these three groups can be clearly distinguished in
phylogenetic analysis (Pittman et al. 1999; Shmidt et al. 2002; Supplementary Figure
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4). SPCAs transport Ca but also Mn with high affinity and they function both in Mn
detoxification via exocytosis and also in providing Mn for Golgi-localised enzymes
(Yadav et al. 2007). In S. cerevisiae, the SPCA, PMR1, functions as a Ca and Mn
transporter at the Golgi, transporting these ions into the secretory pathway. Both ions
can exert distinct functions: Ca supplied by PMR1 is required to sustain vacuolar
protein sorting whereas Mn is required to activate various Mn-requiring enzymes
involved in the addition of complex carbohydrates onto N- and O-linked glycosylated
proteins (Lapinskas et al. 1995; Durr et al. 1998). The fact that on a low Ca medium
ECA3 complements the yeast triple null mutant strain K616 (∆pmr1 ∆pmc1 ∆cnb1),
lacking its endogenous Golgi (PMR1) and vacuolar (PMC1) Ca-pumps, is consistent
with ECA3 functioning as a Ca pump in yeast (Figure 11). K616 also exhibits growth
sensitivity to high levels of extracellular Mn, which results from toxicity of excessive
cellular levels due to the lack of functional endogenous pumps (Lapinskas et al.
1995, Culotta et al. 2005). Expression of ECA3 reduces the Mn-sensitive phenotype
of the triple yeast mutant K616 and also the single pmr1 mutant, indicating that ECA3
could serve a similar function to PMR1 (Figure 11). ECA3 is particularly effective in
reducing the Zn-sensitive phenotype in yeast suggesting that it may have the
capability of transporting zinc. However ECA3 does not seem to play a major role in
Arabidopsis under zinc-deficiency, as wild-type plants and eca3 mutants were
similarly affected under such conditions.
There are SPCAs in fungi, animal and bacteria but they have not been
identified in higher plants. Phylogenetically, AtECA3 clusters with “animal-type
SERCA pumps”, while AtECA1 and AtECA2 cluster more closely with “plant-type
SERCA pumps” (Pittman et al. 1999; Supplementary Figure 4). Our data indicate that
in plants, ECA3-type pumps could have a comparable physiological role to SPCA-
types and function in transporting Ca and Mn into the Golgi-associated secretory
pathway. The partial rescue of metal-dependent growth phenotypes displayed in
yeast mutants would support a transport function for ECA3 but more direct transport
assays would be required to show that ECA3 functions as a Ca and Mn transporter.
Future experiments will be aimed at testing the hypothesis that ECA3 transports Mn
into the Golgi to supply various Mn-requiring enzymes for biochemical processes
required for normal growth of Arabidopsis.
Mn deficiency is a serious nutritional disorder for plants in many areas of the
world, and in arable crops this can lead to reduced yield. Not only is there variability
between plant species in their ability to grow on low Mn, there are also considerable
differences among genotypes of the same species (Pedas et al. 2005). Varieties
displaying tolerance to Mn deficiency have been termed as Mn-efficient (Pedas et al.
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12
2005; Jiang 2006) but the mechanisms underlying this have not been clearly defined.
In barley, differential capacity for high-affinity Mn influx is thought to contribute to Mn
efficiency; in wheat it may be due to improved internal utilization rather than higher
Mn accumulation (Jiang 2006). ECA3 homologues could play a role in the Mn
efficiency of crop species and there is the potential in the future to test whether
expression of AtECA3 could improve Mn efficiency.
In summary, this study has provided evidence that AtECA3 plays a crucial
role in Mn nutrition in Arabidopsis. The Golgi localisation of ECA3 could explain why
disruption of ECA3 does not lead to a major effect on the overall Mn content of the
plant. The severe phenotype observed under Mn deficient conditions may be due to
a reduction in the Mn content of the Golgi. Our observations indicate that other
secretory pathway/endomembrane-localised Mn transporters such as AtMTP11
cannot compensate for the lack of ECA3 under Mn-deficiency. AtECA1, AtCAX2, and
AtMTP11 have previously been reported to confer tolerance to high Mn
concentrations but AtECA3 is the first to be identified that is required for growth
under low Mn conditions.
Materials and Methods
Plant material and growth conditions
Seeds of wild-type and transgenic Arabidopsis thaliana (cv Columbia-8) were
sterilized in 10% (v/v) bleach for 20 min, rinsed five times with sterile water and
inoculated onto plates containing 0.8% w/v agarose (Melford, UK), 1% w/v sucrose
and either half-strength Murashige and Skoog medium (0.5 x MS) (Murashige and
Skoog 1962), or the same medium but with Ca levels reduced from 1.5 mM to 0.1
mM (0.5 x MS*). For experiments investigating the effect of Mn deficiency on
phenotype, Mn was omitted from the 0.5 x MS* medium. Seeds were stratified at 4°C
for 48 h prior to transfer to a controlled environment cabinet (23 °C 16 h light, 18 °C 8
h dark) and plates were incubated vertically or horizontally as indicated. To examine
the development of the plants to maturity, seed were sown in soil (1 part Levingtons
F5 No. 2: 1 part John Innes No. 2: 1 part Vermiperl-graded horticultural vermiculite,
medium grade) sterilized by autoclaving at 121 °C for 15 min at 1 bar pressure and
plants were grown in a controlled-environment cabinet (22 °C 16 h light, 20 °C 8 h
dark cycle) or under glasshouse conditions. To obtain organ material from mature
plants for gene expression analysis, plants were grown hydroponically as described
previously (Mills et al. 2003).
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13
Chlorophyll determination and fresh weight measurements of seedlings
Fresh weight and chlorophyll measurements were determined using seedlings grown
on five separate plates, each plate having 6 wild-type seedlings and 6 mutant
seedlings. The data presented are generally the means from the five plates ± S.E
expressed on a per seedling basis. A Student’s t-test (paired) was used to determine
significant differences between mutant and wild-type plants and * indicates a
significant difference (P<0.05). Chlorophyll was determined following extraction in
N,N-dimethylformamide (DMF) (Moran 1982).
Elemental analysis
Shoots and roots were harvested separately from plants grown on agarose plates
(see above). Plant samples were dried for two days at 70°C and weighed. Samples
were digested at 120°C in 0.5 ml of 65% nitric acid for 4 h. After the solution was
cooled, it was diluted to 10 ml with deionized water and metal content was
determined by atomic absorption.
RNA Isolation, Reverse Transcription-PCR (RT-PCR) and real-time PCR
Total RNA was extracted from different plant organs (roots, leaves, stems,
flowers and siliques) of hydroponically grown plants and from root and shoot tissue
from plants grown on agarose plates (see above) using a phenol/SDS extraction and
LiCl precipitation method based on Verwoerd et al. (1989). The RNA was checked for
purity, integrity and quantity using RNA gel electrophoresis and spectrophotometry
(Sambrook et al. 1989). First strand cDNA synthesis using 2 µg of total RNA was
carried out using Superscript RNase H-reverse transcriptase (Invitrogen, UK)
according to the manufacturer’s instructions with an oligo (dT)12-18 primer. The
expression pattern of AtECA1-4 was determined by semi-quantitative RT-PCR using
primers designed to specifically amplify parts of each sequence. AtECA1 and
AtECA4 forward primer, 5′- GCTACCGCTACTCACCTCGC-3′; AtECA1 reverse
primer, 5′-CCAACGCCGAGGTAAGTAAC-3′; AtECA4 reverse primer, 5′-
GCAAGGATGAGTAAAGCAACAA-3′; AtECA2 forward primer, 5′-
TAGGGACGAGGAAAATGG-3′ and reverse primer, 5′-
GCTCTAAACAACTTCCCTTC-3′; AtECA3 forward primer, 5′-
GCTATGACAGTACTTGTTGTTG-3′ and reverse primer 5′-
GCCTGTATTTCTAGAGAGGA-3′. Control PCR was performed using primers to S16
riosomal gene (40S component): forward primer 5′-ACGCCATCCGTCAGAGTATC-
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14
3′; reverse primer 5′-CGCCACAAGCAGAGTCCTAT-3′. Products were amplified
using Biomix Taq (Bioline, UK) and cycles were chosen that were in the exponential
phase of PCR amplification. Primers were used at a final concentration of 0.2 µM.
PCR conditions were: 94°C for 2 min, the required number of cycles of 94°C for 30
sec, then 65°C (ECA1), 52°C (ECA2) or 60 °C (ECA3, ECA4) or 56°C (S16) for 1
min, then 72 °C for 1 min. Amplified DNA was separated on 1.2% (w/v) agarose gels
and visualised using ethidium bromide. A similar procedure was used for real-time
PCR but using Superscript III reverse transcriptase and oligo(dT)20 primers
(Invitrogen, UK). The same primer pair was used for S16 (reference gene) and
ECA3, and the primer pair 5′-TAGGACCATCGCGTTTGAGAAC-3′ (forward)
with 5′-TCTCCATGGTGGCATCGTTAAG-3′ (reverse) was used for ECA2. Real-time
PCR reactions were performed in a 96-well plate using a Chromo 4 Thermal Cycler
(MJ Research) with the Opticon Monitor III program. SYBR Green (Finnzymes,
Finland) was used to monitor cDNA amplification. For data evaluation, target gene
expression was normalised to reference gene expression by an efficiency calibrated
method using the equation Ratio=(Etarget)∆Ct(control-test)/ (Ereference)
∆Ct(control-test) where E is
PCR efficiency (2 is optimal) and Ct is the number of PCR cycles before the signal
reaches the threshold level (Pfaffl, 2001).
Cloning of AtECA3 and plasmid constructs
p426-ECA3 yeast expression construct
AtECA3 from A. thaliana (L. cv Columbia-0) was cloned in two overlapping parts as
initial attempts to amplify the full-length cDNA failed. The 5′ part was amplified from
clone RZL01a05 (AV554735/AV545775) obtained from Kazusa DNA Research
Institute, Chiba, Japan (Asamizu et al 2000) using the primers ECA3.F (5′-
GCGGATCCAAACCCTTGGCTTTTCCAAC-3′) and ECA3-5′.R (5′-
CATCCCATTGCTGTCAAAGACGGTACCTT-3′). The ECA3.F primer introduced a
BamHI site, allowing the product to be cut with BamHI, and at an EcoRI site within
the ECA3 sequence for ligation into the same sites of p426 (Mumberg et al. 1994)
giving p426-5′-ECA3. The 3′ part of ECA3 was amplified from reverse–transcribed
RNA from leaf tissue using the primers ECA3-3′.F (5′-
TGATGTCGGTGTCTAAGATATGTGT-3′) and ECA3.R (5′-
CGAAGCTTCGAAAAAGCCATCTCTTGCT-3′) and cloned into pGEMTe. The
primer ECA3-3′.F overlaps the end of the 5′ part and the ECA3.R primer introduced a
HindIII site, allowing the ECA3 3′ fragment to be excised with EcoRI and HindIII and
ligated into the same sites of p426-5′-ECA3 to produce the full-length ECA3 cDNA in
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15
p426 (p426ECA3). Sequencing of the final product confirmed that the full-length
ECA3 was identical to the sequence predicted from genome sequencing. The
GenBank accession number for ECA3 cloned in this study is EU082212.
YFP-ECA3 construct
Gateway technology was used to produce the YFP-ECA3 construct. ECA3 cDNA
was PCR-amplified from the p426-ECA3 construct and integrated into the pENTR-
TOPO vector by TOPO cloning (Invitrogen). ECA3 was then sub-cloned into the
pEarleyGate 104 destination vector (Earley et al. 2006), which attaches YFP in-frame
to the N-terminus of ECA3.
ECA3 promoter:GUS construct
For tissue-specific expression studies, 1.5 kb of the 5′-UTR for ECA3 was amplified
by high-fidelity PCR with primers 2548 (5′-
CACCGTGTACAGTTGTATAGTTTACCAAC-3′) and 2549 (5′-
GTTGGAAAAGCCAAGGGTTTCTG-3′). The PCR product was cloned into
pENTR/D-TOPO® (Invitrogen) using the Gateway technology and transferred to
plasmid pMDC162 (Curtis and Grossniklaus, 2003), to generate a construct
containing the open reading frame for a β-glucuronidase (GUS) reporter gene under
the control of the ECA3 promoter region.
Yeast strains, transformation, and growth
The K616 S. cerevisiae strain (MATα pmr1::HIS3 pmc1::TRP1 cnb1::LEU2, ura3;
Cunningham and Fink, 1994) was transformed with p426ECA3 or p426 vector alone
using a Li-acetate method based on Gietz et al. (1992). Yeast transformants were
selected on media lacking uracil. Yeast strain K601/W3031A (MATα leu2, his3, ade2,
and ura3; Cunningham and Fink, 1994) transformed with the empty p426 vector was
used as a positive control.
To compare the growth of K616 cells containing p426ECA3 or p426 alone,
cells were were first grown in liquid culture overnight at 30 °C in synthetic complete
(SC) without uracil (pH 5.0) and containing 20 gl-1 glucose as the carbon source. The
cultures were adjusted to the same OD600 and aliquots were inoculated onto 2% (w/v)
agar plates containing the SC-uracil medium (pH 4.9) with galactose or glucose and
various additions as specified in the results section. Plates were incubated at 30 °C
for 3–5 d.
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16
Transient expression of ECA3 in tobacco
Four-week old tobacco (Nicotiana tabacum cv. Petit Havana) plants grown at 22°C
on a 16h/8h light/dark cycle, were used for Agrobacterium-tumefaciens (strain
GV3101)-mediated transient expression (Batoko et al. 2000). pVKH18En6-GONST1-
GFP (Handford et al., 2004) or PEG104-ECA3-YFP-transformed A. tumefaciens were
cultured at 28°C, until the stationary phase (approximately 24 h), washed, and
resuspended in infiltration medium (MES 50 mM pH 5.6, Glucose 0.5% [w/v], Na3Po4
2 mM, acetosyringone 300 µM (from 750 mM stock in dimethyl sulfoxide). The
bacterial suspension was inoculated using a 1-mL syringe without a needle by gentle
pressure through the stomata on the lower epidermal surface (Brandizzi et al., 2002) .
Transformed plants were then incubated under normal growth conditions for 3 d at
22°C.
Confocal microscopy
Transformed leaves were analyzed 72 h after infection of the lower epidermis.
Confocal imaging was performed using an inverted Zeiss LSM510 META laser
scanning microscope with a 63x oil immersion objective. For imaging expression of
GFP and YFP, excitation lines of an argon ion laser of 458 nm for GFP and 514 nm
for YFP with a 475/525-nm bandpass filter for GFP and a 530/600-nm bandpass filter
for YFP were used alternately with line switching using the multi-track facilities of the
microscope. Appropriate controls were performed to exclude any cross talk and
bleed through of fluorescence.
ECA3-promoter:GUS expression analysis
Agrobacterium tumefaciens strain C5851 (Koncz and Shell, 1986) was transformed
by electroporation and transformants were selected on YEP plates (1% yeast extract,
2% peptone, 1.5% agar) containing 25µg/ml gentamycin and 50 µg/ml kanamycin.
Wild type A. thaliana ecotype cv Columbia-0 plants were transformed by floral
dipping (Clough and Bent, 1998). Seeds were selected on 0.5x MS plates without
sucrose supplemented with 25 µg/ml Hygromycin B under short-day (8-h light)
conditions. After 2 weeks, transformants were tested for GUS activity as described in
Haritatos et al.(2000). For flower and silique staining, plants were selected on plates
as before and then transferred to soil under long-day (16-h light) conditions for two
weeks. Transverse sections of GUS-stained roots were obtained as in Haritatos et al.
(2000), and visualized in a Nikon Eclipse 80i light microscope. At least four
independent transformant lines were tested in each case.
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17
Isolation of insertional mutants for AtECA2 and AtECA3.
Seed for putative T-DNA-insertion lines for AtECA2 and AtECA3 from the SALK
collection (http://signal.salk.edu/) were obtained from the Nottingham Arabidopsis
Stock centre. These were seed lines N576961 (eca2-1), N539146 (eca2-2), N545567
(eca3-1), and N570619 (eca3-2). To isolate eca mutants homozygous for the insert a
similar procedure to that previously described for hma4 mutants was followed (Mills
et al. 2005). Briefly, genomic DNA was isolated from soil-grown plants using the
DNAMITE plant kit (Microzone Ltd, UK). To genotype the plants with respect to the T-
DNA insert, PCR was carried out on genomic DNA using the primer for the T-DNA
left border (Salk LBa1, 5′-TGGTTCACGTAGTGGGCCATCG-3′) and gene-specific
primers ECA2.F (5′ TAGGGACGAGGAAAATGG-3′) for eca2-1 and eca2-2, ECA3.R
(5′-CATCCCATTGCTGTC AAAGACGGTACCTT-3′) for eca3-1, and ECA3.F (5′-
TCCAATCTGCAGAGCATGGTCCTATGA-3′) for eca3-2. PCR conditions were 94°C
for 2 min followed by 40 cycles of 94°C for 30 s, then 54°C (ECA2.F) or 56°C
(ECA3.F) or 58°C (ECA3.R) for 1 min, then 72°C for 1 min (except 2 min for
ECA3.R), then a final elongation step of 72°C for 5 min.
For N576961 (eca2-1), N545567 (eca3-1), and N570619 (eca3-2) sequencing
confirmed the original SALK predicted T-DNA locations, while for N539146 (eca2-2),
the insert location was confirmed at 139 bp 3′ of the predicted location. Mutant alleles
for ECA2 (eca2-1, eca2-2) and ECA3 (eca3-1, eca3-2) were obtained and lines
homozygous for the inserts were selected for phenotypic analysis. The lack of a
transcript was confirmed at the RNA level by RT-PCR using primers which span the
insertion site. For ECA2: ECA2.F (see above) and ECA2RTR (5′-
CTTAAGGATTACTTCTCTGCTA-3′); ECA3: ECA3ex10F (5′-
GCATACACGATTCTATGTTGCAGACAGATGAT-3′) and ECA3SGR (5′-
GTTACCAAATTGACCCACAGAAGT-3′). Amplification of Actin2 was used as a
positive control using primers: Actin2.F 5′-GGT AAC ATT GTG CTC AGT GGT GG-
3′ and Actin2.R 5′- CTC GGC CTT GGA GAT CCA CAT C-3′. After a denaturation at
94°C for 2 min, PCRs were cycled as follows: 24 cycles of (94°C, 30 s; 56°C, 1 min;
72°C, 1 min) for Actin2; 40 cycles of (94°C, 30 s; 50°C, 1 min; 72°C, 1 min) for ECA2,
and 40 cycles of (94°C, 30 s; 55°C, 1 min; 72°C, 2 min) for ECA3. A final elongation
step of 72°C for 5 min was used in all.
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18
Acknowledgements
We thank Nottingham Arabidopsis stock centre for supplying mutant seed lines. This
work was supported by BBSRC (51/P17217) and EU Framework VI programme for
RTD (contract no FOOD-CT-2006-016253). This publication reflects only the authors’
views; the Community is not liable for any use that may be made of the information
contained therein.
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(2003) Expression profiles of Arabidopsis thaliana in mineral deficiencies reveal
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24
Williams, L.E., Pittman, J.K, and Hall, J.L. (2000) Emerging mechanisms for heavy
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FIGURE LEGENDS
Figure 1. Isolation of mutant lines
(A). Determining the genotype of plants from the Salk line 045567. PCR was carried
out on genomic DNA from individual plants (1-3) using gene-specific primers
ECA3Ex10.F and ECA3.R to detect plants that contained a wild-type copy of the
gene; these amplified a band of the predicted size, 2.07 Kb (upper panel). SALK
LBa1 primer for the T-DNA and the gene-specific primer ECA3.R were used to detect
the presence of the T-DNA in ECA3. They amplified a band of the approximate
predicted size, 1.96 Kb (lower panel). The zygosity of plants 1-3 determined from this
analysis is indicated: wt=wild-type, hom = homozygous for the insert and het =
heterozygous for the insert. (B). RT-PCR using RNA isolated from wt and eca
mutants. Primers designed to amplify a product across the insertion site gave
products for ECA2 and ECA3 of 0.67 and 1.67 respectively only in wt plants. Actin 2
primers were used to amplify a product of 0.2 Kb as a positive control in all samples.
(C). Insertion sites for mutants isolated in this study.
Figure 2. Effect of low Ca on growth of Arabidopsis eca2 or eca3 mutants
compared to wild-type plants.
Plants were grown for 17 d on plates in 0.5 x MS media containing varying levels of
Ca: 0.1 mM and 1.5 mM (A), 0 mM (B); white line = 1 cm. Fresh weight values are
shown for eca2-1 (top) and eca3-1 (bottom) compared to wild-type (wt) in A and for
wt and all mutants in B; data represent the means ± S.E. from five separate plates
each having 6 seedlings per genotype per plate (wild-type, black bar; mutant, grey
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25
bar). * indicates a significant difference between mutant and wild-type plant (P<0.05),
Student’s t-test (paired). Results are from representative experiments repeated at
least twice for each mutant.
Figure 3. eca3 mutants show reduced growth and chlorosis on Mn-deficient
media.
Plants were grown on plates in 0.5 x MS* media containing normal levels of Mn (50
µM) or without Mn present (0 µM) for 17d. (A). Seedling phenotype of eca2-1, eca3-
1 and eca3-2 mutants compared to wild-type (wt); black line = 1cm. (B). Fresh weight
and chlorophyll content for eca3-1 and eca3-2 mutants (grey bars) compared to wild-
type (wt) (black bars). (C). Fresh weight and chlorophyll content for eca2-1 mutant
(grey bars) compared to wild-type (wt) (black bars). Results in B and C represent the
means ± S.E. from at least five separate plates each having 6 seedlings per
genotype per plate and * indicates a significant difference between mutant and wild-
type plant (P<0.05), Student’s t-test (paired); results are from a representative
experiment repeated at least three times.
Figure 4. Mn concentration range for growth of eca3-2 mutants.
Plants were grown on plates in 0.5 x MS* media with increasing concentrations of
Mn. Wild-type plants, solid line and square symbols; eca3-2 mutants, dotted line and
circles. Results represent the means ± S.E. from three separate plates each having 6
seedlings per genotype per plate.
Figure 5. eca mutants show a similar response to wild-type plants when grown
in the absence of Zn, Fe or Cu.
(A) Seedling phenotype of eca3-1 and eca3-2 mutants compared to wild-type (wt)
when grown for 27 d on plates in 0.5 x MS* medium with Zn present at the normal
concentration (15 µM) (+Zn) or absent (-Zn). Fresh weight of shoot (top) and
chlorophyll content (bottom) for eca3-1 and eca3-2 mutants (grey bars) compared to
wild-type (wt) (black bars). (B) Wild-type (black bars) and eca mutants (grey bars)
were grown for 19 d on either 0.5 x MS medium (control) or in the absence of either
Fe (-Fe) or Cu (-Cu). Data in A. and B. generally represent means ± S.E. from five
separate plates each having 6 seedlings per genotype per plate. * indicates a
significant difference between mutant and wild-type plant (P<0.05), Student’s t-test
(paired).
Figure 6. Elemental analysis of eca3 mutants
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26
Mn, Ca and Zn concentration in root and shoot tissue of wild-type (black bars), eca3-
1 (grey bars) and eca3-2 mutants (white bars) grown on plates on basal (0.5 x MS*)
or 0 Mn (0.5 x MS* with Mn omitted) for 17 d. Results are means ± S.E. from four
independent pools of plants from different experiments and each pool is from at least
five replicate agarose plates. * indicates a significant difference between mutant and
wild-type plant (P<0.05), Student’s t-test (paired).
Figure 7. ECA expression analysis
(A) Organ distribution of ECAs in hydroponically grown plants determined using RT-
PCR. (B) ECA2 and ECA3 expression in different organs determined from microarray
analysis. Graphs show data (mean and standard error) extracted from
Genevestigator (ATH1: 22k array) for the same organs
(https://www.genevestigator.ethz.ch/).
Figure 8. ECA3 promoter:GUS studies
The GUS reporter gene was expressed in wild type Arabidopsis plants under the
control of the promoter region for ECA3. Different tissues were subsequently tested
for β-glucuronidase activity: (A, B), primary root; (C-E), lateral roots; (F), root vascular
tissue; (G), transverse section of the root; (H), guard cells; (I-J), leaves; (K), flowers;
(L), stamens; (M), petals; (N-P), siliques.
Figure 9. Metal regulation of ECA2 and ECA3 expression
Relative quantification of ECA2 and ECA3 expression by real-time PCR in seedlings
grown for 15 days on plates with 0.5 x MS* medium (control) or on the same medium
without Mn (-Mn) or Zn (-Zn). Results show expression normalised to the reference
gene and relative to expression levels in shoots on control medium. Results are
mean and standard error of results from 3 independent experiments.
Figure 10. Co-localization of ECA3 and GONST1 fluorescent protein fusions in
the Golgi apparatus.
Confocal laser scanning microscopy of a tobacco epidermal leaf cell expressing
ECA3-YFP and GONST1-GFP. (Left) ECA3-YFP marks small motile organelles.
(Middle) GONST1-GFP marks the Golgi apparatus. (Right) Colocalization of the
markers. Scale bar = 5 µm.
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27
Figure 11. AtECA3 expression alleviates Mn, Zn and Ca -dependent growth
defects in yeast
K616 (∆pmr1, ∆pmc1, ∆cnb1) (A and B) and pmr1 (∆pmr1) mutants (C) transformed
with p426ECA3 (K616+ECA3; pmr1+ECA3) or p426 vector (K616+vector;
pmr1+vector) were grown overnight in SC minus uracil with 2% (w/v) glucose. K601
cells are shown as a positive control (wt). A and B: Cultures were adjusted to
OD600=0.25 and used undiluted at this concentration (1) or at a 100-fold dilution
(1/100). 5 µl aliquots were spotted onto SC-Ura plates containing 2% (w/v) galactose
as the carbon source (p426 uses a galactose-inducible promoter), with the following
additions: A: either MnCl2 (0.3 mM) or ZnSO4 (3 mM). B: Ca (20 mM) and EGTA (10
mM) or EGTA (10 mM). C: Cultures were adjusted to OD600=0.4 and used at this
concentration (1) or 10-fold dilution (1/10). 7 µl aliquots were spotted onto SC-Ura
plates 2% (w/v) galactose with 0 mM, 0.3mM or 1mM MnCl2. Plates were incubated
for 3-4 d at 30 oC.
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28
Table I. eca mutant alleles identified in this study.
Introns and exons are numbered so that the first exon begins with the start ATG and
is followed by the first intron.
Allele Salk line Position
eca2-1 0576961 1st of five exons. In large cytoplasmic loop between
phosphorylated aspartate and KGAAE motif.
eca2-2 0539146 1st of four intron. In large cytoplasmic loop between
phosphorylated aspartate and KGAAE motif.
eca3-1 0545567 12th of thirty three introns. Middle of third
transmembrane domain.
eca3-2 0570619 16th of thirty three introns. In large cytoplasmic loop
between phosphorylated aspartate and KGAPE motif
Supplementary Figure legends
Figure 1. Growth of eca mutants is similar to wild-type plants on soil. Plants
were grown in soil in a controlled environment cabinet (22°C 16 h light, 20°C 8 h dark
cycle) and photographed after 55 d.
Figure 2 Phenotype of eca3 mutants grown under Mn-deficient conditions.
Left: eca3-1 seedlings grown on horizontal plates on 0.5 x MS* medium in the
absence of Mn for 10 d. Arrows indicate particular features which are observed: (i)
dark areas are often seen at the tips of developing leaves; (ii) on leaves where the
lamina is expanding, browning is often observed at the junction between the petiole
and the lamina; (iii) some leaves fail to expand further and senescing areas are
observed. Right: eca3-1 mutants grown on vertical plates on 0.5 x MS* medium in
the absence of Mn for 27 d. Leaves stop growing and become bleached.
Figure 3. Elemental analysis of eca3 mutants
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29
Mn, Ca and Zn content of in root and shoot tissue of eca3-1 and eca3-2 mutants
grown on plates in basal (0.5 x MS*) or 0 Mn (0.5 x MS* with Mn omitted) for 17 days
presented as a percentage of levels in wild-type plants. Black bars, shoots; grey
bars; roots. Dashed line represents 100%, the value given to wild-type plants.
Results are mean percentages from four independent pools of plants from different
experiments and each pool is from at least five replicate agarose plates.
Figure 4 Maximum-Likelihood Phylogenetic Tree for P2-type ATPases created
using Tree-puzzle 5.2
Maximum-Likelihood Phylogenetic Tree created using Tree-puzzle 5.2. (ClustalW
alignment input, VT substitution model, 8000 Quartet puzzling steps, neighbour-
joining tree for parameter estimation. Node labels indicate percentage support values
for each branch, and scale indicates maximum likelihood branch lengths on
consensus tree (no clock)).
Sodium ATPase ScENA1 designated as outlier. Archaeon and bacterial Ca-ATPases
boxed orange, P2B Ca-ATPases boxed pink, Pmr1-type P2A Ca-ATPases boxed
purple, plant-type P2A Ca-ATPases boxed light grey, SERCA-type P2A Ca-ATPases
boxed dark grey, AtECA3-type P2A Ca-ATPases boxed white.
Annotations indicate species initials and either common name or accession number.
Full details are: ScENA1 P13587 Saccharomyces cerevisiae; SePacL P37278
Synechococcus elongatus PCC7942; MmQ8PU16 Methanosarcina mazei;
MmAAM31157 Methanosarcina mazei; MgA4QVG4 Magnaporthe grisea;
GzUPI000023E0E5 Gibberella zeae; AfEAL88529 Aspergillus fumigatus;
GgSERCA1 P13585 Gallus gallus; VvA5C4X8 Vitis vinifera; VvA5AP46 Vitis vinifera;
LeLCA1 Q42883 Lycopersicon esculentum; ScPMC1 P38929 Saccharomyces
cerevisiae; DbP54209 Dunaliella bioculata; OlA4RV19 Ostreococcus lucimarinus;
OtQ01C29 Ostreococcus tauri; MtQ8VZX6 Medicago truncatula; HsSERCA1
O14983 Homo sapiens; OcSERCA1B P04191 Oryctolagus cuniculus; OsACA1
PlantsT Protein:64540; OsACA10 PlantsT Protein:64585; OsACA11 PlantsT
Protein:64590; OsACA2 PlantsT Protein:64545; OsACA3 PlantsT Protein:64550;
OsACA4 PlantsT Protein:64555; OsACA5 PlantsT Protein:64560; OsACA6 PlantsT
Protein:64565; OsACA7 PlantsT Protein:64570; OsACA8 PlantsT Protein:64575;
OsACA9 PlantsT Protein:64580; OsECA1 PlantsT Protein:64525; OsECA2 PlantsT
Protein:64530; OsECA3 PlantsT Protein:64535; AtACA1 PlantsT Protein:85361;
AtACA10 PlantsT Protein:85379; AtACA11 PlantsT Protein:85374; AtACA12 PlantsT
Protein:85376; AtACA13 PlantsT Protein:85370; AtACA2 PlantsT Protein:85384;
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30
AtACA4 PlantsT Protein:85369; AtACA7 PlantsT Protein:87038; AtACA8 PlantsT
Protein:64708; AtACA9 PlantsT Protein:64711; AtECA1 PlantsT Protein:85357;
AtECA2 PlantsT Protein:85377; AtECA3 PlantsT Protein:64735; AtECA4 PlantsT
Protein:85358; YlO43108 Yarrowia lipolytica; PaO94117 Pichia angusta; CaPmr1
Q9P872 Candida albicans; ScPmr1 P13586 Saccharomyces cerevisiae; NcPmr1
Q9UUX9 Neurospora crassa; NcNca1 Q9UUY0 Neurospora crassa; NcNca2
Q9UUY2 Neurospora crassa; NcNca3 Q9UUY1 Neurospora crassa; HsSPCA1
P98194 Homo sapiens; HsSPCA2 O75185 Homo sapiens; AfQ4VE13 Aspergillus
fumigatus; CePmr1 Q6LA80 C.elegans; CeQ9XTG6 C.elegans; SpPmr1 O59868
Schizosaccharomyces pombe.
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Figure 1
Plant number
1 2 3
wt hom het
A.
TAA
T-DNAATG TAAeca2-1
1.46 kb 2.08 kb
T-DNAATGeca2-2
1.52 kb 2.01 kb
TAG
TAG
T-DNAATGeca3-1
3.7 kb 7.1 kb
eca3-2ATG
5.5kb 5.3kb
T-DNA
C.
B.
wt eca3-2 eca3-1
wt eca2-1 eca2-2
Actin
Actin
ECA2
ECA3
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eca2-1
eca2-2
0
5
10
15
20
25
[Ca] mM
wt / eca3-1
Fre
sh w
t. pe
r pl
ant (
mg)
0.1 1.5
0
5
10
15
20
25
Fre
sh w
t. pe
r pl
ant (
mg)
0.1 1.5[Ca] mM
wt / eca2-1Figure 2
wt eca
eca3-1
eca3-2
A.
B.
Fre
sh w
t. pe
r pl
ant (
mg)
*
0
0.2
0.4
0.6
0.8
1.0
1.2
eca2
-1
eca3
-2
eca2
-2
eca3
-1
wt
wt
wt
wt
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0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
0
2
4
6
8
10
12
14
0
5
10
15
20
100/0 100/500
5
10
15
20
100/0 100/500
5
10
15
20
100/0 100/50
*
*
*
*
Tot
al c
hlor
ophy
ll (µ
g/se
edlin
g)
wt / eca3-2
wt / eca3-2
wt / eca3-1
wt / eca3-1 wt / eca2-1
wt / eca2-1
B. C.
Ca/Mn (µM) Ca/Mn (µM) Ca/Mn (µM)
Ca/Mn (µM)Ca/Mn (µM) Ca/Mn (µM)
Fre
sh w
t. p
er p
lant
(m
g)
Fre
sh w
t. p
er p
lant
(m
g)
Fre
sh w
t. p
er p
lant
(m
g)
100/0 100/0100/50 100/50
Tot
al c
hlor
ophy
ll (µ
g/se
edlin
g)
Tot
al c
hlor
ophy
ll (µ
g/se
edlin
g)
100/0 100/50
Ca/Mn (µM)100/0 100/50
eca3-2 wt
eca3-1 wt
eca2-1 wt
eca3-2 wt
eca3-1 wt
eca2-1 wt
A. Figure 3
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Fre
sh w
t. pe
r pl
ant (
mg)
0
2
4
6
8
10
12
0 1 2 3 50
[Mn] µM
║
║
Figure 4
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Figure 5
+Zn +Zn-Zn -Zn
0
2
4
6
8
10
10
2
4
6
8
10
1
Tot
al c
hlor
ophy
ll (µ
g/se
edlin
g)
wt eca3-2 wt eca3-2 wt eca3-1 wt eca3-1
+Zn+Zn -Zn -Zn
A.
wt eca3-1 wt eca3-1
0
5
10
15
20
25
30
eca2-1 eca2-2 eca3-3 eca3-1 eca2-1 eca2-2 eca3-3 eca3-1 eca2-1 eca2-2 eca3-3 eca3-1
eca2
-1
eca2
-2
eca3
-2
eca3
-1
control - Fe - Cu
eca2
-1
eca2
-2
eca3
-2
eca3
-1
eca2
-1
eca2
-2
eca3
-2
eca3
-1
*
0
10
20
30
Fre
sh w
t. p
er p
lant
(m
g)
B.
0
20
40
60
80
0
20
40
60
80
wt eca3-2 wt eca3-2
Fre
sh w
t. p
er s
hoot
(m
g)
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Mn
(µ
g/g)
*
Zn
(µ
g/g)
*
Ca
(mg/
g)
**
*
Basal 0 Mn
0
5
10
15
20
25
30
35
wt
eca3
-1
eca3
-2 wt
eca3
-1
eca3
-2 wt
eca3
-1
eca3
-2 wt
eca3
-1
eca3
-2
0
50
100
150
200
250
300
wt
eca3
-1
eca3
-2 wt
eca3
-1
eca3
-2 wt
eca3
-1
eca3
-2 wt
eca3
-1
eca3
-2
0
100
200
300
400
500
wt
eca3
-1
eca3
-2 wt
eca3
-1
eca3
-2 wt
eca3
-1
eca3
-2 wt
eca3
-1
eca3
-2
shoot root shoot root
Figure 6
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Figure 7
ECA1
ECA2
ECA3
ECA4
S16
Root Leaf Flower Silique Stem
OrganA.
B.
0
1500
0
1500 ECA2
ECA3
Sca
led
sign
al in
tens
ity
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Figure 8
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Shoots
Rel
ativ
e ex
pres
sion
rat
io
1
0
2ECA3
ECA2
1
0
3
2
ZIP4
20
40
Control -Mn -Zn
Roots
1
0
2
ECA3
ECA2
1
0
2
ZIP4
20
0
40
Control -Mn -Zn
10
1
Figure 9
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Figure 10
ECA3-YFP GONST1-GFP composite
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Zn Mn
1
Figure 11
A.
K616+
vector
K616+
EC
A3
K616+
vector
K616+
EC
A3
wt+
vector
wt +
vector
1/100
B.
1
1/10
1
1/10
1
1/100
0.3
1.0
[Mn] (mM)C.
EGTA
EGTA+Ca
1
1
K616+
vector
K616+
EC
A3
wt +
vector
1/100
1/100
pmr1+
vector
pmr1+
EC
A3
wt +
vector
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Supplementary Figure 1
wt eca3-2 eca3-1wt eca2-1 eca2-2
Figure 1. Growth of eca mutants is similar to wild-type plants on soil. Plants were grown in soil in a controlled environment cabinet (22°C 16 h light, 20°C 8 h dark cycle) and photographed after 55 d.
Supplementary Figure 2
ii. iii. i.
1cm
Figure 2 Phenotype of eca3 mutants grown under Mn-deficient conditions.
Left: eca3-1 seedlings grown on horizontal plates on 0.5 x MS* medium in the absence of Mn for 10 d. Arrows indicate particular features which are observed: (i) dark areas are often seen at the tips of developing leaves; (ii) on leaves where the lamina is expanding, browning is often observed at the junction between the petiole and the lamina; (iii) some leaves fail to expand further and senescing areas are observed. Right: eca3-1 mutants grown on vertical plates on 0.5 x MS* medium in the absence of Mn for 27 d. Leaves stop growing and become bleached.
Supplementary Figure 3
0
2040
60
80100
120
140160
180
eca3
-1
eca3
-2
eca3
-1
eca3
-2
eca3
-1
eca3
-2
eca3
-1
eca3
-2
eca3
-1
eca3
-2
eca3
-1
eca3
-2
0
20
40
60
80100
120
140
160
180
eca3
-1
eca3
-2
eca3
-1
eca3
-2
eca3
-1
eca3
-2
eca3
-1
eca3
-2
eca3
-1
eca3
-2
eca3
-1
eca3
-2
Basal
0 Mn
Perc
enta
ge o
f wt
Perc
enta
ge o
f wt
Mn Ca Znshoot root shoot root shoot root
Figure 3. Elemental analysis of eca3 mutants
Mn, Ca and Zn content of root and shoot tissue of eca3-1 and eca3-2 mutants grown on plates in basal (0.5 x MS*) or 0 Mn (0.5 x MS* with Mn omitted) for 17 days presented as a percentage of levels in wild-type plants. Black bars, shoots; grey bars; roots. Dashed line represents 100%, the value given to wild-type plants. Results are mean percentages from four independent pools of plants from different experiments and each pool is from at least five replicate agarose plates.
P 2B
0.1
ScENA1MmQ8PU16MmAAM31157
SePacL ScPMC1NcNca2NcNca3
100100
OsACA3OsACA2
AtACA1OsACA1
AtACA2AtACA7
10074
59
100
AtACA11AtACA4
99
OsACA6OsACA7
OsACA4OsACA5
7364
61
96
100
OsACA8AtACA12
AtACA1378
97
AtACA9AtACA10
AtACA877
91
OsACA10OsACA11OsACA9
7790
94
97
99
99
AtECA2MtQ8VZX6VvA5AP46
LeLCA1100
100100
VvA5C4X8AtECA1AtECA4
100100
OsECA1OsECA2
100
99
99
AfEAL88529GzUPI00002MgA4QVG4NcNca1
9897
96
CeQ9XTG6GgSERCA1
HsSERCA1OcSERCA1B
9894
83
OlA4RV19OtQ01C29
94
DbP54209OsECA3AtECA3
8468
68
81
97
CePmr1HsSPCA1
HsSPCA298
93
SpPmr1ScPmr1YlO43108
NcPmr1AfQ4VE13
98
PaO94117CaPmr1
61
51
85
73
92
P 2A
Plant
Fungal+Animal
Plant ECA3-type
Pmr1-type
Archaea + Bacteria
P 2D
Supplementary Figure 4
Figure 4 Maximum-Likelihood Phylogenetic Tree for P2-type ATPases created using Tree-puzzle 5.2
Maximum-Likelihood Phylogenetic Tree created using Tree-puzzle 5.2. (ClustalWalignment input, VT substitution model, 8000 Quartet puzzling steps, neighbour-joining tree for parameter estimation. Node labels indicate percentage support values for each branch, and scale indicates maximum likelihood branch lengths on consensus tree (no clock)).
Sodium ATPase ScENA1 designated as outlier. Archaeon and bacterial Ca-ATPasesboxed orange, P2B Ca-ATPases boxed pink, Pmr1-type P2A Ca-ATPases boxed purple, plant-type P2A Ca-ATPases boxed light grey, SERCA-type P2A Ca-ATPases boxed dark grey, AtECA3-type P2A Ca-ATPases boxed white.
Annotations indicate species initials and either common name or accession number. Full details are: ScENA1 P13587 Saccharomyces cerevisiae; SePacL P37278 Synechococcus elongatus PCC7942; MmQ8PU16 Methanosarcina mazei; MmAAM31157 Methanosarcina mazei; MgA4QVG4 Magnaporthe grisea; GzUPI000023E0E5 Gibberella zeae; AfEAL88529 Aspergillus fumigatus; GgSERCA1 P13585 Gallus gallus; VvA5C4X8 Vitis vinifera; VvA5AP46 Vitis vinifera; LeLCA1 Q42883 Lycopersicon esculentum; ScPMC1 P38929 Saccharomyces cerevisiae; DbP54209 Dunaliella bioculata; OlA4RV19 Ostreococcus lucimarinus; OtQ01C29 Ostreococcus tauri; MtQ8VZX6 Medicago truncatula; HsSERCA1 O14983 Homo sapiens; OcSERCA1B P04191 Oryctolagus cuniculus; OsACA1 PlantsTProtein:64540; OsACA10 PlantsT Protein:64585; OsACA11 PlantsT Protein:64590; OsACA2 PlantsT Protein:64545; OsACA3 PlantsT Protein:64550; OsACA4 PlantsTProtein:64555; OsACA5 PlantsT Protein:64560; OsACA6 PlantsT Protein:64565; OsACA7 PlantsT Protein:64570; OsACA8 PlantsT Protein:64575; OsACA9 PlantsTProtein:64580; OsECA1 PlantsT Protein:64525; OsECA2 PlantsT Protein:64530; OsECA3 PlantsT Protein:64535; AtACA1 PlantsT Protein:85361; AtACA10 PlantsTProtein:85379; AtACA11 PlantsT Protein:85374; AtACA12 PlantsT Protein:85376; AtACA13 PlantsT Protein:85370; AtACA2 PlantsT Protein:85384; AtACA4 PlantsTProtein:85369; AtACA7 PlantsT Protein:87038; AtACA8 PlantsT Protein:64708; AtACA9 PlantsT Protein:64711; AtECA1 PlantsT Protein:85357; AtECA2 PlantsTProtein:85377; AtECA3 PlantsT Protein:64735; AtECA4 PlantsT Protein:85358; YlO43108 Yarrowia lipolytica; PaO94117 Pichia angusta; CaPmr1 Q9P872 Candida albicans; ScPmr1 P13586 Saccharomyces cerevisiae; NcPmr1 Q9UUX9 Neurosporacrassa; NcNca1 Q9UUY0 Neurospora crassa; NcNca2 Q9UUY2 Neurospora crassa; NcNca3 Q9UUY1 Neurospora crassa; HsSPCA1 P98194 Homo sapiens; HsSPCA2 O75185 Homo sapiens; AfQ4VE13 Aspergillus fumigatus; CePmr1 Q6LA80 C.elegans; CeQ9XTG6 C.elegans; SpPmr1 O59868 Schizosaccharomyces pombe.
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