ECA3, a Golgi-localised P2A-type-ATPase, plays a crucial role in manganese nutrition in Arabidopsis

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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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2

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.




3

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




4

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.




5

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




6

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




10

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




11

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.




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).




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-




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




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.




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.




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.




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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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




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




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.




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.




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.





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;




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.




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




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




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




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




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)




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




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




Figure 8




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




Figure 10

ECA3-YFP GONST1-GFP composite




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




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.


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.


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


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


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.

ECA3, a Golgi-localised P2A-type-ATPase, plays a crucial role in manganese nutrition in Arabidopsis

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Transcript of ECA3, a Golgi-localised P2A-type-ATPase, plays a crucial role in manganese nutrition in Arabidopsis