Journal of Experimental Botany Advance Access published February 7, 2014 Journal of Experimental Botany doi:10.1093/jxb/eru020
Research paper
HMA1 and PAA1, two chloroplast-envelope PIB-ATPases, play distinct roles in chloroplast copper homeostasis
1
CNRS, Laboratoire de Physiologie Cellulaire et Végétale, UMR 5168, 17 rue des Martyrs, F-38054 Grenoble, France Université Grenoble Alpes, Laboratoire de Physiologie Cellulaire et Végétale, F-38054 Grenoble, France 3 CEA, Direction des Sciences du Vivant, Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Physiologie Cellulaire et Végétale, F-38054 Grenoble, France 4 INRA, Laboratoire de Physiologie Cellulaire et Végétale, USC1359, 17 rue des Martyrs, F-38054 Grenoble, France 5 Biology Department, Colorado State University, Fort Collins, Colorado 80523, USA 2
* To whom correspondence should be addressed. E-mail: [email protected] Received 20 September 2013; Revised 25 November 2013; Accepted 19 December 2013
Abstract Copper is an essential micronutrient but it is also potentially toxic as copper ions can catalyse the production of free radicals, which result in various types of cell damage. Therefore, copper homeostasis in plant and animal cells must be tightly controlled. In the chloroplast, copper import is mediated by a chloroplast-envelope PIB-type ATPase, HMA6/ PAA1. Copper may also be imported by HMA1, another chloroplast-envelope PIB-ATPase. To get more insights into the specific functional roles of HMA1 and PAA1 in copper homeostasis, this study analysed the phenotypes of plants affected in the expression of both HMA1 and PAA1 ATPases, as well as of plants overexpressing HMA1 in a paa1 mutant background. The results presented here provide new evidence associating HMA1 with copper homeostasis in the chloroplast. These data suggest that HMA1 and PAA1 behave as distinct pathways for copper import and targeting to the chloroplast. Finally, this work also provides evidence for an alternative route for copper import into the chloroplast mediated by an as-yet unidentified transporter that is neither HMA1 nor PAA1. Key words: Arabidopsis, chloroplast, copper, envelope transporter, metal homeostasis, PIB ATPase.
Introduction Copper (Cu) is an essential micronutrient for plants and plays key roles in processes such as photosynthesis (Burkhead et al., 2009). However, Cu can be toxic either by catalysing the production of free radicals damaging for biomolecules or by binding to sulphhydryl groups in proteins, resulting in their inactivation. Therefore, Cu homeostasis must be finely tuned to adapt to Cu availability in environment and to respond to cell requirements in Cu. Cu acquisition in plant cells is mediated by the high-affinity transporter COPT-1 (Sancenon et al., 2004) and maybe the ZIP transporter ZIP2 (Puig et al., 2007a). Within plant cells, Cu is sequestered by metallothioneins or nicotianamine, but
also transported either to organelles or to the extracellular space by COPT transporters, ZIP transporters (Wintz et al., 2003), and PIB-type ATPases (or heavy metal ATPase, HMA). Some ions are captured by Cu chaperones that deliver Cu to specific protein targets to control subcellular Cu distribution (Hänsch and Mendel, 2009). The Arabidopsis genome encodes for over 30 proteins related to yeast and mammalian metallochaperones, but only a few of them have been characterized so far. The Arabidopsis Cu-chaperones Atx1 and CCH, orthologues of the yeast Atx1 chaperone, are thought to interact with the PIB-type ATPases AtHMA5 and AtHMA7 (Himelblau et al., 1998; Andrés-Colás et al., 2006;
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Sylvain Boutigny1,2,3,4, Emeline Sautron1,2,3,4, Giovanni Finazzi1,2,3,4, Corinne Rivasseau1,2,3,4, Annie Frelet-Barrand1,2,3,4, Marinus Pilon5, Norbert Rolland1,2,3,4 and Daphné Seigneurin-Berny1,2,3,4,*
Page 2 of 12 | Boutigny et al. during evolution, allowing the acquisition of diverse metal specificities. Among the eukaryotic PIB-4 ATPases, exclusively found in plants and algae, only AtHMA1 and HvHMA1 from Arabidopsis and barley, respectively, have been studied so far. Previous work by this study group provided evidence for the following: (i) AtHMA1 can transport Cu and Zn in yeast; (ii) lack of AtHMA1 affects chloroplast Cu levels; and (iii) apparent AtHMA1 activity in the chloroplast envelope is enhanced by Cu (Seigneurin-Berny et al., 2006). Later, experiments carried out in yeast concluded that AtHMA1 could effectively transport Cu2+ and Zn2+ but also Ca2+, Cd2+, and Co2+ (Moreno et al., 2008). Another study combining in planta analysis and expression in yeast, suggested that AtHMA1 might export Zn2+ from the chloroplast (Kim et al., 2009). Finally, HvHMA1 was also proposed to export Zn but also Cu from barley plastids (Mikkelsen et al., 2012). From these four studies, it emerges that HMA1 can transport a broad range of divalent cations, probably depending on the physiological conditions. However, the direction of this transport still remains unclear. The phenotype of the paa1 mutant can be partially rescued by Cu supplementation, suggesting that another Cu transporter is present in the chloroplast envelope. HMA1 has been presented as a candidate for mediating this alternative low-affinity Cu pathway (Pilon et al., 2006). Analysis of the hma1 mutant showed that PC function is not impaired (Seigneurin-Berny et al., 2006) suggesting that, in agreement with its defined role, PAA1 supplies Cu for PC. In other words, there may be at least two Cu transporters in the chloroplast envelope. In order to investigate potential functional redundancy between PAA1 and AtHMA1, the current work generated plants in which both ATPases are mutated (hma1 paa1 double mutant). Furthermore, plants overexpressing AtHMA1 in the absence of PAA1 were produced, and the phenotypes of these plant lines were compared to those of wild-type (WT) plants and single hma1 or paa1 mutants.
Materials and methods Plant material and growth conditions Arabidopsis thaliana plants, Wassilevskija (Ws) and Lansberg (Ler) backgrounds, were germinated in Petri dishes containing solidified medium (Murashige and Skoog, MS, 1%, w/v, agarose) for 2 weeks before transfer to soil. Plants were then grown in culture chambers at 22 °C (10/14 light/dark cycle) with a light intensity of 100 μmol m–2 s–1 in standard conditions. Soil cultures were always watered with tap water containing 30 μM CuSO4 except when mentioned. For phenotypic analyses, seeds were germinated in plates containing MS salts in the absence or presence of CuSO4 at various concentrations under different light intensities, with 0.5% (w/v) sucrose in heterotrophic conditions or without sucrose for autotrophic conditions. The Arabidopsis mutant hma1 (line DRC42; Seigneurin-Berny et al., 2006) and the Arabidopsis mutant paa1 (paa1.1; Shikanai et al., 2003) were already available. The hma1 mutant is an insertional T-DNA mutant that carries the nptII selection marker (kanamycin resistance), while the paa1.1 mutant has a nonsense mutation in the eighth exon leading to truncation of the C-terminal region containing the ion-transduction, phosphorylation, and ATP-binding domains.
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Puig et al., 2007b). Plant cells also contain an orthologue of the yeast CCS chaperone, responsible for supplying Cu to Cu/ Zn superoxide dismutases (Cu/ZnSODs) located in the cytosol, the peroxisome, and the chloroplast (Chu et al., 2005; Huang et al., 2012). In Arabidopsis chloroplasts, the major targets for Cu delivery are the stromal Cu/ZnSOD (CSD2), involved in the dismutation of superoxide to hydrogen peroxide, and plastocyanin (PC), a thylakoid protein which is involved in photosynthetic electron transfer. The Arabidopsis genome codes for two PC isoforms, PETE1 and PETE2. PETE2 is the moreabundant isoform and accumulates at high levels in response to Cu feeding, possibly to buffer the excess Cu. PETE1, the less-abundant isoform, is essential for electron transport under Cu-deficient conditions (Abdel-Ghany, 2009). PC is essential for photosynthesis; thus, for plants grown in autotrophic conditions, Cu delivery to the thylakoid lumen is a priority (Weigel et al., 2003). Cu delivery into the chloroplast requires a chloroplast-envelope PIB-type ATPase, AtHMA6/ PAA1 (Shikanai et al., 2003). AtHMA8/PAA2, a second PIBATPase, is found in the thylakoid membrane, where it imports Cu for the maturation of functional PC (Abdel-Ghany et al., 2005). AtHMA1, another chloroplast-envelope PIB-ATPase, could also import Cu into the chloroplast (Seigneurin-Berny et al., 2006). Characterizations of the phenotypes of single paa1, paa2, and hma1 knockouts suggest that: (i) PAA1 could be the main route of Cu supply to CSD2 and to the thylakoid transporter, PAA2, and through this, to PC isoforms; and (ii) AtHMA1 would provide Cu to CSD2 and could play an important role during stress conditions. However, AtHMA1 may also transport other metal ions (Moreno et al., 2008; Kim et al., 2009; Mikkelsen et al., 2012; see Supplementary Table S1 available at JXB online). Based on the presence of conserved residues in transmembrane helices 6, 7, and 8, PIB-type ATPases have been divided into five subgroups of different ionic selectivity (Arguëllo, 2003). The Arabidopsis PIB-ATPases PAA1 and PAA2 belong to the IB-1 subgroup, which comprises prokaryotic and eukaryotic Cu+-ATPases such as CopA from Escherichia coli and Enterococcus hirae or the human ATPases ATP7A and ATP7B (for recent reviews, see Lutsenko et al., 2008; Rosenzweig and Argüello, 2012). In Arabidopsis, the functions of PAA1 and PAA2 were first studied in planta (Shikanai et al., 2003; Abdel-Ghany et al., 2005). A recent study combining heterologous expression in Lactococcus lactis and in vitro enzymic assays, demonstrated that PAA1 is a Cu+ATPase (Catty et al., 2011). AtHMA1, the third chloroplast PIB-ATPase, belongs to subgroup IB-4, which was initially described as gathering putative Co2+-ATPases (Arguëllo, 2003). Some members of this subgroup have been recently characterized and among them, CzcP from Cupriavidus metallidurans (Scherer and Nies, 2009) and CtpD from Mycobacterium smegmatis (Raimunda et al., 2012) showed a broader metal specificity (Zn2+, Cd2+, Co2+, and Ni2+). Raimunda and collaborators (2012) highlighted that PIB-4 ATPases share significant sequence similarity (48–66%), but cluster in distinct phylogenetic branches. This suggests that the genes coding for these ancient proteins diverged early
Chloroplast copper homeostasis | Page 3 of 12 Membrane protein extracts from leaves Leaves were first homogenized in 0.22 mM tetra-sodium pyrophosphate and 1 g l–1 BSA and centrifuged at 100 000 g for 20 min at 4 °C. The pellet was washed with 50 mM Tris-HCl (pH 6.8), resuspended in 50 mM Tris-HCl (pH 6.8) and 1 % (v/v) SDS, and then centrifuged for 10 min at 14 000 g and 4 °C to eliminate insoluble material. Protein content was estimated using the BIO-RAD protein assay reagent.
Construction of vector for stable HMA1 expression in the Arabidopsis paa1.1 mutant The HMA1 cDNA sequence was excised from a pBluescript KS– vector (described in Seigneurin-Berny et al., 2006) by BglII and SalI and inserted into a BamHI–SalI-digested pFP101 binary vector the GFP gene marker for the selection of transformed plants (Bensmihen et al., 2004). The resulting plasmid was prepared using a QIAfilter Plasmid Midi Kit (Qiagen Laboratories, Germany) before being used for Agrobacterium tumefaciens transformation. Arabidopsis transformation paa1.1 mutant plants were transformed according to Clough and Bent (1998) by dipping the floral buds of 4-week-old plants into a surfactant-containing (Silwett L-77) A. tumefaciens C58 inoculum. Transgenic Arabidopsis seeds were selected based on GFP expression driven by the At2S3 seed-specific promoter. Only lines segregating 3:1 for GFP expression and expressing the recombinant protein were selected for further analysis. Primary transformants were then self-pollinated to obtain plants homozygous for the insertion. Production of the hma1 paa1 double mutant The hma1 paa1 double mutant was obtained after pollination of the paa1.1 mutant (Lansberg background) with pollen from the hma1 mutant, DRC42 (Ws background). F1 progeny were grown and allowed to self-fertilize. F2 progeny were grown on kanamycin-supplemented MS medium with sucrose to eliminate all plants containing two WT HMA1 alleles (Supplementary Fig. S1B, available at JXB online). The genotypes of F2 individuals were determined by PCR for HMA1 and PAA1 WT alleles and for the T-DNA insertion in the hma1 mutant allele or the presence of a point mutation for the paa1 allele (primers listed in Supplementary Table S2, available at JXB online). All genotypes were validated by two PCRs performed on two DNA extracts from different leaves of the same line (a total of four PCRs). Analysis of transcripts Total RNA was extracted from leaf tissue using a RNeasy Mini Kit (Qiagen). cDNA was obtained using the Ambion M-MLV Reverse Transcriptase (Applied Biosystems). The sets of primers used to detect HMA1 and PAA1 transcripts are listed in Supplementary Table S2, available at JXB online. Chloroplast purification from Arabidopsis Arabidopsis protoplasts were isolated from Arabidopsis rosette leaves as described by Kunst (1998) with the following modifications. Leaves
Measuring metal ions Aliquots of purified chloroplast fractions corresponding to 10–40 μg of chlorophyll were mineralized and later dissolved in 1% (v/v) HNO3. Elemental analysis was performed on an inductively coupled plasma mass spectrometer (ICP-MS, Hewlett-Packard 4500 Series, Agilent Technologies, Massy, France) equipped with a Babington nebulizer and a Peltier-cooled double-pass Scott spray chamber. The device was calibrated at 24, 25, and 26 m/z for Mg, 43 and 44 m/z for Ca, 54 and 57 m/z for Fe, 63 and 65 m/z for Cu, and 64, 66 and 67 m/z for Zn, using standard solutions in 1% HNO3. ICP-MS analyses were performed in triplicate after appropriate sample dilution. The quantification results for Mg, Ca, Fe, Cu, and Zn were the mean values obtained for isotopes 24, 44, 57, 65, and 66, respectively. In vivo spectroscopic estimation of PC/photosystem I ratios In vivo spectroscopic estimation of PC/photosystem I (PSI) ratios was performed in vivo in WT and mutant lines using a JTS-10 spectrophotometer (Biologic, France). Leaves were illuminated with a far-red source at 720 nm to completely oxidize PSI and its donor pool. Absorption changes were detected by discrete flashes (duration 10 μs) delivered by two light-emitting diodes. Light was filtered to select P700 and PC redox changes. Absorption changes associated with P700 redox changes were computed as ΔI/I 705 nm– ((ΔI/I 870 nm)/2). DI/I PC was computed as ΔI/I 870 nm+((ΔI/I 705 nm)/10), as described previously (Seigneurin-Berny et al., 2006). Superoxide dismutase activity Whole leaves (~80 mg) were ground in liquid nitrogen and suspended in 200 μl of 50 mM KH2PO4 pH 7.8, 1 mM EDTA, and 0.1% (w/v) BSA. The suspension was centrifuged at 14 000 g for 10 min at 4 °C and soluble proteins were recovered in the supernatant. SOD activity was measured using an in-gel assay, as described by McCord (1999) using 10 μg soluble proteins. In the gel, the intensity of each band was measured using Quantity One software (Bio-Rad, Hercules, CA, USA). For each line, statistical analyses were performed on protein extracts from different plants grown in three independent experiments.
Results Characterization of the hma1 paa1 double mutant in Arabidopsis Double mutants were obtained from single hma1 mutant (Ws background) and paa1 mutant (Ler background; Supplementary Fig. S1, available at JXB online). Two homozygous double mutants, dm1 and dm2, whose genotypes were
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SDS-PAGE and Western blot analyses These procedures were carried out as previously described (Seigneurin-Berny et al., 2006), using a rabbit polyclonal antibody raised against amino acids 213–368 of HMA1 (1/1000 dilution) and antibodies against CSD2 and PC (Agrisera AB) used at 1/5000 and 1/10 000, respectively. For each line, statistical analyses were performed on protein extracts from different plants grown in three independent experiments.
(2–3 g) were cut into small pieces before infiltration with 30 ml protoplast isolation buffer containing 1.6% (w/v) cellulase and 0.25% (w/v) pectolyase (both from Kyowa Hakko, Tokyo). Samples were incubated at 28 °C for 2 h. After purification on Percoll, protoplasts were diluted in 10 ml protoplast storage buffer without CaCl2 and centrifuged for 5 min at 100 g. Protoplasts were then diluted in 5 ml protoplast lysis buffer and lysed by passing them through a 15-μm mesh nylon net. The resulting suspension was then layered on top of a cold preformed Percoll gradient (Seigneurin-Berny et al., 2008) and centrifuged at 13,300 g for 10 min. Intact chloroplasts were collected from the gradient (lower band), diluted 3-fold in resuspension buffer (0.4 M sorbitol, 20 mM HEPES-KOH pH 7.6, 2.5 mM EDTA, 10 mM NaHCO3, 0.15%, w/v, BSA) and centrifuged for 90 s at 1465 g. Chlorophyll levels were determined by spectrophotometry (Bruinsma, 1961).
Page 4 of 12 | Boutigny et al.
A
Line 31 lineage
The hma1 mutant exhibits a photosensitivity phenotype under high light (Seigneurin-Berny et al., 2006). In the current study, in MS medium supplemented with sucrose and in the presence of 10 μM Cu, the double mutant could grow under a light intensity of up to 150 μmol m–2 s–1 (data not shown). At higher light intensities, both double mutants and paa1 mutants could only grow if Cu was increased to 20 μM in the medium. In these conditions, the double mutant was more light sensitive than the paa1 mutant (Fig. 2B), as shown by photobleaching of the leaves (variegated phenotype with white areas), a phenotype similar to the one of the hma1 mutants (for light intensity >280 μmol m–2 s–1). In autotrophic conditions (on MS plates without sucrose, or in soil) and under light intensities more than 200 μmol m–2 s–1, the paa1 mutant and the double mutants stopped growing at the cotyledon stage and then died. This was associated with an enhanced photosensitivity phenotype, even in the presence of 20 μM Cu (data not shown). In autotrophic conditions but under low light (50 μmol m–2 s–1), Cu concentrations higher than 5 μM were required to partially restore a WT phenotype for the paa1 and double mutants, compared to the lower Cu concentrations required for these plants to grow in similar but heterotrophic conditions. The partial rescue obtained by using high Cu concentrations (30 μM) was not observed when plants
B
Line 136 lineage
WT Ws
hma1
paa1
hma1 paa1 dm1 dm2
WT Ler
HMA1 PAA1 mut
hma1 paa1 dm2 dm1 pp/hh Pp/hh pp/Hh pp/hh
PAA1 WT Actin 2
paa1Fwd/mutpaa1rev paa1Fwd/wtpaa1rev
C
Tag5/hma1rev
WT Ler
WT Ws
dm1
paa1
hma1
dm2
hma1Fwd/hma1rev
D
E
Hh/Pp
hh/pp
hh/PP
Hh/pp
hh/pp
Hh/pp
Fig. 1. Validation of the genotype of homozygous hma1 paa1 double mutant lines dm1 and dm2. (A) The genotypes of F3 lines obtained by self-crossing two independent heterozygous double mutants (lines 31 and 136) were analysed by PCR using a primer for T-DNA (Tag 5) and a HMA1-specific primer (hma1Rev), or with two HMA1-specific primers (hma1Fwd/hma1Rev), with two pairs of PAA1-specific primers (paa1Fwd/wtpaa1rev or paa1Fwd/ mutpaa1Rev) to detect native and mutated forms of the PAA1 allele. (B) The presence of full-length HMA1 transcripts and mutated PAA1 transcripts was analysed by RT-PCR on RNA extracted from leaves. Primers used for HMA1 detect only the WT transcript since primers are located on either side of the T-DNA insertion. Specific primers for PAA1 detect either the native or the mutated PAA1 transcript. ACTIN2 transcript was used as a control. (C) The absence of the HMA1 protein in homozygous hma1 mutant and hma1 paa1 double-mutated lines (dm1 and dm2) was validated by Western blotting using a specific antibody. Each lane contains 25 μg total membrane proteins. The arrows indicate the position of HMA1 (larger band, below two faint cross-reacting signals). (D and E) Phenotypes of F3 hma1 paa1 double-mutant lines obtained after self-fertilization of hh/pP line 31 (D) and hH/pp line 136 (E). Plants were first grown on MS medium in the presence of kanamycin before transferring to soil. Lines with HH/pp genotype are not resistant to kanamycin and thus are not recovered in the F3 generation.
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validated by PCR (Fig. 1A) were selected for further characterization. As shown in Fig. 1B, HMA1 transcripts were absent from dm1 and dm2 and only the mutated form of the PAA1 transcript was detected in these lines. As expected, HMA1 was not detected in dm1 and dm2 mutants (Fig. 1C). In autotrophic conditions, dm1 and dm2 mutants could grow and produce viable seeds when placed on soil and watered with tap water containing 30 μM CuSO4 (Fig. 1D, E). However, in these conditions, the reproductive cycle of dm1 and dm2 was at least as long as that of the paa1 mutant (i.e. ~4 months), contrasting with the 2-month reproductive cycle of a WT strain. In these conditions, the hma1 mutant behaved like the WT line. In heterotrophic conditions with limited (0.1 μM) Cu and at a light intensity of 100 μmol m–2 s–1, dm mutants had a reduced growth rate and increased photosensitivity compared to WT (Fig. 2A, left). This phenotype could be partially rescued by increasing the Cu concentration in the medium to 5 μM (Fig. 2A, right). In these conditions, the behaviour of the double mutants and paa1 mutants were very similar (Shikanai et al., 2003). However, in the presence of Cu (5 μM), the growth rate of the double mutants was slightly slower than that of the paa1 mutant (Supplementary Fig. S2, available at JXB online).
Chloroplast copper homeostasis | Page 5 of 12
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Fig. 2. Impact of Cu (A) and light (B) on growth of the double hma1 paa1 mutant. Seeds from WT lines (Ws and Ler), hma1 and paa1 mutants, and the two independent double hma1 paa1 mutants were sown on plates containing MS salts plus 0.5 % sucrose (suc) in the presence of various Cu concentrations (0.1 μM, 5, 20 μM). Plates were placed under light intensities ranging from 50 to 200 μmol m–2 s–1.
were grown in soil (Fig. 1D, E), probably because only part of the Cu added to soil cultures remains available for plants. Expression and activity of Cu/ZnSOD and PC were analysed in the different lines grown in soil (autotrophic conditions) containing Cu. Compared to the WT plants, CSD2 protein level significantly decreased in the paa1 mutant (Fig. 3A) but not in the double mutant.
In these growth conditions, Cu/ZnSOD activity decreased significantly in single hma1 and paa1 mutants compared to WT plants (Fig. 3B), although to a lesser degree than previously observed for plants grown in the absence of Cu (Shikanai et al., 2003; Seigneurin-Berny et al., 2006). In the two double mutants, Cu/ZnSOD activity (CSD1+CSD2) was lower than in either WT plants or single mutants (Fig. 3B),
Page 6 of 12 | Boutigny et al.
B 1.2
1.2 1.0
CSD activity (a.u.)
amount CSD2 (a.u.)
A *
0.8 0.6 0.4 0.2 0.0
*
1.0
*
0.8
*
**
dm1
dm2
0.6 0.4 0.2 0.0
WT Ws
hma1
WT Ler
paa1
dm1
dm2
WT Ws
hma1
WT Ler
paa1
hma1 paa1
hma1 paa1
1.4
amount PETE2 (a.u.)
amount PETE1 (a.u.)
1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 WT Ws
hma1
WT Ler
paa1
dm1
dm2
hma1 paa1
1.2 1.0 0.8
*
0.6
* **
0.4
**
*
*
0.2 0.0 WT Ws
hma1
WT Ler
paa1
dm1
dm2
hma1 paa1
D 1.4 PC/P700 (r.u.)
1.2 1.0 0.8 0.6 0.4 0.2 0.0 WT Ler
hma1
paa1
dm1
dm2
hma1 paa1 Fig. 3. Expression and activity of Cu/ZnSOD and PC in the hma1 paa1 double mutant. (A and C) Expression of CSD2 and PC isoforms. Soluble protein extracts were prepared from leaves of the different lines grown in soil in the presence (light grey) or absence (dark grey) of Cu. Proteins (25 μg per extract) were separated by SDS-PAGE. CSD2 and PC expression was analysed by Western blotting using antibodies raised against CSD2 (A) and PC (C). This last antibody detects both PETE1 and PETE2 PC isoforms. Band intensity was quantified using ImageJ software and normalized relative to the signal detected in the WT Ler sample. Values are mean of three experiments. (B) Cu/ZnSOD activity (Cu/ZnSOD CDS1 and CSD2). The same samples (10 μg) were analysed on native PAGE and SOD activity was determined by in-gel assay. The intensity of detected signals was quantified using Quantity One software. To compare activity levels across the different lines, values were normalized relative to the value obtained for the WT Ler sample. Values are mean of four experiments. Error bars indicate standard deviation. Statistical comparison was performed using Student’s t-test. Asterisks denote significant differences between the different lines and the WT Ler line (*P
Research paper
HMA1 and PAA1, two chloroplast-envelope PIB-ATPases, play distinct roles in chloroplast copper homeostasis
1
CNRS, Laboratoire de Physiologie Cellulaire et Végétale, UMR 5168, 17 rue des Martyrs, F-38054 Grenoble, France Université Grenoble Alpes, Laboratoire de Physiologie Cellulaire et Végétale, F-38054 Grenoble, France 3 CEA, Direction des Sciences du Vivant, Institut de Recherches en Technologies et Sciences pour le Vivant, Laboratoire de Physiologie Cellulaire et Végétale, F-38054 Grenoble, France 4 INRA, Laboratoire de Physiologie Cellulaire et Végétale, USC1359, 17 rue des Martyrs, F-38054 Grenoble, France 5 Biology Department, Colorado State University, Fort Collins, Colorado 80523, USA 2
* To whom correspondence should be addressed. E-mail: [email protected] Received 20 September 2013; Revised 25 November 2013; Accepted 19 December 2013
Abstract Copper is an essential micronutrient but it is also potentially toxic as copper ions can catalyse the production of free radicals, which result in various types of cell damage. Therefore, copper homeostasis in plant and animal cells must be tightly controlled. In the chloroplast, copper import is mediated by a chloroplast-envelope PIB-type ATPase, HMA6/ PAA1. Copper may also be imported by HMA1, another chloroplast-envelope PIB-ATPase. To get more insights into the specific functional roles of HMA1 and PAA1 in copper homeostasis, this study analysed the phenotypes of plants affected in the expression of both HMA1 and PAA1 ATPases, as well as of plants overexpressing HMA1 in a paa1 mutant background. The results presented here provide new evidence associating HMA1 with copper homeostasis in the chloroplast. These data suggest that HMA1 and PAA1 behave as distinct pathways for copper import and targeting to the chloroplast. Finally, this work also provides evidence for an alternative route for copper import into the chloroplast mediated by an as-yet unidentified transporter that is neither HMA1 nor PAA1. Key words: Arabidopsis, chloroplast, copper, envelope transporter, metal homeostasis, PIB ATPase.
Introduction Copper (Cu) is an essential micronutrient for plants and plays key roles in processes such as photosynthesis (Burkhead et al., 2009). However, Cu can be toxic either by catalysing the production of free radicals damaging for biomolecules or by binding to sulphhydryl groups in proteins, resulting in their inactivation. Therefore, Cu homeostasis must be finely tuned to adapt to Cu availability in environment and to respond to cell requirements in Cu. Cu acquisition in plant cells is mediated by the high-affinity transporter COPT-1 (Sancenon et al., 2004) and maybe the ZIP transporter ZIP2 (Puig et al., 2007a). Within plant cells, Cu is sequestered by metallothioneins or nicotianamine, but
also transported either to organelles or to the extracellular space by COPT transporters, ZIP transporters (Wintz et al., 2003), and PIB-type ATPases (or heavy metal ATPase, HMA). Some ions are captured by Cu chaperones that deliver Cu to specific protein targets to control subcellular Cu distribution (Hänsch and Mendel, 2009). The Arabidopsis genome encodes for over 30 proteins related to yeast and mammalian metallochaperones, but only a few of them have been characterized so far. The Arabidopsis Cu-chaperones Atx1 and CCH, orthologues of the yeast Atx1 chaperone, are thought to interact with the PIB-type ATPases AtHMA5 and AtHMA7 (Himelblau et al., 1998; Andrés-Colás et al., 2006;
© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: [email protected]
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Sylvain Boutigny1,2,3,4, Emeline Sautron1,2,3,4, Giovanni Finazzi1,2,3,4, Corinne Rivasseau1,2,3,4, Annie Frelet-Barrand1,2,3,4, Marinus Pilon5, Norbert Rolland1,2,3,4 and Daphné Seigneurin-Berny1,2,3,4,*
Page 2 of 12 | Boutigny et al. during evolution, allowing the acquisition of diverse metal specificities. Among the eukaryotic PIB-4 ATPases, exclusively found in plants and algae, only AtHMA1 and HvHMA1 from Arabidopsis and barley, respectively, have been studied so far. Previous work by this study group provided evidence for the following: (i) AtHMA1 can transport Cu and Zn in yeast; (ii) lack of AtHMA1 affects chloroplast Cu levels; and (iii) apparent AtHMA1 activity in the chloroplast envelope is enhanced by Cu (Seigneurin-Berny et al., 2006). Later, experiments carried out in yeast concluded that AtHMA1 could effectively transport Cu2+ and Zn2+ but also Ca2+, Cd2+, and Co2+ (Moreno et al., 2008). Another study combining in planta analysis and expression in yeast, suggested that AtHMA1 might export Zn2+ from the chloroplast (Kim et al., 2009). Finally, HvHMA1 was also proposed to export Zn but also Cu from barley plastids (Mikkelsen et al., 2012). From these four studies, it emerges that HMA1 can transport a broad range of divalent cations, probably depending on the physiological conditions. However, the direction of this transport still remains unclear. The phenotype of the paa1 mutant can be partially rescued by Cu supplementation, suggesting that another Cu transporter is present in the chloroplast envelope. HMA1 has been presented as a candidate for mediating this alternative low-affinity Cu pathway (Pilon et al., 2006). Analysis of the hma1 mutant showed that PC function is not impaired (Seigneurin-Berny et al., 2006) suggesting that, in agreement with its defined role, PAA1 supplies Cu for PC. In other words, there may be at least two Cu transporters in the chloroplast envelope. In order to investigate potential functional redundancy between PAA1 and AtHMA1, the current work generated plants in which both ATPases are mutated (hma1 paa1 double mutant). Furthermore, plants overexpressing AtHMA1 in the absence of PAA1 were produced, and the phenotypes of these plant lines were compared to those of wild-type (WT) plants and single hma1 or paa1 mutants.
Materials and methods Plant material and growth conditions Arabidopsis thaliana plants, Wassilevskija (Ws) and Lansberg (Ler) backgrounds, were germinated in Petri dishes containing solidified medium (Murashige and Skoog, MS, 1%, w/v, agarose) for 2 weeks before transfer to soil. Plants were then grown in culture chambers at 22 °C (10/14 light/dark cycle) with a light intensity of 100 μmol m–2 s–1 in standard conditions. Soil cultures were always watered with tap water containing 30 μM CuSO4 except when mentioned. For phenotypic analyses, seeds were germinated in plates containing MS salts in the absence or presence of CuSO4 at various concentrations under different light intensities, with 0.5% (w/v) sucrose in heterotrophic conditions or without sucrose for autotrophic conditions. The Arabidopsis mutant hma1 (line DRC42; Seigneurin-Berny et al., 2006) and the Arabidopsis mutant paa1 (paa1.1; Shikanai et al., 2003) were already available. The hma1 mutant is an insertional T-DNA mutant that carries the nptII selection marker (kanamycin resistance), while the paa1.1 mutant has a nonsense mutation in the eighth exon leading to truncation of the C-terminal region containing the ion-transduction, phosphorylation, and ATP-binding domains.
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Puig et al., 2007b). Plant cells also contain an orthologue of the yeast CCS chaperone, responsible for supplying Cu to Cu/ Zn superoxide dismutases (Cu/ZnSODs) located in the cytosol, the peroxisome, and the chloroplast (Chu et al., 2005; Huang et al., 2012). In Arabidopsis chloroplasts, the major targets for Cu delivery are the stromal Cu/ZnSOD (CSD2), involved in the dismutation of superoxide to hydrogen peroxide, and plastocyanin (PC), a thylakoid protein which is involved in photosynthetic electron transfer. The Arabidopsis genome codes for two PC isoforms, PETE1 and PETE2. PETE2 is the moreabundant isoform and accumulates at high levels in response to Cu feeding, possibly to buffer the excess Cu. PETE1, the less-abundant isoform, is essential for electron transport under Cu-deficient conditions (Abdel-Ghany, 2009). PC is essential for photosynthesis; thus, for plants grown in autotrophic conditions, Cu delivery to the thylakoid lumen is a priority (Weigel et al., 2003). Cu delivery into the chloroplast requires a chloroplast-envelope PIB-type ATPase, AtHMA6/ PAA1 (Shikanai et al., 2003). AtHMA8/PAA2, a second PIBATPase, is found in the thylakoid membrane, where it imports Cu for the maturation of functional PC (Abdel-Ghany et al., 2005). AtHMA1, another chloroplast-envelope PIB-ATPase, could also import Cu into the chloroplast (Seigneurin-Berny et al., 2006). Characterizations of the phenotypes of single paa1, paa2, and hma1 knockouts suggest that: (i) PAA1 could be the main route of Cu supply to CSD2 and to the thylakoid transporter, PAA2, and through this, to PC isoforms; and (ii) AtHMA1 would provide Cu to CSD2 and could play an important role during stress conditions. However, AtHMA1 may also transport other metal ions (Moreno et al., 2008; Kim et al., 2009; Mikkelsen et al., 2012; see Supplementary Table S1 available at JXB online). Based on the presence of conserved residues in transmembrane helices 6, 7, and 8, PIB-type ATPases have been divided into five subgroups of different ionic selectivity (Arguëllo, 2003). The Arabidopsis PIB-ATPases PAA1 and PAA2 belong to the IB-1 subgroup, which comprises prokaryotic and eukaryotic Cu+-ATPases such as CopA from Escherichia coli and Enterococcus hirae or the human ATPases ATP7A and ATP7B (for recent reviews, see Lutsenko et al., 2008; Rosenzweig and Argüello, 2012). In Arabidopsis, the functions of PAA1 and PAA2 were first studied in planta (Shikanai et al., 2003; Abdel-Ghany et al., 2005). A recent study combining heterologous expression in Lactococcus lactis and in vitro enzymic assays, demonstrated that PAA1 is a Cu+ATPase (Catty et al., 2011). AtHMA1, the third chloroplast PIB-ATPase, belongs to subgroup IB-4, which was initially described as gathering putative Co2+-ATPases (Arguëllo, 2003). Some members of this subgroup have been recently characterized and among them, CzcP from Cupriavidus metallidurans (Scherer and Nies, 2009) and CtpD from Mycobacterium smegmatis (Raimunda et al., 2012) showed a broader metal specificity (Zn2+, Cd2+, Co2+, and Ni2+). Raimunda and collaborators (2012) highlighted that PIB-4 ATPases share significant sequence similarity (48–66%), but cluster in distinct phylogenetic branches. This suggests that the genes coding for these ancient proteins diverged early
Chloroplast copper homeostasis | Page 3 of 12 Membrane protein extracts from leaves Leaves were first homogenized in 0.22 mM tetra-sodium pyrophosphate and 1 g l–1 BSA and centrifuged at 100 000 g for 20 min at 4 °C. The pellet was washed with 50 mM Tris-HCl (pH 6.8), resuspended in 50 mM Tris-HCl (pH 6.8) and 1 % (v/v) SDS, and then centrifuged for 10 min at 14 000 g and 4 °C to eliminate insoluble material. Protein content was estimated using the BIO-RAD protein assay reagent.
Construction of vector for stable HMA1 expression in the Arabidopsis paa1.1 mutant The HMA1 cDNA sequence was excised from a pBluescript KS– vector (described in Seigneurin-Berny et al., 2006) by BglII and SalI and inserted into a BamHI–SalI-digested pFP101 binary vector the GFP gene marker for the selection of transformed plants (Bensmihen et al., 2004). The resulting plasmid was prepared using a QIAfilter Plasmid Midi Kit (Qiagen Laboratories, Germany) before being used for Agrobacterium tumefaciens transformation. Arabidopsis transformation paa1.1 mutant plants were transformed according to Clough and Bent (1998) by dipping the floral buds of 4-week-old plants into a surfactant-containing (Silwett L-77) A. tumefaciens C58 inoculum. Transgenic Arabidopsis seeds were selected based on GFP expression driven by the At2S3 seed-specific promoter. Only lines segregating 3:1 for GFP expression and expressing the recombinant protein were selected for further analysis. Primary transformants were then self-pollinated to obtain plants homozygous for the insertion. Production of the hma1 paa1 double mutant The hma1 paa1 double mutant was obtained after pollination of the paa1.1 mutant (Lansberg background) with pollen from the hma1 mutant, DRC42 (Ws background). F1 progeny were grown and allowed to self-fertilize. F2 progeny were grown on kanamycin-supplemented MS medium with sucrose to eliminate all plants containing two WT HMA1 alleles (Supplementary Fig. S1B, available at JXB online). The genotypes of F2 individuals were determined by PCR for HMA1 and PAA1 WT alleles and for the T-DNA insertion in the hma1 mutant allele or the presence of a point mutation for the paa1 allele (primers listed in Supplementary Table S2, available at JXB online). All genotypes were validated by two PCRs performed on two DNA extracts from different leaves of the same line (a total of four PCRs). Analysis of transcripts Total RNA was extracted from leaf tissue using a RNeasy Mini Kit (Qiagen). cDNA was obtained using the Ambion M-MLV Reverse Transcriptase (Applied Biosystems). The sets of primers used to detect HMA1 and PAA1 transcripts are listed in Supplementary Table S2, available at JXB online. Chloroplast purification from Arabidopsis Arabidopsis protoplasts were isolated from Arabidopsis rosette leaves as described by Kunst (1998) with the following modifications. Leaves
Measuring metal ions Aliquots of purified chloroplast fractions corresponding to 10–40 μg of chlorophyll were mineralized and later dissolved in 1% (v/v) HNO3. Elemental analysis was performed on an inductively coupled plasma mass spectrometer (ICP-MS, Hewlett-Packard 4500 Series, Agilent Technologies, Massy, France) equipped with a Babington nebulizer and a Peltier-cooled double-pass Scott spray chamber. The device was calibrated at 24, 25, and 26 m/z for Mg, 43 and 44 m/z for Ca, 54 and 57 m/z for Fe, 63 and 65 m/z for Cu, and 64, 66 and 67 m/z for Zn, using standard solutions in 1% HNO3. ICP-MS analyses were performed in triplicate after appropriate sample dilution. The quantification results for Mg, Ca, Fe, Cu, and Zn were the mean values obtained for isotopes 24, 44, 57, 65, and 66, respectively. In vivo spectroscopic estimation of PC/photosystem I ratios In vivo spectroscopic estimation of PC/photosystem I (PSI) ratios was performed in vivo in WT and mutant lines using a JTS-10 spectrophotometer (Biologic, France). Leaves were illuminated with a far-red source at 720 nm to completely oxidize PSI and its donor pool. Absorption changes were detected by discrete flashes (duration 10 μs) delivered by two light-emitting diodes. Light was filtered to select P700 and PC redox changes. Absorption changes associated with P700 redox changes were computed as ΔI/I 705 nm– ((ΔI/I 870 nm)/2). DI/I PC was computed as ΔI/I 870 nm+((ΔI/I 705 nm)/10), as described previously (Seigneurin-Berny et al., 2006). Superoxide dismutase activity Whole leaves (~80 mg) were ground in liquid nitrogen and suspended in 200 μl of 50 mM KH2PO4 pH 7.8, 1 mM EDTA, and 0.1% (w/v) BSA. The suspension was centrifuged at 14 000 g for 10 min at 4 °C and soluble proteins were recovered in the supernatant. SOD activity was measured using an in-gel assay, as described by McCord (1999) using 10 μg soluble proteins. In the gel, the intensity of each band was measured using Quantity One software (Bio-Rad, Hercules, CA, USA). For each line, statistical analyses were performed on protein extracts from different plants grown in three independent experiments.
Results Characterization of the hma1 paa1 double mutant in Arabidopsis Double mutants were obtained from single hma1 mutant (Ws background) and paa1 mutant (Ler background; Supplementary Fig. S1, available at JXB online). Two homozygous double mutants, dm1 and dm2, whose genotypes were
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SDS-PAGE and Western blot analyses These procedures were carried out as previously described (Seigneurin-Berny et al., 2006), using a rabbit polyclonal antibody raised against amino acids 213–368 of HMA1 (1/1000 dilution) and antibodies against CSD2 and PC (Agrisera AB) used at 1/5000 and 1/10 000, respectively. For each line, statistical analyses were performed on protein extracts from different plants grown in three independent experiments.
(2–3 g) were cut into small pieces before infiltration with 30 ml protoplast isolation buffer containing 1.6% (w/v) cellulase and 0.25% (w/v) pectolyase (both from Kyowa Hakko, Tokyo). Samples were incubated at 28 °C for 2 h. After purification on Percoll, protoplasts were diluted in 10 ml protoplast storage buffer without CaCl2 and centrifuged for 5 min at 100 g. Protoplasts were then diluted in 5 ml protoplast lysis buffer and lysed by passing them through a 15-μm mesh nylon net. The resulting suspension was then layered on top of a cold preformed Percoll gradient (Seigneurin-Berny et al., 2008) and centrifuged at 13,300 g for 10 min. Intact chloroplasts were collected from the gradient (lower band), diluted 3-fold in resuspension buffer (0.4 M sorbitol, 20 mM HEPES-KOH pH 7.6, 2.5 mM EDTA, 10 mM NaHCO3, 0.15%, w/v, BSA) and centrifuged for 90 s at 1465 g. Chlorophyll levels were determined by spectrophotometry (Bruinsma, 1961).
Page 4 of 12 | Boutigny et al.
A
Line 31 lineage
The hma1 mutant exhibits a photosensitivity phenotype under high light (Seigneurin-Berny et al., 2006). In the current study, in MS medium supplemented with sucrose and in the presence of 10 μM Cu, the double mutant could grow under a light intensity of up to 150 μmol m–2 s–1 (data not shown). At higher light intensities, both double mutants and paa1 mutants could only grow if Cu was increased to 20 μM in the medium. In these conditions, the double mutant was more light sensitive than the paa1 mutant (Fig. 2B), as shown by photobleaching of the leaves (variegated phenotype with white areas), a phenotype similar to the one of the hma1 mutants (for light intensity >280 μmol m–2 s–1). In autotrophic conditions (on MS plates without sucrose, or in soil) and under light intensities more than 200 μmol m–2 s–1, the paa1 mutant and the double mutants stopped growing at the cotyledon stage and then died. This was associated with an enhanced photosensitivity phenotype, even in the presence of 20 μM Cu (data not shown). In autotrophic conditions but under low light (50 μmol m–2 s–1), Cu concentrations higher than 5 μM were required to partially restore a WT phenotype for the paa1 and double mutants, compared to the lower Cu concentrations required for these plants to grow in similar but heterotrophic conditions. The partial rescue obtained by using high Cu concentrations (30 μM) was not observed when plants
B
Line 136 lineage
WT Ws
hma1
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hma1 paa1 dm1 dm2
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hma1 paa1 dm2 dm1 pp/hh Pp/hh pp/Hh pp/hh
PAA1 WT Actin 2
paa1Fwd/mutpaa1rev paa1Fwd/wtpaa1rev
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Tag5/hma1rev
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E
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Fig. 1. Validation of the genotype of homozygous hma1 paa1 double mutant lines dm1 and dm2. (A) The genotypes of F3 lines obtained by self-crossing two independent heterozygous double mutants (lines 31 and 136) were analysed by PCR using a primer for T-DNA (Tag 5) and a HMA1-specific primer (hma1Rev), or with two HMA1-specific primers (hma1Fwd/hma1Rev), with two pairs of PAA1-specific primers (paa1Fwd/wtpaa1rev or paa1Fwd/ mutpaa1Rev) to detect native and mutated forms of the PAA1 allele. (B) The presence of full-length HMA1 transcripts and mutated PAA1 transcripts was analysed by RT-PCR on RNA extracted from leaves. Primers used for HMA1 detect only the WT transcript since primers are located on either side of the T-DNA insertion. Specific primers for PAA1 detect either the native or the mutated PAA1 transcript. ACTIN2 transcript was used as a control. (C) The absence of the HMA1 protein in homozygous hma1 mutant and hma1 paa1 double-mutated lines (dm1 and dm2) was validated by Western blotting using a specific antibody. Each lane contains 25 μg total membrane proteins. The arrows indicate the position of HMA1 (larger band, below two faint cross-reacting signals). (D and E) Phenotypes of F3 hma1 paa1 double-mutant lines obtained after self-fertilization of hh/pP line 31 (D) and hH/pp line 136 (E). Plants were first grown on MS medium in the presence of kanamycin before transferring to soil. Lines with HH/pp genotype are not resistant to kanamycin and thus are not recovered in the F3 generation.
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validated by PCR (Fig. 1A) were selected for further characterization. As shown in Fig. 1B, HMA1 transcripts were absent from dm1 and dm2 and only the mutated form of the PAA1 transcript was detected in these lines. As expected, HMA1 was not detected in dm1 and dm2 mutants (Fig. 1C). In autotrophic conditions, dm1 and dm2 mutants could grow and produce viable seeds when placed on soil and watered with tap water containing 30 μM CuSO4 (Fig. 1D, E). However, in these conditions, the reproductive cycle of dm1 and dm2 was at least as long as that of the paa1 mutant (i.e. ~4 months), contrasting with the 2-month reproductive cycle of a WT strain. In these conditions, the hma1 mutant behaved like the WT line. In heterotrophic conditions with limited (0.1 μM) Cu and at a light intensity of 100 μmol m–2 s–1, dm mutants had a reduced growth rate and increased photosensitivity compared to WT (Fig. 2A, left). This phenotype could be partially rescued by increasing the Cu concentration in the medium to 5 μM (Fig. 2A, right). In these conditions, the behaviour of the double mutants and paa1 mutants were very similar (Shikanai et al., 2003). However, in the presence of Cu (5 μM), the growth rate of the double mutants was slightly slower than that of the paa1 mutant (Supplementary Fig. S2, available at JXB online).
Chloroplast copper homeostasis | Page 5 of 12
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Fig. 2. Impact of Cu (A) and light (B) on growth of the double hma1 paa1 mutant. Seeds from WT lines (Ws and Ler), hma1 and paa1 mutants, and the two independent double hma1 paa1 mutants were sown on plates containing MS salts plus 0.5 % sucrose (suc) in the presence of various Cu concentrations (0.1 μM, 5, 20 μM). Plates were placed under light intensities ranging from 50 to 200 μmol m–2 s–1.
were grown in soil (Fig. 1D, E), probably because only part of the Cu added to soil cultures remains available for plants. Expression and activity of Cu/ZnSOD and PC were analysed in the different lines grown in soil (autotrophic conditions) containing Cu. Compared to the WT plants, CSD2 protein level significantly decreased in the paa1 mutant (Fig. 3A) but not in the double mutant.
In these growth conditions, Cu/ZnSOD activity decreased significantly in single hma1 and paa1 mutants compared to WT plants (Fig. 3B), although to a lesser degree than previously observed for plants grown in the absence of Cu (Shikanai et al., 2003; Seigneurin-Berny et al., 2006). In the two double mutants, Cu/ZnSOD activity (CSD1+CSD2) was lower than in either WT plants or single mutants (Fig. 3B),
Page 6 of 12 | Boutigny et al.
B 1.2
1.2 1.0
CSD activity (a.u.)
amount CSD2 (a.u.)
A *
0.8 0.6 0.4 0.2 0.0
*
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*
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*
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* **
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*
*
0.2 0.0 WT Ws
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WT Ler
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dm2
hma1 paa1
D 1.4 PC/P700 (r.u.)
1.2 1.0 0.8 0.6 0.4 0.2 0.0 WT Ler
hma1
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dm1
dm2
hma1 paa1 Fig. 3. Expression and activity of Cu/ZnSOD and PC in the hma1 paa1 double mutant. (A and C) Expression of CSD2 and PC isoforms. Soluble protein extracts were prepared from leaves of the different lines grown in soil in the presence (light grey) or absence (dark grey) of Cu. Proteins (25 μg per extract) were separated by SDS-PAGE. CSD2 and PC expression was analysed by Western blotting using antibodies raised against CSD2 (A) and PC (C). This last antibody detects both PETE1 and PETE2 PC isoforms. Band intensity was quantified using ImageJ software and normalized relative to the signal detected in the WT Ler sample. Values are mean of three experiments. (B) Cu/ZnSOD activity (Cu/ZnSOD CDS1 and CSD2). The same samples (10 μg) were analysed on native PAGE and SOD activity was determined by in-gel assay. The intensity of detected signals was quantified using Quantity One software. To compare activity levels across the different lines, values were normalized relative to the value obtained for the WT Ler sample. Values are mean of four experiments. Error bars indicate standard deviation. Statistical comparison was performed using Student’s t-test. Asterisks denote significant differences between the different lines and the WT Ler line (*P
