- © 2010 American Society of Plant Biologists
Abstract
Manganese (Mn) is an essential element, acting as cofactor in numerous enzymes. In particular, a Mn cluster is indispensable for the function of the oxygen-evolving complex of photosystem II. Metal transporters of the Natural Resistance-Associated Macrophage Protein (NRAMP) family have the ability to transport both iron and Mn. AtNRAMP3 and AtNRAMP4 are required for iron mobilization in germinating seeds. The results reported here show that, in adult Arabidopsis (Arabidopsis thaliana) plants, AtNRAMP3 and AtNRAMP4 have an important role in Mn homeostasis. Vacuolar Mn accumulation in mesophyll cells of rosette leaves of adult nramp3nramp4 double mutant plants was dramatically increased when compared with the wild type. This suggests that a considerable proportion of the cellular Mn pool passes through the vacuole and is retrieved in an AtNRAMP3/AtNRAMP4-dependent manner. The impaired Mn release from mesophyll vacuoles of nramp3nramp4 double mutant plants is associated with reduced growth under Mn deficiency. However, leaf AtNRAMP3 and AtNRAMP4 protein levels are unaffected by Mn supply. Under Mn deficiency, nramp3nramp4 plants contain less functional photosystem II than the wild type. These data are consistent with a shortage of Mn to produce functional photosystem II, whereas mitochondrial Mn-dependent superoxide dismutase activity is maintained under Mn deficiency in both genotypes. The results presented here suggest an important role for AtNRAMP3/AtNRAMP4-dependent Mn transit through the vacuole prior to the import into chloroplasts of mesophyll cells.
The catalytic centers of numerous enzymes rely on the ability of some transition metals to undergo changes in their redox state. Although manganese (Mn) is involved as a cofactor in a range of additional biochemical pathways, the primary effect of Mn deficiency in photosynthetic organisms is a drop in photosynthetic activity (Marschner, 1995). The process of water splitting and oxygen evolution by PSII, which is a central component of oxygenic photosynthesis, requires a tetra-Mn cluster.
In plants, few of the mechanisms that enable the uptake, distribution, and storage of Mn have been characterized at the molecular level. Hyperpolarization-activated Ca2+ channels and members of the ZIP family have been proposed as pathways for Mn uptake in roots (Korshunova et al., 1999; Vert et al., 2002; White et al., 2002; Pedas et al., 2008). However, to our knowledge, no transporter specifically functioning in Mn uptake from soils has been reported in the root plasma membrane.
Most of the current knowledge on Mn compartmentation in plant cells comes from the analysis of the mechanisms of tolerance to Mn excess. Indeed, when accumulated in excess, Mn can be toxic and has been proposed to be sequestered in vacuoles and Golgi vesicles under these conditions (Marschner, 1995; Pittman, 2005). The analysis of the molecular mechanisms of Mn tolerance in Stylosanthes hamata, a tropical legume tolerant to high Mn, led to the cloning of the Mn-specific transport protein ShMTP8 (Delhaize et al., 2003). When expressed in Arabidopsis (Arabidopsis thaliana), ShMTP8 is targeted to the vacuolar membrane and confers Mn tolerance by mediating the sequestration of excess Mn in this compartment. In Arabidopsis, several pathways for Mn import into the vacuole have been identified. AtCAX2 and AtCAX4 Ca2+/H+ antiporters are able to transport Mn into the vacuole (Hirschi et al., 2000; Pittman et al., 2004; Koren'kov et al., 2006). In addition, AtVIT1 increases the Mn content of the vacuoles when expressed in yeast (Kim et al., 2006). AtVIT1 is thus a good candidate to mediate Mn import into vacuoles. Recently, the functional analysis of AtMTP11 and PtMTP11, the homologues of ShMTP8 in Arabidopsis and poplar (Populus trichocarpa), suggested the existence of an alternative pathway for Mn detoxification by vesicle-mediated transport of this metal (Delhaize et al., 2007; Peiter et al., 2007). In addition, overexpression of the Mn-transporting P2A-type ATPase, AtECA1, localized to the endoplasmic reticulum, increased Mn tolerance (Wu et al., 2002). Whether the vacuole represents an important site for the storage of Mn in plants grown under normal conditions is currently unknown. Moreover, little is known about the mechanisms involved in plant tolerance to Mn deficiency.
Recent work on barley (Hordeum vulgare) confirmed an important decrease in the abundance of functional PSII under Mn deficiency and revealed differences in the fluorescence induction kinetics and state transitions between Mn-efficient and Mn-inefficient genotypes (Husted et al., 2009). In barley, Mn deficiency also leads to increased water loss, likely through alteration of the leaf cuticle (Hebbern et al., 2009). Interestingly, in Chlamydomonas reinhardtii, another photosynthetic organism, in addition to causing a decrease in functional PSII abundance, Mn deficiency also impairs Mn-dependent superoxide dismutase (SOD) activity, leading to an increased sensitivity to oxidative stress (Allen et al., 2007). The cellular pathways for Mn transport and use under Mn-deficient conditions are mostly unknown. A recent study on the Mn-transporting P2A-type ATPase, AtECA3, localized to the Golgi apparatus, demonstrated the importance of Mn import into the Golgi apparatus under Mn deficiency (Mills et al., 2008).
Natural Resistance-Associated Macrophage Proteins (NRAMPs) represent another protein family likely to be involved in Mn transport in plants. Most NRAMPs characterized so far are able to transport a broad range of metals: Fe2+, Mn2+, Zn2+, Cd2+ (for review, see Colangelo and Guerinot, 2006; Nevo and Nelson, 2006). In bacteria, NRAMP homologues, MntH, function primarily as cellular Mn uptake transporters (Makui et al., 2000). Moreover, MntH gene expression is regulated at the transcriptional level through MntR, a Mn-sensing regulatory protein (Que and Helmann, 2000; Patzer and Hantke, 2001). In C. reinhardtii, NRAMP1 is up-regulated upon Mn deprivation (Allen et al., 2007). In yeast, NRAMP homologues, Smf1p and Smf2p, are involved in Mn uptake and recycling. They are coregulated at the posttranslational level by this metal (Liu and Culotta, 1999; Portnoy et al., 2000). The disruption of SMF2 leads to the loss of activity of the mitochondrial MnSOD and of Golgi Mn-dependent enzymes (Luk and Culotta, 2001).
In Arabidopsis, six members of the NRAMP family have been identified. Heterologous expression of the AtNRAMP1, -3, and -4 cDNAs in yeast mutants deficient for metal uptake indicated that these proteins are able to transport iron (Fe), Mn, and cadmium (Cd; Curie et al., 2000; Thomine et al., 2000). AtNRAMP3-GFP and AtNRAMP4-GFP fusion proteins are targeted to the vacuolar membrane (Thomine et al., 2003; Lanquar et al., 2005). In addition, AtNRAMP4 was identified in the vacuolar proteome of Arabidopsis mesophyll cells (Carter et al., 2004). AtNRAMP3 and AtNRAMP4 are strongly expressed during the early stages of germination, and the nramp3nramp4 double mutant displays a strong chlorotic phenotype when seeds are germinated in the absence of Fe supply in the medium (Lanquar et al., 2005). It was thus proposed that AtNRAMP3 and AtNRAMP4 play redundant roles in the export of Fe from the vacuole during seed germination. In this study, a detailed examination of the nramp3nramp4 double mutant adult plants indicated that AtNRAMP3 and AtNRAMP4 crucially contribute to Mn homeostasis. AtNRAMP3 and AtNRAMP4 are required for Mn mobilization from the vacuole of mesophyll cells in adult plants. As a consequence, nramp3nramp4 overaccumulate Mn in vacuoles under standard growth conditions, and nramp3nramp4 growth is strongly impaired under Mn deficiency. This growth phenotype is associated with a decrease in the number of functional PSIIs attributable to a shortage of Mn clusters, whereas mitochondrial MnSOD activity is maintained. The results presented show that AtNRAMP3 and AtNRAMP4 play a major role in the export of vacuolar Mn in photosynthetic tissues of adult plants. These results highlight the importance of Mn transit through vacuoles en route to its main usage site in chloroplasts.
RESULTS
AtNRAMP3 and AtNRAMP4 Are Involved in Mn Retrieval from the Vacuole of Mesophyll Cells
To gain insight into the functions of AtNRAMP3 and AtNRAMP4 in adult plants, the consequences of their absence were investigated in mesophyll cells from plants grown on soil for 6 weeks.
Intact mesophyll protoplasts were released from rosette leaves and, subsequently, the mesophyll protoplast preparation was subjected to gentle lysis and intact vacuoles were recovered. Microscopic observation of the protoplast preparation revealed that only mesophyll protoplasts were present, no other cell types could be recognized, and the vacuole preparation was of high purity. Furthermore, vacuolar pyrophosphatase (V-PPase), a vacuolar membrane protein, was enriched in the vacuole preparation in comparison with the protoplast from which they were extracted (Fig. 1A), whereas E37, a chloroplast envelope protein, was not detected (data not shown; Ferro et al., 2002).
AtNRAMP3 and AtNRAMP4 proteins reside on mesophyll vacuoles and are involved in the control of vacuolar Mn content. A, Immunoblot analysis of proteins from leaves, mesophyll protoplasts, and mesophyll vacuoles from wild-type and nramp3nramp4 mutant plants. The blots were probed with anti-NRAMP3 antibodies, anti-NRAMP4 antibodies, or antibodies raised against the V-PPase. B, Fe, Mn, and Zn concentrations in leaves (left), protoplasts (middle), and vacuoles (right) measured by ICP-AES. Gray bars represent the wild type, and white bars represent nramp3nramp4. Leaf metal concentration is expressed as μg g−1; protoplast and vacuole metal contents are expressed as pg per object. Values are means ± se of six independent experiments for the protoplast preparations and seven independent experiments for vacuoles. Asterisks denote significant differences based on Mann-Whitney U test between genotypes: * P < 0.05, *** P < 0.001. DW, Dry weight. All plants were grown on soil for 6 weeks.
AtNRAMP3 and AtNRAMP4 protein levels were examined in leaves, mesophyll protoplasts, and vacuoles by immunoblot using isoform-specific antibodies (Supplemental Fig. S1; Lanquar et al., 2005). In nramp3nramp4 leaves, protoplasts, or vacuoles, no signal corresponding to AtNRAMP3 or AtNRAMP4 was detected (Fig. 1A), confirming the absence of both proteins. In wild-type plants, AtNRAMP3 and AtNRAMP4 were detected in leaves and in mesophyll protoplasts. There was only a weak signal for AtNRAMP3 in mesophyll protoplasts. This indicates that AtNRAMP3 may be more abundant in other leaf tissues, such as the vasculature, than in mesophyll, in agreement with the AtNRAMP3 expression pattern (Thomine et al., 2003). In mesophyll vacuoles, the signals corresponding to AtNRAMP3 and AtNRAMP4 were about 20 times stronger than in mesophyll protoplasts, indicating an enrichment of these two membrane proteins (Fig. 1A). These results indicate that both AtNRAMP3 and AtNRAMP4 are localized in the vacuolar membrane of mesophyll cells of adult leaves, as reported previously in other cell types (Thomine et al., 2003; Lanquar et al., 2005).
To investigate the functions of AtNRAMP3 and AtNRAMP4 in mesophyll cells, the metal concentrations were measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES) in leaf tissues, mesophyll protoplasts, and mesophyll vacuoles of wild-type and nramp3nramp4 plants grown on soil. The concentrations of Fe, Mn, and zinc (Zn), three possible substrates of NRAMP3 and NRAMP4, were similar in nramp3nramp4 and wild-type leaves (Fig. 1B). This result is consistent with published data on younger seedlings (Lanquar et al., 2005). Similarly, no statistically significant differences were observed between metal contents of nramp3nramp4 and wild-type mesophyll protoplasts for Fe or Zn (Fig. 1B). By contrast, protoplasts isolated from nramp3nramp4 plants contained 2.1 to 2.8 times more Mn than wild-type protoplasts (Fig. 1B). Furthermore, analysis of nramp3nramp4 vacuoles revealed that they contained between 6.5 and 56 times more Mn than wild-type vacuoles (Fig. 1B). An additional experiment employing an independent nramp3nramp4 double knockout mutant in the Columbia accession confirmed the strongly increased vacuolar Mn content in nramp3nramp4 double mutants when compared with the wild type (Ravet et al., 2009b). Microscopic observations did not show any size difference between wild-type and nramp3nramp4 double mutant vacuoles. Furthermore, no statistically significant difference in vacuolar contents of Fe or Zn was detected between the double mutant and the wild type.
These results show that the loss of AtNRAMP3 and AtNRAMP4 proteins greatly enhances the sequestration of Mn in mesophyll vacuoles, while in wild-type plants, AtNRAMP3 and AtNRAMP4 continuously mediate the retrieval of Mn from the vacuole. Furthermore, these results suggest that net Mn storage in the vacuole of mesophyll cells is minor in wild-type Arabidopsis under normal growth conditions (about 10% of protoplast Mn) but that a substantial proportion of cellular Mn transits through the vacuole.
Adult nramp3nramp4 Plants Display a Conditional Growth Defect under Mn Deficiency
The results of this study indicate that in Mn-sufficient nramp3nramp4 mesophyll cells, Mn sequestration in the vacuole is strongly increased. To test whether this alteration in intracellular Mn distribution is associated with a defect in plant growth, plants were grown on perlite and watered either with a control Mn-sufficient nutrient solution containing 5 μm Mn (C) or with a nutrient solution lacking Mn (Mn-deficient conditions [−Mn]). After 9.5 weeks of growth, the biomass of nramp3nramp4, nramp3, and nramp4 mutant rosettes grown under control conditions was not significantly different from that of wild-type plants (Fig. 2, A, C, and D; Table I). The rosettes of adult nramp3nramp4 mutant appeared smaller because their leaves were curled: the leaf margins grow toward the abaxial side of the leaf (Fig. 2, A and B). The effect of Mn deficiency on the wild type was variable between different experiments: wild-type rosette biomass was not reduced in the experiment displayed in Figure 2C and reduced by 45% compared with control conditions in the experiments shown in Figure 2D and Table I. By contrast, nramp3nramp4 consistently displayed a much stronger growth reduction than the wild type under Mn-deficient conditions (63% in Fig. 2C to 86% in Fig. 2D; n = 4 independent biological replicates; Table I). Qualitatively similar results were obtained in hydroponic culture (Supplemental Fig. S3A). Under Mn deficiency, nramp3nramp4 mutant leaves were not curled but their size was reduced in comparison with the wild type (Fig. 2B). The rosette biomass of nramp3 and nramp4 single mutants was indistinguishable from the wild type (Fig. 2C), indicating that AtNRAMP3 and AtNRAMP4 are functionally redundant under Mn-deficient conditions. Measurements of dry biomass gave comparable results, indicating that the water content was similar between wild-type, nramp3, nramp4, and nramp3nramp4 plants grown either under control conditions or under Mn deficiency (data not shown). The growth phenotype of the nramp3nramp4 mutant under Mn deficiency could be rescued by introduction of AtNRAMP3 or AtNRAMP4 wild-type genes in the nramp3nramp4 mutant background (Fig. 2D; Supplemental Fig. S2).
Arabidopsis nramp3nramp4 plants display a growth reduction in the absence of Mn supply in the medium that is rescued by expression of the AtNRAMP3 or AtNRAMP4 gene. Plants were grown on perlite for 8 weeks (A and D) or for 9.5 weeks (B and C) on control medium (C; 5 μm Mn) or −Mn medium (0 μm Mn). A, Photographs of wild-type and nramp3nramp4 rosettes. B, Leaves of the wild type and nramp3nramp4. At left are younger leaves, and at right are older leaves. C, Fresh biomass of wild-type (WT), nramp3 (nr3), nramp4 (nr4), and nramp3nramp4 (nr3nr4) rosettes. Values are means ± se (n = 10–20 plants for each genotype in each condition). D, Fresh biomass of wild-type, nramp3nramp4, nramp3nramp4 AtNRAMP3 (nr3nr4+NR3), and nramp3nramp4 AtNRAMP4 (nr3nr4+NR4) rosettes. Values are means ± se (n = 12–24 plants for each genotype in each condition). Different letters denote statistically significant differences (P < 0.01 based on Kruskal-Wallis test for multiple comparisons).
Results shown correspond to means ± se with n number of plants weighted and n′ number of Fv/Fm measurements on different leaves. Results from one representative experiment out of five are shown. Asterisks denote significant differences based on Mann-Whitney U test: ** P < 0.01, * P < 0.05.
The Growth Defect of the nramp3nramp4 Mutant Is Not Associated with Reduced Mn Concentrations in Leaves or Mesophyll Protoplasts
To test whether the lower growth of nramp3nramp4 mutants under Mn deficiency is associated with a decreased Mn content in leaves or mesophyll cells, metal concentrations in leaves and metal contents of mesophyll protoplasts were measured by ICP-AES in plants grown under controlled mineral nutrition. Plants were watered either with control Mn-sufficient nutrient solution (C) or with a nutrient solution lacking Mn (−Mn). Upon growth under control conditions, no differences in leaf metal concentrations were observed between wild-type and nramp3nramp4 mutant plants for Fe, Mn, and Zn (Fig. 3A). In wild-type and nramp3nramp4 mutant plants, growth under Mn-deficient conditions resulted in a decrease in leaf Mn concentrations by about 90% when compared with plants grown under control conditions; in wild-type plants grown under Mn-deficient conditions, Fe concentrations were unchanged and Zn concentrations were slightly increased (Fig. 3A). Fe, Mn, and Zn concentrations in leaves of the nramp3nramp4 mutant grown under Mn-deficient conditions were significantly higher than in leaves of the wild type in the same conditions. No statistically significant differences were detected between either nramp3 or nramp4 single mutant and wild-type rosette metal concentrations for any of the three metals, Fe, Mn, or Zn (Supplemental Table S1).
Mn concentration in leaves and mesophyll protoplasts is equal or higher in nramp3nramp4 than in the wild type (WT). Wild-type (gray bars) and nramp3nramp4 (white bars) plants were grown on perlite for leaf metal concentration measurements and in hydroponic conditions for protoplast relative metal concentrations under control (C; 5 μm Mn) or Mn-deficient (−Mn) conditions for 8 weeks. A, Mean ± se of Fe, Mn, and Zn concentrations of leaves from plants grown on perlite (n = 3 samples each containing 10 leaves from three to four plants). DW, Dry weight. B, Mean ± se of Fe, Mn, and Zn relative concentrations of protoplasts from plants grown under hydroponic conditions (n = 3 protoplast preparations from independent biological replicates). Results are expressed as relative units (R.U): Fe, Mn, and Zn metal contents were normalized to the geometric mean of all other reliably measured element contents (calcium, copper, Fe, Mg, Mn, molybdenum, phosphorus, and Zn). Note that expressing the results as metal content/protoplast yielded similar results. Asterisks indicate statistically significant differences between the wild type and the nramp3nramp4 mutant (Mann-Whitney U test): ** P < 0.01.
For Fe and Zn, relative metal concentrations in mesophyll protoplasts isolated from plants grown under control or Mn-deficient conditions followed a similar pattern as leaf metal concentrations (Fig. 3B). Mn concentrations in nramp3nramp4 mutant protoplasts were 2- to 3-fold higher than in wild-type protoplasts under control (Mn-sufficient) conditions. This result is in agreement with the result obtained on mesophyll protoplasts from plants grown on soil (Fig. 1B). In contrast, under Mn deficiency, relative Mn concentrations in wild-type and nramp3nramp4 mutant protoplasts were not statistically different (Fig. 3B).
Taken together, these data demonstrate that Mn concentrations in nramp3nramp4 leaves or mesophyll protoplasts, respectively, are equal or higher than in the wild type, under both control and Mn-deficient conditions. This result excludes that a defective Mn supply to nramp3nramp4 mesophyll cells is responsible for the growth defect of this mutant under Mn deficiency.
The Level of Active PSII Is Lower in the nramp3nramp4 Mutant under Mn Deficiency
Mn is an essential cofactor of the water-splitting complex of PSII. Therefore, the growth defect observed in nramp3nramp4 under Mn-deficient conditions could be explained by a defect in the ability to split water, leading to a lower activity of photosynthesis. Since a defect in PSII can be detected through the quantification of chlorophyll fluorescence, the ratio of variable fluorescence to maximal fluorescence (Fv/Fm) was measured in wild-type and nramp3nramp4 plants (Maxwell and Johnson, 2000).
Fv/Fm values close to 0.8 obtained for wild-type, nramp3, nramp4, and nramp3nramp4 plants grown under control (Mn sufficient) conditions are consistent with the ratios reported for Arabidopsis and other higher plants (Björkman and Demmig, 1987; Table I; Supplemental Table S2; Supplemental Fig. S4). In Mn-deficient plants, the ratio dropped to 0.65 for wild-type plants and was further significantly reduced to 0.57 in the nramp3nramp4 plants (Table I). This indicates a decline of the photosynthetic efficiency under Mn deficiency, which is exacerbated in the nramp3nramp4 mutant compared with the wild type. Fv/Fm values were rescued to values equal to or higher than wild-type values in nramp3nramp4 mutant lines complemented by AtNRAMP3 or AtNRAMP4 (Supplemental Table S2). In the same conditions, Fv/Fm values in nramp3 and nramp4 single mutants, respectively, were not significantly lower than in the wild type, confirming the functional redundancy of these two genes (Supplemental Fig. S4). Upon prolonged growth in Mn-deficient conditions for an additional 3 weeks, Fv/Fm dropped to similarly low values for the wild type, nramp3, nramp4, and nramp3nramp4 (Supplemental Fig. S4).
To further investigate the effect of Mn deficiency on PSII, thermoluminescence measurements were performed on intact leaves of plants grown under Mn-deficient and Mn-sufficient conditions on perlite for 8 weeks. In thermoluminescence measurements, the emitted light originates from charge recombinations of trapped charge pairs within PSII (Inoue, 1996). The charge pairs involved can be identified by their emission temperature, which strongly depends on the redox potentials of the charge pairs. The most important thermoluminescence band for investigating the electron transfer within PSII is the B-band. Recombination of the S2 or S3 state of the oxygen-evolving complex at the donor side of PSII with the semireduced secondary quinone acceptor in PSII (QB−) yields the B-band at around 30°C (Rutherford et al., 1982). Figure 4A shows the changes in the intensity of the B-band at 24°C recorded from leaves of dark-adapted plants as dependent on the number of exciting single-turnover flashes. As expected for leaves, the highest intensity of the B-band was observed after the second flash, and the intensity of this band oscillates with a period of 4. This oscillation reflects the cycle of the oxidation states of the Mn cluster (S0–4; Fig. 4A). In nramp3nramp4 leaves, the intensity of the B-band was strongly reduced. A normal oscillation pattern was still observed during the first four flashes. This was dampened almost completely when more flashes were given (Fig. 4A). These results show that the function of the water-splitting complex is normal in nramp3nramp4 but that very few Mn clusters are functional in the nramp3nramp4 double mutant. A low intensity of the flash-induced B-band has been described previously in tobacco (Nicotiana tabacum) plants in which the level of PsbP was severely down-regulated by the RNA interference technique. In these plants, the Mn cluster of PSII is very unstable and disassembles quickly in the dark (Ifuku et al., 2005).
PSII activity and chloroplast Mn content are altered in Mn-deficient nramp3nramp4 plants. A, Intensity of thermoluminescence signals recorded from excised leaf pieces of dark-adapted Mn-deficient wild-type and nramp3nramp4 plants grown on perlite for 8 weeks under Mn-deficient conditions. Black and gray squares represent the amplitude in arbitrary units (a.u.) of the thermoluminescence (TL) B-band in wild-type and nramp3nramp4 leaf segments, respectively. Series of zero to eight single-turnover flashes were given at 1°C after 5 min of dark adaptation of the leaf segment at 20°C. Similar results were obtained in two independent biological replicates. B, Fluorescence recovery after photoinhibition measured on attached leaves of double mutant (left) and wild-type (right) plants grown on perlite for 8 weeks under Mn-deficient conditions. Top, plants grown in the absence of Mn (−Mn); bottom, plants grown in control conditions (C; 5 μm Mn). White arrows indicate the onset/termination of the measuring light, and black arrows indicate the onset/termination of the actinic light (white light at 2,000 μmol quanta m−2 s−1) and dim light (6 μmol quanta m−2 s−1). Similar results were obtained in two independent biological replicates. C, D1 (PsbA) protein levels in leaves of wild-type or nramp3nramp4 plants analyzed by immunoblot (top panel). Five, 2.5, 1.25, and 0.625 μg of total leaf proteins from 9.5-week-old plants grown on perlite under control (C; 5 μm Mn) or Mn-deficient (−Mn) conditions were loaded. Silver staining of proteins on gels run in parallel indicates that equal amounts of proteins were loaded (bottom panel). Similar results were obtained in three independent biological replicates. D, Relative Mn concentrations of intact chloroplasts isolated from wild-type (WT; black bars) or nramp3nramp4 (white bars) plants grown under control (C; 5 μm Mn) or Mn-deficient (−Mn) conditions for 8 weeks in hydroponic conditions. Mn concentrations were normalized to the geometric mean of all other reliably measured element concentrations (copper, phosphorus, and sulfur). Note that additional elements could not be used for normalization because of their concentrations in the chloroplast suspension buffer. Results are expressed as mean ratios ± se, taking the Mn content in chloroplast from wild-type plants grown under control conditions as a reference (n = 3 independent biological replicates).
In Mn-deficient nramp3nramp4 plants, PSII was highly susceptible to illumination with high light (Fig. 4B). When leaves of Mn-deficient nramp3nramp4 plants were exposed to high light intensities (2,000 μmol quanta m−2 s−1) for 1 h, the variable fluorescence was lowered to values below the fluorescence level measured with the low-intensity measuring light on dark-adapted leaves (Fig. 4B, top left). This indicates that the plastoquinone pool was partially reduced in the dark-adapted plants prior to the measurements (Bondarava et al., 2003). During a subsequent period of incubation in dim light for 3 h, the variable fluorescence probed by short flashes of actinic light did not recover, suggesting that the repair of photoinhibited PSII was not achieved. In the presence of Mn, the mutant showed a much lower loss of variable fluorescence (Fig. 4B, bottom left) and the recovery of the variable fluorescence was similar to that of the wild type grown under identical conditions (Fig. 4B, bottom right).
The level of D1 (PsbA), the core protein of PSII that binds the Mn cluster, was analyzed by immunoblotting in wild-type and nramp3nramp4 leaves from plants grown under control and Mn-deficient conditions. In plants grown under Mn-sufficient conditions, D1 protein levels were higher in nramp3nramp4 than in wild-type leaves (Fig. 4C; Supplemental Fig. S5). The amount of D1 was strongly reduced in Mn-deficient wild-type leaves and even further reduced in Mn-deficient nramp3nramp4 mutant plants (Fig. 4C; Supplemental Fig. S5). The decreased abundance of D1 protein in Mn-deficient nramp3nramp4 mutant leaves is in agreement with the observation of a lower number of active PSIIs in these plants (Table I; Supplemental Fig. S4).
The growth defect observed in the mutant under Mn deficiency is thus associated with a severe decrease in the number of functional PSII centers. To test whether the decrease in the number of functional PSIIs is associated with a shortage of Mn, we measured the relative Mn concentrations in chloroplasts isolated from mesophyll cells of wild-type and nramp3nramp4 plants grown under control and Mn-deficient conditions. Relative Mn concentrations were similar in chloroplasts from the wild type and nramp3nramp4 grown under control conditions. The Mn concentrations in chloroplasts of Mn-deficient wild-type plants was reduced to 50% of that of control plants (Fig. 4D). Under Mn deficiency, nramp3nramp4 mutant chloroplasts exhibited merely about half of the Mn concentration found in the wild type. This result suggests that a reduced availability of Mn to form Mn clusters in the chloroplasts is a cause of the lower number of active PSIIs in the mutant under Mn deficiency. Taken together, these data indicate that under Mn deficiency, the defect in Mn export from the vacuole in nramp3nramp4 perturbs Mn allocation to the chloroplast and leads to a lower number of active PSIIs.
Mn-Dependent SOD Activity Is Not Reduced in nramp3nramp4 Mesophyll Cells
As the defect in Mn export from the vacuoles of nramp3nramp4 reduces the number of functional PSIIs in leaves under Mn deficiency, we tested whether the function of the other quantitatively major Mn-dependent protein, MnSOD, is defective in this mutant. In-gel measurements of SOD activity were performed after separation by native PAGE of total protein extracts from leaves of wild-type and nramp3nramp4 mutants grown on perlite under control conditions, Fe deficiency, or Mn deficiency. Three bands were detected that displayed SOD activity (Fig. 5A). On the basis of inhibition by KCN (data not shown) and hydrogen peroxide (H2O2; Fig. 5B), the bands could be assigned to CuZnSOD (KCN and H2O2 sensitive), FeSOD (H2O2 sensitive), and MnSOD (insensitive to KCN and H2O2). Under Fe deficiency, FeSOD activity was strongly reduced in both wild-type and nramp3nramp4 mutant leaves (Fig. 5A). Under Mn deficiency, MnSOD was neither strongly decreased in the wild type nor in the nramp3nramp4 double mutant (Fig. 5B). These data show that in contrast to PSII function, MnSOD activity, which is known to be localized in the mitochondria, is maintained in the nramp3nramp4 mutant under Mn deficiency.
MnSOD activity is maintained in nramp3nramp4 Mn-deficient leaves. In-gel SOD activity staining was performed in the absence (A) or in the presence of 5 mm H2O2 (B). One hundred micrograms of total leaf proteins from 9.5-week-old plants grown on perlite under control (C; 5 μm Mn), Fe-deficient (−Fe), or Mn-deficient (−Mn) conditions was separated by native PAGE before activity staining. Similar results were obtained with two independent biological replicates.
Regulation of AtNRAMP3 and AtNRAMP4 Protein Levels
Previous results indicated that in seedlings, AtNRAMP3 and AtNRAMP4 expression levels are regulated in response to Fe status (Thomine et al., 2003; Lanquar et al., 2004, 2005). The results obtained in this study suggested an important role of AtNRAMP3 and AtNRAMP4 in Mn distribution to PSII under Mn deficiency. Therefore, the regulation of AtNRAMP3 and AtNRAMP4 protein levels was reexamined in adult plants grown under Fe- or Mn-deficient conditions. Wild-type and nramp3nramp4 plants were grown hydroponically under control (C), Fe-deficient (−Fe), and Mn-deficient (−Mn) conditions. In Fe-deficient plants, Fe concentrations were decreased by 85% in shoots and 90% in roots of both genotypes when compared with controls (Supplemental Fig. S3B), while Mn accumulation was increased as described previously (Vert et al., 2002). In Mn-deficient plants, Mn concentrations were decreased by 90% in shoots and 80% in roots when compared with controls (Supplemental Fig. S3B). Concentrations of other cations were unchanged. AtNRAMP3 and AtNRAMP4 protein levels were analyzed by immunoblot in three biological replicates. Under Fe deficiency, AtNRAMP3 protein levels increased in roots (in two out of three experiments) but did not show any regulation in shoots of adult plants (Fig. 6A). Levels of AtNRAMP4 protein increased under Fe deficiency in both shoots (in three out of three experiments) and roots (in two out of three experiments). These results are consistent with previous reports (Thomine et al., 2000; Lanquar et al., 2005). In contrast, these experiments did not reveal any significant regulation of AtNRAMP3 or AtNRAMP4 protein levels in response to Mn deficiency (Fig. 6A). In addition, staining of promoter AtNRAMP3::GUS (Thomine et al., 2003) or promoter AtNRAMP4::GUS (Lanquar et al., 2005) seedlings grown for 7 d on agar-based medium containing either no Mn (−Mn) or 0.1 mm Mn (C) did not exhibit any Mn-dependent regulation of AtNRAMP3 or AtNRAMP4 promoter activity (data not shown; Lanquar et al., 2005).
AtNRAMP3 and AtNRAMP4 protein levels are not regulated in response to Mn deficiency, but ferritins are accumulated in shoots of Mn-deficient nramp3nramp4 plants. A, AtNRAMP3 and AtNRAMP4 protein levels in roots and shoots of wild-type plants analyzed by immunoblot. Extracts of root and shoot proteins from nramp3nramp4 plants grown on control medium were loaded as a negative control. B, FER1 and IRT1 protein levels were monitored by immunoblot on nramp3nramp4 and wild-type plants. The FER1 antibody was used on blots of shoot extracts, and the IRT1 antibody was used on blots of root extracts. In both panels, 15 μg of total proteins from roots or shoots of plants grown for 6 weeks under hydroponic conditions was analyzed. Mn-deficient plants were grown without added Mn in the growth medium for 6 weeks. To induce Fe deficiency, after 4 weeks, plants were transferred in a medium containing 20 μm ferrozine in the absence of added Fe for 2 more weeks.
Ferritin Protein Levels Are Up-Regulated in nramp3nramp4 Mutants under Mn Deficiency
The specific up-regulation of AtNRAMP3 and AtNRAMP4 under Fe deficiency, although they are primarily involved in Mn mobilization from the vacuole in adult plants, is intriguing. To further investigate this apparent link between Fe and Mn homeostasis, the level of ferritin 1 protein, AtFER1, was monitored in leaves of wild-type and nramp3nramp4 plants grown hydroponically under control, Mn-deficient (−Mn), and Fe-deficient (−Fe) conditions. Immunoblotting to detect AtFER1 was performed on four independent biological replicates. Ferritins store Fe in nontoxic form in the stroma of chloroplasts (Briat et al., 1999). In plants grown under control conditions, the levels of AtFER1 in shoots were unchanged in the double mutant (in three out of four experiments; Fig. 6B). Under Fe deficiency, AtFER1 is neither detectable in the wild type nor in the double mutant (in all four experiments), as described previously (Ravet et al., 2009a). Under Mn deficiency, the amount of AtFER1 in wild-type plants was slightly decreased compared with the signal observed in control conditions (in two out of four experiments) or unchanged (in two out of four experiments). Interestingly, in Mn-deficient nramp3nramp4 plants, the amount of FER1 was consistently between 3.5 and seven times higher when compared with wild-type plants grown under the same conditions (in four out of four experiments; Fig. 6B). This increase was consistently observed in plants grown in perlite watered with – Mn medium, in plants grown hydroponically in −Mn medium, and in 2-week-old-plants grown on Mn-deficient agar medium. The up-regulation of AtFER1, a component of Fe homeostasis in the shoots of nramp3nramp4 mutants under Mn deficiency, suggests a higher Fe status in chloroplasts of nramp3nramp4 mutants compared with the wild type under these conditions. This is in agreement with the higher Fe concentrations in leaves and protoplasts from Mn-deficient nramp3nramp4 compared with the wild type (Fig. 3).
To test whether other components of the Fe homeostasis network were also deregulated, the levels of AtFER1 and AtIRT1 were monitored in the roots. Protein extracts from two biologically independent replicates were analyzed. AtIRT1 is the high-affinity Fe transporter, and IRT1 protein levels are up-regulated under Fe deficiency (Connolly et al., 2002; Seguela et al., 2008). IRT1 was detected only in roots of Fe-deficient plants, and protein levels were equivalent in wild-type and nramp3nramp4 plants (Fig. 6B). This is different from the finding in barley, where HvIRT1 is up-regulated in response to both Fe and Mn deficiency (Pedas et al., 2008). AtFER1 protein levels were equivalent in roots of wild-type and nramp3nramp4 plants under control and Mn-deficient conditions (data not shown).
DISCUSSION
The results presented in this study demonstrate that AtNRAMP3 and AtNRAMP4 are involved in Mn homeostasis in the adult plant. The strong accumulation of Mn in nramp3nramp4 mutant mesophyll vacuoles suggests that in this tissue, AtNRAMP3 and AtNRAMP4 participate in the release of Mn from the vacuoles (Fig. 1). Under Mn deficiency, nramp3nramp4 double mutant plant growth is reduced (Fig. 2). This growth defect correlates with a lower number of functional PSIIs, consistent with a shortage of Mn to form Mn clusters (Fig. 4).
AtNRAMP3 and AtNRAMP4 Operate in the Retrieval of Mn from Vacuoles in Leaf Mesophyll Cells
Mesophyll vacuoles of adult nramp3nramp4 plants contain substantially higher amounts of Mn than those of the wild type (Fig. 1B). This result suggests a function of AtNRAMP3 and AtNRAMP4 in the retrieval of Mn from vacuoles. Transfer of Mn into and retrieval from vacuoles thus appears to proceed continuously in the wild type, resulting in the accumulation of, on average, only 10% of protoplast Mn inside vacuoles in the steady state (Fig. 1B). The results obtained here suggest that the mesophyll vacuole is predominantly a compartment for Mn transit rather than for Mn storage. Despite the strongly enhanced accumulation of Mn in vacuoles of the nramp3nramp4 mutant, global leaf Mn concentrations are barely increased when compared with the wild type (Figs. 1 and 3; Supplemental Fig. S3). This could be due to the fact that the mesophyll intracellular Mn pool accounts for only a minor part of total leaf Mn. Mn concentrated in the cell wall or in other leaf cell types, such as vascular bundles or epidermal cells, could mask differences between the mesophyll cells of different genotypes. Alternatively, in the mutant, the increase in mesophyll vacuolar Mn contents could be compensated by a reduction in Mn contents in a different cell type. Lack of AtNRAMP3 and AtNRAMP4 leads to a modification of the subcellular distribution of Mn. While mesophyll vacuoles of wild-type plants grown on soil contain on average 10% of the total Mn pool in mesophyll protoplasts, vacuoles isolated from the mutant contain on average 70% of the intracellular Mn. By contrast, 92% and 80% of the total protoplast Zn pool was associated with the vacuoles of wild-type and mutant mesophyll cells, respectively. The finding that only 10% of the Mn is associated with wild-type vacuoles is surprising in view of other studies indicating that most Mn is stored in the vacuole. Based on Mn broadening of 31P NMR signal or on the analysis of 54Mn radiotracer release kinetics, two groups concluded that most Mn is stored in the vacuoles of maize (Zea mays) and barley root cells, respectively (Quiquampoix et al., 1993; Pedas et al., 2005). The discrepancy with our results may be explained by the use of different experimental approaches, by a different intracellular distribution of Mn in root and mesophyll cells, or by differences in Mn storage between species. González and Lynch (1999) reported that most Mn is stored in the vacuole of bean leaf cells. However, their study addressed Mn storage under Mn excess, a condition in which vacuolar Mn sequestration may be favored.
Fe was also detected in mesophyll vacuoles of wild-type and double mutant plants. Although AtNRAMP3 and AtNRAMP4 are capable of Fe transport (Thomine et al., 2000; Lanquar et al., 2005), no difference between the Fe contents of nramp3nramp4 and wild-type vacuoles was detected (Fig. 1), even when vacuoles were isolated from the mesophyll of Fe-deficient plants (data not shown). Thus, AtNRAMP3 and AtNRAMP4 are not likely to be required for Fe export from the vacuole in this tissue. However, the relatively high variation between biological replicates could mask small differences in Fe contents between wild-type and nramp3nramp4 double mutant vacuoles.
Under Mn Deficiency, Optimal Mn Supply to Chloroplasts Requires AtNRAMP3/AtNRAMP4-Dependent Mn Transit through the Vacuole
When plants are grown in control conditions, the high amount of Mn retained in nramp3nramp4 vacuoles does not lead to any reduction in plant growth (Fig. 2; Supplemental Fig. S3). This suggests that although a large proportion of cellular Mn transits through the vacuole in the mesophyll of wild-type plants, a lack of AtNRAMP3/AtNRAMP4-dependent Mn retrieval from the vacuole does not limit cellular Mn supply. However, under Mn deficiency, nramp3nramp4 plants display a strong growth reduction (Fig. 2). This growth inhibition is not associated with a lower Mn pool in mesophyll cells (Fig. 3) but with an alteration of the intracellular distribution of Mn instead. Chloroplast Mn concentrations of Mn-deficient nramp3nramp4 plants were much lower than in wild-type plants (Fig. 4). The intracellular Mn that is missing in the chloroplasts of nramp3nramp4 plants is likely sequestered in the vacuoles, based on the results obtained using Mn-sufficient plants (Fig. 1B). These results suggest that, under Mn deficiency, transit of Mn through the vacuole becomes a limiting pathway for the distribution of this metal to indispensable Mn-requiring proteins. Mn is an important cofactor of the oxygen-evolving complex of PSII. Compared with the wild type, a lower number of functional PSIIs correlated with an enhanced impact of Mn deficiency on biomass production in the double mutant. By contrast, mitochondrial MnSOD activity was not decreased under Mn deficiency in the wild type or in nramp3nramp4. The results presented suggest that under Mn deficiency, the use of the vacuolar Mn pool is required for optimal photosynthesis and plant growth but not for providing Mn cofactor to MnSOD in mitochondria. In contrast, in C. reinhardtii, Mn deficiency leads to a decrease in both PSII fluorescence and MnSOD activity (Allen et al., 2007). In Arabidopsis mesophyll cells, the oxygen-evolving complex of PSII thus appears as a major Mn-requiring enzyme, and its activity is more sensitive to Mn deficiency than that of MnSOD, in agreement with previous work on other plants (Marschner, 1995). In yeast, SMF2, a membrane protein of the NRAMP family localized in intracellular vesicles, plays a role in providing Mn to mitochondrial SOD (Luk and Culotta, 2001). In Arabidopsis, this role could be played by another Mn transport pathway distinct from AtNRAMP3 and AtNRAMP4. The results presented here underline the importance of Mn transit through the vacuole for Mn supply to the chloroplast under conditions of Mn deficiency, whereas under Mn-sufficient conditions, Mn transit through the vacuole is not required to maintain the primary Mn-dependent chloroplast function. Under Mn deficiency, the cellular Mn uptake is strongly reduced due to low external availability, and we hypothesized that cellular Mn recycling following protein degradation becomes substantially more important. Cellular Mn recycling is likely to involve passage through the vacuole as a compartment known to be involved in organelle autophagy (Wada et al., 2009). We speculate that under Mn deficiency and nonexcess Mn sufficiency, Mn transit through the vacuole is part of a continuous cellular recycling pathway for Mn involving AtNRAMP3 and AtNRAMP4, which becomes essential for the maintenance of photosynthesis and growth under conditions of Mn deficiency. However, as the results presented in Figure 1A do not formally exclude that AtNRAMP3 and AtNRAMP4 may reside on other membranes than the tonoplast, the possibility remains that the nramp3nramp4 phenotype is also related to a defect in Mn transport to or from another intracellular compartment.
AtNRAMP3 and AtNRAMP4 Have Redundant Functions in Mesophyll Cells
Under Mn deficiency, decreases in plant biomass production and maximum quantum yield were only observed in the nramp3nramp4 double mutant, not in nramp3 and nramp4 single mutants (Fig. 2; Supplemental Fig. S4). This result indicates that AtNRAMP3 and AtNRAMP4 genes have redundant functions in Mn homeostasis in leaves. This is in agreement with previous reports on the redundant functions of AtNRAMP3 and AtNRAMP4 in Fe mobilization during seed germination and in Cd tolerance (Lanquar et al., 2005; Oomen et al., 2009). By contrast, AtNRAMP3 and AtNRAMP4 functions in pathogen resistance are additive (Segond et al., 2009). Overlapping but not fully identical localization of AtNRAMP3 and AtNRAMP4 promoter activity (Thomine et al., 2003; Lanquar et al., 2005) and circumstantial evidence for a higher abundance of the AtNRAMP4 protein in mesophyll cells when compared with AtNRAMP3 (Carter et al., 2004) support both the possibility of functional redundancy and of functional differentiation between the two proteins.
Cross Talk between Fe and Mn Homeostasis
Although other NRAMP family members in yeast, C. reinhardtii, and bacteria are up-regulated under Mn deficiency, neither AtNRAMP3 nor AtNRAMP4 is regulated in response to Mn deficiency at the protein level (Portnoy et al., 2000; Patzer and Hantke, 2001; Allen et al., 2007). It is possible that the expression level of AtNRAMP3 and AtNRAMP4 observed under control conditions is enough to support sufficient rates of Mn retrieval from vacuoles also under Mn deficiency. Alternatively, the transport activity of these proteins might be regulated at a different level. In contrast, AtNRAMP3 and AtNRAMP4 protein abundance is up-regulated in response to Fe deficiency (Fig. 6; Lanquar et al., 2005). This suggests that AtNRAMP3 and AtNRAMP4 may play a role during Fe deficiency in adult plants, which remains to be identified. A previous report showed that in nramp3 knockout mutants, Mn accumulation is increased under severe Fe deficiency (Thomine et al., 2003). Conversely, Mn accumulation under Fe deficiency is reduced in 35S-AtNRAMP3 Arabidopsis lines (Thomine et al., 2003). This effect was associated with similar changes in Zn accumulation and other responses to Fe deficiency, suggesting that AtNRAMP3 modulates responses to Fe deficiency (Baxter et al., 2008). In our study, no difference in Mn accumulation was detected between adult wild-type and nramp3nramp4 plants grown under Fe deficiency (Supplemental Fig. S3). In C. reinhardtii, Mn deficiency leads to secondary Fe deficiency (Allen et al., 2007). Under the conditions used in this study, none of the Arabidopsis Fe deficiency-responsive proteins analyzed, AtNRAMP3, AtNRAMP4, or IRT1, was up-regulated under Mn deficiency, indicating that Mn deficiency does not trigger secondary Fe deficiency.
Increased accumulation of FER1 and an increase in Fe concentration in leaves of nramp3nramp4 plants grown under Mn deficiency suggest a coordinated homeostasis of Mn and Fe (Figs. 3 and 6). It was shown that, in C. reinhardtii, Fer1 is up-regulated under Fe deficiency. It has been proposed that CrFer1 functions to buffer the Fe released as a consequence of the degradation of some Fe-containing proteins of the photosynthetic apparatus (Long et al., 2008). In Arabidopsis, ferritins have recently been shown to be necessary to prevent oxidative damage originating from the presence of free Fe in plastids when plants are exposed to excess Fe (Ravet et al., 2009a). In microorganisms, Mn plays a role in protection against oxidative stress (Anjem et al., 2009). During Mn deficiency, increased degradation of photosynthetic proteins leading to the release of free Fe or a decreased Mn protective effect could lead to oxidative stress. The observed increase in ferritin levels could buffer Fe and prevent oxidative damage.
MATERIALS AND METHODS
Plant Material
The generation of the nramp3-1 and nramp4-1 single mutants, the 35S-AtNRAMP3 lines, the nramp3nramp4 double mutant, and the complemented lines nramp3nramp4 AtNRAMP3 and nramp3nramp4 AtNRAMP4 of Arabidopsis (Arabidopsis thaliana) was described previously (Thomine et al., 2003; Lanquar et al., 2005).
Growth Conditions
For perlite growth experiments, perlite (Puteaux Fournitures Horticoles) was washed once with 2.5 mm EGTA and then with about 10 volumes of deionized water. Arabidopsis seeds were sown on perlite and watered with a modified Hoagland solution [0.28 mm KH2PO4, 1.25 mm KNO3, 0.75 mm MgSO4, 1.5 mm Ca(NO3)2, 25 μm H3BO3, 50 μm KCl, 1 μm ZnSO4, 0.1 μm Na2MoO4, 0.5 μm CuSO4, 10 μm Fe-HBED, and 3 mm MES-KOH, pH 5.7]. Fe was provided as Fe3+ chelated to HBED [N,N′-di(2-hydroxybenzyl) ethylene diamine-N,N′-diacetic acid monochloride hydrate; Strem Chemicals]. Fe-HBED was prepared as described (Lanquar et al., 2005). Plants were grown for 6 to 9.5 weeks with (C) or without 5 μm MnSO4 (−Mn).
For the hydroponic growth experiments, seeds were surface sterilized and sown on bottom-cut 0.5-mL Eppendorf tubes filled with 0.8% (w/v) Noble agar. For the first week, six seeds were placed in 500 mL of 0.5× Hoagland medium supplemented with 20 μm Fe-HBED and with (C and −Fe) or without 5 μm MnSO4 (−Mn). Then, plants were grown either for 5 more weeks in 1× Hoagland medium supplemented with 10 μm Fe-HBED and with (C) or without 5 μm MnSO4 (−Mn) or for 3 weeks in medium supplemented with 10 μm Fe-HBED and 5 μm MnSO4 and then transferred into a medium supplemented with 5 μm MnSO4 and 20 μm ferrozine (−Fe, 3-(2-pyridyl)-5,6-bis(4-phenylsulfonic acid)-1,2,4-triazine) for 2 weeks. For culture on soil, on perlite, or in hydroponics, plants were grown in a climate chamber under the following conditions: 9/15-h light/dark; light intensity of 200 μE m−2 s−1; constant temperature of 21°C; 60% relative humidity.
Vacuole Isolation
After removing the abaxial epidermis by rubbing on P600 sandpaper Arabidopsis leaves of plants grown on soil for 6 weeks, protoplasts were isolated by enzymatic digestion of approximately 2 g of leaves in 50 mL of MCP (500 mm sorbitol, 1 mm CaCl2, and 10 mm MES-KOH, pH 5.6) supplemented with 0.03% (w/v) Pectolyase Y23 (Yakult Pharmaceuticals) and 0.75% (w/v) Cellulase R10 (Yakult Pharmaceuticals) at 23°C for 2 h. Protoplasts were filtered through a 75-μm nylon mesh, pelleted (100g, 5 min), and washed twice with MCP; approximately 2 × 108 protoplasts were obtained. Protoplasts were diluted to 15 to 20 × 106 cells mL−1 and were lysed by addition of an equal volume of protoplast lysis buffer (200 mm sorbitol, 10% [w/v] Ficoll 400, 20 mm EDTA, 10 mm HEPES-KOH, pH 8, 0.15% [w/v] bovine serum albumin, and 2 mm dithiothreitol [DTT]) prewarmed to 42°C. Protoplast lysis was monitored microscopically, and lysed protoplasts were kept on ice. Vacuoles were isolated and concentrated using a step gradient prepared on ice (1,500g, 20 min, 4°C): bottom phase, 1 volume of lysed protoplasts; middle phase, 0.8 volume of lysis buffer diluted in vacuole buffer to reach a concentration of 4% (w/v) Ficoll; top phase, 0.2 volume of vacuole buffer (500 mm sorbitol, 10 mm HEPES, pH 7.5 [KOH], 0.15% [w/v] bovine serum albumin, 1 mm DTT, 2 μg mL−1 leupeptin, and 2 μg mL−1 antipain). Vacuoles were recovered at the interface between the middle phase and the top phase. Purity of the vacuole preparation was monitored by microscopy; approximately 2 × 107 vacuoles were obtained in total.
For immunoblot analysis, after isolation, vacuoles were lysed as follows: 1 volume of vacuoles was mixed by inversion with 3 volumes of 50 mm MOPS, pH 7.8, 0.1 mm DTT, and 1× Complete Protease Inhibitor Cocktail Tablets (Roche Applied Science). The vacuolar membrane was collected by ultracentrifugation at 110,000g at 4°C for 90 min, and the pellet was resuspended in the same MOPS-DTT buffer. Protein concentration was determined by the Bradford method.
Chloroplast Isolation
Protoplasts were isolated and lysed as described above for the isolation of vacuoles. Percoll gradients were prepared by ultracentrifugation (40,000g, 55 min, 4°C, without brake): bottom phase, 1 volume of 100% Percoll; top phase, 1 volume of washing medium (300 mm sorbitol, 40 mm Tricine, pH 7.6, 2.5 mm EDTA, and 0.5 mm MgCl2) and kept at 4°C. Chloroplasts were isolated by loading 6 mL of protoplast lysate on the preformed Percoll gradient followed by centrifugation (13,000g, 10 min, 4°C, without brake). Intact chloroplasts were recovered at the bottom of the tube and were washed four times with cold washing medium (Ferro et al., 2002). Purity and integrity of the chloroplast preparation was monitored by microscopy, and the amount of chloroplasts was evaluated by measurement of the chlorophyll content.
Protein Isolation
Total protein extracts from protoplasts, shoots, and roots were prepared by homogenization in 50 mm HEPES-NaOH, pH 7.2, 1.5 mm MgCl2, 1 mm EGTA, 1 mm EDTA, 10% (w/v) glycerol, 1% (v/v) Triton X-100, 150 mm NaCl, 5 mm DTT, and 1× Complete Protease Inhibitor Cocktail Tablets (Roche Applied Science). Samples were then centrifuged for 10 min at 1,000g and 4°C, and supernatants were recovered. Protein concentration was estimated by the Bradford method.
Immunoblot Analysis
Unless specified, 15 μg of vacuolar membrane, protoplast, root, or shoot proteins was separated by SDS-PAGE and immunoblot analysis was performed. Rabbit anti-AtNRAMP3 polyclonal antibodies were raised against two synthetic peptides corresponding to N-terminal and C-terminal regions of AtNRAMP3 protein (N-term, H2N-MPQLENNEPLLINEC-CONH2; C-term, H2N-CTPWPFKAESSH-COOH; Eurogentec). The anti-AtNRAMP3 antibodies were affinity purified against the same peptides.
The antibodies used in this study were diluted as follows: AtNRAMP3, 1:1,000; AtNRAMP4, 1:2,000; AtV-PPAse, 1:20,000 (Sarafian et al., 1992); AtFer, 1:10,000 (Dellagi et al., 2005); AtIRT1, 1:8,000 (Seguela et al., 2008); PsbA/D1, 1:15,000 (Agrisera). Immunoblotting was performed as described previously (Lanquar et al., 2005).
Metal Content Measurements
Leaves, shoots, and roots were harvested separately and washed. Roots were desorbed by incubation in ice-cold buffer containing 5 mm CaCl2 and 1 mm MES-KOH, pH 5.7, for 10 min. The dry biomass of the samples was measured after drying at 60°C. Protoplast and vacuole samples were concentrated in a SpeedVac after determination of the number of vacuoles or protoplasts per sample by counting objects using the microscope. All samples were digested in 65% nitric acid in a MARS5 microwave (CEM) at 200°C at 15 bar for 10 min. After dilution in trace metal-free water, the metal contents of the samples were determined by ICP-AES using an IRIS Advantage Duo ER/S (Thermo Jarrell Ash) as described (Lanquar et al., 2005). The metal concentrations were expressed as mg kg−1 dry weight for tissue samples, pg per object for protoplasts and vacuoles, or relative to the geometric mean of a combination of elements: calcium, copper, Fe, Mg, Mn, molybdenum, phosphorus, and Zn for protoplasts; copper, phosphorus, and sulfur for chloroplasts. Note that other elements could not be used because of contaminant levels in the buffer in which chloroplasts were suspended.
Chlorophyll Fluorescence Measurements
Room temperature chlorophyll fluorescence was measured in vivo using a pulse-amplitude modulation fluorimeter (PAM 101-3; Walz). The intensity of the measuring light (standard PAM 101set) was sufficiently low (integral intensity about 10−8 mol quanta m−2 s−1, frequency of modulated light of 1.6 kHz) to prevent the reduction of plastoquinone. Saturating flashes (1 s) were given to measure the maximum fluorescence. Efficiency of the photochemical electron transport was assayed by calculating Fv/Fm (Maxwell and Johnson, 2000). In all experiments, the Fv/Fm was measured on leaves of dark-adapted plants. As photoinhibitory white light (Fig. 4B), a cold light source was used with an intensity of 2,000 μmol quanta m−2 s−1. Recovery was followed for 3 h in dim white light (5 μmol quanta m−2 s−1).
Thermoluminescence Measurements
Thermoluminescence was measured with a home-built apparatus. To measure the thermoluminescence originating from the S2/3QB− charge recombination (B-band), excised leaf pieces were incubated in the dark at 20°C for 5 min and then flashed with saturation xenon flashes at 1°C (1-s interval between flashes). Samples were heated at a constant rate (0.4°C s−1) from 1°C to 70°C, and the light emission was recorded. Graphical and numerical data analyses were performed as described (Ducruet and Miranda, 1992).
SOD Activity
Total leaf protein extracts were prepared by homogenization in 100 mm potassium phosphate, pH 7, 3.5 mm DTT, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 15% (v/v) glycerol, 1% (w/v) polyvinylpyrrolidone, and 0.1% (v/v) Triton X-100. Samples were centrifuged (10,000g, 5 min, 4°C), and supernatants were recovered. Protein concentration was estimated by the Bradford method. Total protein extracts were separated on a native polyacrylamide gel (12% Precast Gel; Bio-Rad), and SOD activity was detected on the gel as described (Beauchamp and Fridovitch, 1971). FeSOD and CuZnSOD activities were identified by sensitivity to 2 mm KCN for CuZnSOD and sensitivity to 5 mm H2O2 for CuZnSOD and FeSOD. Treatments were performed prior to activity staining.
Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers NM_127879 and NM_126133.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Characterization of an anti-AtNRAMP3 polyclonal antibody.
Supplemental Figure S2. Rescue of the nramp3nramp4 growth defect in the absence of Mn by AtNRAMP3 or AtNRAMP4.
Supplemental Figure S3. Phenotype and metal content of nramp3nramp4 plants grown in hydroponic culture.
Supplemental Figure S4. Time course variations of Fv/Fm in wild-type, nramp3, nramp4, and nramp3nramp4 plants under Mn-sufficient or Mn-deficient conditions.
Supplemental Figure S5. Quantification of D1 immunoblot and silver staining as a loading control.
Supplemental Table S1. Metal concentrations of wild-type and nramp mutant rosettes grown in the presence (control) or absence of Mn (−Mn).
Supplemental Table S2. Chlorophyll fluorescence of complemented nramp3nramp4 plants.
Acknowledgments
We thank Drs. F. Gaymard and C. Curie for sharing FER1 and IRT1 antibodies before publication.
Footnotes
↵1 This work was supported by the Centre National de la Recherche Scientifique and the Agence Nationale pour la Recherche (DISTRIMET grant no. ANR–07–BLAN–0110), by the French Ministry of Research (Ph.D. fellowship to V.L.), and by an Heisenberg Fellowship (no. Kr1967/4–1 to U.K.).
↵2 Present address: Carnegie Institution for Science, Department of Plant Biology, 260 Panama Street, Stanford, CA 94305.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Sébastien Thomine (thomine{at}isv.cnrs-gif.fr).
↵[W] The online version of this article contains Web-only data.
- Received November 13, 2009.
- Accepted February 20, 2010.
- Published February 24, 2010.