Article Figures & Data
Figures
- Figure 1.
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.
- Figure 2.
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).
- Figure 3.
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.
- Figure 4.
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).
- Figure 5.
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.
- Figure 6.
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.
Tables
- Table I. Rosette fresh biomass and chlorophyll fluorescence of 8-week-old wild-type and nramp3nramp4 plants grown on perlite in the presence (control; 5 μm Mn) or absence of Mn (−Mn)
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.
Sample Fresh Biomass (n) Fv/Fm (n′) Control medium Wild type 537 ± 69 (11) 0.798 ± 0.003 (10) nramp3nramp4 582 ± 92 (6) 0.796 ± 0.004 (12) −Mn medium Wild type 279 ± 36 (15) 0.655 ± 0.013 (11) nramp3nramp4 82 ± 24** (10) 0.572 ± 0.017* (9)
Supplemental Data
Supplemental Figures and Tables
Files in this Data Supplement:
- Supplemental Data - Supplemental Figures 1-5
- Supplemental Data - Supplemental Table I
- Supplemental Data - Supplemental Table II
