Rice (Oryza sativa ‘Guara’) and barley (Hordeum vulgare ‘Baroness’) were grown hydroponically in a growth chamber under controlled environmental conditions at 25/20 °C day/night temperatures, 75 ± 5 % relative humidity and a photon flux density of 150 µmol m−1 s−1 photosynthetic active radiation (PAR) at mid-plant height during a 16 h photoperiod. After germination in 1 mm CaSO4 for 10–12 d, seedlings were transferred to nutrient solution with 20 or 30 plants of barley and rice in 5 L pots, respectively. The nutrient solution was constantly aerated only for barley. The composition of the nutrient solution was (μm): Ca(NO3)2 1000 for barley or 500 Ca(NO3)2 and 500 NH4NO3 for rice, KH2PO4 100, K2SO4 375, MgSO4 325, FeEDDHA 20, NaCl 10, H3BO3 8, CuSO4 0·2, ZnSO4 0·2, Na2MoO4 0·05. Barley plants additionally received 0·2 µm and rice plants 1 µm MnSO4 according to their Mn requirements for optimum growth (preliminary experiments). Half of the plants received additional Mn (50 µm, this Mn supply has proved to be most appropriate for the study of Mn toxicity in previous and preliminary studies) for up to 4 d when the fifth leaf was fully developed (approx. 34–38 d after transfer to nutrient solution), whereas the other plants received 0·2/1 µm continously. During pre-culture the nutrient solution was changed every 2–3 d in order to prevent nutrient deficiencies. During the Mn treatment the nutrient solution was changed twice a day to maintain a constant Mn supply.
Harvesting of plant material
Leaf material for physiological and proteomic analyses was harvested from the older (second, fourth) and younger (fourth, sixth) leaves and immeditately frozen in liquid nitrogen. Until further analysis the material was stored at –80 °C. For mineral analysis leaf material was dried at 65 °C.
Apoplastic washing fluid
Apoplastic washing fluid was extracted from leaves using a vaccum infiltration/centrifugation technique. Leaf blades were excised from the plants and cut in the middle. Leaf halves were washed in chilled double-demineralized water (ddH2O) in order to reduce cytosolic contamination. Leaf halves from 20 leaves were combined and infiltrated with chilled ddH2O by reducing the pressure four times for 2·5 min followed by a slow relaxation. The AWF was recovered by centrifugation of the blotted cuttings at 4000 g and 4 °C for 5 min in 500 mL centrifugal devices (Nalgene, Nalge Nunc). The AWF was immediately frozen in liquid nitrogen and, until further analysis, stored at −80 °C. In order to estimate the apoplastic volume leaves were weighed before and after infiltration.
For Mn determination the dried and milled plant material was dry-ashed overnight at 480 °C. Samples were prepared and measured as descibed by Führs et al. (2009)
Total Si was determined following the protocol described by Iwasaki et al. (2002a
) using 10 mg of dried plant material.
For determination of Mn concentrations in the AWF, 50 µL samples were filtered through centrifugal filter devices (0·2 µm, GHP Nanosep Mt centrifugal device, PAL Lifesciences, Ann Arbor, MI, USA) for 10 min at 4000 g. Filtrates were diluted 1 : 50 with ddH2O and measured using inductively coupled plasma-optical emission spectroscopy (ICP-OES; Spektro Analytical Instruments GmbH, Kleve, Germany).
Isolated chloroplasts were dry-ashed overnight at 480 °C. The ash was dissolved in 300 µL of 6 m HCl supplemented with 1·5 % (w/v) hydroxylammonium chloride. Samples were diluted 1 : 10 with ddH2O before analysis using ICP-OES. For the analysis of Mn in the individual chloroplast isolation fractions, 300 µL sub-samples were filtered through centrifugal filter units (see above) and centrifuged for 10 min at 4000 g. Filtrates were diluted 1 : 10 with ddH2O and measured using an ICP-MS (7500CX, Agilent, Böblingen, Germany). Results were expressed as a percentage of the total Mn concentration in the leaf fresh matter.
Chlorophyll fluorescence of the older leaves was determined using a Mini-PAM fluorometer (Waltz, Germany). Measurements were made on dark-adapted plants using the light induction curve program: 1 min after a first saturation pulse actinic light was turned on and from then every 30 s a new saturation pulse was applied over a period of 6·5 min. The yield, ETR, nP, nQ and NPQ values were calculated using the included software. All measurements were repeated four times and submitted to statistical analysis.
Determination of the chlorophyll content
Prior to harvest of the leaves their chlorophyll contents were determined according to Schulte auf'm Erley et al. (2007)
using a portable chlorophyll meter (SPAD). The means of five measurements over the whole leaf blade of the fourth and sixth leaf are shown.
Determination of protein contents
About 100 mg of frozen leaf fresh matter was homogenized in 1 mL of chilled ddH2O in a swinging mill (MM200, Retsch, Germany) with a frequency of 30 s−1 for 2 min. The homogenate was centrifuged at 5000 g for 1 min at 4 °C, and the supernatant was used for protein quantification using the 2D Quant Kit (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer's instructions.
Protein in the AWF was quantified according to the method described by Bradford (1976)
For 2D isoelectric focusing IEF/SDS–PAGE protein was quantified after resolubilization of precipitated proteins using the 2D Quant Kit (GE Healthcare, USA) according to the manufacturer's instructions.
Determination of the phenol concentration in the AWF
Determination of H2O2-consuming guaiacol-POD and H2O2-producing NADH-POD activity in the AWF
-consuming guaiacol-POD and H2
-producing NADH-POD activities were determined as described by Führs et al. (2009)
Determination of the cytosolic contamination of the AWF using malate dehydrogenase (MDH) as marker enzyme
The MDH activity was determined according to the method described by Bergmeyer and Bernt (1974)
including the preceding generation of oxalacetate from aspartate and α-ketoglutarate using glutamic-oxalacetic-transaminase (GOT). The decrease in absorption due to NADH oxidation was determined spectrophotometrically at λ = 340 nm (Uvikon 943 Double Beam, UV/ VIS Spectrophotometer, Kontron Instruments) for 1·2 min. Results were expressed as a percentage of the activity obtained from freshly prepared leaf homogenates.
Isolation of chloroplasts
Chloroplasts were isolated according to Tanaka et al. (2004)
with minor modifications. Leaf material was homogenized in 100 mL of ‘homogenization buffer’ per 10 g of fresh matter [350 mm
sorbitol, 2 mm
EDTA (pH 8·0), 25 mm
HEPES-NaOH, pH 7·0] using a blender for 4 × 3 s. The homogenate was filtered through four layers of gaze (50 µm
mesh size) and centrifuged at 1200 g
for 5 min at 4 °C. The resulting pellet was resuspended in 12–15 mL of ‘homogenization buffer’ containing 0·35 m
sorbitol, 2 mm
EDTA and 25 mm
HEPES (pH 7·0), and loaded onto a discontinuous gradient formed by 10, 30 and 80 % Percoll. The gradient formation and separation of chloroplasts was done by centrifugation at 2600 g
for 40 min at 4 °C. Intact chloroplasts were removed from the 30 %/80 % Percoll boundary layer and subsequently centrifuged at 1800 g
for 5 min at 4 °C. The resulting pellet contained the ‘residue Mn’ fraction. The supernatant was removed and the resulting pellet was resuspended and centrifuged at 1800 g
for 5 min at 4 °C. After weighing the pellet containing the ‘chloroplastic Mn’, it was resuspended in homogenization buffer with a final concentration of 100 mg mL−1
. Sub-samples were taken from each washing step in order to determine the labile bound Mn fraction during the chloroplast isolation procedure (‘soluble Mn’). Isolated chloroplasts and sub-samples were immediately frozen in liquid nitrogen and stored at −80 °C until further analyses.
2D IEF/SDS–PAGE of soluble leaf homogenate and soluble chloroplastic proteins
Proteins from the leaf homogenates were extracted according to Hurkman and Tanaka (1986)
with three biological replicates for both Mn-control and Mn-treated plants. Each replicate comprised a pool of 11 individual plants.
For investigation of the water-soluble chloroplastic proteome, isolated chloroplasts were frozen in liquid nitrogen. After thawing and centrifugation, the supernatant, containing the soluble chloroplastic proteins, was precipitated and used for IEF/SDS–PAGE as described by Führs et al. (2008)
. The recovered protein had to be combined into one sample due to the low chloroplast yields of the isolation procedure in rice.
2D Blue native BN/SDS–PAGE of photosynthetic protein complexes from thylakoid membranes
Proteins from thylakoid membranes of 10 mg of isolated chloroplasts were prepared and photosynthetic protein complexes were separated by 2D BN/SDS–PAGE as described by Heinemeyer et al. (2004)
Staining of 2D gels
2D IEF/SDS–polyacrylamide gels made from water-soluble chloroplastic proteins were stained with silver nitrate using an adapted method described by Heukeshoven and Dernick (1988)
Visualization of gels of 2D IEF/SDS–PAGE and 2D BN/SDS–PAGE
Coomassie colloidal-stained gels were scanned with 300 dpi (Epson Expression 1600 Scanner) and stored as .tiff files. For analysis of 2D IEF/SDS–polyacrylamide gels the files were transformed into .mel files. Subsequently the spots were detected, quantified and the three biological replications statistically analysed using Imagemaster™ 2D PLATINUM Software 6·0 (GE Healthcare, USA). The quantification parameter was the relative spot volume (%Vol, spot volume of one spot in relation to the sum of all detected spots on the gel) in order to eliminate protein loading differences. Only protein spots with a minimum spot volume of 0·01 % were included. Spots of significantly changed volumes (at least P < 0·05 and a spot ratio between the treatments of at least <0·5 or >2·0) were sequenced.
Mass spectrometric protein analysis and data interpretation
Mass spectrometric protein analysis and data interpretation were performed as described by Führs et al. (2008
Statistical analysis, if not mentioned otherwise, was carried out using SAS Release v8·0 (SAS Institute, Cary, NC, USA) or the Multi Experiment Viewer MeV 4·1 (Saeed et al., 2003
). Results from analysis of variance and correlation analysis are given according to their level of significance as ***, ** and * for P
< 0·001, 0·01 and 0·05, respectively. Results of the mean comparisons are given at P
< 0·05 (Tukey).