Supplemental Data
Supplemental Material and Methods
Immunoblotting for analysis of 2-Cys PRX protein expression
Proteins were extracted on ice in 50 mM Tris, pH 8 and 5 mM β-mercaptoethanol with a 1:200 dilution of proteinase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Protein concentrations of supernatants were determined after centrifugation at 20,000g for 10 min at 4°C using Bradford reagent and BSA as a standard (Spector, 1978). Proteins (50µg) were separated using SDS-PAGE, transferred to PVDF membrane (Millipore, Billerica, MA, USA) and incubated with a 1:10,000 dilution of α-AtBAS1 antibody (Broin et al., 2002). Detection of antigen was accomplished with a 1:40,000 dilution of HRP-conjugated α-rabbit IgG (Sigma-Aldrich, Germany) and Immobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA).
Quantification of chlorophyll and amino acids
Chlorophyll extraction and quantification was performed as described (Arnon and Whatley, 1949). Briefly, leaves (50 mg) were homogenized with a mortar and a pestle in 1 mL of acetone:water (4:1, v/v) and incubated at 4°C in the dark for 5 h. After centrifugation (2655g, 3 min), the supernatant was diluted 1:10 with acetone:water (4:1, v/v) and the absorption was measured spectrophotometric at 664 nm and 647 nm.
Amino acid levels were determined by UPLC-ESI-qTOF-MS (Acquity UPLC, Synapt HDMS G2, Waters, Milford, MA, USA) after derivatization with the AccQ Ultra Kit (Waters, Eschborn, Germany) as described (Salazar et al., 2012).
Ascorbate, glutathione and jasmonate quantification
For quantification of reduced ascorbate (AsA) and dehydroascorbate (DHA), leaf material (130 mg) was extracted as described (Page et al., 2012) with some modifications. Briefly, leaves were shock frozen, ground in liquid nitrogen and homogenized in 1 mL of water containing 5% metaphosphoric acid (MPA, Sigma-Aldrich, St. Louis, MO, USA) for 3 min at 20 Hz in a Retsch MM 301 ball mill (Retsch Inc., Germany). The homogenate was centrifuged for 10 min at 15,000g at 4°C. The supernatant was filtered through a glass microfibre filter (Whatman, Internat. Ltd., England). One aliquot (50 µL) was combined with water containing 5% MPA (50 µL) to determine reduced AsA. Another aliquot (50 µL) was combined with 50 µL of water containing 20 mM tris(2-carboxylethyl)phosphine hydrochloride (TCEP) (Sigma-Aldrich, St. Louis, MO, USA) and 5% MPA, and incubated at room temperature for 30 min to reduce DHA to AsA (to assay AsA plus DHA – referred to as total AsA).
Samples (2.5 µL injection volume) were analyzed by ultra high performance liquid chromatography (Waters Acquity UPLC, Milford, MA, USA) coupled to a Waters 2996 Photodiode Array Detector (Waters, Milford, MA, USA). AsA was analyzed on an Acquity BEH Amide column (100 x 2.1 mm, 1.7 µm particle size; Waters; Milford, MA, USA) operated with a flow rate of 0.2 mL min–1 at 25°C using solvent A (acetonitrile:water, 50:50, v/v) and solvent B (acetonitrile:water, 90:10, v/v) both containing 10 mM ammonium acetate and 0,02% acetic acid (adjusted to pH 5). A linear gradient elution from 0.1% to 99.9% solvent B in 10 min was applied. AsA was detected and quantified at 265 nm using an external standard calibration curve. The content of DHA was calculated by subtracting the amount of AsA from total AsA.
Glutathione (GSH) and glutathione disulfide (GSSG) were extracted from 250 mg of leaf material with 500 µL of 0.05% hydrogen chloride in water containing 8.5 mM S-methyl methanethiosulphonate (for protection of the thiol group of GSH from autooxidation) and 30 nmol glutathione ethylester (internal standard). Samples were incubated in a sonic bath for 5 min, further incubated on ice for 20 min and centrifuged. The supernatants were analyzed by using UPLC-ESI-qTOF-MS (Acquity UPLC, Synapt G2 HDMS, Waters, Milford, MA, USA) as described (New and Chan, 2008).
For jasmonate analysis, leaf material (200 mg) was shock frozen and extracted with 1 mL of ethyl acetate:formic acid, (99:1, v/v). Dihydrojasmonic acid (50 ng) and jasmonic acid norvaline conjugate (50 ng) were added as internal standards. Extraction and analysis of jasmonates was performed as described (Stingl et al., 2013).
LITERATURE CITED
Arnon DI, Whatley FR (1949) Is chloride a coenzyme of photosynthesis? Science 110: 554-556
Broin M, Cuine S, Eymery F, Rey P (2002) The plastidic 2-cysteine peroxiredoxin is a target for a thioredoxin involved in the protection of the photosynthetic apparatus against oxidative damage. Plant Cell 14: 1417-1432
New LS, Chan EC (2008) Evaluation of BEH C18, BEH HILIC, and HSS T3 (C18) column chemistries for the UPLC-MS-MS analysis of glutathione, glutathione disulfide, and ophthalmic acid in mouse liver and human plasma. J Chromat Sci 46: 209-214
Page M, Sultana N, Paszkiewicz K, Florance H, Smirnoff N (2012) The influence of ascorbate on anthocyanin accumulation during high light acclimation in Arabidopsis thaliana: further evidence for redox control of anthocyanin synthesis. Plant Cell Environ 35: 388-404
Salazar C, Armenta JM, Cortes DF, Shulaev V (2012) Combination of an AccQ.Tag-ultra performance liquid chromatographic method with tandem mass spectrometry for the analysis of amino acids. Methods Mol Biol 828: 13-28
Spector T (1978) Refinement of the coomassie blue method of protein quantitation. A simple and linear spectrophotometric assay for less than or equal to 0.5 to 50 microgram of protein. Anal Biochem 86: 142-146
Stingl N, Krischke M, Fekete A, Mueller MJ (2013) Analysis of defense signals in Arabidopsis thaliana leaves by ultra-performance liquid chromatography/tandem mass spectrometry: jasmonates, salicylic acid, abscisic acid. Methods Mol Biol 1009: 103-113
Supplemental Table 1: Primers used for genotyping and quantitative gene expression analysis.
2-Cys PRX A (cDNA-5´) / At3g11630 / fw 5´-TCCACTGGTTGGAAACAAG-3´
rev 5´-ATGAGCACTCCGAACGACTT-3´
2-Cys PRX A (cDNA-3´) / At3g11630 / fw 5´-CCAACATTCCACCATCAACA-3´
rev 5´-AGTTTTGGGTCGGGTTTCAT-3´
2-Cys PRX A (GK_295C05) / At3g11630 / fw 5´-CTTCCACTGGTTGGAAACAAG-3´
rev 5´-AATGCCTGCAACATTGAAAAC-3´
2-Cys PRX B (cDNA) / At5g06290 / fw 5´-TAGGGGTCTCTGTCGACAGTG-3´
rev 5´-TGGGGTCAGGTTTCATTGAT-3´
2-Cys PRX B (SALK_17213) / At5g06290 / fw 5´-ATCCGAGTGGAGAATATTCCG-3´
rev 5´-TAGGGGTCTCTGTCGACAGTG-3´
tAPX (WiscDsLox457-460A17) / At1g77490 / fw 5´-TCCCTAAGGTATGTGCACCAG-3´
rev 5´-ATGATTTCACCAAAATGTGCC-3´
LB b1.3 (Left border primer SALK) / 5´-ATTTTGCCGATTTCGGAAC-3´
LB 8409 (Left border primer GK) / 5’-ATATTGACCATCATACTCATTGC-3’
LB p745 (Left border primer Wisc..) / 5´-AACGTCCGCAATGTGTTATTAAGTTGTC-3´
Actin 2/8 / At3g18780 / fw 5´-GGTGATGGTGTGTCT-3´
rev 5´-ACTGAGCACAATGTTAC-3´
BAP1 / At3g61190 / fw 5´-TAAACCGGAGACCCATCAAG-3´
rev 5´-TCGACATTTCTCGTCGATTTT-3´
CHS / At5g13930 / fw 5´-TGAGAACCATGTGCTTCAGG-3´
rev 5´-TTAGGGACTTCGACCACCAC-3´
F3H / At3g51240 / fw 5´-GTGGCGGATATGACTCGTCT-3´
rev 5´-CGTCACTTTCACCCAACCTT-3´
HSFA2 / At2g26150 / fw 5´-CAGCAAGGATCTGGGATGTC-3´
rev 5´-GCTGTTGCCTCAACCTAACT-3´
HSP101 / At1g74310 / fw 5´-TGAGCTAGCTGTGAATGCAG-3´
rev 5´-TCAACTGGTCAACAGCCAAA-3´
OXI1 / At3g25250 / fw 5´-TCATCTACATTGGCCGTGTC-3´
rev 5´-CGTCGCTCCATACAACATCT-3´
PAP1 / At1g56650 / fw 5´-TCTTCGCCTTCATAGGCTTC-3´
rev 5´-CATTGAGATGGTTGCAGTCG-3´
PAP2 / At1g66390 / fw 5´-GTGCATGGACTGCTGAAGAA-3´
rev 5´- ATCGACCAGCAATCAAGGAC-3´
Unknown protein / At1g49150 / fw 5´-GCCGTTTTGGTACTCCTGTC-3´
rev 5´-GACCACCGACGAAAAGACC-3´
ZAT12 / At5g59820 / fw 5´-TGGGAAGAGAGTGGCTTGTTT-3´
rev 5´-TAAACTGTTCTTCCAAGCTCCA-3´
Actin 2/8 / At3g18780 / fw 5´-GGTGATGGTGTGTCT-3´
rev 5´-ACTGAGCACAATGTTAC-3´
Supplemental Figures
Figure S1
Supplemental Figure S1. Isolation of the 2cpa 2cpb double knockout mutant.
(A) Western blot analysis of 2-Cys PRX protein expression in wild type (WT) and in the single T-DNA insertion mutants 2cpa and 2cpb. Lack of 2-Cys PRX in protein extracts (50 µg of protein) from four independent homozygous 2cpa 2cpb double mutant progeny plants. (B) The Coomassie-stained blot is shown as loading control. (C) Intron-exon structures and RT-PCR of the 2CPA and 2CPB genes. Position and directions of the T-DNA insertions in the 2CPA gene, line GK-295C05 (GK), and in the 2CPB gene, line SALK_017213 (SALK), are shown. RT-PCR: 2CPA, 2CPB and actin2/8 (ACT) genes were amplified for 30 cycles resulting in amplification products of 2CPA and 2CPB in the wild type (WT) but not in the mutants.
Figure S2
Supplemental Figure S2. Sucrose sensitivity of wild type (WT), 2cpa, 2cpb and 2cpa 2cpb mutant plants. Plants were grown for 10 days on Murashige-Skoog (MS) medium, MS medium supplemented with 1% sucrose or 3% sorbitol. Bleached cotyledons are indicated (arrowhead).
Figure S3
Supplemental Figure S3. Growth phenotype and lower chlorophyll content of the 2cpa 2cpb mutant.
(A) Rosette diameter of wild type (WT, black bars) and 2cpa 2cpb plants (white bars) was determined under short-day (9 h light/15 h dark cycle) or long-day (16 h light/8 h dark cycle) conditions. (B) Total chlorophyll (black bars), chlorophyll a (light grey bars) and chlorophyll b (dark grey bars) are shown in wild type and 2cpa 2cpb leaves. All data represent the means ± SD (n = 3). Significant differences between WT and mutant plants are indicated by asterisks (*, P < 0.05; **, P < 0.01; **, P < 0.001) using Student’s t test.
Figure S4
Supplemental Figure S4. Leaf pigmentation and aromatic amino acid levels in wild type and 2cpa 2cpb mutant plants.
(A) Purple pigmentation indicating anthocyanin accumulation in wild type (WT), tapx, 2cpa 2cpb and 2cpa 2cpb tapx mutant plants after 1 and 2 d high light (HL, 900 µmol m2 s-1) treatment. (B) Levels of aromatic amino acids in wild type (WT, black bars) and 2cpa 2cpb plants (white bars). All data represent the means ± SD (n = 3). Significant differences between WT and mutant plants are indicated by asterisks (*, P < 0.05; **, P < 0.01) using Student’s t test.
Figure S5
Supplemental Figure S5. Ascorbate, glutathione and jasmonate levels in wild type and 2cpa 2cpb mutant plants. Metabolites were determined in wild type (WT) and 2cpa 2cpb plants after moderate (ML) and high light (HL) treatment. (A) Levels of ascorbic acid (AsA) and dehydroascorbic acid (DHA). (B) Levels of reduced glutathione (GSH) and oxidized glutathione (GSSG). (C) Levels of jasmonic acid (JA) and jasmonic acid isoleucine conjugate (JA-Ile). All data represent the means ± SD (n = 3). Significant differences between WT (black) and mutant plants (white) are indicated by asterisks (*, P < 0.05; **, P < 0.01) using Student’s t test.
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