, Moreover, the percentage of the ratio between activity and capacity of the AOX 200 (previously defined by others as AOX engagement and here calculated from Figure 1C
, After porin levels 209 correction, the total amount of AOX was similar between genotypes but significantly 210 different between redox treatments, being the mean of the total AOX amount in non-211 treated, diamide-treated and DTT-treated samples of 0.57 ± 0.04, 0.13 ± 0.02 and 1.28 ± 212 0.28, respectively. Therefore, the redox treatments significantly affected the 213 mitochondrial protein extracted. The reason for these differences remain uncertain, 214 however, these results do not affect to the evaluation of the AOX redox state among 215 genotypes in the non-treated samples. In the absence of redox chemicals, the AOX was 216 mainly found in its monomeric (reduced) form, thus representing 89 to 93% of the total 217 AOX detected in all genotypes (Figure 3 and Supplemental Figure S2B). Signals of the 218 dimeric forms, one with a lower molecular weight perhaps indicating heterodimers from 219 different isoforms, were only between 7% and 11% of the total AOX, order to further study the AOX regulation, the redox state of the AOX protein was 205 analyzed in isolated leaf membranes with or without the addition of DTT or diamide 206 (Figure 3 and Supplemental Figure S2B, see Material and Methods for details
, solution mixed with 4 mL of scintillation cocktail (Rotizint Eco Plus, ROTH) measured 586 by a liquid scintillation counter, 2015.
, 2006) using 592 approximately 50 mg of frozen-powdered leaf tissue. Derivatization and gas 593 chromatography-time of flight-mass spectrometry (GC-TOF-MS) analyses were carried 594 out as described previously, Golm Metabolome Database, p.36, 2005.
, Data were normalized to the mean value of Col-0 plants in moderate 600 light conditions (i.e. the value of all metabolites for Col-0 at moderate light was set to 1), 2011.
, Values presented are means ± SE of seven replicates which were taken from same rosettes
, Leaves of similar size from the Col-0 and mutant plants were fed via the petiole by placing 606 in a solution containing 10 mM MES-KOH
, light intensities applied during the petiole feeding 610 were lower than in the other experiments in order to avoid leaf wilting induced by the 611 high transpiration occurring during the high light treatment. Four fold-increase in light 612 intensity during the high light treatment was maintained as in the other experiments
, 30 mg of frozen-powdered leaf tissue was extracted and derivatized 615 as described above and analyzed by GC-TOF-MS in parallel with a mixture of authentic 616 standards of several metabolites at four different concentrations, 2014.
, Net CO 2 assimilation (A N ), stomatal conductance (g s ) and chlorophyll fluorescence were 628 measured simultaneously with an open infrared gas-exchange analyser system
,
, Fully expanded leaves were clamped and leaf chamber conditions were set 631 to: 400 ?mol CO 2 mol -1 air (C a ), temperature of 23ºC and photosynthetically active 632 photon flux density (PPFD) of 150 and 600 ?mol m -2 s -1 (provided by the light source of 633 the Li-6400 with 10% blue light) at moderate light and after high light treatment, 634 respectively. After approx. 3 min, gas-exchange and chlorophyll fluorescence 635 measurements were performed in the light. The actual quantum efficiency of the 636 photosystem II (PSII)-driven electron transport (?PSII) and the electron transport rate 637 (ETR) were determined as previously, 2011.
, A N was measured at saturating light conditions (PPFD of 1000?mol m -2 s -1 ) 642 under photorespiratory (21% O 2 ) and non-photorespiratory (approx. 2% O 2 ) conditions 643 in all genotypes and treatments. The percentage of O 2 inhibition of A N was, p.673
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, Figure 1. In vivo mitochondrial electron transport chain activities in trxo1 mutants
, pathway activity (? cyt ) and (C) AOX pathway activity 928 (? alt ) in leaves of Col-0, Total respiration
, AOX capacity (V alt ) and 935 (B) protein levels in leaves of Col-0, trxo1-1 and trxo1-2 plants at moderte light (ML) 936 and after 2 and 4 hours of high light (HL) treatment. Values in A are means ± SE of five 937 replicates. In B, one representative blot is presented and the relative values shown are the 938 mean of the four immunoblot experiments performed with similar results (Supplemental 939 Figure S2). The relative values indicate the intensities of the signals from AOX 940 normalized to those from porin, and then expressed as fold-changes relative to values of loading among genotypes in each (redox) treatment. The table below the 949 image shows the mean values (n=2) of the percentages on the oxidized and reduced forms 950 in the different genotypes after
, TCA cycle and other respiratory fluxes in trxo1 mutants
, C3-4) corresponds to the metabolic flux through glycolysis 957 and TCA cycle, respectively. The 14 CO 2 evolution was measured every hour after the 958 addition of the labeled Glc and summed up to calculate the total 14 C evolved. Each point 959 represent the means ± SE of three replicates, vol.3
, photosynthetic electron transport rate (ETR), 964 stomatal conductance (g s ) and the percentage of O 2 inhibition of photosynthesis in leaves 965 in Col-0, trxo1-1 and trxo1-2 plants grown under ML and after 2 and 4h of high light 966 (HL) treatment. Details on the gas exchange and chlorophyll fluorescence measurements 967 can be found in the Material and Methods section. Values are means ± SE of six (A N , g s 968 and ETR) or four (% O 2 inhibition) replicates and asterisks denote significant differences, Figure 5. Photosynthetic and photorespiratory parameters under high light stress 963 conditions. Net photosynthesis (A N )
, Relative 975 metabolite levels in leaves of Col-0, trxo1-1 and trxo1-2 plants under all light conditions 976 were normalized to the mean level of the Col-0 plants under ML conditions and fold-977 change values were log2 transformed (i.e. the level of all metabolites of Col-0 plants 978 under ML is 0). In this heat map, red and blue colors represent log2 fold-increased and -979 decreased metabolites, respectively. Values are means ± SE of six replicates and asterisks 980 denote significant differences, Figure 6. Metabolite profiling in trxo1 mutants. Heat map showing the relative levels of 972 the GC-MS-analyzed metabolites in Col-0, trxo1-1
, Total 13 C label redistribution into primary metabolites in trxo1 mutants. The 985 total 13 C label redistribution in selected leaf metabolites of Col-0, pp.1-1
, Relative metabolite levels in leaves of Col-0, trxo1-1 and trxo1-999 2 plants under moderate light (ML) and after 2 and 4h of high light (HL) treatment. Data 1000 is presented as means ± SE for six biological replicates normalized to the mean level of 1001 the Col-0 plants under ML. Bold numbers denote significant differences, Supplemental Table S2
, Total 13 C label redistribution in leaf metabolites of Col-0, trxo1-1006 1 and trxo1-2 plants after 2 and 4 hours of 13 C-Glucose labeling under moderate light and 1007 high light conditions. Values are means ± SE of six replicates, and those values bold and 1008 underlined denote significant differences, Supplemental Table S3
, Total 13 C label redistribution in leaf metabolites of Col-0, trxo1-1012 1 and trxo1-2 plants after 2 and 4 hours of 13 C-Malate labeling under moderate light and 1013 high light conditions. Values are means ± SE of six replicates, and those values bold and 1014 underlined denote significant differences
, Supplemental Table S5. Primers used in the qPCR analyses performed in this study, p.1018
, A) qPCR analysis of transcript levels from genes related to mitochondrial TRX 1021 system and AOX1a in leaves trxo1-1 and trxo1-2 plants under moderate light conditions 1022 (ML). Primers used and gene information can be found in Supplemental Table S5. Values 1024 the Col-0 plants; n.d. (not detected). (B) Photograph representative of Col-0, trxo1-1 and 1025 trxo1-2 plants after growing for 6 weeks under ML conditions. (C) Rosette biomass 1026 accumulation of plants grown as in B. Values are means ± SE of twelve replicates and 1027 asterisks denote significant differences
, and trxo1-2 plants at moderate light (ML) and 1031 after 2 and 4 hours of high light (HL) treatment. Three blots of each AOX and porin 1032 proteins are shown, which were used for the AOX amount quantifications, together with 1033 the blot shown in Figure 2B, as described in Material and Methods section. (B) AOX 1034 protein redox state in trxo1 mutants. AOX protein immunodetection in leaf membranes 1035 fractions from Col-0, trxo1-1 and trxo1-2 plants grown under ML conditions (see 1036 Experimental Procedures) and treated with 50 mM DTT (for reduced-monomeric form 1037 detection) or 50 mM diamide, Supplemental Figure S2. AOX protein amount and redox analysis. (A) AOX and porin 1030 protein levels in leaves of Col-0, trxo1-1