Volume 7, Issue 7, Pages 1151-1166 (July 2014) Mitogen-Activated Protein Kinase 4 Is a Salicylic Acid-Independent Regulator of Growth But Not of Photosynthesis in Arabidopsis  Gawroński Piotr , Witoń Damian , Vashutina Kateryna , Bederska Magdalena , Betliński Błażej , Rusaczonek Anna , Karpiński Stanisław   Molecular Plant  Volume 7, Issue 7, Pages 1151-1166 (July 2014) DOI: 10.1093/mp/ssu060 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 Isolation and Characterization of mpk4-2/ics1 Double Mutant. (A) Amplification of ICS1 and MPK4 loci with allele-specific PCR primers. Top panel: band of ˜500bp present in Col-0 and mpk4-2 was obtained with ICS1-specific primers, while band of ˜450bp present in ics1 and mpk4-2/ics1 was amplified from different genomic loci. Bottom panel: amplification with MPK4- and T-DNA-specific primers, bands of ˜1000bp and ˜700bp represent wild-type and mpk4-2 allele, respectively. Transcript abundance of MPK4 and ICS1 was checked by RT–PCR (B) with gene-specific primers as a control; primers for ACTIN2 (ACT2) were used on the same cDNA samples. (C) qRT–PCR quantification of PR1 transcript (SAR marker) abundance. Three biological replicates run in triplicate were used (n = 9). (D) Total salicylic acid (SA) levels in analyzed genotypes (n ≥ 5). (C) and (D) data represent mean values ± SEM. The letters above the bars indicate different homogeneous groups with statistically significant differences (Tukey HSD test, P < 0.05). (E) Morphological phenotype of 5-week-old WT, ics1, mpk4-2, and mpk4-2/ics1. Molecular Plant 2014 7, 1151-1166DOI: (10.1093/mp/ssu060) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 2 Photosynthetic Performance of mpk4-2, ics1, mpk4-2/ics1, and WT Plants. (A) Typical chlorophyll a fluorescence induction curves recorded during 5min light treatment (100 μmol m–2 s–1) followed by 7min of dark relaxation. Data represent an average for at least six separate plants (gray shading indicates 95% confidence intervals for analyzed genotypes). (B) Representative pictures of chlorophyll a fluorescence parameters. The panels display false-color images of quantum yield of PSII (Y(II)) and quantum yield of non-regulated energy dissipation (Y(NO)) of 4-week-old Arabidopsis rosettes. Scale bar = 1 cm. (C–E) Light curves of Y(II), Y(NO), and ETR parameters of WT (diamonds), mpk4-2 (circles), ics1 (squares), and mpk4-2/ics1 (orange triangles). Five-week-old plants were dark acclimated for 30min and exposed to gradually increasing actinic light intensities. Data represent mean values of at least six independent plants ± SD (n ≥ 6). Asterisks indicate significant difference by Tukey HSD test (* P < 0.05; ** P < 0.01; *** P < 0.001). Molecular Plant 2014 7, 1151-1166DOI: (10.1093/mp/ssu060) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 3 Quantification of DRP and BAP1 Transcripts (1O2 Gene Markers (Ramel et al., 2012)) in Analyzed Mutants and Wild-Type. Data are mean values of at least three biological replicates (run in triplicate); error bars indicate ± SEM (n = 9). The letters above the bars indicate different homogeneous groups with statistically significant differences (Tukey HSD test, P < 0.05). Molecular Plant 2014 7, 1151-1166DOI: (10.1093/mp/ssu060) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 4 Fluorescence Transients: O-J-I-P Test. (A) Fluorescence induction under continuous illumination, normalized to F0. (B) Spider-plot of selected parameters calculated from induction curve normalized to the WT. Data represent mean values for at least six rosettes (n ≥ 6). Asterisks indicate significant difference by Tukey HSD test (* P < 0.05; ** P < 0.01; *** P < 0.001). Molecular Plant 2014 7, 1151-1166DOI: (10.1093/mp/ssu060) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 5 Transcript Levels of Major Chloroplast Antioxidative Enzymes in Analyzed Genotypes. (A) Simplified scheme of H2O2 metabolism in chloroplasts. (B) Quantification of genes’ expression involved in ROS metabolism in chloroplasts by quantitative real-time RT–PCR in wild-type and analyzed mutants. Total RNA was extracted from 4-week-old Arabidopsis rosettes and subjected to reverse transcription. Quantitative real-time PCR was performed using gene-specific primers (for sequences, see the ‘Methods’ section). Primers for two housekeeping genes were used: UPL7 and YLS8. Data are mean values of at least three biological replicates (run in triplicate); error bars indicate ±SEM (n = 9). The letters inside the bars indicate different homogeneous groups with statistically significant differences (Tukey HSD test, P < 0.05). Molecular Plant 2014 7, 1151-1166DOI: (10.1093/mp/ssu060) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 6 ROS-Scavenging Enzyme Activity in Analyzed Genotypes. (A) Superoxide dismutase, (B) catalase, (C) ascorbate peroxidase, and (D) activity of different forms of SOD normalized to the WT. Data represent average values of at least three biological and three technical replicates (n = 9) ±SEM. The letters above the bars indicate different homogeneous groups with statistically significant differences (Tukey HSD test, P < 0.05). Molecular Plant 2014 7, 1151-1166DOI: (10.1093/mp/ssu060) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 7 Ultrastructure of Chloroplasts in Analyzed Plants. (A) Wild-type. (B)ics1 mutant. (C–E)mpk4-2. (F–H)mpk4-2/ics1. CW, cell wall; GT, grana thylakoids; PG, plastoglobuli; S, starch; ST, stroma thylakoids; Str, stroma; V, vacuole (n = 4). Bar = 2 μm. Molecular Plant 2014 7, 1151-1166DOI: (10.1093/mp/ssu060) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions