Fig 2: Restoration of catalase activity in transgenic lines and variants form active homo‐tetramers. (a) Catalase activity was measured in leaf extracts from wild type, cat2‐1 mutant and different independent transgenic lines grown under short (8hL 16hD) and long‐day (16hL 8hD) conditions and sampled at the same time relative to start of the light period. For each line, three biological replicates were each measured in triplicate. Grey, black and white bars are data from separate experiments. Percentage of catalase activity relative to wild type in each experiment is indicated at the top of the graph (activities are given in Table S2). Error bars represent the SE. *** Indicates significantly lower catalase activity (p < 0.001) by Student's t‐test compared to the wild type in each experiment. (b) Characterisation of catalase isoforms by native PAGE. Upper panel: Total protein (20 µg per line) extracted from leaves of 4‐week‐old wild type, cat2‐1, CAT2PSI, CAT2WSQV and CAT2ARL was separated on native‐PAGE and blotted on nitrocellulose membrane with catalase antibody (Agrisera, AS09501) which recognises all three catalase isoforms (CAT1, CAT2 and CAT3). Cross reacting protein (triangle) and CAT2 (circle). A shifted band of CAT2ARL is indicated by an arrow. Lower panel: in‐gel activity assay

Fig 3: Catalase C terminal variants rescue growth phenotypes of the cat2‐1 mutant in normal air and under photorespiratory conditions. (a) Transgenic lines were generated by expressing different variants of CAT2 under its own promoter and 3′UTR. (b) 2‐ 3‐ 4‐, and 5‐week‐old plants of wild type, cat2‐1, 3rd independent line of PSI3 and WSQV3, and 2nd independent line of ARL2 grown under short‐day conditions (Light intensity: 190 µmol. m−2. s−1, 8 h light and 16 h dark, 20°C, 60% humidity) for 4 weeks. Scale bars represent 1 cm. All plants photographed at the same magnification once a week for 4 weeks. Plants photographed at the 1st, 2nd, 3rd and 4th weeks (indicated as Week 1, Week 2, Week 3 and Week 4, respectively). The experiment was repeated three times (three independent lines) with similar results. (c) Rosette fresh weight; three independent lines are presented (grey, black and white bars). With each independent line, wild type and cat2‐1 mutant were also grown under identical growth conditions. Plants presented by black and white bars are 5‐week‐old. In comparison, plants presented by grey bars are 6 weeks old. Means were calculated from six plants per experiment. *** Significant difference from wild type at p < 0.001 by Student t‐test. Error bars represent the SE. (d) Phenotypes of wild type, cat2‐1, and transgenic lines under photorespiratory and control conditions. The appearance of plants grown at 16/8 h day/night photoperiod at 22°C for 2 weeks and subjected to 100 µmol m−2 s−1 constant light for 1 week with air‐tight tape (left image) and air‐permeable tape (right image). (e) False‐colour images of Fv/Fm (maximum quantum yield of photosystem II) [Color figure can be viewed at wileyonlinelibrary.com]

Fig 4: SnRK2.4 and SnRK2.10 modulate catalase (CAT) on multiple levels during response to salt stress. Wild type and snrk2 mutants’ seedlings were subjected to treatment with 150 mM NaCl for times indicated. CAT1 expression was determined by RT-qPCR (A), total catalase protein was determined by immunoblot analysis (B), and total catalase activity assay was performed (C); error bars represent SD; stars represent statistically significant differences in comparison with the wt plants (Student t-test) where * p < 0.05; ** p < 0.001; *** p < 0.0001. After exposure, membranes were stripped and reused for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) detection as a loading control. At least two independent biological replicates of the experiment were performed, each with four samples per data point. Results of one representative experiment are shown.

Fig 5: Cell fractionation reveals all catalase forms are targeted to peroxisomes. Subcellular fractionation of 4‐week‐old Arabidopsis thaliana leaves of wild type, cat2‐1 mutant, and transgenic lines (a–f). A crude peroxisome pellet of the indicated lines was separated on a sucrose gradient and hydroxypyruvate reductase (HPR, red) and catalase (black) specific activities were determined in each fraction. The fractions corresponding to the peroxisomes were separated by SDS‐PAGE and immunoblotted with anti‐CAT2 (f, upper panel) and anti‐Atpβ antibodies (f, lower panel). The vertical black line in panel (f) upper panel indicates where a lane was moved to produce the final figure. The original image can be seen in Figure S11C [Color figure can be viewed at wileyonlinelibrary.com]