Nuclear and peroxisomal targeting of catalase

Abstract

Catalase is a well-known component of the cellular antioxidant network, but there have been conflicting conclusions reached regarding the nature of its peroxisome targeting signal. It has also been reported that catalase can be hijacked to the nucleus by effector proteins of plant pathogens. Using a physiologically relevant system where native untagged catalase variants are expressed in a cat2-1 mutant background, the C terminal most 18 amino acids could be deleted without affecting activity, peroxisomal targeting or ability to complement multiple phenotypes of the cat2-1 mutant. In contrast, converting the native C terminal tripeptide PSI to the canonical PTS1 sequence ARL resulted in lower catalase specific activity. Localisation experiments using split superfolder green fluorescent protein revealed that catalase can be targeted to the nucleus in the absence of any pathogen effectors, and that C terminal tagging in combination with alterations of the native C terminus can interfere with nuclear localisation. These findings provide fundamental new insights into catalase targeting and pave the way for exploration of the mechanism of catalase targeting to the nucleus and its role in non-infected plants.

Keywords: ROS; nucleus; peroxisome; redox signalling.

Figure 1

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]

Figure 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, CAT2^PSI, CAT2^WSQV and CAT2^ARL 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 CAT2^ARL is indicated by an arrow. Lower panel: in‐gel activity assay

Figure 3

Redox balance and oxidative signalling are restored in complemented lines. Wild type, cat2‐1 mutant, and transgenic lines were grown under short (SD; 8hL 16hD) for 4 weeks and then transferred into long‐day (LD; 16h L8hD) conditions for 1 week and measurements made of reduced and oxidised ascorbate (a) and glutathione (b). In each case, black bars represent the reduced form and red bars the oxidised form. ***p < 0.001 by Student's t‐test compared with the wild type (Content of the reduced form). GSSG and GSH ratio was also calculated (SD; 10:1, 2:1, 12:1, 7:1, 15:1 for wild type, cat2‐1, CAT2^PSI2 CAT2^WSQV2 and CAT2^ARL1, respectively); (LD; 17:1, 7:1, 18:1, 11:1, 21:1 for wild type, cat2‐1, CAT2^PSI2 CAT2^WSQV2 and CAT2^ARL1, respectively). Data are means of three independent extracts of three different plants. Error bars represent the SE. Black asterisks (content of GSH) or red (content of GSSG). (c) RT‐qPCR analysis of oxidative marker transcripts from plants grown under SD conditions for 4 weeks. The mean value of three replicates was normalised using ACTIN 2 as the internal control. The expression level in the wild type was assigned a value of 1. (*p < 0.05) and (***p < 0.001) by Student's t‐test compared to the wild type [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

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]

Figure 5

C terminal tagged wild type CAT2 and short‐form target to peroxisomes, wild type can also target to nucleus. CAT2 variants tagged with sfGFP11 at the C terminus transfected into protoplasts of transgenic Arabidopsis expressing sfGFP1‐10^OPT targeted to peroxisome (top) and nucleus (bottom). (b,f) Positive control: PX‐mCherry‐11and NU‐mCherry‐11 for peroxisome and nucleus, respectively. (b,g) Negative control (no plasmid). (c,h) CAT2^PSI‐sfGFP11. (d,i) CAT2^WSQV‐sfGFP11. (f,j) CAT2^ARL‐sfGFP11. Scale bars = 10 µm (top) and 20 µm (bottom). Representative images reflecting the results of 3 (top) and 2 (bottom) independent experiments are shown. Protoplasts were incubated for 27–29 h in light and scanned using confocal laser scanning microscopy [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6

N terminal tagged CAT2 variants target to nucleus and peroxisomes. CAT2 variants tagged with sfGFP11 at the N terminus transfected into protoplasts of transgenic Arabidopsis expressing sfGFP1‐10^OPT targeted to peroxisome (a–j) and nucleus (k–o). The middle row (f–j) shows a magnified image of the protoplast indicated by a white dotted box in the panel above. Positive control: PX‐mCherry‐11 (a,f) and NU‐mCherry‐11 (k) for peroxisome and nucleus, respectively. (b,g,l) Negative control (no plasmid). (c,h,m) sfGFP11‐CAT2^PSI. (d,l,n) sfGFP11‐CAT2^WSQV. (e,j,o) sfGFP11‐CAT2^ARL. Scale bars = 20 µm top and bottom 5 μm middle. Representative images reflecting the results of 1 (top) and 2 (bottom) independent experiments are shown. Protoplasts were incubated for 27–29 h in light and scanned using confocal laser scanning microscopy [Color figure can be viewed at wileyonlinelibrary.com]