1. Volume 9, Issue 5, Pages 737-748 (May 2016)
Association–Dissociation of Glycolate Oxidase with Catalase in Rice: A Potential Switch to Modulate Intracellular H2O2 Levels 
Zhisheng Zhang, Yuanyuan Xu, Zongwang Xie, Xiangyang Li, Zheng-Hui He, Xin-Xiang Peng 
Molecular Plant 
Volume 9, Issue 5, Pages (May 2016)
DOI: /j.molp
Copyright © 2016 The Author Terms and Conditions

2. Figure 1
Expressions of CAT Isoforms and the Interaction between GLO and CAT In Vitro.
(A) mRNA transcript abundance of the three CAT genes was determined by qRT–PCR. Rice seedlings at the five-leaf stage were used for RNA extraction. Values are means ± SD of three biological replicates (n = 3).
(B) In-gel activity staining of CAT isozymes expressed in yeast and extracted from rice leaves. CAT isozymes were separated in gradient CN–PAGE (3%–20%). R-CAT represents crude enzymes extracted from rice leaves. Y-CATA, Y-CATB, Y-CATC represents the enzyme extracted from yeast expressing pYES2-CATA, pYES2-CATB, pYES2-CATC, respectively. Y-CK represents the enzyme extracted from wild-type yeast cells. The number above each lane indicates the activity unit of enzyme loaded (1 U = 1 μmol H2O2 min−1). The result is representative of three independent experiments.
(C) Subcellular localization of CAT isoforms. The GFP-tagged CAT isoform and CFP-tagged PTS1 fusion construct were transiently expressed in rice protoplasts. Cells are imaged by a confocal microscope at 14–16 h after transfection; the CFP-tagged PTS1 was used as the peroxisomal marker. The red signals represent chlorophyll autofluorescence. The result is representative of three independent experiments. Scale bar, 4 μm.
(D) GLO-interacting proteins identified by in vitro pull-down assay. NHisGLO1-interacting proteins were affinity pulled down from the total protein extracted from rice leaves (at the five-leaf stage). The samples were then further separated by SDS–PAGE and silver-stained. The bands specific to the NHisGLO1 were identified by mass spectrometry.
(E) Identification of the protein bands isolated in (D) by LC-MS/MS. The identified CATC peptides are highlighted in yellow.
(F) Interactions between each GLO and each CAT in yeast. Either NHisGLO1 or NHisGLO4 was co-expressed with CATA, CATB, or CATC in yeast cells. Proteins were affinity pulled down from yeast total protein extracts and fractionated by SDS–PAGE; GLO and CAT proteins were detected by western blotting. Different combinations of GLO and CAT isoforms expressed in yeast cells are indicated at the top of each panel (NHisGLO1 + CATA; NHisGLO1 + CATB; NHisGLO1 + CATC; NHisGLO4 + CATA; NHisGLO4 + CATB; NHisGLO4 + CATC). The result is representative of three independent experiments.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

3. Figure 2 GLO and CAT Interact In Vivo.
(A) Leaf tissues of five-leaf stage transgenic rice plants carrying NHisGLO1 were homogenized to generate a protein extract. Total protein and proteins affinity purified from the protein extracts by Ni-IDA (Pull-down) were fractionated by SDS–PAGE and silver-stained. Molecular weight ladders (M) were loaded on the left lane.
(B) The pull-down protein samples were separated by SDS–PAGE (as in Figure 2A) and subjected to western blotting analysis with both of the two antibodies indicated (GLO, CAT). The result is representative of three independent experiments.
(C) Interaction between GLO and CAT detected by BiFC analysis. NYFP-GLO and CYFP-CAT were co-expressed in rice protoplasts. Fluorescence was detected by confocal microscopy. Pairwise co-transfected constructs are labeled at the top of each panel. NYFP without any GLO fusions was used as a control. The red signals represent chlorophyll autofluorescence. Scale bar, 4 μm.
(D) The frequency of the BiFC-positive protoplasts in each pairwise transfection was calculated on basis of 200 protoplast cells observed according to Akamatsu et al. (2013). The data represent means ± SD of five biological replicates (n = 5). Different lowercase letters in the same column indicate significant differences at P < 0.05.
(E) GLO-interacting proteins as identified by Co-IP analysis. Proteins immunoprecipitated by the GLO antibody were subjected to gradient CN–PAGE (3%–20%, as in Figure 1B) and analyzed by western blotting with a CAT antibody. YFP antibody was used as the control (control IP). The result is representative of three independent experiments.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

4. Figure 3
Effects of Various Chemicals on the Interaction between GLO and CAT as Detected by BiFC.
(A) BiFC fluorescence of the rice protoplasts treated with various chemicals (ABA, 100 μM; chitin, 10 nM; SNP, 100 μM; 3-AT, 2 mM; GLC, 5 mM; HPMS, 500 μM; SA, 100 μM). The red signals represent chlorophyll autofluorescence. Scale bar, 4 μm.
(B) The frequency of the BiFC-positive protoplasts was calculated on the basis of 200 protoplast cells observed according to Akamatsu et al. (2013).
(C) NYFP-GLO and CYFP-CAT protein syntheses in rice protoplasts were not affected by the chemicals tested. Levels of NYFP-GLO and CYFP-CAT were detected by western blotting with an YFP antibody.
The data represent means ± SD of five biological replicates (n = 5). Different lowercase letters in the same column indicate significant differences at P < 0.05.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

5. Figure 4 Effect of SA and GLC on H2O2 Accumulation in Rice Leaves.
(A) H2O2 accumulation in rice leaf segments (20 mm2) treated with 2 mM SA, or 5 mM GLC, or 2 mM SA + 5 mM GLC, as labeled. The data represent means ± SD of five biological replicates (n = 5). Different lowercase letters in the same column indicate significant differences at P < 0.05.
(B) H2O2 DAB staining in rice leaves (10-cm long). Rice leaves were fed through the cut end with 2 mM SA, or 5 mM GLC, or 2 mM SA + 5 mM GLC, respectively. The result is representative of three independent experiments.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

6. Figure 5
A Dynamic Switch Model for the Association–Dissociation of Glycolate Oxidase and Catalase in Plants.
The scheme was formed by referring to Zhang (2011). According to the substrate channeling mechanism, when the GLO and CAT complex is dissociated, the product H2O2 will be increased in the bulk medium. Diffusions of GLC and H2O2 are supposed to be directional.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions