Volume 10, Issue 1, Pages 131-142 (January 2017) Contribution of the Alternative Respiratory Pathway to PSII Photoprotection in C3 and C4 Plants  Zi-Shan Zhang, Mei-Jun Liu, Renate Scheibe, Jennifer Selinski, Li-Tao Zhang, Cheng Yang, Xiang-Long Meng, Hui-Yuan Gao  Molecular Plant  Volume 10, Issue 1, Pages 131-142 (January 2017) DOI: 10.1016/j.molp.2016.10.004 Copyright © 2017 The Author Terms and Conditions

Figure 1 Characterization of Leaves from aox1a Mutant and Wild-Type Arabidopsis Plants. (A, B, and D) Total respiration (R) rate, the COX pathway respiration rate (the sensitivity of O2 uptake to 2 mM KCN in the presence of 20 mM SHAM), the AOX pathway respiration rate (the sensitivity of O2 uptake to 20 mM SHAM), and the AOX pathway respiration rate/total respiration rate ratio (AOX/total R rate) as well as the initial and total activities and activation state of NADP-MDH in the leaves of aox1a mutant and wild-type (WT) plants in 2 h of darkness (D) or under high light (600 μmol m−2 s−1; L). (C) Maximum quantum yield of PSII (Fv/Fm) in the leaves of aox1a mutant and wild-type plants exposed to 600 μmol m−2 s−1 light at 25°C for the indicated times. Prior to high-light treatment, the leaves were infiltrated with 0 or 1 mM SHAM solution. B on the x axis of plot (C) indicates before infiltration with reagent. Means ± SD, n = 10–18. Different letters indicate significant differences between different treatments (P < 0.05). See also Supplemental Figures 1 and 2. Molecular Plant 2017 10, 131-142DOI: (10.1016/j.molp.2016.10.004) Copyright © 2017 The Author Terms and Conditions

Figure 2 PSII Photoinhibition and Photorespiration in Leaves of aox1a Mutant and Wild-Type Arabidopsis Plants. (A and B) Maximum quantum yield of PSII (Fv/Fm) and PSII excitation pressure (1 − qP) in the leaves of aox1a mutant and wild-type plants after exposure to 600 or 1200 μmol m−2 s−1 light at 25°C for 2 h under normal (400 μL L−1), low (50 μL L−1), or high (1500 μL L−1) CO2 and normal (210 mL L−1) or low (20 mL L−1) O2. N indicates normal environment (600 μmol m−2 s−1 light, 400 μL L−1 CO2, and 210 mL L−1 O2). Means ± SD, n = 10–18. (C–E) Ratio of the abundances of glycine and serine (Gly/Ser), post-illumination CO2 burst (PIB), and O2 inhibition of Pn ([Pn2%O 2 − Pn21%O2]/Pn2%O2) in the leaves of aox1a mutant and wild-type plants under 600 μmol m−2 s−1 light. Means ± SD, n = 3–6. Different letters indicate significant differences between different treatments (A and B) or between WT and mutant plants (C–E) (P < 0.05). See also Supplemental Figures 3–5. Molecular Plant 2017 10, 131-142DOI: (10.1016/j.molp.2016.10.004) Copyright © 2017 The Author Terms and Conditions

Figure 3 Activation of the AOX Pathway and Contribution of the AOX Pathway to PSII Photoprotection in C3 and C4 Leaves under High Light. (A–G) Total respiration (R) rate, the COX pathway respiration rate (the sensitivity of O2 uptake to 2 mM KCN in the presence of 20 mM SHAM), the AOX pathway respiration rate (the sensitivity of O2 uptake to 20 mM SHAM), the AOX pathway respiration rate/total respiration rate ratio (AOX/total R rate), relative expression of AOX genes, and immunoblot analysis of AOX protein in leaves of C3 (Cucumis sativus) and C4 plants (Zea mays) after 0, 2, and 5 h high-light (600 μmol m−2 s−1) treatments. (H and I) Maximum quantum yield of PSII (Fv/Fm) in Cucumis sativus and Zea mays leaves exposed to 600 μmol m−2 s−1 light at 25°C for the indicated times. Before high-light treatment, leaves were infiltrated with 0 or 1 mM SHAM solution. B on the x axis in plot (C) indicates before infiltration with reagent. The loading controls of plot (G) are shown in Supplemental Figure 1C. Means ± SD, n = 10–18 or 3 (real-time PCR). Different letters indicate significant differences between different treatments (P < 0.05). See also Supplemental Figures 1, 2, and 6. Molecular Plant 2017 10, 131-142DOI: (10.1016/j.molp.2016.10.004) Copyright © 2017 The Author Terms and Conditions

Figure 4 Relationship of the AOX Pathway and Photorespiration in C3 and C4 Leaves. (A and B) Maximum quantum yield of PSII (Fv/Fm) in C3 (Cucumis sativus) and C4 plant (Zea mays) leaves after exposure to 600 or 1200 μmol m−2 s−1 light at 25°C for 2 h under normal (400 μL L−1), low (50 μL L−1), or high (1500 μL L−1) CO2 and normal (210 mL L−1) or low (20 mL L−1) O2 in the absence or presence of 1 mM SHAM. N indicates normal environment (600 μmol m−2 s−1 light, 400 μL L−1 CO2, and 210 mL L−1 O2). Means ± SD, n = 10–18. (C and D) Post-illumination CO2 burst (PIB), O2 inhibition of Pn (O2-Pn; (Pn2%O2 − Pn21%O2)/Pn2%O2), and ratio of the abundances of glycine and serine (Gly/Ser) in C3 and C4 plant leaves in the absence or presence of 1 mM SHAM. Means ± SD, n = 3–6. Different letters indicate significant differences between different treatments (P < 0.05). See also Supplemental Figures 3–5. Molecular Plant 2017 10, 131-142DOI: (10.1016/j.molp.2016.10.004) Copyright © 2017 The Author Terms and Conditions

Figure 5 Influence of Photorespiration Inhibitor and CO2 Concentration on the Activity of NADP-MDH in Leaves of Cucumis sativus under High-Light Conditions. (A and B) Initial activity, total activity and activation state of NADP-MDH in leaves of Cucumis sativus exposed to high light (600 μmol m−2 s−1; L) for 2 h under (A) normal atmosphere (400 μL L−1 CO2 and 21% v/v O2) in the absence (water) or presence of inhibitors of AOX (SHAM) or photorespiration (aminoacetonitrile, AAN); or under (B) normal (400 μL L−1), low (50 μL L−1), or high (1500 μL L−1) CO2 atmosphere. Means ± SD, n = 5. Different letters indicate significant differences between different treatments (P < 0.05). Molecular Plant 2017 10, 131-142DOI: (10.1016/j.molp.2016.10.004) Copyright © 2017 The Author Terms and Conditions

Figure 6 Effect of SHAM on PSII Photoinhibition in Three C3 and Five C4 Species. Maximum quantum yield of PSII (Fv/Fm) in leaves of three C3 species (Nicotiana tabacum, Triticum aestivum, and Salix babylonica), three NADP-ME type C4 plants (Zea mays, Sorghum bicolor, and Euchlaena mexicana), one NAD-ME type C4 plant (Portulaca oleracea), and one PPCK-type C4 plant (Salvia farinacea) exposed to 1200 μmol m−2 s−1 light at 25°C for the indicated times. Leaves were collected from a campus or farm. Prior to high-light treatment, the leaves were infiltrated with 0, 0.5, 1, or 2 mM SHAM solution. The B on the x axis indicates before SHAM infiltration. Means ± SD, n = 10. Different letters indicate significant differences between the different treatments (P < 0.05). Molecular Plant 2017 10, 131-142DOI: (10.1016/j.molp.2016.10.004) Copyright © 2017 The Author Terms and Conditions

Figure 7 Scheme of Relationships between the AOX Pathway and PSII Photoprotection. When the AOX pathway is inhibited, or in the aox1a mutant (1), NADH accumulates (2). The accumulation of NADH (2) inhibits GDC complex activity (3), resulting in the accumulation of Gly, the substrate of the GDC complex (4). The accumulation of Gly (4) then feedback inhibits the upstream metabolism of photorespiration, resulting in the accumulation of glycolate (5) and NADPH (6). The accumulation of glycolate (5) inhibits D1 protein turnover, causing PSII photoinhibition. The accumulation of NADPH (6) decreases photosynthetic electron transport, aggravating PSII photoinhibition. Simultaneously, NADPH accumulation increases NADP-MDH activation (7) and advances the export of reducing equivalents. 3-PGA, 3-phosphoglycerate; AOX, alternative oxidase; COX, cytochrome c oxidase; GA, glyceric acid; GDC, glycine decarboxylase; Gly, glycine; Mal, malate; NAD-MDH, NAD-malate dehydrogenase; NADP-MDH, NADP-malate dehydrogenase; OAA, oxaloacetate; OH-Pyr, hydroxypyruvate; RuBisCO, ribulose 1,5-bisphosphate carboxylase/oxygenase; RuBP, ribulose 1;5-bisphosphate; Ser, serine; UQ, ubiquinone. Molecular Plant 2017 10, 131-142DOI: (10.1016/j.molp.2016.10.004) Copyright © 2017 The Author Terms and Conditions