Supplementary Materials. The biochemical basis for thermoregulation in heat-producing flowers

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1 Supplementary Materials The biochemical basis for thermoregulation in heat-producing flowers Yui Umekawa, Roger S. Seymour 2, *, and Kikukatsu Ito United Graduate School of Agricultural Science, Iwate University, -- Ueda, Morioka, Iwate, 2-, Japan. 2 School of Biological Sciences, University of Adelaide, Adelaide, SA, Australia. Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University, -- Ueda, Morioka, Iwate 2-, Japan. * Corresponding author ( kikuito@iwate-u.ac.jp)

2 2 Plant Plant 2 Plant Plant y = -.x 2 +.2x +. R² =. y = -.2x 2 -.x +. R² =.2 y = -.x 2 +.x +. R² =.2 y = -.x 2 +.2x +. R² = T Term ( /K) Supplementary Figure S. Curve fitting of individual spadix respiration rate using a modified Arrhenius model. Individual data in Fig.a are unified and presented.

3 2 2 Plant y =.2x R² = /T ( /K) 2 2 Plant 2 y =.22x +. 2 R² = /T ( /K) 2 2 Plant y =.x +. 2 R² = /T ( /K) 2 2 Plant y =.x +. 2 R² = /T ( /K) Supplementary Figure S2. Curve fitting of individual spadix respiration rates using a classical Arrhenius model. Data are derived from Seymour et al..

4 a b Aconitase activity (nmol isocitrate/min/mg protein) Aconitase ICDH activity (nmol NAD(P)H/min/mg protein) 2 ICDH NAD-IDH + -ICDH NADP-IDH + -ICDH Supplementary Figure S. Temperature effects on citrate-driven mitochondrial respiration. (a) Metabolism and respiration in mitochondria. Citrate is metabolized to 2OG through isocitrate mediated by aconitase and ICDH. In the presence of rotenone and malonate, produced NAD(P)H is oxidized to NAD(P) + via NDA. Externally added NADH is oxidized by external NADH dehydrogenase NDB. I-V: Complex I-V; ICDH; isocitrate dehydrogenase; 2OG: 2-oxoglutarate; Suc-CoA: Succinyl-CoA; Suc: Succinate; Fum: Fumarate; Mal: Malate; OAA: Oxaloacetate; OM: outer membrane; IM: Inner membrane; IMS: Intermembrane space. (b) Temperature response of enzyme activities of aconitase and ICDH (n = ). There are two types of mitochondrial ICDH: NAD + -type (black) and NADP + - type (white).

5 k (NDA) 2NADPH + 2H OG + CO 2 + UQ 2NADP + + Isocitrate + UQH 2 H 2 O k (ICDH) k 2 (AOX & COX) /2O 2 k, k, E a E a NADPH k 2, H ΔG o =2 kj/mol NADP + E a O2 AOX & COX ΔG o =.2 kj/mol H 2 O (ground state) Reaction coordinate Supplementary Figure S. A model for pre-equilibrium reaction mediated by mitochondrial NADPH-NDA/ICDH and terminal oxidases (AOX and COX). Intra-mitochondrially produced NADPH by ICDH show pre-equilibrium reaction by NDA and following oxidation by terminal oxidases (AOX and COX). Rate constants (k, k and k 2 ) and activation energy are depicted as E a, E a and E a. Gibbs free energy changes (ΔG o ) for each reaction is also shown. 2-OG: 2-oxoglutarate; UQ: Ubiqunone; UQH 2 : Ubiquinol.

6 a. (nmol O 2 /min/mg protein) AOX (-Pyr) COX y = -.2x 2 +.x +. R² =. y = -.x 2 +.x +.2 R² =. b T Term ( /K) 2 ( o C) Respiratory control range AOX (-Pyr) COX E o (kj/mol) 2-2. o C 2. o C y = -22.x y = -22.x T Term ( /K) Supplementary Figure S. Comparison of the relationship between temperature and E o for mitochondrial respiration mediated by the AOX- and COX-pathways. (a) Curve fitting of the mitochondrial respiration via AOX - (red) or COX- (blue) pathway (n = ). NADPH-NDA/ICDHmediated oxygen consumption is analysed. (b) Determination of the temperature response of E o for AOX- (red) and COX- (blue) pathways (n = ). NADPH-NDA/ICDH-mediated oxygen consumption is analysed.

7 Supplementary Table S. Respiration variables derived from modified Arrhenius model. ln R REF ( o C, 2K) (nmol O 2 /min/g spadix) E o ( o C, 2K) (kj/mol) ( o C, 2K) ( K 2 ) Temperature at E o = ( o C) Total mass (g) Plant Plant Plant Plant Mean ± SD.±..±. -2.2±..2±.2.±. Values for ln R REF (respiration rate), E o (overall activation energy) and (temperature sensitivity) with T REF at o C are shown. Temperatures at which E o shows zero and total mass of each spadix are also indicated.

8 Supplementary Table S2. Variables of AOX- or COX-mediated respiration pathway. ln R REF ( o C, 2K) (nmol O 2 /min/mg protein) E o ( o C, 2K) (kj/mol) AOX.±..2±. COX.2±. 2.±. NDA-mediated oxygen consumptions for AOX and COX capacities were indicated. Values for ln R REF and E o with T REF at o C were calculated from modified Arrhenius model. Data are means ± SD (n = ).

9 Supplementary reference. Seymour, R. S., Lindshau, G. & Ito, K. Thermal clamping of temperature-regulating flowers reveals the precision and limits of the biochemical regulatory mechanism. Planta 2, 2- (2).