s -1 (A) and (B) 2 s 1 in WT, riq1, and the riq1 mutant complemented by

Transcription

1 A s -1 B s -1 Supplemental Figure 1. The NPQ Phenotype in riq Mutants Was Complemented by Introduction of RIQ Genes. (A) and (B) 2 s 1 in, riq1, and the riq1 mutant complemented by the RIQ1 gene (A), and in, riq2, and the riq2 mutant complemented by the RIQ2 gene (B). After 30 min of dark adaptation, AL was applied for 5 min (white boxes); this was followed by a 4-min dark period for relaxation of qe (black boxes). Data are means ± SD (n = 3). 1

2 260 s Supplemental Figure 2. Reduced NPQ Was Observed in riq Mtuants Under Red Light.. 2 s 1 riq1 riq2 and riq1 riq2. n 2

3 (kda) riq1 riq2 riq1 riq2 riq1 riq2 riq1 riq2 (kda) Supplemental Figure 3. Similar Signals of RIQ1 Were Detected Using Two Types of Antibodies. Immunodetection of RIQ1 in and in riq mutants using two types of antibodies that recognized the predicted mature form of the RIQ1 sequence (left) and 14 amino acid residues of the RIQ1 sequence lane. Molecular weight markers are shown next to the panels. 3

4 A B Supplemental Figure 4. Linear Electron Transport Activity Was Similar in and riq Mutants. (A) and (B) Light-intensity dependence of ETR (A) and 1-qL (B) in, riq1, riq2, and riq1 riq2. Data are means ± SD (n = 4). 4

5 Supplemental Figure 5. Proton Motive Force Was Normally Formed in riq Mutants. ECS (electrochromic shift) signals were monitored in leaves of, pgr5, riq1, riq2, and riq1 riq2. After 2 s 1 for 5 min, a dark pulse (1 s) was applied to record the light-dark change of the signal by using Dual PAM. ECS t represents the total size of the proton motive force (pmf) formed in the light. pmf thylakoid membrane. ECS t was normalized against ECS ST, namely the ECS change induced by a single turn-over flash; this change depends on the charge separation of the photosystems. Data are means ± SD (n = 3). **P 5

6 1/8 1/4 1/2 riq1 riq2 riq1 riq2 curt1a npq4 RIQ1 RIQ2 CURT1A Lhcb1 Lhcb2 Lhcb3 Lhcb4 (CP29) Lhcb5 (CP26) Lhcb6 (CP24) PsbD PsbO PsbS PsaA Cyt f AtpF LHCII PSII PSI Cyt b 6 f CF 0 of ATPase Supplemental Figure 6. Accumulation of Major Thylakoid Proteins Was Unaffected in riq Mutants. Immunodetection of various thylakoid proteins. A chloroplast membrane protein extract corresponding to 2 6

7 Supplemental Figure 7. Xanthophyll Cycle Was Normally Operated in riq Mutants. Carotenoids were extracted from leaves of, riq1, riq2, curt1a, riq1 curt1a, and riq2 curt1a before or 2 s 1 for 5 min and then analyzed by HPLC. V; violaxanthin, A; antheraxanthin, Z; zeaxanthin. De-epoxidation state (DEPS) was calculated as (0.5*A +Z)/(V+A+Z). Data are means ± SD (n = 3). **P 7

8 CP43 - D2 D1 - LHCII - (kda) 50 - State1 riq1 riq2 stn7 State2 riq1 riq2 stn7 pthr 25 - CBB 15 - Supplemental Figure 8. riq Mutants. riq1 riq2 stn7 8

9 A B riq1 9

10 C riq2 D riq1 riq2 10

11 E F riq1 G riq2 H riq1 riq2 Supplemental Figure 9. Additional Electron Micrographs of Chloroplasts of and riq Mutants. (A) to (D) Chloroplast ultrastructures of (A), riq1 (B), riq2 (C), and riq1 riq2 (D), taken from the same samples as shown in ones of (A), riq1 (B), riq2 (C), and riq1 riq2 (D) in Figure 7, respectively. Bars (E) to (H) Magnified images of thylakoid ultrastructures of (E), riq1 (F), riq2 (G), and riq1 riq2 (H), as defined by the dotted squares in Figures 7 (A) to (D) 11

12 A CURT1A(AT4G01150) curt1a 123 ATG 200 bp TAG B curt1a CURT1A Actin1 C curt1a curt1a + CURT1A-HA HA D s CURT1A Cyt f Supplemental Figure 10. A Novel Allele of curt1a Mutant. (A) Schematic structural models of CURT1A. Black and gray boxes represent UTR and cording regions of curt1a (SK24234) is indicated. (B) CURT1A transcripts in and curt1a. Actin1 (AT2G37620) mrna was detected as a control. (C) Immunodetection of CURT1A in, curt1a, and its complementation line, which was generated by the introduction of CURT1A (D) curt1a, and its complementation line, as measured at photons m 2 s 1 n = 3) 12

13 A B riq1 13

14 C riq2 D curt1a 14

15 E riq1 curt1a F riq2 curt1a * * 15

16 G H riq1 I riq2 J curt1a K riq1 curt1a L riq1 curt1a M riq2 curt1a N riq2 curt1a 16

17 Supplemental Figure 11. Additional Electron Micrographs of Chloroplasts of and riq and curt1a Mutants. (A) to (F) Chloroplast ultrastructures of (A), riq1 (B), riq2 (C), curt1a (D), riq1 curt1a (E), and riq2 curt1a (F), taken from the same samples as shown in ones of (A), riq1 (B), riq2 (C), curt1a (D), riq1 curt1a (E), and riq2 curt1a (F) (F), abnormally aggregated thylakoids and wavy envelopes are indicated by black and white arrowheads, respectively. Unusual holes in the stroma (reflecting dents in the envelopes) are indicated by asterisks. (G) to (N) Magnified images of thylakoid ultrastructures of (G), riq1 (H), riq2 (I), curt1a (J), riq1 curt1a (K) and (L), and riq2 curt1a (M) and (N), as defined by the dotted squares in Figures 9 (A) to (F), 17

18 Genotype Chl a+b/fw (μg/mg) Chl a/b Wild type 1.38 ± ± 0.04 riq ± ± 0.04 riq2 1.5 ± ± 0.04 riq1 riq2 1.6 ± ± 0.01 Supplemental Table 1. Chlorophyll Content and Chlorophyll a/b Ratio. Chlorophyll (Chl) content and a/b ratio were determined as described by Porra et al. (1989). FW; fresh weight. Data are means ± SD (n = 3). 18

19 Supplemental Table 2. List of Primers Used in This Study. 19