The chloroplast 2-cysteine peroxiredoxin functions as thioredoxin oxidase in redox regulation of chloroplast metabolism

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Figure 2—source data 1

Values from the FBPase activity tests shown in Figure 2B and D.

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FBPase activity in the presence of different Trx-f1 concentrations.

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Values of the MDH activity test shown in Figure 4B.

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Altered MDH and PRK activities in 2cysprxAB mutants after light-dark transitions.

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Fresh weight for two fluctuating light schemes (40 s L/5 s H; 80 s L/10 s H) including complementation lines.

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Tables

Table 1

Light cycle	WT [mg]	2cysprxAB [mg]	Ratio WT/2cysprxAB
constant	372 ± 74	117 ± 20	3.19
40 s/5 s	26 ± 5	22 ± 4	1.21
80 s/10 s	21 ± 6	24 ± 5	0.87
120 s/15 s	14 ± 3	11 ± 2	1.34

Appendix 1—table 1

Component	Start value [µM]	Addition t = 100 [µM]	Addition t = 200 [µM]
FBPaseox FBPasered	0 4.70756e-3	0 0	0 0
Trxf1ox	0	0	0
Trxf1red	5	0	5
2-CysPrxox 2-CysPrxred	0 0	0 0	2.5–20 0
FBP	0	600	0
Fru-6-P	0	0	0

Appendix 1—table 2

Parameter	Value	Reference/comment
k1	2.9616e-2 µM−1s−1	Calculated from Collin et al. (2003)
k2	1.84e-3 µM−1 s−1	Collin et al. (2003)
KM(FBP)	0.028 µM	Pilkis et al., 1987
kcat_3	0.1 s−1	Villadsen and Nielsen, 2001
Keq_1	0.5697	calculated
Keq_2	0.3856	calculated
FBPasetotal	4.70756 µM	calculated
2-CysPrxtotal	0–20 µM	Enzyme test concentration
Trxf1total	5 µM	Enzyme test concentration

Appendix 1—table 3

Reaction number	Reaction
1	${\displaystyle Trxf{1}_{red}+FBPas{e}_{ox}A\underset{k_{1^{-}}}{\overset{k_{1^{+}}}{\rightleftharpoons }}BTrxf{1}_{ox}+FBPas{e}_{red}}$
2	${\displaystyle Trxf{1}_{red}+2-CysPr{x}_{ox}A\underset{k_{2^{-}}}{\overset{k_{2^{+}}}{\rightleftharpoons }}BTrxf{1}_{ox}+2-CysPr{x}_{red}}$
3	${\displaystyle FBP\stackrel{FBPas{e}_{red}}{\to }F6P+P_i}$

Appendix 1—table 4

Reaction number	Reaction
1	${\displaystyle v_1=k_1\ast \left(\left[Trxf{1}_{red}\right]\ast \left[FBPas{e}_{ox}\right]-\frac{\left[Trxf{1}_{ox}\right]\ast \left[FBPas{e}_{red}\right]}{Ke{q}_{\mathrm{\_}1}}\right)}$
2	${\displaystyle v_2=k_2\ast \left([Trxf{1}_{red}\ast \left[2-CysPr{x}_{ox}\right]-\frac{\left[Trxf{1}_{ox}\right]\ast \left[2-CysPr{x}_{red}\right]}{K_{eq\mathrm{\_}2}}\right)}$
3	${\displaystyle v_3=\frac{kca{t}_3\ast \left[FBPas{e}_{red}\right]\ast \left[FBP\right]}{\left[FBP+K{m}_{FBP}\right]}}$${\displaystyle v_3=\frac{kca{t}_3\ast \left[FBPas{e}_{red}\right]\ast \left[FBP\right]}{\left[FBP+K{m}_{FBP}\right]}}$${\displaystyle v_3=\frac{kca{t}_3\ast \left[FBPas{e}_{red}\right]\ast \left[FBP\right]}{\left[FBP+K{m}_{FBP}\right]}}$

Appendix 2—table 1

component	start value [µM]	Reference/comment
FTRox	0	Assumption in light 100% reduced
FTRred	4.7727	calculated from Yoshida and Hisabori (2017)
Trxf1ox	3.798e-1	20% oxidized
Trxf1red	1.5192	80% reduced
FBPaseox FBPasered	1.426528 5.706112	20% oxidized 80% reduced

Appendix 2—table 2

parameter	value	Reference/comment
k1	7.7047e−2 µM−1µM−1s−1	Fitted
k2	6.819e−2 µM−1s−1	Fitted
k3	2.9616e−2 µM−1s−1	Calculated from Collin et al. (2003)
k4	1.84e−3 µM−1s−1	Collin et al. (2003)
Keq_Trxf1FBPase	0.5697	Calculated
Keq_Trxf12CP	0.3856	Calculated
Fdtotal	69 µM	calculated from Peltier et al. (2006)
Fdred_fix	34.5 µM	Estimated (50% reduced)
FTRtotal	4.7727 µM	calculated from Yoshida and Hisabori (2017)
Trxf1total	1.899 µM	calculated from Peltier et al. (2006)
FBPasetotal	7.13267 µM	calculated from Peltier et al. (2006)
2-CysPrxtotal	63.3 µM	Calculated from Peltier et al. (2006)
2-CysPrxred_fix	21.522 µM	Calculated
2-CysPrxox_fix	41.778	Calculated

Appendix 2—table 3

reaction number	reaction
1	${\displaystyle F{d}_{red}+F{d}_{red}+FT{R}_{ox}\underrightarrow{k_1}F{d}_{ox}+F{d}_{ox}+FT{R}_{red}}$
2	${\displaystyle FT{R}_{red}+Trxf{1}_{ox}\underrightarrow{k_2}FT{R}_{ox}+Trxf{1}_{red}}$
3	${\displaystyle Trxf{1}_{red}+FBPas{e}_{ox}A\underset{k_3^{-}}{\overset{k_3^{+}}{\rightleftharpoons }}BTrxf{1}_{ox}+FBPas{e}_{red}}$
4	${\displaystyle Trxf{1}_{red}+2-CysPr{x}_{ox}A\underset{k_2^{-}}{\overset{k_2^{+}}{\rightleftharpoons }}BTrxf{1}_{ox}+2-CysPr{x}_{red}}$

Appendix 2—table 4

reaction number	Reaction
1	${\displaystyle v_1=k_1\ast \left[F{d}_{red}\right]\ast \left[F{d}_{red}\right]\ast \left[FT{R}_{ox}\right]}$
2	${\displaystyle v_2=k_2\ast [FT{R}_{red}\ast \left[\mathrm{T}\mathrm{r}\mathrm{x}\mathrm{f}{1}_{ox}\right]}$
3	${\displaystyle v_3=k_3\ast \left(\left[Trxf{1}_{red}\right]\ast \left[FBPas{e}_{ox}\right]-\frac{\left[Trxf{1}_{ox}\right]\ast \left[FBPas{e}_{red}\right]}{K_{eq\mathrm{\_}Trxf1FBPase}}\right)}$${\displaystyle v_3=k_3\ast \left(\left[Trxf{1}_{red}\right]\ast \left[FBPas{e}_{ox}\right]-\frac{\left[Trxf{1}_{ox}\right]\ast \left[FBPas{e}_{red}\right]}{K_{eq\mathrm{\_}Trxf1FBPase}}\right)}$${\displaystyle v_3=k_3\ast \left(\left[Trxf{1}_{red}\right]\ast \left[FBPas{e}_{ox}\right]-\frac{\left[Trxf{1}_{ox}\right]\ast \left[FBPas{e}_{red}\right]}{K_{eq\mathrm{\_}Trxf1FBPase}}\right)}$
4	${\displaystyle v_4=k_4\ast \left([Trxf{1}_{red}\ast \left[2-CysPr{x}_{ox}\right]-\frac{\left[Trxf{1}_{ox}\right]\ast \left[2-CysPr{x}_{red}\right]}{K_{eq\mathrm{\_}Trxf12CP}}\right)}$

Additional files

Supplementary file 1

Primers used for cloning.

for: forward primer, rev: reverse primer.

https://doi.org/10.7554/eLife.38194.024

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Transparent reporting form

https://doi.org/10.7554/eLife.38194.025

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