Figure 1. Current concepts of how drought increases the generation of reactive oxygen species (ROS) in photosynthesis. A. Cartoon of leaf section in well-watered plants in which relatively high intercellular CO2 concentrations (Ci) allow efficient regeneration of terminal oxidants and limit RuBP oxygenation. B. Drought-induced stomatal closure restricts CO2 uptake, favoring photorespiratory production of H2O2 in the peroxisome (1) and possibly favoring production of superoxide and H2O2 (2) or singlet oxygen (3) by the photosynthetic electron transport chain.

NADPH

NADP+

RuBP

GlycolatePGA

Sugar-P

Glycolate

Glyoxylate

H2O2

O2

PSII

PSI

CO2 O2

Peroxisome

O2

1O2O2

O2.

H2O2

Chloroplast

NADPH

NADP+

RuBP

GlycolatePGA

Sugar-P

Glycolate

Glyoxylate

H2O2

O2

PSII

PSI

CO2O2

Chloroplast

Peroxisome

O2

1O2

O2

O2.

H2O2

CO2

CO2

CO2

CO2CO2

CO2CO2

CO2CO2

CO2

CO2

CO2

CO2

CO2

CO2

CO2CO2

CO2

CO2

Epidermis

Leaf interior

CO2CO2 CO2

A B3

2

1

CYTOSOL APOPLAST

NADPH

NADP+

O2H2O2

Figure 2. Multiple ROS-producing enzymes at the cell surface/exterior. Enzymes are shown in blue and their redox cofactors are indicated in yellow. Class III peroxidases may accept electrons from several types of compound to generate superoxide, but in many cases their physiological reductant is not established (reviewed by O’Brien et al., 2012).

Superoxide dismutase

CuZn

O2

NADPH oxidase

Flavocytochrome

PlasmaMembrane

O2.-

H2O2

PutrescineCadaverine

e-

O2H2O2Superoxide

dismutase

CuZn

Class III peroxidase

HemeO2

.-

Amine oxidase

Cu

Polyamine oxidase

FADSpermidineSpermine

O2

H2O2

Oxalate oxidase

MnOxalate

O2

H2O2

Figure 3. The 15 of the 302 redox-linked genes that respond > 2-fold in the same direction in both drought 1 and drought 2 datasets and their response in related conditions. Data extracted from Genevestigator are shown as log2 values compared to controls. Red and green indicate induction and repression. Genes are ordered from top according to the number of conditions in which they respond. The full list of genes and their expression values are given in Supplemental Table S1. For details of experiments, see Supplemental Table S2.

AGI Gene name Redox class Drought 1 Drought 2 Salt ABA Osmotic

At2g29460 GSTU4 glutathione S-transferase TAU4 glutathione 1.49 4.36 1.99 3.78 2.28At1g17020 SRG1 senescence related gene 1 (ascorbate oxidase) ascorbate 1.11 4.94 3.92 3.47 0.60At5g35790 G6PD1 glucose-6-phosphate dehydrogenase 1 NADPH -1.02 -1.83 -1.27 -2.24 -0.57At1g06830 ROXY6 CC-type glutaredoxin glutathione -3.24 -1.23 -2.39 0.41 -2.65At1g35720 ANNAT1 annexin 1, peroxidase activity ROS 1.33 1.80 1.20 0.01 0.61At2g28190 CSD2 CuZn superoxide dismutase ROS -2.72 -1.77 -2.18 -0.15 -0.59At1g08830 CSD1 CuZn superoxide dismutase ROS -1.49 -1.18 -1.36 0.49 0.11At5g17220 GSTF12 glutathione S-transferase PHI12 glutathione 4.76 3.96 1.90 0.13 0.52At4g02520,At2g02930 GSTF2,GSTF3 glutathione S-transferases PHI2, PHI3 glutathione -1.31 -3.25 -1.55 -0.28 0.73At5g06290 2-cys PrxB 2-cys peroxiredoxin subunit B thioredoxin -1.56 -2.13 -1.06 -0.48 -0.12At1g56500 thioredoxin family protein, suppressor of quenching 1 thioredoxin -1.05 -1.13 -0.21 0.21 -0.43At4g39640 GGT1 g-glutamyl transpeptidase 1 glutathione -1.12 -2.44 -0.64 -0.24 0.02At1g78370 GSTU20 glutathione S-transferase TAU20 glutathione -2.27 -2.29 -0.03 -0.81 -0.08At3g03190 GSTF11 glutathione S-transferase TAU11 glutathione -2.21 -1.38 0.53 0.99 0.18At5g18600 ROXY10 CC-type glutaredoxin glutathione -1.27 -1.21 0.27 1.06 -0.48

Figure 4. Peroxide-removing enzymes: roles as antioxidants, in signaling, or both? Cartoon of the best characterized peroxide-metabolizing enzymes in plants. Other mechanisms are possible and for ease of display reactions are not shown stoichiometrically. APX, ascorbate peroxidase. CAT, catalase. DHAR, dehydroascorbate reductase. GRX, glutaredoxin. GST, glutathione S-transferase. MDHAR, monodehydroascorbate reductase. NTR, NADPH-thioredoxin C. PRXII, type II peroxiredoxin. 2CPRX, 2-cys PRX. TRX, thioredoxin.

Thiol-independent peroxide metabolismAntioxidative systems participate in signaling

by their effects on peroxide levels

Thiol-dependent peroxide metabolismSignaling is also possible through antioxidative activity per se

H2O2

H2O2 2 H2O

CAT

O2ASC

H2O2 2 H2O

APX

MDHA

NAD(P)H

MDHAR

NAD(P)+

ASCDHA

GSH

DHAR

GSSG

GST

GSH GSSG

ROOH ROH

PRXII

GSH GSSG

ROOH ROH

SH SH

PRXIISHSOH

PRXIIGSS SH

+ GSH

2CPRX

ROOH ROH

SH SH SHSOH

S S

GRXSH SH

GRXSH

2CPRX

2CPRX

GSS

NTR/TRX

SHSH S S

NTR/TRX

Dismutation by catalase

APX functioning independently of

glutathione

Ascorbate-glutathione pathway

GST glutathione peroxidase

function

Glutathione-linked peroxiredoxin

Thioredoxin-linked peroxiredoxin

Figure 5. Analysis of drought-inducible gene expression in responses to redox perturbation. A. Heatmap of drought-induced genes extracted from Genevestigator and the response of these genes to ABA or oxidative stress (top), and histogram showing expression of oxidative stress marker genes after the different treatments (bottom). Data are shown as log2 values compared to Col-0 (wild-type or untreated). Experimental details are given in Supplemental Table S2. Red and green on the heatmap indicate induction and repression according to the color scale shown at the top. The five genes for which data are shown in the bottom histogram are (left to right) APX1, GSTU24, UGT75B1, UGT73B5, and GPX6 (for values, see Supplemental Table S3B). B. Overlap of induced genes (cut-off 2-fold) in the two drought experiments. 375 genes were induced >2-fold in at least one of the experiments (Supplemental Table S3A). C. Number of these 375 genes that were induced > 2-fold by the different oxidative stresses (indicated in red circles within the outer blue circles).

B

66 272 37

C

Total number of drought-induced genes = 375

External H2O2

(extracellular)

cat2: H2O2

(peroxisome)

flu: singlet O2

(chloroplast)

375

360

15 21

580

317

354

0

2

4

6

8

Exte

rnal

H2O

2

(ext

race

llula

r)

DRO

UG

HT

1

DRO

UG

HT

2

Para

quat

: O2- ,

H2O

2

(chl

orop

last

)

cat2

: Hig

h CO

2

cat2

: H2O

2

(per

oxis

ome)

flu :

sing

let O

2

(chl

orop

last

)

AO

xida

tive

stre

ss m

arke

r tra

nscr

ipts

(log 2 t

reat

ed/c

ontr

ol)

ABA

Paraquat: O2, H2O2

(chloroplast)

-

129

ABA

cat2: high CO2

(negative control)

3

372

246