the high light response in arabidopsis involves aba ... · the high light response in arabidopsis...

The High Light Response in Arabidopsis Involves ABASignaling between Vascular and Bundle Sheath Cells W

Gregorio Galvez-Valdivieso,a,1 Michael J. Fryer,a,1 Tracy Lawson,a Katie Slattery,a William Truman,b NicholasSmirnoff,b Tadao Asami,c William J. Davies,d Alan M. Jones,e Neil R. Baker,a and Philip M. Mullineauxa,2

a Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, United Kingdomb School of Biosciences, University of Exeter, Exeter EX4 4QD, United KingdomcDepartment of Applied Biological Chemistry, University of Tokyo, Bunkyo, Tokyo 113-8657, Japand Department of Biological Sciences, Lancaster Environment Centre, University of Lancaster, Lancaster LA1 4YQ, United

KingdomeDepartments of Biology and Pharmacology, University of North Carolina, Chapel Hill, North Carolina 27599-3280

Previously, it has been shown that Arabidopsis thaliana leaves exposed to high light accumulate hydrogen peroxide (H2O2)

in bundle sheath cell (BSC) chloroplasts as part of a retrograde signaling network that induces ASCORBATE PEROXIDASE2

(APX2). Abscisic acid (ABA) signaling has been postulated to be involved in this network. To investigate the proposed role of

ABA, a combination of physiological, pharmacological, bioinformatic, and molecular genetic approaches was used. ABA

biosynthesis is initiated in vascular parenchyma and activates a signaling network in neighboring BSCs. This signaling

network includes the Ga subunit of the heterotrimeric G protein complex, the OPEN STOMATA1 protein kinase, and

extracellular H2O2, which together coordinate with a redox-retrograde signal from BSC chloroplasts to activate APX2

expression. High light–responsive genes expressed in other leaf tissues are subject to a coordination of chloroplast

retrograde signaling and transcellular signaling activated by ABA synthesized in vascular cells. ABA is necessary for the

successful adjustment of the leaf to repeated episodes of high light. This process involves maintenance of photochemical

quenching, which is required for dissipation of excess excitation energy.

INTRODUCTION

In the natural environment, plants are frequently exposed to

fluctuating light intensities and often absorb more light energy

than can be consumed by photosynthetic metabolism and thus

require that excess excitation energy be dissipated.Many abiotic

and biotic stresses limit photosynthesis, which causes further

increases in excess excitation energy needing to be dissipated

(Long et al., 1994; Asada, 1999; Baker 2008). Failure to dissipate

excitation energy results in overreduction of the photosynthetic

chain components that direct linear electron flux (LEF) fromwater

to NADPH (Baker et al., 2007). Part of the absorbed light energy is

dissipated as heat in the light-harvesting complexes of photo-

system II (PSII) through non photochemical quenching (NPQ;

Horton et al., 1996; Muller et al., 2001). Additional dissipation of

excitation energy is also achieved by photochemical quenching

(Baker et al., 2007; Baker, 2008) and reflects that action of

processes such as the reduction of molecular oxygen at photo-

system I by the Mehler reaction (Asada, 1999; Ort and Baker,

2002; Baker et al., 2007) and through photorespiration (Asada,

1999;DouceandNeuberger, 1999). These twoprocessesproduce

reactive oxygen species (ROS) that are scavenged by lipid-

and water-soluble low molecular weight antioxidants and anti-

oxidant enzymes (Mittler et al., 2004; Apel and Hirt, 2004; Van

Breusegem et al., 2008). Sustained exposure to very high light

intensities, well in excess of light intensities optimal for growth

(hereafter called excess light), will exceed the antioxidant and

excitation energy dissipation capacity of the leaf and cause

oxidative damage to the photosynthetic apparatus (Aro et al.,

1993; Asada, 1999; Krieger-Liszkay, 2005; Triantaphylides et al.,

2008), photobleaching, and cell death (Karpinski et al., 1999;

Muhlenbock et al., 2008).

In excess light–stressed plants, damaged chloroplasts initiate

retrograde signaling to the nucleus (Nott et al., 2004; Pogson

et al., 2008) to downregulate the expression of photosynthetic

genes and upregulate stress defense genes to mitigate oxidative

stress (Rossel et al., 2002, 2007; Kimura et al., 2003; Koussevitzky

et al., 2007; Muhlenbock et al., 2008). In contrast with ex-

cess light treatments, Arabidopsis thaliana leaves that are ex-

posed to a moderate increase (typically <10-fold) over growth

light intensity (hereafter called high light [HL]) do not suffer

oxidative stress (Fryer et al., 2003; Davletova et al., 2005) or

irreversible photoinhibition (Russell et al., 1995; Karpinski et al.,

1997; Fryer et al., 2003). However, these plants do accumulate

H2O2 in the chloroplasts of bundle sheath cells (BSCs) and

neighboring mesophyll cells (Fryer et al., 2003; Mullineaux et al.,

2006). BSCs in Arabidopsis form a single layer of elongated cells

around the vasculature (Kinsman and Pyke, 1998; Leegood,

1 These authors contributed equally to this work.2 Address correspondence to [email protected] author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Philip M.Mullineaux ([email protected]).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.108.061507

This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been

edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online

reduces the time to publication by several weeks.

The Plant Cell Preview, www.aspb.org ã 2009 American Society of Plant Biologists 1 of 20

2008). Limitations in the supply of CO2 to such cells (Morison

et al., 2005)may cause them tomore readily produce ROS via the

photoreductionofO2 (Hibberd andQuick, 2002; Fryer et al., 2003;

Leegood, 2008). Theaccumulation ofH2O2 inBSCchloroplasts is

associated with the rapid induction of the antioxidant gene

ASCORBATE PEROXIDASE2 (APX2; Fryer et al., 2003; Ball

et al., 2004; Karpinski et al., 1997, 1999). HL-mediated induction

of APX2 requires LEF, redox signals from reduced glutathione,

and abscisic acid (ABA; Karpinski et al., 1999; Fryer et al., 2003;

Ball et al., 2004; Chang et al., 2004). These latter features are

common to amajority of other HL-responsive genes examined to

date (Rossel et al., 2002; Ball et al., 2004; Bechtold et al., 2008).

However, in contrast with most HL-responsive genes so far

examined, the induction of APX2 expression also requires extra-

cellular H2O2 (Karpinski et al., 1999; Bechtold et al., 2008). The

BSC-specific expression of APX2 in response to HL allows this

gene to be used as a BSC-specific reporter (Mullineaux et al.,

2006), in contrast with the expression of many HL-responsive

genes that may not be confined to a single leaf tissue (Bechtold

et al., 2008).

The induction of APX2 expression and increased capacity to

dissipate excitation energy in BSCs can be prevented if the leaf is

exposed to high humidity (Fryer et al., 2003). This observation

suggests that at low humidity, HL-exposed BSCs experience a

loss of water (Fryer et al., 2003). Water loss leads to the

accumulation of ABA, which plays a central role in the regulation

of plants’ water status (Davies et al., 2002; Christmann et al.,

2006). HL-mediated APX2 induction is attenuated in the ABA

signaling mutants abi1-1 and abi2-1 (Fryer et al., 2003). In the

mutant altered in APX2 expression8-1 that constitutively ex-

presses APX2, ABA content is threefold elevated under non-

stress conditions, providing a correlation between ABA

accumulation and APX2 expression (Rossel et al., 2006). Supply

of ABA to plants under low light conditions has shown that many

HL-expressed genes are responsive to ABA (Fryer et al., 2003;

Rossel et al., 2006; Bechtold et al., 2008).

Based on the requirement of APX2 induction for an extracel-

lular source of H2O2 in BSCs (Karpinski et al., 1999; Bechtold

et al., 2008), we speculate that an ABA-regulated plasma mem-

brane NADPH oxidase as shown in guard cells (Pei et al., 2000;

Murata et al., 2001;Mustilli et al., 2002; Kwak et al., 2003; Li et al.,

2006) may be a source of ROS in BSCs (Mullineaux et al., 2006).

BSCs may contain additional ABA signaling components that

regulate extracellular ROS, such as the heterotrimeric G protein

complex (Suharsono et al., 2002; Booker et al., 2004; Joo et al.,

2005; Li et al., 2006), the OPEN STOMATA1 (OST1) protein

kinase (Mustilli et al., 2002; Xie et al., 2006), and ABI1 (Murata

et al., 2001).

ABA is synthesized in response to a reduction in water po-

tential (Davies et al., 2002; Nambara and Marion-Poll, 2005;

Christmann et al., 2006, 2007). ABA biosynthesis is partitioned

between plastids and the cytosol (Qin and Zeevaart, 1999;

Nambara and Marion-Poll, 2005). The oxidative cleavage of the

precursor carotenoid 99-cis-neoxanthin to xanthoxin, catalyzed

by the plastidial enzyme 99-cis-epoxycarotenoid dioxygenase

(NCED), is the committed step for ABA biosynthesis (Nambara

and Marion-Poll, 2005). Xanthoxin is then exported from the

plastid, and the two remaining biosynthetic steps to ABA, cat-

alyzed by enzymes coded by ABA DEFICIENT2 (ABA2) and ABA

ALDEHYDE OXIDASE3 (AAO3), occur in the cytosol (Nambara

and Marion-Poll, 2005).

In this study, we set out to establish how ABA, secreted from

vascular parenchyma cells (Endo et al., 2008), regulates HL-

responsive gene expression and integrates into H2O2- and

redox-mediated retrograde signaling from chloroplasts in

BSCs. We conclude that in HL-exposed leaves at ambient or

lower humidity (1) paracrine (i.e., cell-to-nearby-cell) signaling

occurs between vascular parenchyma cells and BSCs, (2) ABA

signaling integrates into H2O2- and redox-mediated retrograde

signaling from BSC chloroplasts, and (3) ABA is required for an

effective physiological response of leaves to a fluctuating light

environment.

RESULTS

Exposure to Moderate HL Treatment Does Not Induce

Signaling Pathways Associated with Exposure to

Excess Light

To determine the effects of exposure to HL in the BSCs, we

measured the expression of the antioxidant gene APX2 and

chlorophyll fluorescence as an indicator of photosynthetic per-

formance and photoinhibition. The response of leaves was

determined to a fivefold increase in PPFD over their growth

PPFD (150 mmol m22 s21), hereafter referred to as HL. Exposure

to HL caused detectable accumulation of APX2 transcript after

20 min, which continued to rise throughout the duration of the

experiment (Figure 1A). The exposure of leaves to HL resulted in

a decline in the chlorophyll fluorescence parameter Fv/Fm, which

defines themaximumquantum efficiency of PSII photochemistry

(see Methods; Baker, 2008; see Supplemental Figure 1 online).

Fv/Fm values reverted to pre-HL exposure values when plants

were returned to their growth conditions (see Supplemental

Figure 1 online). Under these conditions, the expression of genes

controlled by retrograde signaling pathways responsive to ex-

cess light (Danon et al., 2005; Koussevitzky et al., 2007; Pryzbyla

et al., 2008) showed no significant change (see Supplemental

Figure 2 online). Taken together, these data indicate that the

plant’s responses to HL exposure did not induce permanent

damage to leaves and did not activate retrograde signaling

associated with such photooxidative stress.

ABAAccumulation inHL-ExposedLeaves IsDue toChanges

in Leaf Water Status

To examine the possible role of ABA in the response of leaves to

HL, the ABA content of leaves was determined. ABA content

increased in attached and detached leaves by 65 and 60%,

respectively, when exposed to HL at 25% RH (hereafter called

low humidity; see Methods). This indicates that the ABA accu-

mulation is leaf autonomous; no other source of ABA, such as

from roots (Nambara and Marion-Poll, 2005), is involved. The

increase in ABA was observed after 15 min in HL and was

followed by an increase in APX2 expression (Figure 1A). At 80%

2 of 20 The Plant Cell

Figure 1. ABA Levels and APX2 Expression in HL-Exposed Leaves and Osmotically Stressed Petioles.

(A) Increase in ABA content (closed symbols, dashed line) and APX2 transcript levels (open diamonds, solid line) in leaves attached to the rosette and

exposed to HL (750 mmol m�2 s�1 PPFD) and 25% RH. Each leaf (one per plant) was clamped into a CIRAS leaf chamber to control humidity and

temperature (see Methods). NCED3 transcript levels are shown (closed squares, dotted line). ABA levels were determined using a radioimmunoassay

(seeMethods) from fully expanded outer leaves attached to rosettes during HL exposure. The data presented, expressed asmg per gram dry weight (gm

DWt�1), are the means (6SE; n = 6) from one expanded leaf from each of three plants for each time point in two experiments. Foliar transcript levels were

determined by quantitative real-time PCR on single-stranded cDNA prepared from total leaf RNA (see Methods) harvested at each time point. Each data

point is the mean cDNA level (6SE) relative to the zero time point, low-light cDNA level. Transcript levels were normalized with respect to CYCLOPHILIN

transcript levels, which do not respond to HL (Rossel et al., 2006).

(B) Foliar ABA content in detached leaves exposed to HL (PPFD of 750 mmol m�2 s�1) for 45 min at either low humidity (25% RH) or high humidity (80%

RH) or kept at the growth PPFD of 150 mmol m�2 s�1 (LL). The difference in ABA levels between the mean (6SE) HL and LL samples at low humidity was

significant (P = 0.024 from t test; n = 8 from two experiments). One fully expanded leaf from each rosette was used and clamped into a CIRAS leaf

chamber to control humidity and temperature (see Methods).

(C) APX2LUC expression in osmotically stressed petioles is light dependent. Fully expanded leaves of APX2LUC/Col-0 plants (Karpinski et al., 1999)

were detached and infiltrated with PEG-400 between 0 and 0.5 M for 2 h at a PPFD of 20 mmol m�2 s�1 and then petioles were detached and bathed in

the same concentrations of PEG-400 plus 1 mMD (�) luciferin for a further 1.5 h either at growth PPFD (low light) or in the dark. At the end of this period,

ABA and Redox Signaling in High Light 3 of 20

RH (hereafter termed high humidity; see Methods), HL did not

increase the ABA level (Figure 1B).

In the first 10 min of exposure to HL under low humidity, the

transpiration rate increases (Fryer et al., 2003), which transiently

lowers leaf water status and provides the necessary cue to

initiate ABA accumulation. However, the sudden increase in light

intensity may also directly contribute (a) signal(s) that triggers

ABA accumulation in leaves. To distinguish between these two

possibilities, ABA accumulation was measured in petioles of

Columbia-0 (Col-0) plants harboring an APX2 promoter LUCIF-

ERASE gene fusion (APX2LUC; Karpinski et al., 1999) subjected

to a range of osmotic stress treatments under low-light condi-

tions and in the dark. Petioles contain BSCs, which express

APX2 (Fryer et al., 2003; Ball et al., 2004), and vascular paren-

chyma that carry out ABA biosynthesis in the Arabidopsis leaf

(Endo et al., 2008). Petioles of Col-0/APX2LUC were chosen as

an experimental system because they could be bathed in solu-

tions of different osmotic potential, and on exposure to light,

APX2 expression could be rapidly monitored by imaging the

luminescence produced by luciferase (Figure 1C). Incubation of

isolated APX2LUC petioles in a range of polyethylene glycol

(PEG-400) solutions of increasing osmotic potential under low-

light conditions activated APX2 expression (Figure 1C), with

osmotic potential thresholds of 20.5 to 20.8 MPa (calculations

based on Money, 1989). Similar observations were made when

petioles were treated with sorbitol or mannitol solutions of

equivalent osmotic potential (data not shown). The 0.4 M PEG-

400 that inducedAPX2 expression also caused a decrease in the

chlorophyll fluorescence parameters Fq9/Fm9 and Fv/Fm and an

increase in qL andNPQ (Table 1). Fq9/Fm9 provides an estimate of

the quantum efficiency at which PSII operates under a given

PPFD (Baker, 2008). qL and NPQ provide information of the

redox state of the primary quinone electron acceptor of PSII (QA)

and NPQ, respectively (Baker, 2008). Similar changes in these

characteristics accompany the induction of APX2 expression

(Karpinski et al., 1997, 1999; Fryer et al., 2003; Chang et al.,

2004).

ABA levels in petioles increased in low light and in the dark by

30 and 27%, respectively, when 0.4MPEG-400, a concentration

above the threshold for APX2 expression, was applied (Figure

1D). These data indicate that ABA accumulation in petioles is not

dependent on a light-associated signal in addition to a change in

water status. Similarly, treatment of HL-exposed leaves with the

LEF inhibitor DCMU (Duysens, 1972) did not inhibit ABA accumu-

lation (Figure 1E), confirming the observations in dark-incubated

osmotically stressed petioles.

A Capacity for Foliar ABA Biosynthesis Is Required for

Induction of HL-Responsive Genes

ABA has been implicated in the induction of BSC-specific APX2

expression and some additional HL-responsive genes whose

expression occurs in several leaf tissues (Fryer et al., 2003;

Rosseletal., 2006;Bechtoldetal., 2008).Toexamine the roleof the

foliar ABA biosynthetic pathway, the compound ABAmineSG,

a specific inhibitor of NCED activity that is involved in the

synthesis of ABA (Kitahata et al., 2006), was applied to detached

HL-illuminated leaves of Col-0/APX2LUC. ABAmineSG inhibited

the rise in foliar ABA levels and the increase in APX2 expression

in leaves exposed to HL at low humidity (Figure 2A).

The predominantNCED gene expressed in Arabidopsis leaves

is NCED3 (Iuchi et al., 2001; Tan et al., 2003; Ruggiero et al.,

2004; Endo et al., 2008).When rosettes of a null mutant ofNCED3

(nced3-2; see Supplemental Figure 3 online) were exposed to

HL, induction of APX2 expression was attenuated (Figure 2B)

consistent with the data obtained with ABAmineSG (Figure 2A).

Leaves from nced3-2 plants did not show an increase in ABA

content under HL (see Supplemental Figure 3 online), although

prestress levels of ABA were no different from those of wild-type

leaves (see Supplemental Figure 3 online). A second T-DNA

insertion mutant allele of NCED3, sto1-1, which has reduced

expression of the gene (Ruggiero et al., 2004; see Supplemental

Figure 3 online), also showed attenuated induction of APX2

expression in HL-exposed leaves (Figure 2B), although not to the

samedegree as nced3-2 (Figure 1D). Null mutant alleles ofABA2,

aba2-11, and aba2-14, which have low levels of foliar ABA under

a range of conditions (Gonzalez-Guzman et al., 2002; Barrero

et al., 2006), failed to induce APX2 expression in response to HL

(Figure 2B).NCED3 expression did not increase uponHL and low

humidity treatment (Figure 1A), suggesting that the mechanism

of action is posttranscriptional.

Figure 1. (continued).

luciferase activity (top panels) and reflected light (bottom panels) were imaged using a CCD camera and false color images for luciferase activity

generated (see Methods). The experiment shown was typical of replicates of these treatments.

(D) ABA levels in osmotically stressed petioles in the low light and the dark. Petioles of APX2LUC leaves were treated with 0.4 M PEG-400 or water as

described in the legend for (C) and then their ABA content determined as in the legend for (A). Some additional petioles of low light–incubated petioles

were imaged for luciferase activity, as shown in (C), to confirm their response to the PEG-400 treatment. The values are the means (6SE) for three

separate experiments from eight petioles pooled in each experiment from four plants.

(E) DCMU has no effect on HL-induced foliar ABA accumulation. Detached leaves were infiltrated with 30 mM DCMU for 2 h (see Methods) prior to

exposure to HL (750 mmol m�2 s�1) at 25% RH. Leaf ABA content was then determined as in the legend for (A). These data are the means (6SE) of two

experiments with four leaves each from a separate plant (n = 8).

Table 1. Chlorophyll Fluorescence Parameters for Water- and 0.4 M

PEG-400–Treated Petioles.

Parameter

Water-Treated

Petioles

0.4 M PEG-400–

Treated Petioles P

Fv/Fm 0.71 6 0.003 0.65 6 0.005 <0.001

Fq9/Fm9 0.34 6 0.012 0.29 6 0.01 0.013

NPQ 1.38 6 0.024 1.51 6 0.05 0.030

qL 0.91 6 0.01 1.09 6 0.014 <0.001

One petiole per plant was used from an outer rosette leaf (n = 4).


Figure 2. The Expression of HL-Responsive Genes Require a Foliar Capacity for ABA Biosynthesis.

(A) APX2LUC expression in leaves treated with the NCED inhibitor ABAmineSG (Kitahata et al., 2006). Image of luciferase activity from Col-0/APX2LUC

infiltrated with ABAmineSG and D (�) luciferin (1 mM) or luciferin only for 2 h prior (see Methods) prior to exposure to HL (750 mmol m�2 s�1) at 25% RH

for 45 min. Luciferase activity was imaged using a CCD camera system and image processing to color code luciferase activity (see Methods). Red

indicates regions of highest luciferase activity. The effect of ABAmine on foliar ABA levels (Kitahata et al., 2006) was confirmed in a single experiment

using four leaves from separate APX2LUC/Col-0 plants for each treatment as described in the legend of Figure 1 and Methods.

(B) APX2 transcript levels in HL-exposed leaves of ABA biosynthesis mutants. Whole rosettes of the wild type (Col-0), nced3-2, aba2-11, and aba2-14

were exposed to HL (750 mmol m�2 s�1) for 45 min at growth humidity (see Methods). APX2 expression was determined as transcript levels in the

mutants relative to the wild type using real-time quantitative RT-PCR (see Methods and legend of Figure 1). Data were normalized to cDNA levels of

CYCLOPHILIN, which shows no significant variation in expression under HL or where ABA levels differ between genotypes (Rossel et al., 2006). All data

shown are the means (6SE) combined from two biological replications of six plants. All differences between mutants and the wild type were significant

(P < 0.001 from t test).

(C) Transcript levels of five HL-responsive genes in leaves of ABA biosynthesis mutants. The genes used in this study are to compare with the BSC-

specific expression of APX2. The conditions and methods are as in legend for (B). Asterisks show where differences between mutants and the wild type

were significant (P # 0.05 from t test).

(D)Hierarchical clustering of shared gene expression responses to HL exposure and exogenous ABA application. A total of 816 genes were identified as

representing a significant overlap between HL and ABA responses and were clustered with data from the Gene Expression Omnibus (GEO) and

NASCARRAYS databases. The 30 min, 1 h, and 3 h ABA data come from NASCARRAYS-176, 4 h ABA fromGSE7112, 3 h ABA #2 from GSE6171, and 3

h HL from GSE7743. In this TREEVIEW representation, red indicates upregulation relative to mock or control treatment, while green indicates

downregulation. The scale bar indicates log (base 2) ratios of treatment to control for the heat map.

Additional HL-responsive genes (Bechtold et al., 2008) were

tested for their expression in HL-exposed ABA biosynthesis-

deficient mutants. All mutants caused a significant reduction in

the expression of the test genes compared with the wild-type

controls (Figure 2C). A meta-analysis of publicly available micro-

array data for treatment of seedlings with ABA (Goda et al., 2008)

compared with data from HL-exposed seedlings (Kleine et al.,

2007; see Methods) revealed that 816 genes were coresponsive

to ABA and HL (P value < 0.00001). Expression of 496 genes was

induced under both conditions, while 320 were suppressed in

response to both treatments (see Supplemental Data Set 1 on-

line). When expression data for these genes were clustered with

other publicly available ABA treatment data (Figure 2D), a strong

correlation was observed between 3 h of HL exposure and plants

3 or 4 h after ABA application at a variety of concentrations

(uncentered correlation = 0.780). Thus, a significant number of

HL-responsive genes are also responsive to ABA levels and may

require foliar ABAbiosynthesis, implying an important role for this

hormone in the response of the leaf to its prevailing light envi-

ronment.


Response to a Change in Light Intensity

Based on the above observations on the correlation betweenHL-

and ABA-responsive gene expression, we hypothesized that a

capacity to synthesize foliar ABA would be important for the

leaf’s ability to adjust to changing light conditions. To test this

hypothesis, nced3-2 was subjected to HL and lowered humidity

for 60 min at the same time each day for 5 d, and chlorophyll

fluorescence parameterswere imaged before, immediately after,

and 2 h after each HL episode. The treatment protocol and times

at which fluorescence parameters were imaged are shown in

Figure 3A (see Methods).

After a single HL treatment, only minor differences in dark-

adapted Fv/Fm between mutant and wild-type plants were ob-

served (Figures 3B, top panel and 3C). However, after 5 d, the

outer, fully expanded leaves of nced3-2 rosettes had lower Fv/Fmvalues than the equivalent wild-type leaves (Figures 3B, bottom

panel, and 3C), indicating that the mutant plants, unlike the wild

type, experienced photoinhibition of PSII that was not recover-

able during a 20-min dark period or after 22 min back in growth

conditions. Measurements of the PSII operating efficiency at the

growth and HL treatment PPFDs (Figure 3D) confirmed that

nced3-2, but not wild-type leaves, experienced an increase in

photoinhibition with increasing days of treatment. There were no

significant differences in NPQ between the mutant and the wild

type during the course of the HL treatments, but there were

decreases in qL values for the mutant (Figure 3D). Thus, the

photoinhibition observed in nced3-2 was attributable to a re-

duced ability for photochemical quenching since QA was more

reduced in the mutant than in the wild type. This would imply that

the mutant is less able than the wild type to use the products of

LEF, ATP, and reductants. Consequently, nced3-2 leaves expe-

rienced increasing photoinhibition and, presumably, photodam-

age to PSII, with increasing periodic exposure to HL.

The same experiments were also conducted on aba2-11 and

aba2-14, but the leaves wilted severely during the HL exposures,

which made the accurate determination of chlorophyll fluores-

cence parameters impossible.

Taken together, these data indicate that induction of ABA

biosynthesis is a key factor in the response of fully expanded

leaves to repeated HL episodes at low RH.

HL Response Is Associated with Biphasic Accumulation of

Extracellular H2O2 in Vein-Associated Cells and the

Leaf Lamina

While previous studies reported a role for the production of

extracellular ROS in the regulation of HL-responsive genes,

direct observation of ROS accumulation during HL exposurewas

not reported. Real-time measurement of H2O2 accumulation in

leaves can be achieved using a sensitive derivative of the H2O2-

specific fluorogenic probe 10-acetyl-3,7-dihydroxyphenoxazine,

called Amplex Red Ultra (ARU; seeMethods). ARU is oxidized by

H2O2 in a peroxidase-catalyzed reaction to resorufin (Zhou et al.,

1997). ARU-resorufin fluorescence reports accumulation of ex-

tracellular H2O2 in leaf lamina tissues (Snyrychova et al., 2008). At

high magnification, ARU fluorescence was observed as a diffuse

overlay of tissues of the veins and immediately adjacent leaf

lamina (see Supplemental Figure 4 online), confirming the ob-

servations of Snyrychova et al. (2008). Laser scanning confocal

microscopy could not be used to establish more precise location

of extracellular H2O2 in leaf lamina and periveinal tissue, since the

laser light source used induced H2O2 production, rapidly caused

bleaching of tissues and quenching of resorufin fluorescence.

To simultaneously image resorufin accumulation in periveinal

and adjacent lamina, initial experiments relied on the colocaliza-

tion of APX2LUC expression with ARU-resorufin fluorescence

(Figure 4A). These experiments showed that 30 min after HL

exposure at low humidity, elongated cells displayed readily

detectable resorufin fluorescence compared with adjacent lam-

ina tissue (Figure 4A). Since APX2LUC expression is confined to

BSCs of the periveinal region in HL-exposed leaves (Fryer et al.,

2003; Ball et al., 2004), we conclude that extracellular H2O2

accumulation is associated with these cells, although we do not

rule out that other vein-associated tissues would also produce

extracellular H2O2.

In a series of experiments with leaves exposed to HL at low

humidity (Figures 4B and 4C), ARU-resorufin fluorescence in

periveinal tissue was detected at 15 min after exposure to HL,

although the peak of fluorescence varied between 15 and 30min

(cf. Figures 4B and 4C). A weaker secondary burst of resorufin

fluorescence was detected at ;90 min after HL exposure. In

adjacent lamina tissue, a similar biphasic burst of fluorescence

was detected; the second burst was of the same or greater

magnitude than the first (Figure 4C). The biphasic changes in

fluorescence were inhibited if leaves were placed in HL at high

humidity (Figure 4C) or if the leaves were pretreated with

ABAmineSG prior to HL exposure at low humidity (Figure 4B).

To verify accumulation of extracellular H2O2 in HL-exposed

leaves, we used transmission electron microscopy to visualize

cerium perhydroxide deposits, formed by reaction of infiltrated

cerium trichloride (CeCl3) with H2O2 (Bestwick et al., 1997; Hu

et al., 2005). Dark cerium perhydroxide deposits were detected

in the apoplast adjoining cells of the bundle sheath and the


Figure 3. A Foliar Capacity for ABA Biosynthesis Is Required for Fully Expanded Leaves to Adjust to Repeated Episodes of HL at Growth Humidity.


vascular parenchyma (Figure 4D, right panel; see Supplemental

Figure 5 online). At 150 mmols m22 s21 PPFD, weaker and less

extensive deposits were observed, showing that the dense

cerium perhydroxide accumulation was due to theHL treatments

(Figure 4D, left panel; see Supplemental Figure 5 online). How-

ever, no staining was detected in the apoplast on the mesophyll

side of BSCs nor in the apoplast surrounding mesophyll cells of

the leaf lamina (Figure 4D; see Supplemental Figures 5 and 6

online). Previous work has shown that the electron-dense cerium

perhydroxide deposits in Arabidopsis leaf cell walls are almost

entirely caused by H2O2 (Solyu et al., 2005).

HL-Induced Accumulation of H2O2 in BSCs in Response to

HL Is Rapid, Humidity Insensitive, and Directly Implicated in

Signaling to the Nucleus

Previous studies showed that the chloroplasts principally of

periveinal cells, but not exclusively of BSCs, accumulate H2O2 in

response to HL (Fryer et al., 2003; Mullineaux et al., 2006).

Infiltration of leaves using 39 39 diaminobenzidine (DAB) pene-

trates cells and stains for H2O2 accumulation in chloro-

plasts (Fryer et al., 2003; Liu et al., 2007; Driever et al., 2008;

Snyrychova et al., 2008), appearing as a brown precipitate

(Thordal-Christiansen et al., 1997). Using DAB, H2O2 accumula-

tion in chloroplasts of periveinal cells was visualized after 10 min

exposure (Figure 5A), and the intensity of DAB staining in the

veins of HL-exposed leaves was not susceptible to prevailing

humidity (Figure 5B). Therefore, it was concluded that there was

no direct role for leaf water status in the HL-induced H2O2

accumulation in the chloroplasts of periveinal cells, such as

BSCs and flanking mesophyll cells.

BSC-specific APX2 expression was elevated in the sulfiredoxin

1 mutant srx1 and the vitamin C-1 deficient mutant vtc1, which

are both partly defective in ROS scavenging capacity in the

chloroplasts (Conklin et al., 1997; Rey et al., 2007; see Discus-

sion) (Figure 5C). There was no greater degree of photoinhibition

nor was it any less reversible than in wild-type plants (see

Supplemental Figure 1 online). Other HL-expressed genes used

in this study were unaffected in their expression in thesemutants

(see Supplemental Table 1 online). These observations show that

while the accumulation of H2O2 in HL-exposed BSC chloroplasts

plays no role in the activation of ABA accumulation, H2O2 has a

BSC-specific function in the induction of APX2 expression under

HL. The specificity of the effect of these mutants on APX2

expression may reflect the propensity for BSC chloroplasts to

accumulate H2O2 more than other leaf tissues under HL condi-

tions (Figure 5A; Fryer et al., 2003) and therefore affect BSC-

specific gene expression more than genes expressed elsewhere

in the leaf.

ABA Signaling Is Associated with Extracellular ROS

Production and HL-Responsive Gene Expression

ABA signaling in guard cells involves the production of ROS at

the plasmamembrane (Murata et al., 2001; Kwak et al., 2003). At

least two components of the guard cell ABA signaling network

were proposed to be involved: OST1 (Mustilli et al., 2002; Li et al.,

2006) and heterotrimeric G proteins (Joo et al., 2005; Li et al.,

2006). Null mutant alleles of ost1 conferred a more than twofold

reduction in APX2 expression under HL (Figure 6A), but no

consistent effect of the loss of OST1 was observed upon the

expression of the five ABA- and HL-responsive genes studied in

Figure 2C (see Supplemental Table 1 online). Throughout the HL

treatment, stomatal conductance values for ost1-1 leaves were

fourfold higher than for wild-type plants (see Supplemental

Figure 7 online), which enhanced transpiration and a more rapid

change in leaf water status than in the wild type.

Total foliar H2O2 content in ost1-1 did not increase upon HL

exposure, in contrast with an increase of 175% in wild-type

plants (Figure 6G). Staining of HL-exposed leaves with DAB


(A) The daily HL regime applied to and chlorophyll fluorescence measurements taken on nced3-2 and Col-0 plants. This daily regime was set up and run

in a chlorophyll fluorescence imager programmed to apply the procedure automatically for a total of 130 min with plants transferred to the machine 2 h

into their photoperiod. After transfer of plants into the Fluorimager cabinet, actinic lights were switched on and provided a PPFD equivalent to that in

growth conditions (150 mmol m�2 s�1) andmeasurements of Fq9/Fm9made every 5min until unchanging values were obtained, typically after 20min. The

modulated (light adapted) fluorescence parameters required to calculate qL and NPQ (see Methods) were collected at point 1 before HL. Then the lights

were switched off for a period of 20 min, at the end of which (point 2) dark-adapted fluorescence parameters were collected to calculate NPQ for point

1 and Fv/Fm (at point 2). The plants were then exposed for 60 min to a 10-fold increased light intensity with the same light source (PPFD of 1500 mmol

m�2 s�1) and the response of plants monitored by measurements of Fq9/Fm9 every 10 min. At the end of the HL exposure (point 3), the modulated

fluorescence parameters were measured to calculate qL and NPQ. Finally, a second 20-min dark period was applied, at the end of which (point 4) dark-

adapted fluorescence parameters, required to calculate NPQ for point 3 and Fv/Fm (at point 4), were measured. After point 4, plants were returned to

their growth environment.

(B) Outer leaves of nced3-2 show lower dark-adapted Fv/Fm values than Col-0 after five daily 60-min HL exposures. Whole rosette images for Fv/Fm are

shown for one mutant and wild-type individual on day 1 before the first HL exposure (point 2 in [A]) and the same individuals at the end of the last HL

exposure (point 4 in [A]) on day 5. Note how uniform Fv/Fm is across the rosettes at the start of the experiment on day 1. By contrast, on day 5, outer

leaves of nced3-2 show lower values in at least half the leaves and overall more variation than the wild type. Note also that Col-0 has increased its

rosette area in the 5-d period in contrast with the mutant.

(C) Daily dark-adapted Fv//Fm values after HL (point 4 in [A]) in nced3-2 and Col-0. The values are the means (6SE) of two separate experiments each

consisting of values collected daily from two outer leaves of three plants of each genotype (n = 30). The asterisks indicate a significant difference (P #

0.05 from t test) between mutant and the wild type at the time point.

(D) Daily Fq9/Fm9, qL, and NPQ values before and after the HL exposure at points 2 and 4, respectively, in (A). The data are the means (6SE) from the

same experiments, and the asterisks denote the same threshold of significance as described for (C).


Figure 4. Extracellular H2O2 Production in Veins and Adjacent Lamina Tissue in HL-Exposed Leaves Is Sensitive to Prevailing Humidity and the ABA

Biosynthesis Inhibitor ABAmine.


showed brown precipitate predominantly in periveinal tissue to

equal levels in both ost1-1 and the wild type (see Supplemental

Figure 8 online).We conclude thatOST1positively regulates both

HL-induced APX2 expression and an increase in nonchloro-

plastic H2O2 levels, although this regulation may be confined to

BSCs.

Null mutants (gpa1-3 and gpa1-4; Ullah et al., 2001; Jones

et al., 2003) in genes encoding the Arabidopsis Ga subunit (G

PROTEIN ALPHA1 [GPA1]) and the mutants agb1-2 (Jones et al.,

2003), and agb1-9 in the geneARABIDOPSISGPROTEINBETA1

(AGB1), encoding the Gb subunit of the heterotrimeric G protein

complex, all showed three- to fivefold stimulation of APX2 ex-

pression under HL conditions (Figure 6B). Furthermore, a gpa1-4

agb1-2 double mutant (Jones et al., 2003) showed the same

increase inAPX2 expression as gpa1-3 and gpa1-4 plants (Figure

6B). The HL-responsive genes coding for lipocalin, HSP17.6C-

C1, HSP17.6B-C1, and RD20, showed a similar pattern of re-

sponse to HL in the G protein mutants (Figures 6C to 6F). ELIP1

gene expressionwas not consistently significantly affected by the

G protein null mutants (see Supplemental Table 1 online). There

was no elevated expression of any of the test genes at ambient

light intensity in the tested mutants, and during HL exposure, all

tested mutants maintained stomatal conductance similar to that

of wild-type plants (see Supplemental Figure 7 online).

Taken together, these data suggest that the heterotrimeric G

protein complex is a negative regulator of HL-responsive gene

expression in wild-type plants. Measurement of total foliar H2O2

content under low-light conditions revealed a 2.5-fold increase in

the gpa1 null mutant (Figure 6H), and there was no increased

DAB staining in the veins of low-light leaves of gpa1-4 compared

to Col-0 (data not shown). We conclude that GPA1 negatively

regulates the levels of extraplastidial H2O2 independent of any

increase in light intensity. Since under HL conditions, the null

mutants showed no differences from wild-type controls in foliar

H2O2 content (data not shown) and DAB staining of periveinal

chloroplasts (see Supplemental Figure 8 online), we conclude

that HL acts by relieving the inhibitory effect by GPA1.

DISCUSSION

Rapid Initiation of Foliar ABA Biosynthesis in Response to

HL under Low Humidity Conditions

We postulate that a rapid transient decline in leaf water content

upon exposure to HL, at low humidity, initiates ABA biosynthesis

and is a key step in coordinating the response of the leaf to these

conditions. This is because leaves experience a sudden increase

in transpiration rate brought about by exposure toHL (Fryer et al.,

2003). This sudden increase in transpiration rate could cause a

transient decline in leaf water potential since leaves have a high

degree of hydraulic resistance to water flow, and hydraulic

conductance is responsive to many environmental condi-

tions, including prevailing light intensity and humidity (Sack and

Holbrook, 2006; Levin et al., 2007; Sellin et al., 2008; Kim and

Steudle, 2009).

In Arabidopsis leaves, NCED3, ABA2, AAO3, and ABA4 are

prominently expressed in leaf veins (Nambara and Marion-Poll,

2005; Christmann et al., 2006; North et al., 2007), and recent

immunolocalization studies showed that vascular parenchyma


(A) Colocalization of APX2 expression and H2O2 accumulation in the veinal tissue of HL-exposed leaves. A detached leaf from a Col-0/APX2LUC plant,

previously infiltrated with ARU and luciferin (see Methods), was clamped into a CIRAS leaf chamber to maintain a constant temperature and low

humidity (25% RH; see Methods). The leaf was then exposed to HL (PPFD 750 mmol m�2 s�1 for 45 min), after which images of reflected light from the

leaf (a), resorufin fluorescence to locate H2O2 accumulation (b), and photon emission from luciferase activity from expression of APX2LUC (c) were

captured (see Methods). In (a), the focal plane highlights the vein (V) and the surrounding darker area is the leaf lamina (L). In (b), the false red color

denotes areas of high resorufin fluorescence, which is mainly in the vein but also may be immediately adjacent lamina; hence, the term periveinal is

used. Note that APX2LUC expression in (c) is confined more tightly to the veins and includes the elongated cells seen in the reflected light image (a),

consistent with expression of this gene in BSCs (Fryer et al., 2003; Ball et al., 2004). The images are from a single typical experiment. The yellow bar in

the right panel denotes 100 mm.

(B) Resorufin fluorescence in HL leaves inhibited for foliar ABA biosynthesis. Col-0 detached leaves were infiltrated with 50 mMABAmineSG and 40 mM

ARU or ARU alone (closed and open circles, respectively) as in Methods. All leaves were then exposed to HL (750 mmol m�2 s�1 PPFD) for 60 min at low

humidity in a leaf chamber as described for (A) and in Methods. Resorufin fluorescence from the veinal focal plane was quantified as described in

Methods. The data are combined from three independent sets of measurements (6SE; n = 3). The relative fluorescence values were normalized to the

starting low light values for resorufin fluorescence in each leaf.

(C) Images of resorufin fluorescence in HL (750 mmol m�2 s�1) exposed leaves at low humidity (25% RH) and high humidity (80% RH) sealed in a leaf

chamber as described for (A) and in Methods. The images are from a typical single experiment and leaves were infiltrated with ARU only as described

for (A) and in Methods. The numbers under each panel refer to the time in minutes from the beginning of exposure to HL. In this focal plane, the

periveinal region (V) and the adjacent leaf lamina (L) are as described for (A). False color yellow indicates resorufin fluorescence, and red color denotes

higher resorufin fluorescence. The graphs show the mean values at each time point (6SE; n = 3) for resorufin fluorescence, normalized to starting low

light values, in the periveinal and lamina focal planes of HL-exposed leaves at low humidity (closed circles) and high humidity (open circles).

(D) Transverse sections of the mesophyll (M), bundle sheath (BS), and vascular parenchyma (VP; Kinsman and Pyke, 1998; Evert, 2006) of HL-exposed

leaves at34000 magnification (the bar on each panel denotes 5 mm) infiltrated with CeCl3 prior to fixation, sectioning, and examination by transmission

electron microscopy (see Methods). Cerium perhydroxide deposition (yellow arrows highlight prominent examples in the apoplast) denotes

accumulation of H2O2. The leaves were exposed to HL (750 mmol m�2 s�1 PPFD) for 45 min at low humidity (right panel) or kept at growth PPFD at

low humidity (left panel) as in the legend of (A), except that the leaves (one per plant) remained attached to the rest of plant when clamped into the leaf

chamber. The two panels show typical images. More examples from two separate batches of plants can be seen in Supplemental Figure 5 online.


tissue is the site of foliar ABA synthesis (Endo et al., 2008).

Petioles contain both vascular parenchyma cells and BSCs

(Kinsman and Pyke, 1998; Evert, 2006; Endo et al., 2008) and

have the capacity to synthesize ABA and express APX2 in these

cell types, respectively (Fryer et al., 2003; Endo et al., 2008). ABA

levels in osmotically stressed petioles in both low light and in the

dark increased by a similar amount (Figure 1D; see Results), and

DCMUhad no effect on ABA accumulation in HL-exposed leaves

at low humidity (Figure 1E). From these experiments, we con-

clude that in HL at low humidity, increased ABA accumulation

requires a decline in leaf water content, but not active LEF or

other light-associated signals.

Figure 5. Accumulation of Chloroplastic H2O2 in the Veins of HL-Exposed Leaves Is Humidity Independent but Does Direct Expression of APX2.

(A) DAB staining for H2O2 accumulation in veins of HL-exposed leaves at low humidity is visible after 10 min exposure to HL. Leaves were infiltrated with

5 mMDAB for 2 h prior to being clamped into a leaf chamber and exposed to HL (750 mmols m�2 s�1 PPFD) at low humidity (25% RH; see Methods and

legend of Figure 4A; Fryer et al., 2002, 2003) for the times indicated. At the end of the HL exposure, leaves were fixed, destained, and photographed.

The dotted line indicates the boundary between HL and LL exposed parts of the leaf.

(B) Higher magnification images from intact leaves shown in (A). The bar in the left panel denotes 20 mm. The elongated files of cells are BSCs (Kinsman

and Pyke, 1998; Fryer et al., 2003) with stained chloroplasts clearly visible between 10 and 45 min of HL exposure.

(C) DAB staining in the veins of HL-exposed leaves is no different in low or high humidity. The leaves were treated as described in (A), except that they

were kept at either 25% RH or 80% RH when clamped into the chamber and exposed to HL.

(D) APX2 expression is higher in mutants with compromised plastidial H2O2 scavenging capacity. APX2 transcript levels in srx1-1, srx1-2 (Rey et al.,

2007), and vtc1-1 (Conklin et al., 1997) plants relative to the wild type (Col-0) exposed to HL (750 mmol m�2 s�1 PPFD) at growth humidity. APX2 cDNA

levels were normalized against CYCLOPHILIN cDNA levels as described in Figure 1A and Methods. The data, expressed as normalized cDNA levels in

the mutant relative to the wild type, are the means (6SE) of three biological replicates totaling nine plants for Col-0, srx1-1, and vtc1-1 and 13 plants for

srx1-2. The differences between mutants and the wild type were significant (P = 0.02, 0.058, and 0.022 from t tests for srx1-1, srx1-2, and vtc1-1,

respectively).


Figure 6. HL-Responsive Gene Expression in ABA Signaling Mutants.

(A) Whole rosettes of ost1-1 and ost1-2 and wild-type plants (Landsberg erecta [Ler]) were exposed to HL (750 mmol m�2 s�1 for 45 min) at growth

humidity, and APX2 cDNA levels were normalized with respect to those of CYCLOPHILIN (see legend of Figure 1A) and expressed as mutant relative to

the wild type. All data shown (means6 SE) are of two biological replications with a total of six to eight plants. The differences between the mutants and

the wild type were significant; P = 0.03 and 0.02 from t tests for ost1-1 and ost1-2, respectively.

(B) APX2 transcript levels in HL exposed heterotrimeric G protein null mutants compared with wild-type plants. Rosettes of null mutants of GPA1 (gpa1-3

and gpa1-4), AGB1 (agb1-2 and agb1-9), and a double mutant (gpa1-4 agb1-2) were exposed to HL at growth humidity along with Col-0 and assayed

for relative APX2 cDNA levels as described in (A). All data shown (means 6 SE) are of two biological replications with a total of six to eight plants. The

differences between the mutants and the wild type are significant (P # 0.05 from t test).

(C) to (F) Transcript levels of HL-responsive genes encoding lipocalin (C), RD20 (D), HSP17.6B-C1 (E), and HSP17.6C-C1 (F) assayed in HL-exposed

rosettes (see legend of [A]) of gpa1-3, gpa1-4, agb1-2, and gpa1-4 agb1-2 relative to Col-0. All cDNA levels were normalized with respect to those of

CYCLOPHILIN and expressed as mutant relative to the wild type. All data shown (means6 SE) are of two biological replications with a total of six to eight

plants. The histograms marked with asterisks are significant (P # 0.05 from t test) between mutant and the wild type.

(G) Foliar H2O2 levels in low light and HL-exposed ost1-1 and wild-type (Ler) leaves. H2O2 levels were determined from cell-free acid extracts of fully

expanded leaves of plants exposed to HL at growth humidity as described in (A) or from plants kept at growth PPFD and humidity (LL). The H2O2

amount in each sample was determined using an amplex red-based in vitro assay (seeMethods). Data are themeans (6SE) of two separate experiments

with four plants per experiment (n = 8). Three replicate determinations were carried per sample. The differences between the mutants and the wild type

are significant (P # 0.05 from t test).


The rise in foliar ABA levels in intact leaves exposed to HL and

lowhumidity occurredat a rate;10-fold lower than indehydration-

stressed detached leaves (calculated from Endo et al., 2008

and data in Figure 1A). This difference is reflected in the strong

induction of NCED3 expression during dehydration stress (Iuchi

et al., 2001; Tan et al., 2003; Endo et al., 2008). By contrast,

NCED3 transcript levels did not rise in HL-exposed leaves at

growth or lower humidity (Figure 1A). However, in our experi-

ments, NCED3 transcripts could be readily detected in well-

watered plants at low light (see Supplemental Figure 3 online)

using a similar RT-PCR technique (see Methods) to that of Endo

et al. (2008), who could not detect this transcript under fully turgid

conditions. This may reflect a difference in watering regimes,

although it should be noted that in nced3-2, the levels of ABA

underwell-watered, low-light conditionswere higher than inwild-

type plants (see Supplemental Figure 3 online), suggesting that

ABA levels can be maintained under nonstress conditions by

NCED3-independent means. By contrast, under HL, foliar ABA

levels did not rise in the mutant (see Supplemental Figure 3

online), underscoring the requirement for an extant capacity for

foliar ABA biosynthesis for many aspects of the HL response

(Figures 2 to 4).

The lack ofNCED3 induction (Figure 1A), but the observed rise

in foliar ABA levels (Figures 1A and 1B) and the requirement for an

extant foliar ABA biosynthetic capability once HL is applied

(Figures 2 to 4), suggest that NCED3 could also be subject to

posttranslational regulation. This notion is supported by the

observation that two different sized NCED3 isoforms of 64 and

58 kDwere detected in dehydration-stressed Arabidopsis leaves

(Endo et al., 2008), showing that posttranslational modification of

NCED3 can occur. Posttranscriptional activation of foliar NCED

by ethylene in cleavers (Gallium aparine) is proposed to occur

concomitant with a slower induction of NCED transcription by

auxin (Kraft et al., 2007).

Under high humidity conditions, ABA accumulation did not

occur (Figure 1B). This could be due to a failure to activate ABA

biosynthesis, since a transient change in leaf water content

would be required. However, we cannot rule out that ABA

catabolism is activated, since recently it has been shown that

the vascular-located ABA 8’hydroxylase gene CYP707A3 is

induced in 10 min of exposure to high humidity conditions

(Okamoto et al., 2009).


Maintenance of Photochemical Quenching andAcclimation

to HL

The maximum expression of HL-responsive genes requires ABA

(Figures 2A to 2C; Bechtold et al., 2008). This requirement may

implicate >800HL- andABA-coresponsive genes (Figure 2D; see

Supplemental Data Set 1 online), suggesting that ABA biosyn-

thesis and signaling in different leaf tissues should be important

for the response of the whole leaf to HL. This was the case since

nced3-2was unable to adjust to a daily exposure to 60min of 10-

fold HL (Figure 3A) and suffered a degree of irreversible photo-

inhibition (Figures 3B and 3C). NPQwas unaffected in themutant

(Figure 3D), but it displayed a lower capacity for photochemical

quenching throughout these treatments (Figure 3D; see Results).

Therefore, in wild-type plants, an extant capability for foliar ABA

biosynthesis is required to maintain or induce additional meta-

bolic capacity to dissipate excitation energy. Failure to do so

results in damage to the photosynthetic apparatus, increased

photooxidative stress, and accelerated foliar senescence. These

symptoms were clearly observed in nced3-2 (Figure 3B) and are

consistent with changes in chlorophyll fluorescence in senescing

leaves (Jenkins et al., 1981). Increases in photochemical quench-

ing observed in BSCchloroplasts after 30min exposure toHL are

humidity sensitive (Fryer et al., 2003), supporting the suggestion

that the ABA-mediated induction of additional electron sink

capacity in this tissue is occurring.

These observations provide a new role for foliar ABA biosyn-

thesis in addition to those described for dehydration stress

(Christmann et al., 2007; Endo et al., 2008), stomatal responses

to low humidity (Xie et al., 2006), and exploitation by the bacterial

pathogen Pseudomonas syringae to subvert host defenses (de

Torres-Zabala et al., 2007).

ABA Signaling in Veins and Lamina in Response to HL at

Low Humidity

For ABA to participate in the regulation of HL-expressed genes in

a range of leaf tissues, it must be secreted from the vascular

parenchyma and induce signaling responses in other cell types.

The interaction of ABA with BSCs is demonstrated by the ABA

biosynthesis- and humidity-dependent biphasic production of

extracellular H2O2 associated with these cells (Figure 4). This

begins at 15 min for the first phase and a much weaker but

evident second burst at 90 min after onset of HL (Figures 4B and

4C). This extracellular H2O2 production may be more precisely

located in the intercellular spaces between BSCs and vascular

parenchyma cells (Figure 4D; see Supplemental Figure 5 online).

These observations are consistent in different experimental

systems with descriptions of ABA-, excess light-, and ozone-

stimulated signaling in guard cells, epidermal cells, and other leaf

tissues requiring plasma membrane NADPH oxidase isoforms

(Kwak et al., 2003; Davletova et al., 2005; Joo et al., 2005; Li et al.,

2006). Moreover, APX2, which is inducible by exogenous ABA

(Fryer et al., 2003; Rossel et al., 2006; Bechtold et al., 2008),

is also inducible by provision of exogenous H2O2 to leaves

(Karpinski et al., 1999; Bechtold et al., 2008), and is completely

inhibited in excess light stressed leaves preinfiltrated with cat-

alase (Karpinski et al., 1999), shows lowered expression in a


(H) Foliar H2O2 levels in fully expanded outer leaves of gpa1-3, gpa1-4, and wild-type (Col-0) rosettes under low light and growth humidity conditions

(see Methods). The methods were as in (G). Data are the means (6SE) of two separate experiments with five plants per experiment (n = 10). Three

replicate determinations were carried per sample. The differences between the mutants and the wild type are significant (P # 0.05 from t test).


double null mutant defective for NADPH oxidases D and F

(Bechtold et al., 2008) and where NADPH oxidase activity is

inhibited (Volkov et al., 2006). In other plant species, these

observations are consistent with ABA treatment of maize (Zea

mays) leaves, which causes an increase in apoplastic H2O2 levels

(Hu et al., 2005; Jiang and Zhang, 2003) and similar distribution

patterns of extracellular vascular H2O2 levels preceded by foliar

ABA accumulation in the Mediterranean shrub Cistus albidus

subject to summer drought (Jubany-Mari et al., 2009).

Mutants defective in OST1 protein kinase function (Mustilli

et al., 2002) showed marked attenuation of APX2 induction in

response to HL (Figure 6A), suggesting that OST1 positively

regulates induction of APX2 expression under HL conditions.

These observations are consistent with the ABI1 protein phos-

phatase 2C (PP2C)-mediated positive regulation of APX2 induc-

tion (Fryer et al., 2003) and its interaction with and activation of

OST1 protein kinase (Yoshida et al., 2006). OST1 alsomay have a

positive role in the production of extracellular H2O2 in HL-

exposed leaves, similar to its proposed role in the production

of ROS at the plasma membrane of guard cells (Mustilli et al.,

2002). This is because total foliar H2O2 levels did not rise in

response toHL treatment, in contrast with a near doubling inCol-0

(Figure 6G), but accumulation of H2O2 in BSC chloroplasts,

estimated by DAB staining, appears to be no different between

mutant and wild-type plants (see Supplemental Figure 8 online).

Therefore, we suggest that OST1 positively regulates extracel-

lular H2O2 production upon activation by ABA synthesized under

HL conditions. In addition, in HL-exposed ost1-1, the normal

induction of the HL-responsive genes (see Supplemental Table

1 online), other than APX2, and accumulation of H2O2 in BSC

chloroplasts (see Supplemental Figure 8 online) rules out any

pleiotropic affect in this mutant caused by enhanced stomatal

conductance (see Supplemental Figure 7 online; Mustilli et al.,

2002).

The induction of APX2 expression was enhanced by four- to

fivefold over wild-type plants in HL-exposed GPA1 and AGB1

null mutants (Figure 6B). In the double mutant gpa1-4 agb1-2,

there was no additive effect of these combinedmutations (Figure

6B). These data suggest that in BSCs, the heterotrimeric G

protein complex is a negative regulator of APX2 expression.

Similar results were reported forRab18 gene expression (Pandey

et al., 2006). Under low-light conditions, the GPA1 null mutants

had elevated levels of total foliar H2O2 (Figure 6H) in the absence

of any increase in vein chloroplast H2O2 content (see Supple-

mental Figure 8 online). Thus, GPA1, and potentially theGprotein

complex, also negatively regulates extracellular H2O2 levels in

veins under ambient light conditions.

In contrast with the situation in animal cells, activation of the

Arabidopsis Ga subunit does not require a G-protein coupled

receptor (i.e., specifically a receptor having guanine nucleotide

exchange factor function since GPA1 is a rapid nucleotide

exchanger) (Johnston et al., 2007; Temple and Jones, 2007).

Indeed, the hydrolysis of GTP is probably the rate-limiting step in

the plant G protein cycle. Therefore, induction of APX2 by ABA

would include triggering a deactivation of some or all of the BSC

GPA1 complement, which would release downstream signaling

from negative regulation. This could include a release of extra-

cellular H2O2 production from negative control, which could

induce APX2 expression. RGS1, a seven-transmembrane-

domain GTPase activating protein, controls the activation state

of GPA1 (Chen et al., 2003). Based on the current body of

evidence, control of the GTPase activating protein activity of

RGS1 occurs upon glucose binding or by binding of some other,

but related, photosynthate product.

H2O2- and Redox-Mediated Retrograde Signaling from

Chloroplasts Is Independent of Humidity

The above considerations show that ABA and its biosynthesis in

leaves are necessary, but not sufficient, for the induction of HL-

responsive genes. BSC chloroplasts and those of flanking me-

sophyll cells accumulate H2O2 within 10 min of exposure of the

leaf to HL (Figures 5A and 5B), and this accumulation is inde-

pendent of prevailing humidity (Figure 5C). This suggests that

any ABA signal from vascular parenchyma cells does not affect

the accumulation of H2O2 in BSC chloroplasts, which is likely to

result from the photoreduction of O2 at photosystem I (Fryer

et al., 2003).

The induction of APX2 expression by mild osmotic stress of

isolated petioles requires light (Figure 1C) and is associated with

a lowering of the oxidation state of QA and the operating

efficiency of PSII photochemistry (Table 1). The change in these

parameters and the repeated observation that the LEF inhibitor

DCMU suppresses the induction of a large majority of nuclear-

encoded HL-responsive genes (Karpinski et al., 1997; Rossel

et al., 2002; Yabuta et al., 2004; Bechtold et al., 2008), including

APX2 expression in petioles (Chang et al., 2004), is consistent

with a requirement for LEF to provide additional redox and ROS

signals sourced from BSC chloroplasts.

Null mutants of SRX1, which codes for sulforedoxin, a key

enzyme component of the plastidial peroxiredoxin-based H2O2

scavenging system in Arabidopsis leaves (Rey et al., 2007),

showed a significant elevation of APX2 expression (Figure 5D).

Similarly, vtc1-1, which has depleted levels of ascorbic acid

(Conklin et al., 1997), also showed a marked stimulation of only

APX2 expression upon exposure to HL (Figure 5C). Neither set of

mutants showed any significantly increased sensitivity to the HL

exposure as determined by chlorophyll fluorescence measure-

ments (see Supplemental Figure 1 online). This discrepancy is

explained by the observation that partial loss of ROS scavenging

capacity occurs in every tissue, but under these moderate HL

treatments, an effect is only noted for redox- and ROS-responsive

APX2 in BSCs which readily accumulate chloroplastic H2O2.

The other HL-responsive genes used in this study did not give

a consistent response across the mutants (see Supplemental

Table 1 online), but this is consistent with the lack of accumu-

lation of H2O2 in any other leaf tissue under these mild HL

conditions. No doubt more extreme photooxidative stress would

provoke alterations in the expression of a much wider group of

ROS- and redox-responsive genes. Nevertheless, many of these

HL-responsive genes still require a chloroplast-sourced redox- or

LEF-associated signal (Karpinski et al., 1997; Rossel et al., 2002;

Ball et al., 2004; Bechtold et al., 2008).

In summary, along with previous observations (Karpinski et al.,

1997, 1999; Fryer et al., 2003; Ball et al., 2004; Chang et al.,

2004), the data presented here confirm that in BSCs, APX2


expression requires an increased chloroplast oxidative state and

active LEF to activate its expression, alongwith a requirement for

an extracellular ABA signal.

Interdependence of a Transcellular ABA Signal and

ChloroplastRetrogradeSignals inLeavesResponding toHL

In the BSCs of HL exposed leaves, all the key signaling events

involving H2O2 sourced from chloroplasts and ABA secreted

from neighboring vascular parenchyma cells is complete within

30 min of exposure. This series of signaling events in HL-

exposed BSCs supports the generic hypothesis proposed by

Pfannschmidt et al. (2009), who suggested that retrograde

signals from chloroplasts may typically act by merging with an

external tissue-sourced signal.

In other leaf tissues, the topology of combined retrograde

signaling and ABA signaling networks may be different. This is

evidenced by less extensive and slower extracellular H2O2

production across leaf tissues (Figure 4B), which may reflect a

later or less responsive interaction with external ABA, and no

evidence for an involvement of OST1 in signaling and differing

responses to G protein mutants (Figure 6). In addition, most leaf

cell types do not accumulate chloroplastic H2O2, but most HL-

responsive genes require an active LEF, supporting the argu-

ment that retrograde redox signals may vary from tissue to tissue

(Ball et al., 2004).

METHODS

Growth of Plants

Arabidopsis thaliana genotypes (Col-0, Ler, or C24) and mutants used

were grown in a peat-based compost in a controlled environment room

under an 8/16-h light/dark cycle at a PPFD of 150 mmol m22 s21, 228C6

18C, and a RH of 50%. All plants studied were at 5 to 6 weeks after

germination.

Genotypes

The mutants used and their ecotype background are as follows: APX2-

LUC (Col-0; Karpinski et al., 1999); nced3-2 (Col-0; see below); sto1-1

(C24; Ruggiero et al., 2004); aba2-11 and aba2-14 (Col-0; Gonzalez-

Guzman et al., 2002; Barrero et al., 2006); srx1-1 and srx1-2 (Col-0; Rey

et al., 2007); vtc1-1 (Col-0; Conklin et al., 1997), ost1-1 and ost1-2 (Ler;

Mustilli et al., 2002); gpa1-3 and gpa1-4 (Col-0; Jones et al., 2003); agb1-2

and agb1-9 (Col-0; Jones et al., 2003; see below); and gpa1-4 agb1-2

(Jones et al., 2003). All genotypes were confirmed.

Isolation of Mutants nced3-2 and agb1-9

The nced3-2 mutant line (N331021) for NCED3 was obtained from the

Nottingham Arabidopsis Stock Centre. The T-DNA insertion site is in the

coding region of the unique exon of NCED3 (see Supplemental Figure 3

online) and is identical to the mutant recently published by Urano et al.

(2009).

The mutant agb1-9 was isolated from a screen of Arabidopsis display-

ing agb1-2 null mutant phenotypes. agb1-9 contains a W313-to-stop

codon mutation that causes premature termination of AGB1 synthesis,

rendering the truncated protein nonfunctional.

Exposure of Leaves to HL

Fully expanded outer leaves detached from the plant were exposed to a

PPFD of 750 (650) mmol m22 s21 at a RH of 25% (low humidity) or 80%

(high humidity) using a fiber optic white light source of 1 cm diameter as

described previously (Fryer et al., 2003). The temperature (278C 6 18C)

and humidity during HL exposure was kept constant using a CIRAS leaf

chamber (PP Systems) as described previously (Fryer et al., 2003). Prior

to clamping in the chamber, detached leaveswere infiltratedwith luciferin

(see below), DCMU (30 mM; Sigma-Aldrich), DAB, or ARU for staining

H2O2 (see below) via their transpiration stream for 2.5 h in low-light

conditions as described previously (Fryer et al., 2002).

Whole rosettes were exposed to a PPFD of 750 (6100)mmolm22 s21 at

ambient (growth) RH. This was done using lamps and water filters as

described previously (Karpinski et al., 1999). Under the lamps, leaf

temperatures increased by #58C over 90 min, the maximum time of

exposure. Temperature increases of <68C do not induce expression of

any of the genes in this study (Bechtold et al., 2008).

Osmotic Stress Treatment of Petioles

Solutions of (0.1 to 0.5 M) PEG-400, sorbitol, mannitol, or water were

infiltrated into detached fully expanded leaves of APX2LUC/Col-0

(Karpinski et al., 1999) for 2 h as described below for the infiltration of

ARU. Then, an;15 to 20mm length of petiole was cut from each leaf and

immersed for a further 1.5 h in the same solution also containing 1 mM D

(2) luciferin under low light or in the dark. At the end of this period,

luciferase activity was imaged and the petioles harvested for determina-

tion of their ABA content.

Chlorophyll Fluorescence Imaging

Chlorophyll fluorescence parameters weremeasured using a Flourimager

chlorophyll fluorescent imaging system (Technologica). The application

of preprogammed regimes of actinic growth light exposure times, satu-

rating light pulses, and dark periods and the calculation and imaging of

the parameters Fv/Fm, Fq9/Fm9, Fv9/Fm9, and NPQ (Baker, 2008) were

performed automatically by the Fluorimager’s software. qL was calcu-

lated postmeasurement from images of Fq9/Fm9, Fo9, and F9.

Visualization of H2O2 Accumulation and Its Measurement in Total

Foliar Extracts

Use of ARU

ARU (a proprietary derivative of 10-acetyl-3,7-dihydroxyphenoxazine;

Invitrogen; Zhou et al., 1997) was used to detect accumulation of ex-

tracellular H2O2 in the veins and periveinal regions ofArabidopsis leaves.

It should be noted that this dye is different in its penetration properties

compared with Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine)

and depending on which tissues in the leaf are being examined

(Snyrychova et al., 2008). ARU was dissolved in dimethylsulfoxide and

then diluted into 50 mM sodium phosphate buffer, pH 7.5, to give a final

concentration of 40 mM for infiltration into leaves. A detailed description

of how dyes can be infiltrated into leaves via the transpiration stream is

given in two publications (Fryer et al., 2002; Driever et al., 2008).

Infiltration was performed into fully expanded detached leaves for

ARU for 2 h under low light (20 to 50 mmol m22 s21) conditions. The

leaveswere cut from the plant under liquid to ensure no airlock formed at

the cut petiole surface. At the end of the infiltration period, the leaf was

then clamped into the CIRAS leaf chamber and exposed to the HL

conditions with the 500- to 570-nm region of the spectrum being

removed using a Rose Pink filter (Lee Filters). This was to prevent

absorption of these wavelengths by ARU during the course of the


experiment and to prevent photodegradation of ARU. Resorufin

(7-hydroxy-3H-phenoxazin-3-one) derivative, the fluorescent product

obtained when ARU reacts with H2O2 (Zhou et al., 1997), was imaged

using a Peltier-cooled CCD camera (Wright Instruments) with a custom-

built LED lighting system (Technologica) that provides a constant and

homogenous excitation light with a maximum at 466 nm. Detection of

fluorescence emission from resorufin was achieved by placing a 590-nm

band-pass filter (Edmund Optics) in front of the lens of the CCD camera.

The camera was controlled by FluorImager V1.01 software (Technolog-

ica), which was purposely designed for image acquisition (576 3 384

pixels) and control of exposure time, which was 5 s for all experiments.

Acquired images were processed using ImageJ software (Abramoff

et al., 2004).

Use of DAB

DAB (5mMat pH 3.8; Fryer et al., 2002; Driever et al., 2008) was infiltrated

into detached leaves as described above for ARU. The leaves were

placed in the CIRAS leaf chamber and subjected to HL at low or high

humidity. At the end of the experiment, leaves were infiltrated with lacto-

glycerol-ethanol to fix them and remove chlorophyll (Fryer et al., 2002;

Driever et al., 2008) prior to imaging.

Use of CeCl3

CeCl3 reacts with hydrogen peroxide to form cerium perhydroxides,

which forms electron-dense deposits that can be visualized by transmis-

sion electron microscopy (Bestwick et al., 1997). Control and HL-

exposed leaves at low humidity were rapidly sliced into ;10-mm strips

and immediately vacuum infiltrated with 5 mM CeCl3 in 50 mM MOPS

buffer, pH 7.2, and incubated for 2 h. Leaf segments (33 3mm)were then

prefixed in 2.5% (v/v) glutaraldehyde buffered with 0.1 M cacodylate

buffer, pH 7.2, at 48C overnight followed by three 10-min rinses in 0.1 M

Na cacodylate buffer, pH 7.2. The leaves were then postfixed in 1% (v/v)

buffered osmium tetroxide for 1 h at 48C followed by three washes in

distilledwater. The leaveswere then stained in 1% (w/v) uranyl acetate for

1 h followed by dehydration in an ethanol series (30, 50, 70, 90, and 100%

ethanol for 10 min each). They were then treated with propylene oxide for

30 min prior to transfer to TAAB LV resin (TAAB Laboratories Equipment):

propylene oxide (1:1) for 12 h in sealed vials. The leaves were then

transferred to 100% TAAB LV for 2 h followed by fresh TAAB LV for 24 h.

Finally, the leaf segments were transferred to fit molds with TAAB LV and

cured for 16 h at 608C. Sections (90 nm) were cut with a diamond knife,

stained with lead citrate, and viewed with a Jeol JEM-1400 electron

microscope at an accelerating voltage of 80 kV.

Total Foliar H2O2 Measurements

Hydrochloric acid (0.1M) extracts from100mgof freshArabidopsis tissue

ground in liquid nitrogen were prepared and purified over activated

charcoal as described by Creissen et al. (1999). H2O2 concentrations in

purified extracts were determined using an Amplex Red assay kit

(Invitrogen) according to the manufacturer’s instructions.

Imaging of Luciferase Activity in Leaf Veins

High-magnification imaging of HL-induced luciferase activity in Col-0/

APX2LUC was as previously described (Fryer et al., 2003). Plants were

sprayed with 1 mM D (2) luciferin (Biosynth), or it was infiltrated into

detached leaves as described above for ARU and for HL experiments.

Images were taken using the same CCD camera system and processed

as described for resorufin (see above) using an image acquisition time of

2 min.

ABAMeasurements

All measurements of foliar ABA content were performed on leaves

clamped in the CIRAS chamber, and only one leaf per plant was used.

After treatments, leaves were frozen in liquid nitrogen, freeze-dried, and

ABA content measured using a radio-immunoassay procedure (Quarrie

et al., 1988), as described previously (Xie et al., 2006). Following exposure

to osmotic stress, individual petioles were immediately frozen in liquid

nitrogen and similarly assayed for ABA content.

RNA Extraction and Quantitative Real-Time RT-PCR

RNA was extracted from 100 to 200 mg of fully expanded leaves using

Triazol reagent (Sigma-Aldrich) according to the manufacturer’s instruc-

tions, except that an additional ethanol precipitation step was included at

the end of the procedure to ensure the RNA was of appropriate quality

(A260/A280 » 2.0). RNA (3 mg) was treated with RNase-free DNase1

(Ambion) and the absence of contaminating genomic DNA confirmed

using a PCR test as described previously (Bechtold et al., 2008). RNA (2

mg)was used tomake random-primed cDNAaspreviously described (Ball

et al., 2004), except that theMuMLV reverse transcriptasewas purchased

from Fermentas. Quantitative real-time PCRwas performed as described

previously (Ball et al., 2004; Bechtold et al., 2008) using a cybergreen-

fluorescence based procedure with reagents purchased in kit form from

Sigma-Aldrich. Relative cDNA levels between two sets of threshold cycle

(Ct) values were calculated using the DDCT method (Kubista et al., 2006)

and normalized with respect to relative cDNA levels for CYCLOPHILIN.

This reference gene was chosen because it shows unchanging transcript

levels in excess light-exposed leaves and in conditions where foliar ABA

content varies (Rossel et al., 2006). The primers used in this study for

quantitative RT-PCR are given in Supplemental Table 2 online.

Bioinformatics

Data for Affymetrix ATH1 GeneChips were downloaded from the

GEO repository (http://www.ncbi.nlm.nih.gov/sites/entrez?db=gds) and

the NASCArrays database (http://affymetrix.Arabidopsis.info/narrays/

experimentbrowse.pl). The HL exposure data (GEO accession number

GSE7743) and ABA treatment data (NASCARRAYS-176) were normal-

ized using GCRMA procedures in the Bioconductor package within the R

statistical environment (Gentleman et al., 2004; Wu et al., 2004). Values

for replicate arrays were averaged and ratios calculated between treat-

ment and control; these ratios were used to rank genes for the responses

to 3 h HL exposure and 3 h following ABA treatment. Significant similar-

ities between these two ranked lists were then calculated using the

Ordered List Bioconductor package (Lottaz et al., 2006). The genes

commonly upregulated and commonly downregulated that contributed

to the significant weighted similarity score were clustered together with

other ABA treatment time points from the NASCARRAYS-176 data set

and additional ABA treatment data sets (GSE7112 and GSE6171). Hier-

archical clusteringwas performed usingCLUSTER (Eisen et al., 1998) and

visualized with the program TREEVIEW (Eisen et al., 1998). Complete

linkage clustering using an uncentered Pearson correlation was applied

after the genes were first ordered by self-organizing maps to produce

better-arranged clusters.

Accession Numbers

The Arabidopsis Genome Initiative locus identifiers of genes used or

mentioned in this study are as follows: AAO3, At2g27150; ABA2,

At1g52340; ABA4, At1g67080; ABI1, At4g26080; ABI2, At5g57050;

AGB1, At4g34460; APX2, At3g09640; BAP1, At3g61190;CYCLOPHILIN,

At2g29960; ELIP1, At3g22840; HSP17.6B-C1, At2g29500; HSP17.6C-

C1, At1g53540; GPA1, At2g26300; LIPOCALIN, At5g58070; LHCB1.2,


At1g29910; NCED3, At3g14440; OST1, At4g33950; PDF1.2, At5g44420;

PR1, At2g14610; RD20, At2g33380; SRX1, At1g31170; VTC1,

At2g39770; ZAT10, At1g27730; and ZAT12, At5g59820.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Dark-Adapted Fv/Fm Measurements on Col-0,

srx1-1, srx1-2, and vtc1-1 before, Immediately after HL Exposure,

and 24 h Later.

Supplemental Figure 2. Expression under HL Conditions of Genes

Controlled by the GUN1/ABI4 and 1O2 - Related Chloroplast-to-

Nucleus Retrograde Signaling Pathways.

Supplemental Figure 3. Characterization of nced3-2.

Supplemental Figure 4. Resorufin Fluorescence from the Periveinal

Region of Amplex Red Ultrainfiltrated Leaves Exposed to Fivefold HL

at Low Humidity.

Supplemental Figure 5. Further Electron Micrographs of CeCl3-

Stained High Light and Control Leaf Sections through Vein Tissue.

Supplemental Figure 6. Further Electron Micrographs of CeCl3-

Stained High Light and Control Leaf Sections through Vein Tissue.

Supplemental Figure 7. Stomatal Conductance of ABA Signaling

Mutants.

Supplemental Figure 8. DAB Staining Showing H2O2 Accumulation

in the Veins of ost1-1 and gpa1-4 Compared with Wild-Type Controls.

Supplemental Table 1. HL-Responsive Gene Expression in Mutants

Used in This Study Where No Significant Effect Was Discerned.

Supplemental Table 2. A List of the Primers and Their Target Genes

Used for qRT-PCR in This Study.

Supplemental Data Set 1A. A List of Genes Whose Expression Is

Upregulated by Both High Light and ABA Treatment.

Supplemental Data Set 1B. A List of Genes Whose Expression Is

Downregulated by Both High Light and ABA Treatment.

ACKNOWLEDGMENTS

We thank Jeffrey Leung (Institut Science Vegetale, Gif-sur-Yvette, France),

Hisashi Koiwa (University of Texas A&M), Jose Luis Micol (Universidad

Miguel Hernandez, Alicante, Spain), Pascal Rey (Centre National de la

Recherche Scientifique, Commissariat a l’Energie Atomique (CEA),

Universite de la Mediterranee, France), and the Nottingham Arabidopsis

Stock Centre for the mutants used in this study and Nobutaka Kitahata

(University of Tokyo, Japan) for the provision of ABAmineSG. We thank

Julian Theobald (Lancaster University) for skilled technical assistance in

the ABA measurements. Electron microscopy was carried out by Peter

Splatt and Gavin Wakley at the Exeter Bioimaging Centre. A.M.J. thanks

Paul Reeves for isolating the agb1-9 mutant. This work was supported by

a grant to N.R.B., W.J.D., and P.M.M. from the UK Biotechnology and

Biological Sciences Research Council (BBSRC). G.G.-V. acknowledges

the support of a Ministerio de Educacion y Ciencias (Spain; EX2005-1107)

and a European Union Marie Curie Fellowship (041283). K.S. acknowl-

edges the support of a BBSRC Research Studentship. This work was also

supported by grants from National Institute of General Medical Sciences

(R01GM065989), the Department of Energy (DE-FG02-05er15671), and

the National Science Foundation (MCB-0723515 and 0718202) to A.M.J.

Received June 16, 2008; revised July 1, 2009; accepted July 8, 2009;

published July 28, 2009.

REFERENCES

Abramoff, M.D., Magalhaes, P.J., and Ram, S.J. (2004). Image pro-

cessing with ImageJ. Biophotonics Int. 11: 36–42.

Apel, K., and Hirt, H. (2004). Reactive oxygen species: Metabolism,

oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55:

373–399.

Aro, E.M., Virgin, I., and Andersson, B. (1993). Photoinhibition of

photosystem II - Inactivation, protein damage and turnover. Biochim.

Biophys. Acta 1143: 113–134.

Asada, K. (1999). The water-water cycle in chloroplasts: Scavenging of

active oxygens and dissipation of excess photons. Annu. Rev. Plant

Physiol. Plant Mol. Biol. 50: 601–639.

Baker, N.R. (2008). Chlorophyll fluorescence: A probe of photosynthe-

sis in vivo. Annu. Rev. Plant Biol. 59: 89–113.

Baker, N.R., Harbinson, J., and Kramer, D.M. (2007). Determining the

limitations and regulation of photosynthetic energy transduction in

leaves. Plant Cell Environ. 30: 1107–1125.

Ball, L., et al. (2004). Evidence for a direct link between glutathione

biosynthesis and stress defense gene expression in Arabidopsis.

Plant Cell 16: 2448–2462.

Barrero, J.M., Rodriguez, P.L., Quesada, V., Piqueras, P., Ponce, M.R.,

and Micol, J.L. (2006). Both abscisic acid (ABA)-dependent and

ABA-independent pathways govern the induction of NCED3, AAO3 and

ABA1 in response to salt stress. Plant Cell Environ. 29: 2000–2008.

Bechtold, U., Richard, O., Zamboni, A., Gapper, C., Geisler, M.,

Pogson, B., Karpinski, S., and Mullineaux, P.M. (2008). Impact of

chloroplastic- and extracellular-sourced ROS on high light-responsive

gene expression in Arabidopsis. J. Exp. Bot. 59: 121–133.

Bestwick, C.S., Brown, I.R., Bennett, M.H.R., and Mansfield, J.W.

(1997). Localization of hydrogen peroxide accumulation during the

hypersensitive reaction of lettuce cells to Pseudomonas syringae pv

phaseolicola. Plant Cell 9: 209–221.

Booker, F.L., Burkey, K.O., Overmyer, K., and Jones, A.M. (2004).

Differential responses of G-protein Arabidopsis thaliana mutants to

ozone. New Phytol. 162: 633–641.

Chang, C.C.C., Ball, L., Fryer, M.J., Baker, N.R., Karpinski, S., and

Mullineaux, P.M. (2004). Induction of ASCORBATE PEROXIDASE 2

expression in wounded Arabidopsis leaves does not involve known

wound-signaling pathways but is associated with changes in photo-

synthesis. Plant J. 38: 499–511.

Chen, J.-G., Willard, F.S., Huang, J., Liang, J., Chasse, S.A., Jones,

A.M., and Siderovski, D.P. (2003). A seven-transmembrane RGS pro-

tein that modulates plant cell proliferation. Science 301: 1728–1731.

Christmann, A., Moes, D., Himmelbach, A., Yang, Y., Tang, Y., and

Grill, E. (2006). Integration of abscisic acid signaling into plant

responses. Plant Biol. 8: 314–325.

Christmann, A., Weiler, E.W., Steudle, E., and Grill, E. (2007). A hydraulic

signal in root-to-shoot signaling of water shortage. Plant J. 52: 167–174.

Conklin, P.L., Pallanca, J.E., Last, R.L., and Smirnoff, N. (1997).

L-ascorbic acid metabolism in the ascorbate-deficient Arabidopsis

mutant vtc1. Plant Physiol. 115: 1277–1285.

Creissen, G.P., Firmin, J., Fryer, M., Kular, B., Leyland, N., Reynolds,

H., Pastori, G., Wellburn, F., Baker, N., Wellburn, A., and

Mullineaux, P. (1999). Elevated glutathione biosynthetic capacity in

the chloroplasts of transgenic tobacco plants paradoxically causes

increased oxidative stress. Plant Cell 11: 1277–1292.

Danon, A., Miersch, O., Felix, G., Camp, R.G., and Apel, K. (2005).

Concurrent activation of cell-death regulating signaling pathways by

singlet oxygen in Arabidposis thaliana. Plant J. 41: 68–80.

Davies, W.J., Wilkinson, S., and Loveys, B. (2002). Stomatal control by

chemical signaling and the exploitation of this mechanism to increase

water use efficiency in agriculture. New Phytol. 153: 449–460.


Davletova, S., Rizhsky, L., Liang, H.J., Zhong, S.Q., Oliver, D.J.,

Coutu, J., Shulaev, V., Schlauch, K., and Mittler, R. (2005). Cyto-

solic ASCORBATE PEROXIDASE1 is a central component of the

reactive oxygen gene network of Arabidopsis. Plant Cell 17: 268–281.

de Torres-Zabala, M., Truman, W., Bennett, M.H., Lafforgue, G.,

Mansfield, J.W., Egea, P.R., Bogre, L., and Grant, M. (2007).

Pseudomonas syringae pv. tomato hijacks the Arabidopsis abscisic

acid signaling pathway to cause disease. EMBO J. 26: 1434–1443.

Douce, R., and Neuberger, M. (1999). Biochemical dissection of

photorespiration. Curr. Opin. Plant Biol. 2: 214–222.

Driever, S.M., Fryer, M.J., Mullineaux, P.M., and Baker, N.R. (2008).

Imaging of reactive oxygen species in vivo. In Plant Signal Transduc-

tion, Vol. 149, Methods and Protocols, T. Pfannschmidt, ed (New

York: Humana Press), pp. 109–116.

Duysens, L.N.M. (1972). O-(3,4-dichlorophenyl)-1,1-dimethylurea

(DCMU) inhibition of system II and light-induced regulatory changes

in energy-transfer efficiency. Biophys. J. 12: 858–863.

Eisen, M.B., Spellman, P.T., Brown, P.O., and Botstein, D. (1998).

Cluster analysis and display of genome-wide expression patterns.

Proc. Natl. Acad. Sci. USA 95: 14863–14868.

Endo, A., et al. (2008). Drought induction of Arabidopsis 9-cis-

epoxycarotenoid dioxygenase occurs in vascular parenchyma cells.

Plant Physiol. 147: 1984–1993.

Evert, R.F. (2006). Esau’s Plant Anatomy: Meristems, Cells, and Tissues

of the Plant Body: Their Structure, Function and Development, 3rd ed.

(Hoboken, NJ: John Wiley & Sons).

Fryer, M.J., Ball, L., Oxborough, K., Karpinski, S., Mullineaux, P.M.,

and Baker, N.R. (2003). Control of Ascorbate Peroxidase 2 expres-

sion by hydrogen peroxide and leaf water status during excess light

stress reveals a functional organisation of Arabidopsis leaves. Plant J.

33: 691–705.

Fryer, M.J., Oxborough, K., Mullineaux, P.M., and Baker, N.R. (2002).

Imaging of photo-oxidative stress responses in leaves. J. Exp. Bot.

53: 1249–1254.

Gentleman, R.C., et al. (2004). Bioconductor: Open software development

for computational biology and bioinformatics. Genome Biol. 5: R80.

Goda, H., et al. (2008). The AtGenExpress hormone and chemical

treatment data set: Experimental design, data evaluation, model data

analysis and data access. Plant J. 55: 526–542.

Gonzalez-Guzman, M., Apostolova, N., Belles, J.M., Barrero, J.M.,

Piqueras, P., Ponce, M.R., Micol, J.L., Serrano, R., and Rodriguez,

P.L. (2002). The short-chain alcohol dehydrogenase ABA2 catalyzes

the conversion of xanthoxin to abscisic aldehyde. Plant Cell 14: 1833–

1846.

Hibberd, J.M., and Quick, W.P. (2002). Characteristics of C4 photo-

synthesis in stems and petioles of C3 flowering plants. Nature 415:

451–454.

Horton, P., Ruban, A.V., and Walters, R.G. (1996). Regulation of light

harvesting in green plants. Annu. Rev. Plant Physiol. Plant Mol. Biol.

47: 655–684.

Hu, X.L., Jiang, M.Y., Zhang, A.Y., and Lu, J. (2005). Abscisic acid-

induced apoplastic H2O2 accumulation up-regulates the activities of

chloroplastic and cytosolic antioxidant enzymes in maize leaves.

Planta 223: 57–68.

Iuchi, S., Kobayashi, M., Taji, T., Naramoto, M., Seki, M., Kato, T.,

Tabata, S., Kakubari, Y., Yamaguchi-Shinozaki, K., and Shinozaki,

K. (2001). Regulation of drought tolerance by gene manipulation of

9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid

biosynthesis in Arabidopsis. Plant J. 27: 325–333.

Jenkins, G.I., Baker, N.R., Bradbury, M., and Woolhouse, H.W.

(1981). Photosynthetic electron transport during senescence of the

primary leaves of Phascolus vulgaris L.: III. Kinetics of chlorophyll

fluorescence emission from intact leaves. J. Exp. Bot. 32: 999–1008.

Jiang, M., and Zhang, J. (2003). Cross-talk between calcium and

reactive oxygen species originated from NADPH oxidase in abscisic

acid-induced antioxidant defence in leaves of maize seedlings. Plant

Cell Environ. 26: 929–939.

Johnston, C.A., Taylor, J.P., Gao, Y., Kimple, A.J., Grigston, J.C.,

Chen, J.G., Siderovski, D.P., Jones, A.M., and Willard, F.S. (2007).

GTPase acceleration as the rate-limiting step in Arabidopsis G

protein-coupled sugar signaling. Proc. Natl. Acad. Sci. USA 104:

17317–17322.

Jones, A.M., Ecker, J.R., and Chen, J.G. (2003). A re-evaluation of the

role of the heterotrimeric G protein in coupling light responses in

Arabidopsis. Plant Physiol. 131: 1623–1627.

Joo, J.H., Wang, S.Y., Chen, J.G., Jones, A.M., and Fedoroff, N.V.

(2005). Different signaling and cell death roles of heterotrimeric G

protein alpha and beta subunits in the Arabidopsis oxidative stress

response to ozone. Plant Cell 17: 957–970.

Jubany-Mari, T., Munne-Bosch, S., Lopez-Carbonell, M., and

Alegre, L. (2009). Hydrogen peroxide is involved in the acclimation

of the Mediterranean shrub, Cistus albidus L., to summer drought.

J. Exp. Bot. 60: 107–120.

Karpinski, S., Escobar, C., Karpinska, B., Creissen, G., and Mullineaux,

P.M. (1997). Photosynthetic electron transport regulates the expression

of cytosolic ascorbate peroxidase genes in Arabidopsis during excess

light stress. Plant Cell 9: 627–640.

Karpinski, S., Reynolds, H., Karpinska, B., Wingsle, G., Creissen, G.,

and Mullineaux, P. (1999). Systemic signaling and acclimation in

response to excess excitation energy in Arabidopsis. Science 284:

654–657.

Kim, Y.X., and Steudle, E. (2009). Gating of aquaporins by light and

reactive oxygen species in leaf parenchyma cells of the midrib of Zea

mays. J. Exp. Bot. 60: 547–556.

Kimura, M., Yamamoto, Y.Y., Seki, M., Sakurai, T., Sato, M., Abe, T.,

Yoshida, S., Manabe, K., Shinozaki, K., and Matsui, M. (2003).

Identification of Arabidopsis genes regulated by high light-stress

using cDNA microarray. Photochem. Photobiol. 77: 226–233.

Kinsman, E.A., and Pyke, K.A. (1998). Bundle sheath cells and cell-

specific plastid development in Arabidopsis leaves. Development 125:

1815–1822.

Kitahata, N., Han, S.Y., Noji, N., Saito, T., Kobayashi, M., Nakano, T.,

Kuchitsu, K., Shinozaki, K., Yoshida, S., Matsumoto, S., Tsujimoto,

M., and Asami, T. (2006). A 9-cis-epoxycarotenoid dioxygenase

inhibitor for use in the elucidation of abscisic acid action mechanisms.

Bioorg. Med. Chem. 14: 5555–5561.

Kleine, T., Kindgren, P., Benedict, C., Hendrickson, L., and Strand,

A. (2007). Genome-wide gene expression analysis reveals a critical

role for CRYPTOCHROME1 in the response of Arabidopsis to high

irradiance. Plant Physiol. 144: 1391–1406.

Kubista, M., Andrade, J.M., Bengtsson, M., Forootan, A., Jonak, J.,

Lind, K., Sindelka, R., Sjoback, R., Sjogreen, B., Strombom, L.,

Stahlberg, A., and Zoric, N. (2006). The real-time polymerase chain

reaction. Mol. Aspects Med. 27: 95–125.

Koussevitzky, S., Nott, A., Mockler, T.C., Hong, F., Sachetto-

Martins, G., Surpin, M., Lim, I.J., Mittler, R., and Chory, J. (2007).

Signals from chloroplasts converge to regulate nuclear gene expres-

sion. Science 316: 715–719.

Kraft, M., Kuglitsch, R., Kwiatkowski, J., Frank, M., and Grossmann,

K. (2007). Indole-3-acetic acid and auxin herbicides up-regulate 9-cis-

epoxycarotenoid dioxygenase gene expression and abscisic acid

accumulation in cleavers (Galium aparine): Interaction with ethylene.

J. Exp. Bot. 58: 1497–1503.

Krieger-Liszkay, A. (2005). Singlet oxygen production in photosynthe-

sis. J. Exp. Bot. 56: 337–346.

Kwak, J.M., Mori, I.C., Pei, Z.M., Leonhardt, N., Torres, M.A., Dangl,


J.L., Bloom, R.E., Bodde, S., Jones, J.D.G., and Schroeder, J.I.

(2003). NADPH oxidase AtrbohD and AtrbohF genes function in ROS-

dependent ABA signaling in Arabidopsis. EMBO J. 22: 2623–2633.

Leegood, R. (2008). Roles of the bundle sheath cells in leaves on C3

plants. J. Exp. Bot. 59: 1663–1673.

Levin, M., Lemcoff, J.H., Cohen, S., and Kapulnik, Y. (2007). Low air

humidity increases leaf-specific hydraulic conductance of Arabidopsis

thaliana (L.) Heynh (Brassicaceae). J. Exp. Bot. 58: 3711–3718.

Li, S., Assmann, S.M., and Albert, R. (2006). Predicting essential

components of signal transduction networks: A dynamic model of

guard cell abscisic acid signaling. PLoS Biol. 4: 1732–1748.

Liu, Y., Ren, D., Pike, S., Pallardy, S., Gassmann, W., and Zhang, S.

(2007). Chloroplast-generated reactive oxygen species are involved in

hypersensitive response-like cell death mediated by a mitogen-acti-

vated protein kinase cascade. Plant J. 51: 941–954.

Long, S.P., Humphries, S., and Falkowski, P.G. (1994). Photoinhibition

of photosynthesis in nature. Annu. Rev. Plant Physiol. Plant Mol. Biol.

45: 633–662.

Lottaz, C., Yang, X., Scheid, S., and Spang, R. (2006). OrderedList – A

bioconductor package for detecting similarity in ordered gene lists.

Bioinformatics 22: 2315–2316.

Mittler, R., Vanderauwera, S., Gollery, M., and Van Breusegem, F.

(2004). Reactive oxygen gene network of plants. Trends Plant Sci. 9:

490–498.

Money, N.P. (1989). Osmatic pressure of aqueous polyethylene glycols.

Relationship between molecular weight and vapor pressure deficit.

Plant Physiol. 91: 766–769.

Morison, J.I.L., Gallouet, E., Lawson, T., Cornic, G., Herbin, R., and

Baker, N.R. (2005). Lateral diffusion of CO2 in leaves is not sufficient

to support photosynthesis. Plant Physiol. 139: 254–266.

Muhlenbock, P., Szechynska-Hebda, M., Plaszczyca, M., Baudo, M.,

Mateo, A., Mullineaux, P.M., Parker, J.E., Karpinska, B., and

Karpinski, S. (2008). Chloroplast signaling and LESION SIMULATING

DISEASE1 regulate crosstalk between light acclimation and immunity

in Arabidopsis. Plant Cell 20: 2339–2356.

Muller, P., Li, X.-P., and Niyogi, K.K. (2001). Non-photochemical

quenching. A response to excess light energy. Plant Physiol. 125:

1558–1566.

Mullineaux, P.M., Karpinski, S., and Baker, N.R. (2006). Spatial

dependence for hydrogen peroxide-directed signaling in light-

stressed plants. Plant Physiol. 141: 346–350.

Murata, Y., Pei, Z.M., Mori, I.C., and Schroeder, J. (2001). Abscisic

acid activation of plasma membrane Ca2+ channels in guard cells

requires cytosolic NAD(P)H and is differentially disrupted upstream

and downstream of reactive oxygen species production in abi1-1 and

abi2-1 protein phosphatase 2C mutants. Plant Cell 13: 2513–2523.

Mustilli, A.C., Merlot, S., Vavasseur, A., Fenzi, F., and Giraudat, J.

(2002). Arabidopsis OST1 protein kinase mediates the regulation of

stomatal aperture by abscisic acid and acts upstream of reactive

oxygen species production. Plant Cell 14: 3089–3099.

Nambara, E., and Marion-Poll, A. (2005). Abscisic acid biosynthesis

and catabolism. Annu. Rev. Plant Biol. 56: 165–185.

North, H.M., De Almeida, A., Boutin, J.P., Frey, A., To, A., Botran, L.,

Sotta, B., and Marion-Poll, A. (2007). The Arabidopsis ABA-deficient

mutant aba4 demonstrates that the major route for stress-induced

ABA accumulation is via neoxanthin isomers. Plant J. 50: 810–824.

Nott, A., Jung, H.-S., Koussevitsky, S., and Chory, J. (2004). Plastid-

to-nucleus retrograde signaling. Annu. Rev. Plant Biol. 57: 739–759.

Okamoto, M., Tanaka, Y., Abrams, S.R., Kamiya, Y., Seki, M., and

Nambara, E. (2009). High humidity inducedabscisic acid 8’-hydroxylase

in stomata and vasculature to regulate local and systemic abscisic acid

responses in Arabidopsis. Plant Physiol. 149: 825–834.

Ort, D.R., and Becker, N.R. (2002). A photoprotective role for O2 as an

alternative electron sink in photosynthesis? Curr. Opin. Plant Biol. 5:

193–198.

Pandey, S., Chen, J.G., Jones, A.M., and Assmann, S.M. (2006).

G-protein complex mutants are hypersensitive to abscisic acid reg-

ulation of germination and post-germination development. Plant

Physiol. 141: 243–256.

Pei, Z.-M., Murata, Y., Benning, G., Thomine, S., Klusener, B., Allen,

G.J., Grill, E., and Schroeder, J. (2000). Calcium channels activated

by hydrogen peroxide mediate abscisic acid signaling in guard cells.

Nature 406: 731–734.

Pfannschmidt, T., Brautigem, K., Wagner, R., Dietzel, L., Schroter,

Y., Steiner, S., and Nykytenko, A. (2009). Potential regulation of gene

expression in photosynthetic cells by redox and energy state: Ap-

proaches towards better understanding. Ann. Bot. (Lond.) 103:

599–607.

Pogson, B.J., Woo, N.S., Forster, B., and Small, I.D. (2008). Plastid

signaling to the nucleus and beyond. Trends Plant Sci. 13: 602–609.

Pryzbyla, D., Gobel, C., Imboden, A., Hamberg, M., Feussner, I., and

Apel, K. (2008). Enzymatic, but not non-enzymatic, 1O2-mediated

peroxidation of polyunsaturated fatty acids forms part of the EXE-

CUTER1-dependent stress response program in the flu mutant of

Arabidopsis thaliana. Plant J. 54: 236–248.

Qin, X., and Zeevaart, J.A.D. (1999). The 9-cis-epoxycarotenoid cleav-

age reaction is the key regulatory step of abscisic acid biosynthesis in

water-stressed bean. Proc. Natl. Acad. Sci. USA 96: 15354–15361.

Quarrie, S.A., Whitford, P.N., Appleford, N.E.J., Wang, T.L., Cook,

S.K., Henson, I.E., and Loveys, B.R. (1988). A monoclonal antibody

to (S)-abscisic acid: its characterization and use in a radioimmuno-

assay for measuring abscisic acid in crude extracts of cereal and lupin

leaves. Planta 173: 330–339.

Rey, P., Becuwe, N., Barrault, M.-B., Rumeau, D., Havaux, M.,

Biteau, B., and Toledano, M.B. (2007). The Arabidopsis thaliana

sulfiredoxin is a plastidial cysteine-sulfinic acid reductase involved in

the photooxidative stress response. Plant J. 49: 505–514.

Rossel, J.B., Walter, P.B., Hendrickson, L., Chow, W.S., Poole, A.,

Mullineaux, P.M., and Pogson, B.J. (2006). A mutation affecting

ASCORBATE PEROXIDASE 2 gene expression reveals a link between

responses to high light and drought tolerance. Plant Cell Environ. 29:

269–281.

Rossel, J.B., Wilson, I.W., and Pogson, B.J. (2002). Global changes in

gene expression in response to high light in Arabidopsis. Plant

Physiol. 130: 1109–1120.

Rossel, J.B., Wilson, P.B., Hussain, D., Woo, N., Gordon, M.J.,

Mewett, O.P., Howell, K.A., Whelan, J., Kazan, K., and Pogson,

B.J. (2007). Systemic and intracellular responses to photo-oxidative

stress in Arabidopsis. Plant Cell 19: 4091–4110.

Ruggiero, B., Koiwa, H., Manabe, Y., Quist, T.M., Inan, G., Saccardo,

F., Joly, R.J., Hasegawa, P.M., Bressan, R.A., and Maggio, A.

(2004). Uncoupling the effects of abscisic acid on plant growth and

water relations. Analysis of sto1/nced3, an abscisic acid-deficient but

salt stress-tolerant mutant in Arabidopsis. Plant Physiol. 136: 3134–

3147.

Russell, A.W., Critchley, C., Robinson, S.A., Franklin, L.A., Seaton,

G.C.R., Chow, W.S., Anderson, J.M., and Osmond, C.B. (1995).

Photosystem II regulation and dynamics of the chloroplast D1 protein

in Arabidopsis leaves during photosynthesis and photoinhibition. Plant

Physiol. 107: 943–952.

Sack, L., and Holbrook, N.M. (2006). Leaf hydraulics. Annu. Rev. Plant

Biol. 57: 361–381.

Sellin, A., Ounapuu, E., and Kupper, P. (2008). Effects of light intensity

and duration on leaf hydraulic conductance and distribution of resis-

tance in shoots of silver birch (Betula pendula). Physiol. Plant. 134:

412–420.


Snyrychova, I., Ayaydin, F., and Hideg, E. (2008). Detecting hydrogen

peroxide in leaves in vivo - A comparison of methods. Physiol. Plant.

135: 1–18.

Solyu, S., Brown, I., and Mansfield, J.A. (2005). Cellular reactions in

Arabidopsis following challenge by strains of Pseudomonas syringae:

From basal resistance to compatibility. Physiol. Mol. Plant Pathol. 66:

232–243.

Suharsono, U., Fujisawa, Y., Kawasaki, T., Iwasaki, Y., Satoh, H.,

and Shimamoto, K. (2002). The heterotrimeric G protein alpha

subunit acts upstream of the small GTPase Rac in disease resistance

of rice. Proc. Natl. Acad. Sci. USA 99: 13307–13312.

Tan, B.C., Joseph, L.M., Deng, W.T., Liu, L., Li, Q.B., Cline, K., and

McCarty, D.R. (2003). Molecular characterization of the Arabidopsis

9-cis epoxycarotenoid dioxygenase gene family. Plant J. 35: 44–56.

Temple, B.R.S., and Jones, A.M. (2007). The plant heterotrimeric

G-protein complex. Annu. Rev. Plant Biol. 58: 249–266.

Thordal-Christiansen, H., Zhang, Z., Wei, Y., and Collinge, D.B.

(1997). Subcellular localization of H2O2 in plants. H2O2 accumulation

in papillae and hypersensitive response during the barley-powdery

mildew interaction. Plant J. 11: 1187–1194.

Triantaphylides, C., Krischke, M., Hoeberichts, F.A., Ksas, B.,

Gresser, G., Havaux, M., Van Breusegem, F., and Mueller, M.J.

(2008). Singlet oxygen is the major reactive oxygen species involved

in photooxidative damage in plants. Plant Physiol. 148: 960–968.

Ullah, H., Chen, J.G., Young, J., Im, K.-H., Sussman, M.R., and

Jones, A.M. (2001). Modulation of cell proliferation by heterotrimeric

G-protein in Arabidopsis. Science 292: 2066–2069.

Urano, K., Maruyama, K., Ogata, Y., Morishita, Y., Takeda, M.,

Sakurai, N., Suzuki, H., Saito, K., Shibata, D., Kobayashi, M.,

Yamaguchi-Shinozaki, K., and Shinozaki, K. (2009). Characteriza-

tion of the ABA-regulated global responses to dehydration in Arabi-

dopsis by metabolomics. Plant J. 57: 1065–1078.

Van Breusegem, F., Bailey-Serres, J., and Mittler, R. (2008). Unrav-

eling the tapestry of networks involving reactive oxygen species in

plants. Plant Physiol. 147: 978–984.

Volkov, R.A., Panchuk, I.I., Mullineaux, P.M., and Schoffl, F. (2006).

Heat stress-induced H2O2 is required for effective expression of heat

shock genes in Arabidopsis. Plant Mol. Biol. 61: 733–746.

Wu, Z., Irizarry, R.A., Gentleman, R., Martinez-Murillo, F., and

Spencer, F. (2004). A model-based background adjustment for

oligonucleotide expression arrays. J. Am. Stat. Assoc. 99: 909–917.

Xie, X.D., Wang, Y.B., Williamson, L., Holroyd, G.H., Tagliavia, C.,

Murchie, E., Theobald, J., Knight, M.R., Davies, W.J., Leyser, H.M.

O., and Hetherington, A. (2006). The identification of genes involved

in the stomatal response to reduced atmosphere relative humidity.

Curr. Biol. 16: 882–887.

Yabuta, Y., Maruta, T., Yoshimura, K., Ishikawa, T., and Shigeoka, S.

(2004). Two distinct redox signaling pathways for cytosolic APX

induction under photooxidative stress. Plant Cell Physiol. 45: 1586–

1594.

Yoshida, R., Umezawa, T., Mizoguchi, T., Takahashi, S., Takahashi,

F., and Shinozaki, K. (2006). The regulatory domain of SKRE2/OST1/

SnRK2.6 interacts with ABI1 and integrates abscisic acid (ABA) and

osmotic stress signals controlling stomatal closure in Arabidopsis.

J. Biol. Chem. 281: 5310–5318.

Zhou, M.J., Diwu, Z.J., Panchuk, V.N., and Haugland, R.P. (1997). A

stable nonfluorescent derivative of resorufin for the fluorometric

determination of trace hydrogen peroxide: Applications in detecting

the activity of phagocyte NADPH oxidase and other oxidases. Anal.

Biochem. 253: 162–168.


DOI 10.1105/tpc.108.061507; originally published online July 28, 2009;Plant Cell

Smirnoff, Tadao Asami, William J. Davies, Alan M. Jones, Neil R. Baker and Philip M. MullineauxGregorio Galvez-Valdivieso, Michael J. Fryer, Tracy Lawson, Katie Slattery, William Truman, Nicholas

Sheath Cells Involves ABA Signaling between Vascular and BundleArabidopsisThe High Light Response in

This information is current as of August 3, 2020

Supplemental Data /content/suppl/2009/07/10/tpc.108.061507.DC1.html

Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

eTOCs http://www.plantcell.org/cgi/alerts/ctmain

Sign up for eTOCs at:

CiteTrack Alerts http://www.plantcell.org/cgi/alerts/ctmain

Sign up for CiteTrack Alerts at:

Subscription Information http://www.aspb.org/publications/subscriptions.cfm

is available at:Plant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY © American Society of Plant Biologists

https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X

http://www.plantcell.org/cgi/alerts/ctmain

http://www.plantcell.org/cgi/alerts/ctmain

http://www.aspb.org/publications/subscriptions.cfm

the high light response in arabidopsis involves aba ... · the high light response in arabidopsis...

Documents