Retrograde Signaling by the PlastidialMetabolite MEcPP Regulates Expressionof Nuclear Stress-Response GenesYanmei Xiao,1 Tatyana Savchenko,1 Edward E.K. Baidoo,4,5 Wassim E. Chehab,6 Daniel M. Hayden,7 Vladimir Tolstikov,2

Jason A. Corwin,3 Daniel J. Kliebenstein,3 Jay D. Keasling,4,8 and Katayoon Dehesh1,*1Department of Plant Biology2Genome and Biomedical Sciences Facilities3Department of Plant SciencesUniversity of California Davis, Davis, CA 95616, USA4Joint BioEnergy Institute, Emeryville, CA 94608, USA5Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA6Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77005, USA7Complete Genomics, Mountain View, CA 94043, USA8Department of Chemical and Biomolecular Engineering, Department of Bioengineering, University of California, Berkeley, Berkeley,

CA 94720, USA*Correspondence: kdehesh@ucdavis.edu

DOI 10.1016/j.cell.2012.04.038

SUMMARY

Plastid-derived signals are known to coordinateexpression of nuclear genes encoding plastid-local-ized proteins in a process termed retrograde sig-naling. To date, the identity of retrograde-signalingmolecules has remained elusive. Here, we showthat methylerythritol cyclodiphosphate (MEcPP), aprecursor of isoprenoids produced by the plastidialmethylerythritol phosphate (MEP) pathway, elicitsthe expression of selected stress-responsivenuclear-encoded plastidial proteins. Genetic andpharmacological manipulations of the individualMEP pathway metabolite levels demonstrate thehigh specificity of MEcPP as an inducer of these tar-geted stress-responsive genes. We further demon-strate that abiotic stresses elevate MEcPP levels,eliciting the expression of the aforementioned genes.We propose that the MEP pathway, in addition toproducing isoprenoids, functions as a stress sensorand a coordinator of expression of targeted stress-responsive nuclear genes via modulation of thelevels of MEcPP, a specific and critical retrograde-signaling metabolite.

INTRODUCTION

Plastids, in addition to their role as biochemical factories synthe-

sizing a variety of fundamental compounds, also function as

sensors to coordinate regulatory mechanisms that are essential

for plant responses to environmental and developmental cues.

Plastids originated by endosymbiosis of free-living cyanobacte-

ria by eukaryotic cells but contain only a remnant of the ancestral

eubacterial genome, with most genes having been transferred to

the nucleus, where they acquired sequence motifs necessary

both for expression and targeting to their final destinations

(Kleine et al., 2009). Of �3,000 plastid proteins, <5% are en-

coded and synthesized by the plastids themselves (Abdallah

et al., 2000). This arrangement therefore requires coordination

of expression of nuclear genes encoding plastid-localized

proteins with plastid development and function. This coordina-

tion is achieved by bidirectional signaling cascades that control

information flow from nucleus to plastids (anterograde signaling)

and by signaling factors from plastids to nucleus (retrograde

signaling) (Jung and Chory, 2010).

Retrograde signaling was first inferred from two barley

mutants whose defects in plastid functions result in downregula-

tion of nuclear-encoded plastid proteins (Bradbeer et al., 1979).

Since this finding, the function of retrograde signaling in plastid

development through the coordination of chlorophyll biosyn-

thesis with the expression of nuclear genes encoding plastid-

localized chlorophyll-binding proteins has been intensively

examined. The genomes uncoupled (gun) are the best studied

retrograde signaling pathway mutants, in which the expression

of the gene-encoding light-harvesting chlorophyll a/b-binding

protein (LHCB) is uncoupled from the functional state of chloro-

plast (Nott et al., 2006; Susek et al., 1993). Thus far, four different

putative sources of retrograde signals have been implicated,

including components of tetrapyrrole biosynthetic pathway

intermediates (Ankele et al., 2007; Larkin et al., 2003; Mochizuki

et al., 2001; Strand et al., 2003; Susek et al., 1993), protein

synthesis (Koussevitzky et al., 2007), the redox state of the

plastids (Bonardi et al., 2005; Heiber et al., 2007; Pesaresi

et al., 2007; Piippo et al., 2006), and the levels of reactive oxygen

species in plastids (Laloi et al., 2006; Lee et al., 2007; op den

Camp et al., 2003; Wagner et al., 2004). None of these proposed

candidates to date have been unequivocally verified (Dynowski

et al., 2008; Mochizuki et al., 2008; Moulin et al., 2008; Pogson

Cell 149, 1525–1535, June 22, 2012 ª2012 Elsevier Inc. 1525

Figure 1. Isolation and Map-Based Cloning of ceh1

(A) Bright-field (top) and dark-field (bottom) image analysis of representative parent (P) plants expressing transcriptionalHPL::LUC fusion before (UW) and 90min

after wounding (W) exhibit wound-inducible transcriptional regulation of HPL. The color-coded bar displays the intensity of LUC activity.

(B) Dark field images of P, ceh1, and complemented (CP) lines exhibit high and constitutive LUC activity in ceh1 and complementation of this phenotype in

CP lines.

(C) Intensity of LUC bioluminescence in independent P, ceh1, and CP lines presented in a log2 scale, quantified using Andor Solis image analysis software.

Data are mean ± SEM; n = 20.

(D) Total RNA was extracted from 3-week-old P, ceh1, and CP lines and was subjected to RT-qPCR analysis. AtHPL transcripts were first normalized to the

At4g34270 and At4g26410 measured in the same samples and to the transcript levels in the P lines. Data are mean of fold difference ± SEM; n = 6.

(E) Map-based cloning placed ceh1 on chromosome 5 between the markers CER457311 and CER457380, which expands a 65.5 kb region on BACs MUF9 and

MUP24. The interval contains 17 genes illustrated as arrows. Sequence analysis of the interval genes in conjunctionwith complementation assays (B–D) identified

CEH1 as HDS gene.

(F) Schematic illustration of the exon-intron structure of HDSwith the solid boxes representing exons. The mutations in the ceh1 and its mutant alleles are shown

in red, and the actual nucleotide and amino acid changes are displayed underneath.

(G) The MEP pathway for isoprenoids biosynthesis in plastids. Pathway enzymes are in bold, and the substrates are displayed in blue. Available Arabidopsis

mutant lines in individual genes are in italics.

1526 Cell 149, 1525–1535, June 22, 2012 ª2012 Elsevier Inc.

et al., 2008).Most recently however, studies of a gain-of-function

mutant of Arabidopsis (gun6-1D) has identified heme, specifi-

cally produced by the plastid ferrochelatase (FC1), as the most

promising retrograde signal candidate potentially used to coor-

dinate photosynthesis-associated nuclear gene (PhANG)

expression with chloroplast development (Beale, 2011; Wood-

son et al., 2011). In addition, a newly identified retrograde

mechanism through the SAL1-PAP pathway, which relies on

chloroplastic SAL1 enzyme-regulating PAP (30-phosphoadeno-sine 50-phosphate) levels, is reported (Estavillo et al., 2011).

There is also evidence for retrograde signaling in plant adap-

tations to stress. The free endosymbiont progenitors of plastids

possessed diverse adaptive stress-responsive networks. There-

fore, plastids, in addition to acquisition of new roles, conceivably

retained some of these pre-endosymbiosis stress-related

functions. If so, plastid-to-nucleus retrograde signaling is

likely to have emerged as essential for plant adaptive respon-

siveness to the environment in order to coordinate expression

of stress-responsive nuclear genes—encoding plastid-localized

proteins—with the prevailing conditions.

The methylerythritol phosphate (MEP) pathway genes,

responsible for plastidial isoprenoid production, are among

the group of genes inherited from endosymbiotic ancestors

and subsequently relocated to the nuclear genome (Bouvier

et al., 2005; Phillips et al., 2008). This essential pathway is

present in most eubacteria and in apicomplexa that biosynthe-

size isoprenoids in their nongreen plastid-type apicoplast

(Jomaa et al., 1999; Rohdich et al., 2004). In plants, the MEP

pathway has remained essential despite the presence of an

independent parallel cytosolic mevalonate (MVA) pathway that

also generates the universal five-carbon precursors of isopre-

noids, isopentenyl diphosphate (IPP), and dimethylallyl diphos-

phate (DMAPP). A deficiency of plastid isoprenoids cannot

be efficiently compensated by the cytosolic mevalonate pre-

cursors (Bouvier et al., 2005; Gutierrez-Nava et al., 2004; Lange

et al., 1998; Rodrıguez-Concepcion and Boronat, 2002),

indicating that these two biosynthetic pathways are largely

independent.

We report here results from a genetic screen designed to

identify genes involved in the regulation of hydroperoxide lyase

(HPL), a stress-inducible nuclear gene encoding a plastid-

localized protein in the oxylipin pathway. A mutant ceh1

(constitutively expressing HPL) was identified that highly and

constitutively expresses HPL transcripts. CEH1 encodes 1-

hydroxy-2-methyl-2-(E)-butenyl4-diphosphate synthase, which

catalyzes the conversion of methylerythritol cyclodiphosphate

(MEcPP) to hydroxymethylbutenyl diphosphate (HMBPP), the

bottleneck step in the MEP pathway (Ostrovsky et al., 1998;

Rodrıguez-Concepcion, 2006). We present experimental

evidence that implicate the CEH1 substrate MEcPP as a retro-

grade-signaling metabolite that induces expression of selected

stress genes.

G3P, glyceraldehyde 3-phosphate; DXP, deoxyxylulose 5-phosphate; MEP, m

MEP, CDP-ME phosphate; MEcPP, methylerythritol cyclodiphosphate; HBMPP

dimethylallyl diphosphate; DXS, DXP synthase; DXR, DXP reductoisomerase; CM

HMBPP synthase; HDR, HMBPP reductase; and IDI, IPP isomerase.

RESULTS

The ceh1 Mutant Phenotype Is Caused by a Mutationin HDS, a MEP Pathway GeneHPL is a stress-inducible nuclear gene encoding a plastidial

enzyme in the HPL branch of the oxylipin pathway. The HPL is

responsible for production of a range of cognate metabolites,

predominantly C6 aldehydes and their derivatives, implicated

as intra- and interplant stress-responsive signaling molecules

(Chehab et al., 2006, 2008). To dissect the upstream signal

transduction machinery responsible for stress induction of

HPL, we devised a genetic screen to identify mutations that

result in constitutive expression of HPL. As the basis of the

screen, transgenic plants were established that contain the

HPL promoter region fused to the firefly luciferase (PHPL::LUC)

that, for simplicity herein, are designated parent (P) plants.

Wounded P plants display a stronger LUC bioluminescence

intensity than unwounded controls (Figure 1A), confirming the

previously reported stress regulation of HPL (Chehab et al.,

2006, 2008). Seeds of the homozygous P plants were ethylme-

thylsulfonate (EMS) mutagenized and used to establish M2

mutants for screening (Experimental Procedures). More than

12,000 independent M2mutants were screened, and 20 putative

mutants with elevated LUC expression were identified. Among

these constitutively LUC-expressing mutants, one line with

a miniature stature displayed the strongest bioluminescence

intensity. On the basis of the strong bioluminescence, this

mutant was selected for detailed analysis and named ceh1 (Fig-

ure 1B).Ceh1 displayed a�100-fold higher LUC activity than the

P control (Figure 1C). Correspondingly, qRT-PCR analysis

showed�17-fold higher levels of the endogenousHPL transcript

in ceh1 as compared to controls (Figure 1D).

To test the genetics of the ceh1mutation, theM3 generation of

this mutant was backcrossed to the P line. F1 plants exhibited

levels of bioluminescence similar to P plants, whereas 25% of

the segregating F2 lines displayed the ceh1 mutant biolumines-

cence phenotype, indicating that a single recessive mutation

confers the constitutive and elevated expression of HPL in

ceh1. Next, ceh1 was mapped to a 65.5 kb region between the

markers CER457311 and CER457380 on the BACs MUF9 and

MUP24 on chromosome 5 (Figure 1E). Sequencing of the 17

genes in the interval revealed a single missense mutation, result-

ing in a leucine-to-phenylalanine substitution within the 19th

exon of the At5g60600 gene (Figure 1F). A 6238 bp fragment

of the genomic region containing a wild-type At5g60600 com-

plemented all ceh1 phenotypes, including notable reduction

of the LUC activity, normal expression levels of HPL, and the

reversion of the miniature stature of the ceh1 plants to the

wild-type size (Figures 1B–1D). This data confirms that

At5g60600 defines CEH1, a single copy gene that encodes

a plastidial nonmevalonateMEP pathway enzyme, HDS, respon-

sible for the conversion of MEcPP to HMBPP (Figure 1G).

ethylerythritol phosphate; CDP-ME, diphosphocytidyl-methylerythritol; CDP-

, hydroxymethylbutenyl diphosphate; IPP, isopentenyl diphosphate; DMAPP,

S, CDP-ME synthase; CMK, CDP-ME kinase; MDS, MEcPP synthase; HDS,

Cell 149, 1525–1535, June 22, 2012 ª2012 Elsevier Inc. 1527

Figure 2. ceh1 Contains Elevated Levels of

SA Biosynthetic Gene and Metabolite and

Intact Levels of JA Biosynthetic Gene and

Metabolite

(A) Free SA levels are �40-fold higher in the ceh1

as compared to the P and CP lines. The gray line

indicates a change of scale on the y axis. Data are

the mean ±SD; n = 9.

(B) Expression level of ICS1 is enhanced in ceh1.

Total RNA was extracted from P, ceh1, and CP

lines and subjected to RT-qPCR analysis. AtICS1

transcripts were normalized as described in Fig-

ure 1D. Data are mean fold difference ±SEM

of three biological, with three technical replicates

for each.

(C) Enhanced resistance of ceh1 to Pst. Bacterial

growth in the P, ceh1, and three independent

CP lines inoculated with Pst was recoded

2 days postinoculation by bioluminescence, as

photon counts per second (CPS). Data are

mean ±SD; n = 20.

(D) Similar levels of JA metabolite in the P, ceh1,

and CP lines. The p value shows insignificant

differences between the P and ceh1lines. Data are

the mean ±SD; n = 9.

(E) Similar levels of AOS transcripts in the P, ceh1,

and CP lines. Total RNA was extracted from the P,

ceh1, and CP lines and subjected to RT-qPCR

analysis. The AOS transcripts were normalized as described in Figure 1D. The p value shows insignificant differences between the lines. Data are mean fold

difference ±SEM of three biological with three technical replicates for each.

(F) Lesion diameter measurements 2 days post spot inoculation with conidia of Botrytis isolate B05.10 show enhanced susceptibly of the ceh1 as compared to P

and CP lines. Data are mean ±SD; n = 20.

Furthermore, F1 progeny generated from a cross of ceh1mutant

to SALK_017595/clb4-2 heterozygous plants containing a

T-DNA insertion in At5g60600 (Figure 1F) failed to complement

the ceh1 mutation, further verifying that ceh1 is a mutant allele

of At5g60600.

The ceh1 Mutant Contains High Salicylic Acid Levelsand Is Resistant to Infection by Biotrophic PathogensPrevious evidence implicates CEH1 in specific stress responses,

as a previously identified ceh1 mutant allele, initially designated

csb3 and later renamed clb4-3 (Figures 1F and 1G), has stunted

growth and constitutively expresses subtilisin 3, a gene whose

promoter in wild-type tomato and in Arabidopsis is strongly

induced by SA and by a biotrophic pathogen Pseudomonas

syringae (Pst) pv. tomato DC3000 (Gil et al., 2005). Moreover,

the clb4-3 line exhibits enhanced resistance to Pst, supported

in part by �11-fold increase in the levels of free SA in response

to the infection, but displays wild-type levels of susceptibility

to the necrotrophic fungal pathogen Botrytis cinerea (Gil et al.,

2005).

To compare the ceh1 to the reference allele clb4-3, we exam-

ined the resistance of ceh1 to Pst and Botrytis infection and

determined the levels of salicylic acid (SA) and jasmonic acid

(JA) metabolites and the corresponding biosynthetic pathway

genes. The basal level of free SA in ceh1 plants was �40-fold

higher than that of the P and complementation lines (Figure 2A).

This higher SA level in ceh1 was accompanied by an �4-fold

increase in transcript levels of ICS1, a stress-inducible nuclear

gene encoding a key plastidial enzyme in the SA-biosynthetic

1528 Cell 149, 1525–1535, June 22, 2012 ª2012 Elsevier Inc.

pathway (Wildermuth et al., 2001) (Figure 2B). When challenged

with Pst, similar to clb4-3 (Gil et al., 2005), ceh1 lines displayed

enhanced resistance to infection as compared to P and com-

plemented lines (Figure 2C).

In contrast to elevated levels of ICS1 transcripts and SA

metabolites, the expression levels of allene oxide synthase

(AOS), a stress-inducible nuclear gene encoding a key plastid-

localized protein in the JA-biosynthetic pathway, and the

corresponding JA metabolites were comparable between ceh1

and control lines (Figures 2D and 2E). However, contrary to

clb4-3 (Gil et al., 2005), the ceh1 lines were more susceptible

to Botrytis infection as compared to controls (Figure 2F).

The AOS is in a branch parallel to the HPL branch of the

oxylipin pathway (Chehab et al., 2008). Yet, despite the elevated

expression levels of HPL, the AOS transcript levels in ceh1

remained at the wild-type levels, illustrating the selective induc-

tion of stress-responsive genes in the ceh1 mutant. In addition,

the enhanced susceptibility of ceh1 to Botrytis despite its

wild-type levels of JA, a metabolite known to mediate responses

to infection with necrotrophic pathogens including Botrytis, is

suggestive of modulation of other stress-responsive pathways

in the mutant plant.

ceh1 Phenotypes Do Not Reflect a GeneralStress ResponseThe MEP pathway, comprised of seven enzymatic steps, begins

with the condensation of pyruvate and glyceraldehyde-3-

phosphate (G3P) to form 1-deoxy-D-xylulose 5-phosphate

(DXP) and ends with the formation of the universal isoprenoid

Figure 3. High LUC Activity and Increased Free SA Levels Are

Associated with Elevated MEcPP Levels

(A) Bright-field (top) and dark-field (bottom) images of a representative of

parent (P), ceh1, and RNAi (as) lines; cosuppressed HDS line (csHDS); and

P line transformed with empty vector (EV) as control. Lines with altered

expression levels of the MEP pathway genes display variegation phenotype.

Dark-field image analyses display the constitutive LUC bioluminescence.

Strong LUC bioluminescence detected only in the ceh1 and csHDS lines.

(B) LUC activity was quantified as in Figure 1C. The gray line indicates

a change of scale on the y axis. Data are mean ±SD; n R 10.

(C) High free SA levels are detected exclusively in the ceh1 and csHDS lines.

Data are mean ±SD; n = 6.

(D) Highest MEcPP levels are present in the ceh1 followed by the csHDS lines.

Thegray line indicatesa changeof scaleon the y axis.Dataaremean±SD;n=3.

See also Figures S1and S2A–S2F.

precursors IPP and DMAPP (Rodrıguez-Concepcion, 2004)

(Figure 1G). In plants, the MEP pathway enzymes are plastid

localized and encoded by nuclear genes that are all single

copy (Phillips et al., 2007; Rodrıguez-Concepcion, 2006), with

the exception of DXS, reported to have two functional isogenes

(Walter et al., 2002).

The elevated SA levels and the high and constitutive LUC

activity phenotypes displayed by ceh1 could be the result of

general stress caused by the disruption of the MEP pathway.

To test this hypothesis, expression levels of each of the indi-

vidual MEP pathway genes were altered in the P background

(Figure 1G). RNA interference (RNAi) or cosuppression trans-

genic lines with reduced transcript levels were established for

each gene. This approach was taken to avoid lethal phenotypes

resulting from disruption of this essential pathway, as evidenced

by the albino seedling lethal phenotypes of the homozygous T-

DNA insertion lines of theMEPpathway genes (Figure 1G) (Bouv-

ier et al., 2005; Gil et al., 2005; Gutierrez-Nava et al., 2004).

We generated RNAi lines for the MEP pathway genes, except

for those lines with altered HDS expression levels obtained by

the cosuppression approach (csHDS). These transgenic lines

displayed varied expression levels of the targeted genes, ranging

from a complete knockout to a partial knockdown. Nonseedling

lethal lines expressing 5%–25% of the wild-type levels of the

respective gene were selected for additional study (Figure S1

available online). All of these lines display variegated (green

and albino leaf sectors) phenotypes (Figure 3A).

Examination of these transgenic established that the HPL-

driven LUC activity and SA levels are only altered in lines with

enhanced levels of MEcPP as compared to controls. Specifi-

cally, the negligible increases in MEcPP levels (%1-fold) de-

tected in asMDS and asHDR lines resulted in slightly higher

LUC activity (up to �1-fold) without elevation of SA levels,

whereas csHDS lines with substantially higher MEcPP contents

mimic high and constitutive HPL-driven LUC activity and

elevated SA levels of the ceh1 mutant (Figures 3A–3D).

Collectively, these data not only display a strong correlation

between MEcPP content and the levels of SA and HPL-driven

LUC activity, but also demonstrate that induction of SA and

HPL stress pathways is not a response to a general stress

syndrome resulting from MEP pathway perturbation.

Accumulation of MEcPP Is Responsible for High SAand Constitutive Expression of HPLTo determine the impact of reduced expression levels of the

individual genes on the substrate levels of their respective en-

coded enzyme, we determined the level of the corresponding

individual MEP pathway metabolite in the transgenic lines, with

the exception of 4-diphosphocytidyl-methylerythritol phosphate

(CDP-MEP) and HMBPP, for which there are no available stan-

dards (Figures S2A–S2F). We also did not examine the levels

of the two core metabolites, G3P and pyruvate, because these

metabolite pools are utilized for various biological processes

other than the MEP pathway. To determine the impact of

the downregulation of MDS on the MEP pathway metabolic

signature, we therefore analyzed the levels of 4-diphosphocy-

tidyl-methylerythritol (CDP-ME), the immediate precursor of

CDP-MEP (Figure S2D). These data show a strong correlation

Cell 149, 1525–1535, June 22, 2012 ª2012 Elsevier Inc. 1529

Figure 4. Induction of HPL Expression Is Independent of SA Levels

(A) High free SA level is exclusively present in the ceh1 mutant lines. The gray

line indicates a change of scale on the y axis. Data are the mean ±SD; n R 6.

(B) High LUC bioluminescence is detected in the ceh1 and ceh1/eds16-1 lines,

but not in the peds16-1 or P plants. LUC bioluminescence presented in a log2scale was quantified as in Figure 1C. Data are mean ±SEM; n R 6.

between the targeted downregulation of the MEP pathway

genes and the enhanced accumulation of the isoprene interme-

diates catalyzed by the corresponding encoded enzyme (Figures

S2A–S2E). Among the metabolites examined, MEcPP was the

most drastically altered, with levels elevated up to �500- and

700-fold in the csHDS and the ceh1 lines, respectively (Figures

3D and S2E), reflecting HDS as a limiting point in the MEP

pathway.

In summary, high and constitutive HPL-driven LUC activity

and elevated SA levels were only found in ceh1 and csHDS lines

and not in mutants for other MEP pathway genes, thus refuting

the hypothesis that the ceh1 reflects general stress caused by

misregulation of the MEP pathway. In addition, MEcPP is the

sole MEP pathway intermediate that, when elevated, results in

activation of the HPL stress response, suggesting that accumu-

lation of this plastidial metabolite is responsible for induction of

HPL expression.

High and Constitutive Expression of HPL Is Independentof SA LevelsThe high and constitutive HPL expression in ceh1 plants could

be caused by elevated basal levels of SA. To address this possi-

bility, double mutants were constructed for ceh1 and eds16-1,

a SA-deficient mutant encoding a dysfunctional ICS1 (Wilder-

muth et al., 2001). As a control, we also crossed eds16-1 to

P (here designated peds16-1). Subsequent to PCR-based

genotype verification using primers against eds16-1 and ceh1

genes, we analyzed free SA levels in the parental, the single,

and the double homozygous mutant lines (Figure 4A). These

data illustrate that, whereas ceh1 has elevated SA levels, all of

the other lines containing the eds16-1 mutations contain negli-

gible basal levels of SA. However, quantitative measurements

of the LUC bioluminescence in these lines verified similarly

high and constitutive LUC activity levels detected exclusively

in the ceh1/eds16-1 and ceh1mutants, and not in the P and

peds16-1 lines (Figure 4B). The high LUC activity, despite the

negligible levels of SA in the ceh1/eds16-1 line, is compelling

evidence that HPL induction in ceh1 is independent of SA levels.

1530 Cell 149, 1525–1535, June 22, 2012 ª2012 Elsevier Inc.

MEcPP Is a Specific Inducer of the HPL ExpressionTo test whether MEcPP could act directly as a signal that

induces HPL expression, we exploited a combination of genetic

and pharmacological manipulations. The genetic manipulations

were carried out by crossing HPL::LUC lines carrying ceh1 with

each RNAi line with reduced expression of MEP pathway

genes. Measurement of LUC activity showed that high and

constitutive HPL expression was only found in the ceh1 and

ceh1/asHDR lines. Because HDR is downstream, whereas

all other enzymes tested are upstream of HDS, this result

suggests that downregulation of genes upstream of the HDS

reduce the substrate flux for MEcPP synthesis and abolish

the high LUC bioluminescence in the respective ceh1/RNAi

lines (Figure 5A).

To examine the specificity of MEcPP as an inducer of

the HPL expression, we exogenously treated the P lines

with water as the control or with MEcPP before and immedi-

ately after wounding, followed by qRT-PCR analysis in

conjunction with quantitative LUC bioluminescence measure-

ments 90 min after these applications (Figures 5B, 5C, and

S3A–S3C). These measurements establish that the HPL

expression is induced in response to exogenous application

of MEcPP before or after wounding. However, the highest

levels of HPL induction are displayed by plants treated with

MEcPP postwounding, suggesting that ease of penetration

through the wound site potentially enhanced accessibility of

this metabolite (Figure 5B). Importantly, lack of any detectable

changes in the expression levels of AOS in response to MEcPP

treatment (Figure 5C) is compelling evidence for the selectivity

of this metabolite in inducing targeted stress responses and

is suggestive of the existence of other eliciting signals yet to

be identified.

Abiotic Stresses Robustly Modulate MEcPP Levelsand Induce the HPL ExpressionNext, we analyzed wounded and high-light-treated plants to

determine whether the endogenous content of MEcPP and

the HPL expression levels are modulated in response to abiotic

stresses. The selection of these two stress stimuli was based

on the established role of wounding in induction of HPL ex-

pression (Chehab et al., 2008; Chehab et al., 2006) and the

well-known role of high light in increasing levels of stress

response genes (Jung and Chory, 2010). These data show

increased levels of MEcPP as well as elevated HPL expression

in plants treated with these abiotic stresses as compared to

untreated controls, albeit at different degrees (Figures 5D–

5G). High-light treatment resulted in a notably stronger induc-

tion of MEcPP and HPL expression levels as compared to

wounding, caused by inherent differences between plant

responses to these two stimuli, by the incompatible degree of

stimulus applied, or by the inevitable patchiness of wounding

as opposed to the even exposure of plants to high light treat-

ment. Nonetheless, these results clearly display the correlation

between the endogenous levels of MEcPP and fold induction

of HPL expression.

In summary, our data clearly show that exogenous application

or stress-induced endogenous accumulation of MEcPP results

in selective induction of HPL expression.

Figure 5. Genetic and Pharmacological Manipulations Establish

Specificity of MEcPP as an Inducer of HPL Expression

(A) Among the F2 generation resulted from the crosses between the ceh1

and the RNAi (as) lines, only ceh1/asHDR lines mimic the ceh1 high LUC

activity. LUC bioluminescence was quantified as in Figure 1C. Data are

mean ±SEM; n = 6.

CEH1 Is Distinct from GUNsTo determine whether ceh1 affects similar mechanisms in plastid

development to those affected by the gunmutants, we examined

the expression levels of LHCB in wild-type (WT) and ceh1 seed-

lings grown in the presence of norflurazon (NF), an inhibitor

of phytoene desaturase, a critical enzyme in the carotenoid

biosynthesis pathway (Oelmuller et al., 1986). These data

demonstrate that NF-treated WT and ceh1 seedlings are both

photo bleached and have reduced levels of LHCB transcripts

(Figures 6A–6C), as distinct from the gun mutants, which have

high levels of LHCB expression on NF-containing medium

(Susek et al., 1993). In addition, the ceh mutants do not alter

gene expression among PhANGs (Figure 6C and Table S1) that

are regulated in the gun-signaling pathway (Woodson et al.,

2011). Thus, ceh1 does not appear to define the retrograde

signaling mechanism(s) directly involved in gun-perturbed

plastid development.

DISCUSSION

A fundamental question in plant biology is how information flows

between endosymbiotic plastids and the nucleus to coordinate

biochemical, structural, and stress responses. In the work re-

ported here, we identify a small plastid-derived metabolite as

a stress-induced signal capable of eliciting specific stress

responses, including changes in nuclear gene expression and

accumulation of salicylic acid.

Plastid-to-nucleus retrograde signaling has been most thor-

oughly described for roles in coordinating the proper expression

of nuclear genes encoding plastid-localized proteins involved in

plastid development, but retrograde signaling has also been

implicated in stress responses. However, the molecular nature

of stress-mediated signaling mechanisms has remained uncer-

tain. Plastid-derived small metabolites are thought to serve as

signals to communicate perturbations in the subcellular environ-

ment to nucleus (Jung and Chory, 2010; Kimura et al., 2003;

Shulaev et al., 2008), but thus far, their identities have remained

elusive. Using a genetic approach, we have identified ceh1,

a retrograde signaling mutant that unlike gun mutants (Beale,

2011; Woodson et al., 2011) does not coordinate expression of

PhANGs. Instead, this ceh1 mutant shows changes in expres-

sion of selected stress-responsive genes, increases in stress-

related salicylic acid, and increased resistance to the biotrophic

pathogen Pseudomonas. Ceh1 is caused by a mutation in the

HDS gene of the MEP pathway, which results in accumulation

of high levels of the intermediate metabolite, MEcPP. Abiotic

(B and C) Exogenous application of MEcPP selectively induces HPL expres-

sion. RT-qPCR analysis of expression levels of HPL(B) and AOS (C) 90 min

after wounded P plants were treated with water (control; gray bars) or 10 mM

MEcPP (black bars). Data are mean ±SD; n = 12.

(D and E) Wounding elevates levels of MEcPP and induces HPL expression.

Levels of MEcPP (D) and relative expression of HPL (E) before (control; gray

bars) and 90 min after wounding (wounded; black bars) of P plants. Data are

mean ±SD; n = 6.

(F and G) High light elevates levels of MEcPP and induces HPL expression.

Levels of MEcPP (F) and relative expression of HPL (G) in control (gray bars)

and 90 min after high light treatment (black bars) of P plants. Data are

mean ±SD; n = 6. See also Figure S3A–S3C.

Cell 149, 1525–1535, June 22, 2012 ª2012 Elsevier Inc. 1531

Figure 6. ceh1 Is Distinct from gun

(A) Comparison of 6-day-old wild-type (WT) and

ceh1 seedlings grown in absence or presence of

NF (5 mM).

(B) Relative expression levels of LHCB in WT and

ceh1 seedlings grown in absence (black bar) or

presence (white bar) of NF. RT-qPCR analyses

were performed on the total RNA extracted from

6-day-old seedlings, and data were normalized

as described in Figure 1D. Data are mean fold

difference ±SEM of three biological with three

technical replicates for each.

(C) Mean expression levels (log2) of the PhANG

genes in genotypes with WT CEH (black bar)

versus mutant CEH (white bar). NS stands for

nonsignificant differences in average gene ex-

pression between the genotypes after ANOVA

with a genomewide FDR of 0.2. See also Table S1.

stresses such as high light and wounding also elevate MEcPP

levels, eliciting the expression of the aforementioned genes.

We propose that this stress-mediated robust induction of

the MEcPP levels serves as a specific and critical retrograde

signal eliciting the expression of selected stress-responsive

nuclear-encoded plastid-localized proteins. Collectively, our

findings establish that the MEP pathway, in addition to pro-

ducing isoprenoids, functions as a stress sensor and a coordi-

nator of expression of stress-responsive pathway genes through

modulation of the levels of MEcPP, a retrograde signaling

metabolite.

Intriguingly, stress-mediated induction of MEcPP levels is

also found in bacterial cultures exposed to oxidative stress

(Ostrovsky et al., 1992, 1998). This similarity in modulations in

the MEcPP levels in response to abiotic stresses suggests at

least some conservation of function of this metabolite as

a stress sensor in regulating selected stress-responsive

processes in eubacteria and plants. Though the exact nature

of the signaling mechanism coupling MEcPP levels to changes

in gene expression is unknown, possible mechanisms are sug-

gested to be via its direct involvement in chromatin remodeling,

as evidenced by its function in nucleoid decondensation in chla-

1532 Cell 149, 1525–1535, June 22, 2012 ª2012 Elsevier Inc.

mydial cultures, through disruption of

association of histone-like proteins with

DNA (Grieshaber et al., 2004, 2006).

Similarly, in plants, MEcPP may alter

the functional organization of nuclear

domains in response to stress, leading

to changes in nuclear architecture and

functional dynamics and resulting in the

expression of the targeted stress-

responsive genes (Figure 7). This model

necessitates a MEcPP plastid-nuclear

mode of transport. Future studies will

be required to unravel the mode of trans-

port and establish the mechanisms in

which MEcPP participates to effect

changes in gene expression and stress

response.

The results presented here provide insights as well as new

questions about the coevolution of endosymbiotic plastids with

eukaryotic cells. The potential use of a conserved stress

signaling mechanism from bacteria in retrograde signaling

begs the question of how this mechanism was modified to allow

communication between plastid and nucleus.

EXPERIMENTAL PROCEDURES

Plant Growth Conditions and Treatment

Arabidopsis plants were grown in a 16 hr light/8 hr dark photoperiod at 22�C.Wounding and pathogen resistance assays were performed as described

previously (Walley et al., 2007, 2008). External applications were performed

using MEcPP purchased from Echelon-Inc (Cat. #I-M054) diluted in water

with 0.01% silwet-77. High-light treatments were at 1,100 mmol m�2 sec�1.

NF treatment was according to the described method (Susek et al., 1993).

Cloning of PHPL::LUC and Plant Transformation

The HPL1 promoter characterized as the 2 Kb upstream region of the HPL1

translational start site was amplified from the genomic DNA of Arabidopsis

(Col-0) using listed primers (Table S2) and was cloned into pCR 2.1-TOPO

Vector (Invitrogen) and subsequently into pAtMDU provided by Stacy Harmer

(University of California, Davis). The SmaI digest of this construct ligated

into pML-BART, used to transform Agrobacterium tumefaciens strain

EHA101 strain that is subsequently employed for transformation of wild-type

Figure 7. Model of MEcPP Mechanism of Action

Stress-mediated induction of MEcPP levels functions as a sensor and

a communication signal to the nucleus that induces selected stress-respon-

sive genes through alteration of nuclear architecture and functional dynamics.

Col-0 plants by floral dip (Clough and Bent, 1998). Basta-resistant transgenic

plants that contained the PHPL::LUC gene as determined by PCR analysis of

the genomic DNA (Table S2) were selected, treated with luciferin, and imaged

as described (Walley et al., 2007).

EMS Mutagenesis and Mutant Screening

Seeds (�200 mg) of homozygous lines with a single copy of the PHPL::LUC

transgene were treated with 0.2% (v/v) EMS, washed with 0.1 M sodium

thiosulfate, and subsequently rinsedwithwater. M1 plants were grown in pools

of �40 plants each, and a total of �12,000 M2 seeds from 70 independent

pools were screened and ceh1 mutant line selected. Luciferase imaging and

activity analysis of mutant lines grown on plates containing 1/2 MS were

carried out as described (Walley et al., 2007).

Map-Based Cloning of ceh1

The map-based cloning of the ceh1 mutation was performed as described

previously (Jander et al., 2002). In brief, theM3 generation of ceh1was crossed

to wild-type Landsberg erecta, and the F2 generation was screened for the

recessive high LUC activity phenotype. The first-pass mapping established

that F2 lines were flanked by the indel markers CER456385 and 454021 on

chromosome 5. A total of 915 F2 plants containing reporter gene were

analyzed with the two markers, and 76 recombination events were identified.

For fine mapping, additional markers (Table S3) between CER456385 and

454021 were designed based on the Cereon Arabidopsis Polymorphism

Collection. Subsequent analysis of phenotypes combined with genotypes

using the 17 markers on F3 generations identified CER457311 and

CER457380 spanning a 65.5 kb region as the closest ceh1 locus flanking

markers. Sequencing of the 17 genes in this region revealed a missense muta-

tion in the gene (At5g60600) that encodes HDS.

Complementation of ceh1 Mutant

Wild-type CEH1 gene including the promoter region was amplified from

genomic DNA of the Col-0 plants using the listed primers (Table S2). The

PCR product was directionally cloned into Gateway Entry Vector pENTR/

D-TOPO and then subcloned into the Gateway destination vector pMDC100

(Curtis and Grossniklaus, 2003). Upon sequence verification, this construct

was introduced into Agrobacterium tumefaciens strain GV3101 for transfor-

mation by the floral dip method (Clough and Bent, 1998). Complementation

(CP) lines were confirmed by PCR-based genotyping using the listed primers

(Table S4).

Generation of the RNAi Line of the MEP Pathway Genes

RNAi lines for all of theMEP pathway genes were generated by overexpression

of the antisense genes in the P line, with the exception of csHDS lines obtained

by the cosuppression of the HDS. Using listed primers (Table S2), antisense or

sense MEP pathway genes were amplified from wild-type Col-0 cDNA and

cloned into Gateway Entry Vector (pENTR/D-TOPO) and subcloned to the

Gateway destination vector pGWB5 (Nakagawa et al., 2007). Transformation

was by floral dip method (Clough and Bent, 1998), and selection was on Kana-

mycin and Hygromycin. All lines were genotyped by PCR analysis using the

listed primers (Table S4), and the gene expression levels were tested by RT-

qPCR using the listed primers (Table S5).

Generation of ceh1/RNAi Lines

To obtain lines containing both the RNAi construct and ceh1, weak RNAi

alleles of the individual MEP pathway genes were crossed to ceh1, and the

presence of transgene in F1 plants was confirmed by PCR reactions using

the 35S promoter primer paired with the gene-specific primers and the point

mutation of the ceh1 gene (Table S4). The resulted F3 seedlings were geno-

typed again subsequent to luciferase imaging.

Generation of ceh1/eds16-1 Double Mutants

To generate eds16-1 and ceh1 double mutants, the P and ceh1 lines were

crossed to eds16-1 as the mother. The presence of mutations was identified

in the F2 populations using listed primers (Table S4) representing eds16-1

and ceh1 mutations. Ceh1 is distinguished from the wild-type by the MboI

restriction site present only in CEH1.

Gene Expression

The expression analyses were performed by quantitative real time-PCR

(qRT-PCR), and At4g34270 and At4g26410 were used as the internal controls

for transcript normalization as previously described (Walley et al., 2008),

using listed primers (Table S5). Expression of PhANGs was measured using

Affymetrix ATH1 microarrays with three independent biological replicates for

wild-type (WT), ceh1complementation line (CP), ceh1 mutant and the cosup-

pression line (csHDS). Expression profiles were log2 transformed and RMA

normalized across all arrays. A nested ANOVA was utilized to test the differ-

ence in genotypes with WT CEH1 function (WT and CP) versus genotypes

with reduced CEH1 function (ceh1 mutant and csHDS) (Sønderby et al.,

2010). Significance was determined after adjusting for a genome-wide FDR

of 0.2 (Storey, 2002) (Table S1).

LC-MS Analysis of MEP Pathway Metabolites

To analyze the MEP pathway metabolites, plant tissue was harvested by flash

freezing and was subsequently ground in liquid nitrogen and extracted at 4�Cwith 13 mM ammonia acetate buffer adjusted to pH 5.5 with acetic acid.

Extracts were spun, and the clear supernatant was freeze dried and subse-

quently reconstituted in 100 ml acetonitrile water (50:50, v/v) containing 10 mM

of the appropriate deuterated standards. Chromatography was performed

on an Agilent Technologies 1200 Series HPLC system, using an Aminex

HPX-87H (BIO-RAD, Hercules, CA, USA) ion exclusion column for separations.

Injection volume was set to 10 ml. Mass spectrometry was performed on

a coupled Agilent Technologies 6210 time-of-flight mass spectrometer

(Agilent Technologies, Santa Clara, CA, USA). Electro spray ionization (ESI)

was set as previously described (Eudes et al., 2011). Data acquisition, pro-

cessing, and analysis were performed by the MassHunter software package.

SA and JA Measurements

Analysis of JAs (MeJA and JA) and free salicylic acid (SA) was carried out by

GC-MS as previously described (Savchenko et al., 2010).

SUPPLEMENTAL INFORMATION

Supplemental Information includes three figures and five tables and can be

found with this article online at doi:10.1016/j.cell.2012.04.038.

Cell 149, 1525–1535, June 22, 2012 ª2012 Elsevier Inc. 1533

ACKNOWLEDGMENTS

We thank Dr. Andrew Grover for his insightful comments on the manuscript.

This project is supported by the National Science Foundation grant IOS-

1036491 (K.D.). This work was performed as part of ENIGMA, a Scientific

Focus Area Program supported by the US Department of Energy, Office of

Science, Office of Biological and Environmental Research, Genomics: GTL

Foundational Science through contract DE-AC02-05CH11231 between

Lawrence Berkeley National Laboratory and the U.S. Department of Energy

(E.E.K.B. and J.D.K).

Received: June 18, 2011

Revised: November 5, 2011

Accepted: April 2, 2012

Published: June 21, 2012

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