Plastid signalling to the nucleus and beyond

Plastid signalling to the nucleus andbeyondBarry J. Pogson1, Nick S. Woo1, Britta Forster1 and Ian D. Small2

1 Australian Research Council Centre of Excellence in Plant Energy Biology, School of Biochemistry and Molecular Biology,

The Australian National University, Canberra, ACT 0200, Australia2 Australian Research Council Centre of Excellence in Plant Energy Biology, MCS Building M316,

University of Western Australia, Crawley, WA 6009, Australia

Review

Communication between the compartments or orga-nelles of cells is essential for plant growth and devel-opment. There is an emerging understanding of signalsgenerated within energy-transducing organelles, suchas chloroplasts and mitochondria, and the nuclear genesthat respond to them, a process known as retrogradesignalling. A recent series of unconnected break-throughs have given scientists a glimpse inside the‘black box’ of organellar signalling thanks to the identi-fication of some of the factors involved in generatingand propagating signals to the nucleus and, in someinstances, systemically throughout photosynthetictissues. This review will focus on recent developmentsin our understanding of retrograde and systemicsignals generated by organelles, with an emphasis onchloroplasts.

Why do organelles need to signal their status?Feedback loops and membrane barriers

Much of the biochemistry of the energy-transducing path-ways within plant cells occurs within organelles, notablythe complex energetic reactions linked to photosynthesisand respiration that take place within chloroplasts andmitochondria, respectively. The electron transport chainsare comprised of multimeric complexes containing bothnuclear- and organelle-encoded polypeptides and a varietyof cofactors whose synthesis and assembly must be tightlycontrolled and able to respond to developmental and exter-nal stimuli. This control is achieved by a series of feedbackloops activated by aspects of organelle activity, includingsignals generated during biogenesis, changes in functionalstate or damage [1–5].

Because the components of the energy-transducing sys-tems are encoded by organelle and nuclear genomes, geneexpression in these separate compartments needs to beregulated. This imposes severe constraints on signallingbecause the pathway must traverse one or more lipidbilayers that are impermeable to almost all proteins, smallmolecules and ions unless specific transporters are pre-sent. Furthermore, organelle genomes code for almost noregulatory proteins or RNAs (see: http://chloroplast.cbio.p-su.edu/; [6]), which necessitates import of organellar regu-latory factors. This means that feedback loops affectingorganellar protein synthesis might well require changes insynthesis of nuclear-encoded regulatory factors.

Corresponding author: Pogson, B.J. ([email protected]).

602 1360-1385/$ – see front matter � 2008 Elsevier L

Signals from the organelle to the nucleus are collectivelyreferred to as retrograde signals and can largely begrouped into two categories: (i) biogenic control – develop-mental control of organelle biogenesis needs to be appro-priately staged and the required subunits and cofactorsneed to be present in correct stoichiometry for accurateassembly (Boxes 1 and 2); and (ii) operational control –

rapid adjustments are made to energy metabolism inresponse to environmental and developmental constraintsto maintain optimal production and both limit and repairdamage induced by oxidative stress. Although some ofthese controls are post-translational modifications, thisreview concentrates solely on those controls that operatevia changes in gene expression and rates of proteinsynthesis (Box 3).

Biogenic control – regulating synthesis and assemblyControl via epistasy of synthesis

The stoichiometric synthesis and assembly of the respir-atory and photosynthetic machinery could be achieved inseveral ways. The least complex method would be to pro-duce all required polypeptides in abundance and degradethe excess. However, such a wasteful system would beunsustainable, especially because the most abundant pig-ments (chlorophylls and carotenoids), soluble protein(Rubisco) and membrane-bound proteins (light-harvestingproteins) in nature are localized to the chloroplast. A moreconservative method would be to synthesize polypeptidesat preset, coordinated rates. However, this over-simplifiedsystem would be too inflexible to deal with changing devel-opmental and environmental impositions. A more flexiblesystem that keeps waste to a minimum has been demon-strated in Chlamydomonas chloroplasts, where excess,unincorporated polypeptides block further translation oftheir own messages (Box 1). The process has been termed‘control by epistasy of synthesis’ (CES) because the syn-thesis of one subunit becomes dependent on the synthesisof another [4,7]. In a similar manner, cofactors thataccumulate in excess of their binding proteins can regulateboth their own synthesis and that of the proteins.

In Chlamydomonas, production of large chloroplastcomplexes seems to be regulated by a cascade of CESinteractions between various subunits [7,8]. The elegantCES theory is consistent with recent observations concern-ing the assembly of Rubisco in higher plants [9] and withevents in the synthesis and construction of yeast mitochon-

td. All rights reserved. doi:10.1016/j.tplants.2008.08.008 Available online 1 October 2008

Box 2. PPR proteins – control factors in organellar gene

expression

The PPR family of proteins found in all eukaryotes consists of 450

members in Arabidopsis and had remained unidentified until the

Arabidopsis genome was sequenced. Although abundant in higher

plants, algae and multicellular animals, PPR gene families are small

[83,84]. Nearly all PPR proteins are targeted to mitochondria or

plastids, and most of those characterized so far are involved in

organelle gene expression, with known functions in RNA editing,

splicing and translation [85]. Because evidence suggests that PPR

proteins have transcript-specific RNA-binding properties, they are

ideal candidates for regulatory factors in signalling pathways within

organelles; hence, the involvement of a PPR protein in CES is

entirely consistent with the function of this class of proteins [12].

However, they also have a role in controlling gene expression

beyond organelles, as suggested by the role of the PPR protein

GENOMES UNCOUPLED 1 (GUN1) [18] in plastid gene expression

(PGE) retrograde signalling pathways that affect nuclear gene

expression (see Figure 1 in main text) [86].

Figure I. Example of control of nuclear gene expression by epistasy of

synthesis. During organelle biogenesis (a), nuclear-encoded regulatory

factors (hatched) activate synthesis of organelle-encoded subunits (dark

green) which, along with nuclear-encoded subunits (light green) are

assembled into organellar protein complexes. Homeostasis (b) occurs when

there is a sufficiency of organelle-encoded subunits and further production of

these subunits is prevented, probably through inhibition of the regulatory

factors by the excess subunits. Environmental stimuli or organelle signals

(white arrows) exert operational control (c) that reactivates expression of

nuclear-encoded organelle factors and reactivates organelle gene expression

and complex assembly. The thick line represents the organellar genome; a

green box indicates a transcriptionally active gene and a red box indicates a

transcriptionally inactive gene.

Box 1. Control by epistasy of synthesis (CES)

During conditions of expansion of organellar capacity driven by

innate developmental programs (biogenic drive, Figure Ia), orga-

nelle-encoded components of photosystem or respiratory com-

plexes are synthesized and assembled. Synthesis depends on

imported regulatory factors and assembly depends on imported

subunits of the same complex. Once optimal capacity has been

achieved (homeostasis, Figure Ib), organelle-encoded components

are no longer synthesized. The block on synthesis might be due to

inhibition of the regulatory factors by unassembled subunits, as

shown here. The block on synthesis can be released as soon as more

imported subunits are brought in (operational control, Figure Ic) in

response to signals from ailing plastids or environmental triggers.

Once the stimulus ends, the plastids will return to homeostasis. In

this way, regulated expression of nuclear genes encoding subunits

of organelle complexes indirectly drives appropriate, coordinated

expression of organelle-encoded subunits of the same complex. CES

has been demonstrated for several subunits of the photosystem

complexes in Chlamydomonas chloroplasts but remains to be

rigorously tested in land plants.

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drial complexes [10], and in our opinion is likely to prove acore paradigm for understanding the control of organellegene expression. It is crucial to note that CES is confinedwithin organelles because itsmode of action requires direct

protein–protein, protein–cofactor and protein–RNA inter-actions; thus, at present, no instances of CES involvingnuclear-encoded subunits have been suggested. Thismightalso explain why certain genes have been preferentiallyretained in organelle genomes, because those that encodeCES-regulated proteins would be unable to retain thismode of control if transferred to the nucleus [11]. CESfeedback loops involve RNA-binding factors that arerequired for the expression of specific organellar genes;one of the factors recently implicated in control of theproduction of the Chlamydomonas cytochrome f subunitis the nuclear-encoded factor MATURATION/STABILITYFACTOR FOR PETA mRNA (MCA1), a pentatricopeptiderepeat (PPR) protein [12] (Box 2).

Plastid gene-expression-dependent retrograde

signalling

Nuclear and organellar gene-expression systems aretightly coordinated, such that inhibiting organelle geneexpression has strong effects on expression of specificnuclear-encoded genes (Figure 1). Inhibition of plastidgene expression (PGE) by tagetitoxin, a specific inhibitorof plastid RNApolymerase, results in the failure of severalnuclear genes to be induced during normal development,

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Figure 1. Biogenic control of nuclear gene expression via tetrapyrrole- and plastid-

gene-expression-dependent retrograde signalling. (a) Under conditions where

there is no disruption of PGE or tetrapyrrole biosynthesis, nuclear gene expression

is unimpaired. (b) Inhibition of PGE alters the state of GUN1, activating the GUN1-

mediated signal [18,19]. The tetrapyrrole pathway converges to some degree with

the GUN1 pathway in the chloroplast [18] and/or cytosol [23]. The retrograde signal

is transmitted to the nucleus via the action of one or more unidentified factors

(represented by broken lines) and triggers repression of nuclear-encoded

chloroplast genes through activation of regulatory factors such as ABI4.

Box 3. Operational control of nuclear gene expression

Organellar electron transport chains are major sites for the

generation of reactive oxygen species (ROS), such as 1O2, H2O2

and O2�, and are also coupled to a variety of redox-sensitive

metabolites and proteins, such as thioredoxin (Trx). In the

chloroplast, the generation of these redox-active species within

the thylakoids is linked to photosynthetic electron transport rates

and has been connected with changes in expression of nuclear-

encoded genes [34]. The mechanisms by which the accumulation of

redox-active species in the chloroplast is communicated to the

nucleus remains mostly unknown, although given the limited half-

lives and diffusive capacity of many of these species, it is likely that

signalling is mediated through activation of second messengers,

such as the EXECUTER proteins (EX1, EX2) involved in 1O2-

dependent nuclear gene regulation [47]. Chloroplast-derived signals

might be modulated by interaction with components of other

signalling pathways, particularly those involved in responses to

abiotic stresses. Furthermore, evidence now exists for the genera-

tion of intercellular signals within the chloroplast that are capable of

inducing systemic responses throughout the plant [56,58]. Whether

the second messengers responsible for this intercellular commu-

nication are chloroplastic, cytosolic or nuclear in origin is unclear

(indicated by question mark). Figure I provides a schematic

visualization of known and predicted signalling networks involved

in operational control of nuclear gene expression.

Figure I. Photosynthesis-dependent operational control of nuclear gene

expression. Accumulation of redox-active compounds such as ROS within

the chloroplast is coupled to rates of photosynthetic electron transport. These

redox-active species activate retrograde signals that might be transduced via,

or interact with, known abiotic stress-signalling pathways and secondary

messengers or metabolites in the cytosol. Photosynthesis-derived signals

might also alter nuclear gene expression intercellularly through the generation

of systemic signals in a process known as systemic acquired acclimation.

Examples of components of the pathways are given. Abbreviations: AOX1a,

mitochondrial alternative oxidase; Chl, chlorophyll; CHS, chalcone synthase;

Fd, ferredoxin; LHC, light-harvesting complex; SOD, superoxide dismutase.

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including the genes encoding the light-harvesting complexprotein LHCB1.2 and the small subunit of Rubisco [13].Likewise, mutations in genes encoding vital componentsof the organelle gene-expression machinery, such as ami-

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noacyl-tRNA synthetases, inhibit expression of a largesuite of nuclear genes, as do inhibitors of plastid trans-lation, such as chloramphenicol and lincomycin [14]. Theimplication is that there is a plastid-derived PGE signalthat is dependent on plastid transcription and/or trans-lation and that is essential for chloroplast biogenesis.However, the suppressive effects of these inhibitors onnuclear gene expression are only exhibited if treatment isapplied within approximately three days of germination[15].

PGE has also been investigated by infiltrating plantswith the carotenoid biosynthetic inhibitor norflurazon,which caused photobleaching, impaired chloroplast differ-entiation and repressed expression of nuclear genes suchas LHCB1.2. Several Arabidopsis (Arabidopsis thaliana)mutants (designated genomes uncoupled [gun]) that accu-mulated LHCB1.2 mRNA despite photobleaching wereisolated [16]. A more recent screen identified othermutants, some of which are allelic to known light-signal-lingmutants, for example cryptochrome1 [17]. Onemutant,gun1, shows de-repression of nuclear genes in plants trea-ted with plastid translation inhibitors, thereby implicatingGUN1 in PGE-dependent retrograde signalling [18,19].The signal or signals that require GUN1 are different from(but partially redundant with) those that require the light-signalling networks [17]. GUN1 is a PPR protein (Box 2)with highest similarity to PLASTID TRANSCRIPTION-ALLY ACTIVE CHROMOSOME PROTEIN 2 (PTAC2).Mutants lacking PTAC2 show defects attributed to a lossof transcription by the plastid-encoded RNA polymerase,and it is proposed that PTAC2 is part of a large multi-protein complex associated with plastid transcription [20].Presumably, GUN1 has a similar function in mediating

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expression of a plastid gene or genes. A PGE signal could begenerated depending on translational activity in the plas-tid sensed or mediated by GUN1 (Figure 1). Under con-ditions where nuclear-encoded chloroplast gene expressionis uninhibited, onemight hypothesize that GUN1 is associ-ated with RNA and/or plastid-encoded transcriptionalcomplexes in the plastid, which causes a state of GUN1that does not allow the generation of the PGE signal.Initiation of the PGE signal for LHCB1.2 repression thendepends on a specific change in the state of GUN1, whichoccurs in the absence of plastid transcription or translation(i.e. lincomycin treatment). Complete lack of the GUN1protein, as in the gun1 mutant, would therefore eliminateinitiation of this plastid signal irrespective of PGE.

Tetrapyrroles and genomes uncoupled mutants in

retrograde signalling

The gun2, gun3, gun4 and gun5 mutations perturb orinteract with the tetrapyrrole biosynthetic pathway, whosemajor products are chlorophyll and haeme (reviewed in[16]) (Figure 1). The gun5mutation alters the H-subunit ofMg-chelatase (CHLH) [21] that catalyses the conversion ofprotoporphyrin IX (ProtoIX) to Mg-ProtoIX, thereby impli-cating Mg-ProtoIX in GUN signalling [3,21]. Similarly, theGUN4 protein interacts with the Mg-chelatase complexand has an affinity for both ProtoIX and Mg-ProtoIX [22],and exogenous addition of Mg-ProtoIX to isolated proto-plasts modulates nuclear gene expression [3]. However,despite exhibiting reduced Mg-ProtoIX in a similar way togun5 mutants, mutants in the I-subunit of Mg-chelataseshow wild-type-like suppression of LHCB1.2 after norflur-azon treatment. This led to speculation that the interactionwith CHLH and Mg-ProtoIX is required for the signal [21].In a recent paper, a novel strategy was used to detectfluorescence emission spectra consistent with ProtoIX andMg-ProtoIX in the chloroplast and cytosol, leading theauthors to suggest that active transport of tetrapyrrolesout of the chloroplast is possible [23]. However, the photo-toxicity and structure of Mg-ProtoIX suggest that itsaccumulation within the cytosol might become problematicunder natural light conditions. Alternatively, it has alsobeen proposed that the tetrapyrrole-mediated retrogradepathwaymight act via the chloroplast-localized GUN1 [18].Thus, exactly howandwhere the tetrapyrrole-related signalis perceived and transmitted through the cytosol and, ulti-mately, to the nucleus remains worthy of further investi-gation.

Once perceived by the nucleus, recent research has indi-cated that ABI4, a transcription factor involved in theresponse to abscisic acid (ABA), participates in both thePGE- and tetrapyrrole-related pathways [18] (Figure 1).Fully 89% of genes that fail to be repressed in gun1 andgun5plants afternorflurazon treatmentare common tobothmutantsandmanyof thesecontainABA-responseelements.Additionally, the ABA-insensitive mutant abi4 exhibits aweakgunphenotype that is abrogated in lincomycin-treatedArabidopsis seedlings [16,18]. Developmental controlsmight also influence PGE- and tetrapyrrole-related GUNsignalling because studies in barley (Hordeum vulgare) andArabidopsis suggest that it might only function in the earlystages of chloroplast development [24,25].

Biogenic control operates during phases of expansion ofplastid function, most notably during early development asplants move to a reliance on photosynthesis for their mainenergy requirements. During these phases, nuclear andorganellar gene expression is coordinated at least in partby the mechanisms described above. Future research islikely to concentrate on discovering whether or not CES isimportant in regulating gene expression in land plantorganelles – to date almost no suitable data have beenobtained to test this – and on completing our understand-ing of the biogenic retrograde pathways that controlnuclear gene expression. The GUN proteins clearly haveimportant roles under the artificial conditions used in thegun screen, but how important are they under more phys-iological conditions? For instance, there are markedchanges in transcript profile in gun1 mutants versus wildtype when both are treated with lincomycin, but in theabsence of inhibitors, only one transcript varies betweenwild type and gun1 seven-day-old seedlings, whether in thelight or dark [19]. What other proteins are involved? Dothey also function in feedback regulation later in devel-opment? The evidence to date suggests that other mech-anisms become predominant once chloroplast function isestablished; this operational control is the topic of the restof this review.

Operational control – optimizing photosyntheticperformancePhotosynthetic electron transport signalling

Typically, three types of chloroplast redox signals havebeen postulated (Box 3): (i) the redox states of componentsof the photosynthetic electron transport (PET) chain, prim-arily plastoquinone (PQ); (ii) redox-active thiol-group-con-taining proteins and antioxidants coupled to PET; and (iii)the generation of reactive oxygen species (ROS) (discussedbelow) [5,26]. Common to all is that a shift in the redoxstate of a compound initiates mostly unknown intra-plas-tidic retrograde signals that are transmitted across thechloroplast, activating similarly elusive cytosolic signaltransduction pathways.

The PQ pool, the electron acceptor of PSII, has long beenimplicated as a specific site for redox sensing and signalinitiation resulting in long-term photoacclimation in partby regulation of expression of plastid and nuclear genessuch as LHCB [1]. However, regulation of nuclear geneexpression by the redox state of the PQ pool has proven tobe highly variable depending on species, gene, develop-mental stage and experimental conditions. Furthermore, apractical challenge has been to distinguish responsesspecific to the PQ redox state as opposed to general lightsignals such as those mediated by photoreceptors. Evi-dence is clearer in green algae, where LHCB gene tran-scription is altered depending on oxidation or reduction ofthe PQ pool [27]. In contrast to algae, PQ redox poise seemsto be more integrated with other signals in higher plants[28], serving as a fine-tuning process [1,26]. Regulation ofnuclear gene expression by PQ redox poise has beendemonstrated for genes such as PETE (plastocyanin),ASCORBATE PEROXIDASE 2 (APX2) and EARLYLIGHT-INDUCIBLE PROTEIN 2 (ELIP2), but not photo-system I (PSI) genes, such as PSAD and PSAF [28–32]. In

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fact, of a group of 2000 light-responsive genes in Arabi-dopsis, only 54 genes were strictly regulated by PQ redoxpoise [33,34]. Furthermore, only part of LHCB expressionis coregulated with PQ redox status [35] because transthy-lakoid membrane potential and ATP synthesis might alsocontribute to the redox state. The binding of transcrip-tional enhancers (LF1, -2 and -3 complexes) and repressors(HLF complex) to the LHCB promoter is altered by thiol-modifying agents [36]. Therefore, in addition to the PSIIstatus, the redox state of the PSI acceptor site has beensuggested as a second PET site, generating a signal bymodulation of the redox state of thioredoxin (Trx) viaelectron transfer from ferredoxin [37]. Trxs can be viewedas redox signals because they modify the redox state andthereby the activity of many chloroplast enzymes andredox-sensitive processes. Trxs therefore partially controlthe generation of ROS by competing with the electrontransfer to molecular oxygen [26].

Reactive oxygen species and operational retrograde

signalling

The energy-transducing organelles are major sources ofvarious ROS in plant cells (Box 3). Adverse conditions suchas drought, temperature extremes or excessive light canaccelerate the generation of ROS, causing oxidative stresswhen detoxification capacity is exceeded [38–40]. Excitedchlorophyll molecules in the triplet state might then inter-act with 3O2 to form singlet oxygen (1O2) at PSII [41,42]. Onthe acceptor side of PSI, electron transfer to O2 can resultin the production of superoxide (O2

�), hydrogen peroxide(H2O2) and hydroxyl radicals (OH�) [43]. The differentchemical properties and sites of generation of individualROS implicate them as possible retrograde signals activat-ing responses specific to the particular stress that inducedtheir production. However, until recently it has proveddifficult to associate changes in nuclear gene expressionwith specific ROS because there are few ways to stimulateaccumulation of one ROS alone [44–46].

1O2 in operational retrograde signalling

Chloroplast-derived 1O2 might have an important regulat-ory role in retrograde signalling [47]; however, due to itsshort half-life and limited diffusion [41], intracellular 1O2

is an unlikely long-distance signal. Instead, oxylipinsgenerated by 1O2-responsive lipoxygenases are possiblecandidates for stable second messengers for 1O2-mediatedgene induction [44]. The fluorescent (flu) mutant of Arabi-dopsis was employed to study 1O2-mediated signalling:FLU is a negative regulator of tetrapyrrole metabolism,such that flu mutants overaccumulate the photosensitizerprotochlorophyllide (PChlide) in the dark and con-sequently generate 1O2 once illuminated [48]. Light-exposed flu plants show suppressed vegetative growthand induction of programmed cell death in leaves. Sup-pressor screens of flu enabled the identification of a novelthylakoid-localized protein, EXECUTER1 (EX1), and itshomologue, EXECUTER2 (EX2) [47,49]. EX1 and EX2have been identified as putative 1O2-signalling com-ponents based on suppression of the flu phenotype in theex1 flu double and the ex1 ex2 flu triple mutants. ex1 fluoveraccumulatesPChlide in the darkandupon illumination

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releases 1O2butdoesnotundergo cell death, suggesting thatEX1 is involved in a 1O2-sensitive programmed-cell-deathresponse [49]. Inaddition, ex1 ex2fluplants lack inductionofalmost all 1O2-responsive genes. The phenotype and effecton ROS signalling of ex1 and ex2 mutants in a wild-typebackground would be of great interest.

H2O2 and O2� in operational retrograde signalling

H2O2 has a comparatively longer half-life and lowertoxicity compared to other ROS [50] and is thus favouredas an intra- and intercellular messenger (reviewed in [39]).It is also the primary ROS generated in plant mitochondria[51], peroxisomes [52], chloroplasts, plasma membranesand cell walls [39,53]. Many stress conditions stimulateproduction of H2O2 in the photosynthetic apparatus, andmany light-stress-induced changes in gene expression areattributable to H2O2 [30,54–56].

Research into ROS-mediated signalling has largelyfocused on the induction of endogenous reporter genes,including the high light (HL)-inducible cytosolic ascorbateperoxidases APX1 and APX2, the zinc finger transcriptionfactors ZAT10 and ZAT12 and the chlorophyll-bindingprotein ELIP2. Both ZAT10 and ZAT12 mediate differentsubsets of the HL-inducible or -repressible gene set, in-cluding APX2 and APX1, respectively [56,57]. In fact,twenty per cent of the HL-set is coexpressed in plantsoverexpressing ZAT10 [56]. Exogenous application ofH2O2 stimulates expression of APX2, ZAT10 and ZAT12,whereas application of catalase diminishes expression ofAPX2 and ZAT10 [56–58]. Additionally, APX2 and ZAT10are induced by ABA, changes in glutathione metabolism,drought and changes in PQ redox poise [29,59,60]. A recentarticle has proposed an interaction of H2O2- and 1O2-directed signals that might take place through modulationof the redox state of the PQ pool [61]. It has been shownthat H2O2 promotes oxidation of the primary PQ electronacceptor, QA, as reflected by elevated levels of both theyield of PSII fluorescence (FPSII) and the coefficient ofphotochemical quenching (qP) after treatment withexogenous H2O2 [62]. This increase in photosynthetic elec-tron transport would be expected to alleviate the accumu-lation of excited species within PSII during exposure toHL,thereby reducing the likelihood of 1O2 formation [61]. Thus,many H2O2-inducible genes are induced by a large numberof signalling pathways.

To function as a signallingmolecule, H2O2 needs to crossthe inner and outer envelopes of the chloroplast and othermembranes, yet its polar nature might limit its capacity todiffuse through hydrophobic membranes unassisted.Recent evidence proposes that H2O2 transport might bemediated by aquaporin channels, such as ArabidopsisTIP1.1 [63], and that aquaporin activity is reversiblyregulated by OH�-mediated oxidation in Zea mays [64].The necessity for aquaporins for H2O2 movement in vivo isyet to be determined.

Evidence for O2�-mediated signalling is derived from

gene expression arrays [46] and O2�-accumulating plants

with deficiencies in chloroplastic copper-zinc superoxidedismutase (CuZn-SOD), which exhibit higher expressionlevels of the majority of chloroplast-encoded genes [65].Because many of these are not induced by other ROS, this

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might indicate the specific sensitivity of chloroplastic genesto O2

� accumulation within the thylakoids. Nuclear-encoded genes involved in anthocyanin biosynthesis arealso upregulated in CuZn-SOD-deficient plants [45].Recently, the specific generation of O2

� in the absence ofH2O2 accumulation enabled the identification of a subset ofnuclear-encoded genes that are likely to be specific to anO2� signalling pathway [46].Not surprisingly, operational control is initiated by a

combination of factors, including redox poise, photosyn-thetic electron transport, different ROS and other factorsnot considered here. Within this complexity, researchershave at times tantalizingly identified factors specific toindividual pathways, such as EXECUTER in singlet ox-ygen signalling, but in many respects our understanding isonly moving forward incrementally. The challenge is todesign physiologically relevant strategies that activatespecific pathways.

Cytosolic and systemic responses to operationalretrograde signalsThe cytosol and nucleus

How retrograde signals are perceived in the cytosol andcommunicated to the nucleus is largely a part of the ‘blackbox’. At some level, the retrograde signals, particularlythose involved in stress responses, are presumed to inter-act with abiotic stress-signalling pathways (Box 3). Abioticstress pathways can involve heterotrimeric G proteins,phospholipase C (PLC), which generates inositol-3-phos-phate (IP3) to open Ca2+ channels, generation of Ca2+/calmodulin (CaM) complexes and activation of CaM-de-pendent kinase (CDPK) (for reviews, see [66–68]).Although there are few details known about these pro-cesses, evidence for the convergence of retrograde signalsand abiotic stress pathways includes the forementionedeffects on expression of HL-inducible genes [50–56], theinfluence of non-chloroplastic ROS on gene expression [39],the rax1 HL-signalling mutant that is perturbed in cyto-solic glutathione metabolism [59] and the identification ofseveral altered expression of APX2 (alx) HL-signallingmutants that are also altered in drought- and ABA-signal-ling pathways [69]. The contribution of glutathione syn-thesis and its redox state to operational retrogradesignalling has been reviewed recently [70]. Additionally,inhibition of PLC, CaM and protein kinases preventedinduction of chlorophyll a oxygenase and LHCB geneexpression in low light [71], and a cytoplasmic serine/threonine protein kinase C proved to be essential forregulation of light-dependent expression of the PSAF sub-unit of PSI in tobacco [72]. Also, induction of GUN signal-ling requires the transcription factor ABI4 [18] and ROSsignalling involves the transcription factors ZAT10 andZAT12, all of which are known to be induced by a range ofbiotic and abiotic stresses [57,60,73]. Finally, a knockout ofthe mitochondrial alternative oxidase (AOX1a) alters thestress-response networks of the cell, making them moresusceptible to stresses that are primarily targeted to thechloroplast, namely excess light and drought [74]. Thissuggests that components of one organelle can alter theredox balance of the cell and thus perturb stress responsesand signalling in another organelle [74]. Thus, there is

little doubt that aspects of operational retrograde signal-ling utilize known cytosolic and nuclear signalling path-ways, but the complexity of signalling networksnecessitates novel and detailed experimentation to deter-mine the relative contribution of each component. Further-more, analysis of the effects of known cytosolic stress-signalling mutants on chloroplastic retrograde signallingtogether with the characterization of new operational con-trol mutants, such as alx and rax, is required.

Systemic response to operational retrograde signals

Systemic acquired acclimation (SAA) is a process that wasfirst described in 1999. In that study, an increase in APX2expression and acclimatory changes in photochemistrywere observed in distal, shaded leaves of an Arabidopsisplant when it was partially exposed to excess light [58]. Arationale for systemic signalling is that as the sun tracksfrom east to west, different parts of the canopy are exposedto full sunlight, with SAA providing a mechanism for pre-acclimating shaded leaves before potentially damaging fullsun exposure [56,58]. Other genes have also been shown tobe inducible by SAA, including PR1, FSD1 and GPX7[59,75,76]. Recently, it has been demonstrated that up to86% of genes upregulated in exposed leaves are coex-pressed in distal, shaded leaves and, as a consequence,both exposed and shaded leaves have enhanced toleranceto oxidative stress [56]. This might have implications forour understanding of HL and ROS retrograde signalling.That is, given the marked overlap in gene expressionprofiles in exposed and shaded leaves [56], it is clear thatthe retrograde signal initiated by HL is either directlycommunicated to the distal leaves or perhaps, and thisis more likely, activates a second messenger that is trans-mitted to distant cells, wherein a very similar signallingpathway is activated (see Figure I in Box 3). Withinstressed tissues, there are some commonalities betweenbiotic and abiotic stress responses, such as salicylic acidcontributing to the HL acclimatory response, interactionbetween wounding and APX2 induction and the multipleroles of LESION SIMULATING DISEASE 1 (LSD1) in HLresponses, SAA, aerenchyma formation and cell death inHL-exposed tissues [75,77–79]. These responses will com-prise a combination of common and separate pathways; thewound-induction of APX2, for example, is not mediated byjasmonic acid (JA)-dependent or known JA-independentpathways [77]. Intriguingly, from the perspective of mod-ulating different signals from different compartments ofthe cell, LSD1 is localized to the cytosol and can inhibit themovement of a bZIP transcription factor, AtbZIP10, fromthe cytosol to the nucleus [80]. With respect to SAA, inshaded leaves the majority of the genes induced are eithermethyl jasmonate, H2O2- and/or ABA-responsive genes.Yet, significantly, SAA is still detectable in several signal-lingmutants and transgenics that alter glutathionemetab-olism, pathogen-related systemic acquired resistance,jasmonates, salicylates, ABA synthesis and/or perception,including abi1, abi2, aba2-3, rax1-1, jar1-1, jin1, sgt1b-3,aos, npr1 andNahG. Considerable work is required in thisarea to determine the nature and function of this short-term SAA signal. To what extent is it distinct to acclima-tory changes in developing leaves induced by long term

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light, temperature or CO2 treatments of mature leaves[81,82]? Is the signal a known compound or a completelynovel secondary messenger, and to what extent does itfunction within stressed tissues in addition to distal tis-sues? That is, does the SAA signal also function in retro-grade signalling?

Conclusions and future perspectivesThe CES theory provides a sophisticated model of howorganellar gene expression can be coupled to the abundanceof imported ‘driver’ proteins. The identification of one of theChlamydomonas CES factors as a PPR protein will help inthe search for equivalent regulatory loops in higher plants.The fact that GUN1, which is involved in retrograde signal-ling, is also a PPR protein suggests intriguing parallelsbetween intra- and inter-organelle signalling pathways.The principal unknowns remain the nature of the feedbacksignals exiting the organelles and the way they are trans-mitted to the rest of the cell. The majority of studies to datehave identified components localized within the plastid andthe transcription factors within the nucleus that respond,yet there is a glaring lack of understanding of transmissionof the signal through the cytosol. Is systemic signallingmoreamenable to study than intracellular signalling and if so,would it be a good model for deconvoluting cytosolic aspectsof retrograde signalling? Considering that there are hun-dreds of chloroplasts per cell, in addition to the influence ofmitochondria and peroxisomes on ROS levels and redox inthe cell, what are the relative contributions of the organellesversus the cytosol in controlling retrograde signalling?

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