Long-distance CO2 signalling in plants

Janice A. Lake, F. Ian Woodward and W. Paul Quick1

Department of Animal and Plant Sciences, University of Sheffield, Western Bank, Sheffield S10 2TN, UK

Received 25 September 2001; Accepted 24 October 2001

Abstract

Stomatal numbers are tightly controlled by environ-mental signals including light intensity and atmo-spheric CO2 partial pressure. This requires control ofepidermal cell development during the early phase ofleaf growth and involves changes in both the densityof cells on the leaf surface and the proportion of cellsthat adopt a stomatal fate. This paper reviews thecurrent understanding of how stomata develop anddescribes recent advances that have given insightsinto the regulatory mechanisms involved usingmutant Arabidopsis plants that implicates a role forlong-chain fatty acids in cell-to-cell communication.Evidence is presented which indicates that long-distance signalling from mature to newly developingleaves forms part of the mechanism by whichstomatal development responds to environmentalcues. Analysis of mutant plants suggests that theplant hormones abscisic acid, ethylene and jasmo-nates are implicated in the long-distance signallingpathway and that the action may be mediated byreactive oxygen species.

Key words: ABA, Arabidopsis, cell development, CO2,

ethylene, jasmonates, leaf development, mutants, plant

hormones, ROS, signalling, stomata.

Introduction

On an annual basis 30031015 g of carbon pass throughstomata (40% of the carbon in the atmosphere) and12031015 g are assimilated as gross primary production(Ciais et al., 1997). The pathway of CO2 moving into aplant via stomata is almost universal in vascular landplants, with only few notable exceptions such as Stylitesandicola with no stomata (Keely et al., 1984) andspecies where stomata have been modified for otherfunctions such as the extrafloral nectaries of Cuscuta

reflexa (Hibberd et al., 1998). A plentiful supply of CO2

is required as the enzyme responsible for photosyntheticCO2 fixation (Rubisco) has a low affinity for CO2 and isalso able to catalyse a second competing and wastefulreaction that uses the much more abundant gas oxygen ina process known as photorespiration. Increased concen-trations of CO2, therefore, enhance the photosyntheticrate by increasing the rate of Rubisco carboxylation anddecreasing Rubisco oxygenation.

A consequence of having open pores on the surface ofa plant is that there is a high rate of water loss, frequentlyat more than 100 times the rate of CO2 fixation. About30–4031018 g of water vapour are transpired annuallythrough stomata, and this exceeds the atmospheric watercontent of 1331018 g (Peixoto and Oort, 1992). Waterlost through the stomata has to be replaced by uptakefrom the soil and this, in turn, is totally dependent onwater availability that, in many environments, is scarce.The aperture of the stomatal pore must therefore reflect acompromise between the photosynthetic requirement forCO2 and the availability of water (Jarvis and Davies,1998). This regulation occurs by a variety of mechanismsthat allow the stomatal aperture to respond to changes inlight intensity, CO2 concentration and water availability.The nature of these signal transduction pathways isoutside the scope of this paper but the reader is referredto an excellent recent review (Schroeder et al., 2001).

Stomatal development and cellular signalling

A second and more recent observation is that the numberand pattern of stomata within the epidermis of photo-synthetic tissues is also tightly controlled (Croxdale,2000). Leaves develop from cells at the margin of theshoot apical meristem (SAM). Initially, a swelling of thisregion occurs which is thought to require alterations ofcell wall properties to allow cell expansion and ‘slippage’.In dicotyledonous plants, three layers of cells arise andare maintained within the leaf meristem by periclinal cell

1 To whom correspondence should be addressed. Fax: q44 (0)114 222 0002. E-mail: p.quick@sheffield.ac.uk

Journal of Experimental Botany, Vol. 53, No. 367, pp. 183–193, February 2002

� Society for Experimental Biology 2002

divisions. These are referred to as L1, L2 and L3 indicotyledonous plants (McHale, 1993). For mono-cotyledonous plants this is reduced to two cell layers(L1 and L2). The epidermis is derived from the L1 layerof the leaf meristem, itself derived from peripheral cells ofthe shoot apical meristem. Control of the planes of celldivision lead to an expansion of the leaf meristem and aflattening of the structure, forming an early flattenedblade-like structure. After this stage leaf morphogenesisproceeds and sub-domains or sub-organ identities beginto develop. These follow the three major axes of the leaf;abaxial–adaxial, apical–basal and proximal or distal (leafmargin) to the central mid-rib. The adaxial and abaxialleaf-surfaces arise from leaf polarity that develops veryearly in leaf development (McConnell and Barton, 1998).The abaxial surface (surface facing away from the axis)grows faster and differentiates earlier than the adaxialsurface causing the leaf blade to curl around the apicalmeristem. This curling of developing leaves around themeristem and other developing leaves restricts exposureof these leaves to the atmosphere and, as discussed later,potentially reduces the ability of developing leaves tointerpret the surrounding environment correctly. Mutantsexist where leaves are ad- or abaxialized. The phantasticamutation of Antirrhinum produces radially symmetricalleaves that are thought to arise due a loss of leaf polarityand hence are completely abaxialized. It is proposed thatthis gene encodes a product that confers the adaxialidentity necessary for the initiation of the leaf-bladestructure (Waites and Hudson, 1995). More recently,two genes (phabulosa and phavoluta) have been implicatedin determining abaxialuadaxial leaf polarity; mutationsof these genes transform abaxial leaf fate into adaxialleaf fate by altering the predicted sterolulipid-bindingdomains of two proteins (McConnell et al., 2001).This separation of leaf surfaces is important for sub-sequent stomatal development as both numbers anddensity of stomata can vary between the upper andlower surface of a leaf.

Cells within the leaf epidermis divide to maintain asheet-like structure of simple pavement cells that willeventually form the flattened interlocking cells character-istic of the leaf surface. These cells occasionally acquiremeristemoid mother cell identity and divide asymmetric-ally giving rise to a large and a small daughter cell. Thesmaller is a meristemoid cell, as it now has the abilityto differentiate into other cell types including stomata(Croxdale, 2000). In monocotyledonous plants thesmaller cell invariably divides symmetrically to form thepair of guard cells that form the stoma; additionally,the four adjacent pavement cells on all sides are inducedto divide asymmetrically and the smaller cells form thesubsidiary cells that make up the stomatal complex. Theinitial asymmetric division orientates the smaller daughtercell towards the apex of the leaf causing a uniform pattern

of complexes to emerge, giving rise to the files of stomatatypical of monocotyledonous leaves (Croxdale, 2000).This pattern of cell division also ensures that stoma arealways separated by at least one cell and are neveradjacent. Pavement cells always divide in a transverseorientation and the pattern of stomatal development islargely fixed so the number of stomata initials withinthe leaf surface is determined at an early and particularstage of leaf differentiation when epidermal cells becomespecified as stomatal progenitors. In dicotyledonousplants the pattern of development is more complex andvariable, but is always initiated by an asymmetric divisionof an epidermal cell. The smaller cell can then undergofurther asymmetric divisions. Only one of the progeny(the smaller cell) can subsequently form a guard cell pairafter a further symmetrical division. Rarely do stomataappear adjacently on the epidermis. It is proposed thatthis results from cell-to-cell communication (Geisler et al.,2000), which prohibits cells adjacent to two stomata ortheir precursors from assuming meristemoid mother cellidentity. In situations where two smaller daughter cells doappear next to each other then a correction procedure isemployed, whereby subsequent divisions are orientatedsuch that guard cells do not develop adjacent to eachother or the fate of one meristemoid mother cell is alteredand it differentiates as a pavement cell. These data havebeen suggested to infer cell-to-cell communication of cellfate (Fenoll and Serna, 2001) and the large number ofmutants that have been identified further support thisview (see below).

Mutants in stomatal development

Several mutants exist in stomatal development, all ofwhich result in increased stomatal number. The mutantsflp and tmm (Yang and Sack, 1995) have distinctphenotypes of clustered stomata, suggesting the spatialcues that normally prevent the formation of adjacentstomata are in some way defective. Two further mutants(hic and sdd1), however, may result from lesions inanother stomatal-development signalling pathway. HICencodes a fatty acid elongase that is involved in thecontrol of stomatal development and, particularly, in theresponse to environmental signals such as CO2 concen-tration (Gray et al., 2000). SDD1 encodes a subtilisin-likeserine protease which, when mutated, causes up to a4-fold increase in stomatal density (Berger and Altmann,2000). Interestingly, subtilisins are also known to catalysethe cleavage of systemin from prosystemin during woundsignalling in solanaceous plants (although this pathwaydoes not appear to operate in wound signalling inArabidopsis) and in the processing of CLAVATA3 thatrepresses cell division in the Arabidopsis shoot apicalmeristem (Fletcher et al., 1999).

184 Lake et al.

Stomatal density is regulated byCO2 concentration

Stomatal development on leaves is measured both asstomatal numbers per unit area (density) and as thefraction of all epidermal cells (index). It has beendemonstrated that increases in atmospheric CO2 sincethe onset of the industrial revolution have occurred inparallel with a decrease in leaf stomatal density(Woodward, 1987). This decrease in density can bereplicated experimentally by an increase in atmosphericCO2 concentration of the growth environment(Woodward, 1987; Woodward and Bazzaz, 1988).Stomatal density responses have now been quantifiedfor over 100 species (Woodward and Kelly, 1995). Thisdata set indicates a broad range of responses fromdecreases to increases in density with CO2 enrichment,although the average response is for a reduction of 30%with a doubling of CO2 concentration (Woodward et al.,2001). The mechanism of this response has remainedunknown until recently when a gene (HIC; high carbondioxide) was identified that was involved in the signaltransduction pathway that controls the response ofstomatal development to changing CO2 concentration inArabidopsis (Gray et al., 2000). When the expression ofHIC is reduced, plants respond to increasing CO2 bya large increase in stomatal density and index. Thiscontrasts with decreases in stomatal density for wild-typeplants grown at elevated CO2. Several other mutants suchas tmm and flp (Yang and Sack, 1995) have alteredstomatal numbers, however, these invariably result fromthe formation of stomatal clusters due to impairmentof the mechanism that controls stomatal spacing (seeabove). Changes in density brought about by alteredgrowth CO2 concentration do not result from theformation of stomatal clusters. This suggests that themechanism for the regulation of stomatal density bythe environment is mechanistically different from thosecell-to-cell mechanisms that determine stomatal spacing.Perturbation of the HIC gene, which alters the stomataldensity response to environmental CO2 concentration,also does not result in the formation of stomatal clusters(Gray et al., 2000) suggesting that this gene may beinvolved in a separate regulatory pathway.

The CO2 signal is systemic

The question arises as to how and where the CO2 signalis detected and how this integrates with the control ofepidermal-cell fate. Although stomatal developmentcontinues late into leaf development the vast majority ofstomatal formation is complete well before full leafexpansion. It is argued that the generally enclosed natureof developing leaves (see above) precludes them fromreliably monitoring atmospheric CO2 concentration as

restricted air movement, reduced light intensity, alteredhumidity, and high respiratory activity would signific-antly alter the CO2 environment from that encounteredby a mature leaf. Mature leaves should best experienceand communicate information concerning the nature ofthe environment to young developing leaves and thiswould require a systemic signal for the developing leaf.Several long-distance environmental signalling pathwayshave been identified in plants and include systemicalterations in gene expression in response to high light(Karpinski et al., 2000), wounding (Ryan and Pearce,1998; Bowles, 1998) and the control of flowering by daylength and temperature (Reeves and Coupland, 2000).Systemic signals induced in response to high light signalsresult in changes of gene expression and biochemicalacclimation and are thought to be mediated by theproduction of hydrogen peroxide (Karpinski et al., 2000).The systemic wound signal is a short peptide, systemin,which is released from prosystemin by a proteolyticcleavage. Systemin is able to travel through the plant andcauses production of jasmonate that, together withethylene, is thought to bring about the wound response(Pearce et al., 1991; O’Donnell et al., 1996). It has recentlybeen demonstrated that a systemic signal is involved in theCO2-mediated changes of stomatal density in Arabidopsis(Lake et al., 2001). Using a gas-tight cuvette system itwas possible to maintain mature leaves in environmentscontaining CO2 concentrations different from the newlydeveloping leaves. The stomatal density (Fig. 1a, b) andindex (Fig. 1c, d) of newly developed leaves reflectedthe CO2 concentration experienced by the mature leaves,rather than the environment in which they had developed.This treatment had no effect on leaf expansion (datanot shown) or the density of epidermal cells (Fig. 1e, f)indicating that the signal regulating stomatal develop-ment controlled, primarily, the extent to which epidermalcells acquired a stomatal fate. The CO2 concentrationexperienced by the young developing leaves exerted nosignificant impact on their stomatal index. These dataprovide clear evidence of a novel long-distance signal thatis produced by mature leaves in response to elevated CO2

and that travels to newly developing leaves, affecting theirsubsequent development. These observations have beenverified using a second species, Sinapis alba (Lake, 2001),though it still remains to be established whether thisresponse is common to all plants.

Biochemical bases of the long-distancestomatal responses to CO2

The roles of phytohormones (ethylene, abscisic acid,gibberellins, cytokinins) in regulating responses toenvironmental stimuli have been known for some time(Taiz and Zeiger, 1991). Many studies have elucidated

CO2 signalling in plants 185

the mechanism by which phytohormones integrate withbiochemical signal transduction pathways to regulateplant processes (Kende, 1993; Bleecker and Kende, 2000;Keiber, 1997; Leung and Giraudat, 1998; Richards et al.,2001; Mok and Mok, 2001). In addition, a whole rangeof new signalling molecules has also been discovered,for example, NO3 (Stitt, 1999), PO4, (Sadka et al., 1994)and sugars (Sheen, 1990). These signalling moleculesand the regulatory systems that bring them into playcan be cell specific (Chrispeels et al., 1999), involve shortdistance cell-to-cell communication (Zambryski andCrawford, 2000; Fletcher and Meyerowitz, 2000), andlonger distance signalling, for example, leaf-to-leaf orroot-to-shoot (Bowles, 1998; Havelange et al., 2000).

The use of mutants showing resistance or susceptibilityto a particular stress or environmental cue, throughdefective or deficient biochemistry, have been usefulfor identifying pathways involved in environmental anddevelopmental responses of plants (Smeekens, 2000).More recently, molecular and genetic techniques havebeen applied, at very fine levels, to identify the intricacyof the processes involved, such as by transcription andtranslation of specific genes. A large number of signaltransduction pathway mutants of Arabidopsis thaliana

have been characterized that have lesions in particularcomponents of a range of signal transduction pathways.As a first step to elucidating the biochemical basis ofthe systemic CO2 signalling pathway the screening of aselection of these mutants to identify those pathways thatmay be critical for the long-distance signalling of stomatalresponses to changes in CO2 was undertaken. The avail-able mutants were chosen specifically to cover severalaspects of the major biochemical pathways involved inlong-distance environmental or developmental responses,but were by no means comprehensive.

Some mutations that do not influencestomata

Changes in stomatal index can arise from changes indensity of stomatal complexes alone, changes in epidermalcell density alone, or by a combination of changes in bothstomatal and epidermal cell densities, each of which mayrepresent a different response. A number of mutantstested showed no effect on the long-distance signallingpathway, however, several mutants did interfere withstomatal density responses to elevated CO2 (Lake, 2001).

Fig. 1. Stomatal density (a), index (c) and epidermal cell density (e) of Arabidopsis Col-0 newly expanded leaves (insertions 16 to 19) when matureleaves (insertions 5 to 13) are supplied with ambient (360 ppm) CO2 concentrations (white bars) or elevated (720 ppm.) CO2 concentrations(black bars). Air external to the cuvettes and surrounding the expanding leaves is ambient (360 ppm.) CO2. w***P-0.0005, *P¼ 0.05, Student’s t-test,bar¼ SE mean, n¼ 150x. The experiment above was repeated with the atmospheric CO2 concentrations reversed (expanding leaves at elevated CO2) todemonstrate that the systemic signal was reversible. Stomatal density (b), index (d) and epidermal cell density (f) of Arabidopsis Col-0 newly expandedleaves (insertions 16 to 19) when mature leaves (insertions 5 to 13) are supplied with ambient (360 ppm) CO2 concentrations (white bars) or elevated(720 ppm.) CO2 concentrations (black bars). Air external to the cuvettes and surrounding the expanding leaves is elevated (720 ppm.) CO2.w***P-0.0005, Student’s t-test, bar¼ SE mean, n¼ 150x. NS¼ not significantly different.

186 Lake et al.

A summary of these results is provided in Table 1. Themutants fah-1, jar-1 and ed5 show no effect on the long-distance CO2 signalling pathway when tested using thisexperimental design. The fah-1 gene encodes the enzymeferulate-5-hydroxylase that is required for the synthesisof sinapate esters, particularly sinapoylmalate, thataccumulate in the adaxial epidermis of leaves and provideresistance against UV-B irradiation (Chapple, 1998).These intermediates are also involved in lignin bio-synthesis that is frequently linked with plant defenceresponses and involved in systemic signalling. jar-1 is ajasmonate-insensitive mutant. Jasmonates are knownto regulate a variety of plant developmental responses(Creelman and Mullet, 1997) and also to be induced bypathogen attack or wounding which often leads to thegeneration of oxidative bursts and the generation ofreactive oxygen species (ROS) (Lamb and Dixon, 1997).The systemic nature of plant wounding responses and thevolatile nature of jasmonates have linked this compoundwith systemic signalling, though the precise mechanismfor long-distance transport remains unclear. ed5 is anuncharacterized ozone-sensitive mutant (P Conklin,personal communication). Several mutants (vtc) havebeen identified that are ozone-sensitive and these largelyhave reduced ascorbic acid concentrations (Smirnoffet al., 2001).

Involvement of the jasmonate signallingpathway

fad-4, a chloroplastic fatty-acid desaturase-deficientmutant has a mutation in the bi-directional pathway

controlling the generation of jasmonates via lipoxygenase(LOX) from linolenic acid. The mutant is devoid of LOXand is therefore jasmonic acid-deficient. Results of thelong-distance signalling experiments with the mutantfad-4 suggest that the major LOX-controlled jasmonatecascades, associated with defence and pathogen attack,are involved in the long-term stomatal responses to CO2.This contrasts with the jar-1 mutant which exhibits wild-type stomatal responses to CO2, thereby suggesting thatinsensitivity to jasmonate and LOX levels does not affectthe capacity of plants to acclimate to elevated CO2, withrespect to stomatal numbers. The fad-4 mutant, however,does implicate the LOX-controlled pathway by showinga lack of stomatal index response, of both leaf surfaces,indicating a loss of normal responses. As a LOX andtherefore, jasmonate-deficient mutant, this suggests thata fully functioning jasmonate pathway may be necessaryfor these particular responses. The fad-4 mutant alsoimplicates the linolenic acid-derived part of the jasmonatefatty acid pathway, mediated by HPL (hydroperoxidelyase). An interesting study of the fate of fatty acidcomposition in isolated chloroplasts of Nicotianatabaccum grown at 700 ppm. CO2, shows that the degreeof saturation of fatty acids is generally increased com-pared to controls; in particular, the accumulation of C16 : 0

(palmitic acid) and the transition of C16 : 0 to C16 : 1

(hexadecanoic acid, the function of the fad-4 desaturase;McCourt et al., 1985) is increased (Radunz et al., 2000).The results here suggest that the fad-4 mutation in someway disrupts this pattern of fatty acid metabolism atelevated CO2, which may affect developmental signallingand processes. The two component parts of fatty-acidmetabolism (JA and HPL) have recently been shown to

Table 1. Stomatal density and index and epidermal cell density response of Arabidopsis thaliana (Col-0) leaves to growth in elevatedCO2(720 ppm)

�ve indicates a significant reduction, qve indicates a significant increase (Student’s t-test, P-0.005, n¼ 150) in density or index relative to similarplants grown at ambient CO2 (360 ppm). Those responses that deviate from the wild type (wt) are highlighted in blue, diminished but significantresponses highlighted in red; *, indicates Landsberg erecta background ecotype of Arabidopsis. Biochemical signalling pathway mutants (Col-0 parentalbackground): the mutant plants used were; fah-1: ferulate-5-hydroxylase-deficient, phenylpropanoid pathway mutant (Chapple et al., 1992); fad-4:chloroplastic fatty acid desaturase-deficient, jasmonate pathway mutant (Hugly et al., 1991; McCourt et al., 1985); jar-1: jasmonate-resistantuinsensitive, lypoxygenase activation-deficient, jasmonate pathway mutant (Staswick et al., 1992); vtc-1y: vitamin C (ascorbate)-deficient,ascorbate pathway mutant (Conklin et al., 1996, 1997); ed5: uncharacterized ozone-sensitive mutant (PL Conklin, personal communication);ein-2: ethylene-insensitive, ethylene pathway mutant (Guzman and Ecker 1990; Chao et al., 1997; Lawton et al., 1994); aba-1: abscisic acid-deficient,ABA pathway mutant (Duckham et al., 1991).

Plant type Stomatal density Stomatal index Epidermal density

Abaxial Adaxial Abaxial Adaxial Abaxial Adaxial

wt �ve �ve �ve �ve No response No responsefah-1 �ve �ve �ve �ve No response No responseed5 �ve �ve �ve �ve No response No responsejar-1 �ve �ve �ve �ve No response No responsefad-4 �ve �ve No response No response �ve �vevtc-1y �ve No response �ve No response No response No responseein-2 �ve �ve No response �ve �ve �veaba-1* qve qve qve qve No response No response

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act independently during an investigation of volatilecompound release in lime bean in response to wounding,and also that the initial steps of the octadecanoidpathway (lipid peroxidation) occur within chloroplasts(Koch et al., 1999). One part of the pathway may actsecondarily to the other in order to differentiate betweenparticular response mechanisms. Interestingly, the recentwork by Gray et al., also implicates fatty acid metabolismin the long-term stomatal responses to CO2 enrichment,with the negatively regulating gene, HIC, encoding fora component of the guard cell fatty-acid condensingenzyme (Gray et al., 2000). Other wax biosynthesismutants (cer1, cer6) also have altered stomatal index(Gray et al., 2000). The fad-4 mutant exhibits a wild-typestomatal density response to CO2, but a lack of responseof both leaf surfaces via an epidermal cell density-inducedchange in stomatal index. This indicates that epidermalcell development has been disrupted. The hic mutantexhibits an increase in stomatal index with elevated CO2

without a concomitant change in epidermal cell density,suggesting that the HIC gene affects stomatal develop-ment independently of epidermal cell development,supporting the hypothesis of differential control of thesecell types in response to CO2. The fdh (fiddlehead) mutantof Arabidopsis produces aberrant epidermal cell develop-ment and is also thought to encode for condensingenzymes of fatty acid metabolism (Yephremov et al.,1999), providing further evidence for the role of fattyacids in regulating epidermal cell development. Severalketoacyl CoA synthase (KCS) genes have now beenidentified that are associated with fatty acid metabolismand while some KCS gene products appear to beassociated with the production of waxes, for example,KCS1, and seed storage lipids (e.g. FAE1) it is becomingclear that other KCS genes, including HIC, play a rolein developmental regulation. For example, FDH isinvolved in controlling the number of trichomes thatdevelop in the epidermis (Yephremov et al., 1999), andCUT1 is necessary for normal stem morphology andpollen fertility (Millar et al., 1999).

Involvement of the ethylene signalling pathway

The mutant ein2 is a strongly ethylene-insensitive mutant,lacking the normal ethylene-induced triple response andshowing increased susceptibility to pathogen attack. Thismutant is also insensitive to abscisic acid (ABA) (Woestand Keiber, 1998). The ein2 component of the pathwayis thought to be membrane bound and central to ethylene-induced and ethylene cross-talking responses, althoughthe cellular location remains unknown (Alonso et al.,1999). Ethylene signalling has been implicated in thedevelopment of root epidermal characteristics (Dolanand Roberts, 1995) and in the stomatal patterning of

Arabidopsis (Serna and Fenoll, 1997). Results of the ein2long-distance signalling experiments (Table 1) show awild-type response for stomatal density, but a lack ofresponse in stomatal index on the abaxial leaf surfaceonly. Stomatal index of the adaxial leaf surface showsa wild-type response. Epidermal cell density, as in themutant fad-4, is reduced on both leaf surfaces. A lack ofstomatal index response on the abaxial surface suggeststhat no change in stomatal numbers has occurred relativeto epidermal cell numbers, i.e. a lack of stomatalresponse. A reduction in stomatal index on the adaxialsurface suggests the presence of a stomatal response aswell as a reduction in epidermal cell numbers. The resultsobtained with the ein2 mutant therefore indicate thatethylene signalling has an effect on epidermal cellnumbers, causing a reduction when mature leaves aresupplied with elevated CO2, to both leaf surfaces, butmore specifically, ethylene is required for normal stomatalresponses on the abaxial leaf surface, independently of anadaxial response.

Differential responses of abaxial and adaxial leafsurfaces, with respect to both development and physio-logical activity are known. Studies involving mutants ofArabidopsis (Bowman, 2000; McConnell and Barton,1998) and Antirrhinum majus (Waites and Hudson, 1995)illustrate the independent control of development of eachleaf surface. Stomatal aperture control is also independ-ent for each leaf surface, as shown in the amphistomatousspecies, Rumex obtusifolius, when grown at ambientand elevated CO2 (Pearson et al., 1995). These findingssupport the view that abaxial and adaxial stomataldevelopment may also be under independent control.Evidence for ethylene-regulated responses specific to theabaxial leaf surface comes from a study of the accumu-lation of ethylene-induced chitinase and b-1,3-glucanaseproteins, associated with fungal and pathogen attackand normal floral development. Distribution of theseproducts following exogenous ethylene application wasconfined to the lower epidermis and along vascularstrands in bean leaves (Mauch et al., 1992). Bergerand Altmann suggested that ethylene may play aregulatory role in stomatal developmental responses,as Arabidopsis seedlings treated with ACC (the ethyleneprecursor) caused clustering of stomata (Serna andFenoll 1997), which may be the result of disruptedepidermal cell development, rather than stomatal devel-opment (Berger and Altmann, 2000). One ethylene-induced developmental response is the inhibitionof cell expansion in leaves (Johnson and Ecker, 1998),which the ein2 phenotype does not exhibit, therefore,the reduction in epidermal cell number in ein2, atelevated CO2, may be due to a lack of inhibition ofcell expansion (i.e. cells continue to expand) and impliesethylene-regulation of epidermal cell expansion in thewild-type response.

188 Lake et al.

Involvement of ascorbate and the ROS signallingpathway

The vtc-1 (ascorbate-deficient) mutant exhibits two inter-esting results when mature leaves are supplied withelevated CO2. The first shows diminished stomatal density(7% decrease compared to 21% decrease in the wild type)and index (4% decrease compared with 8% in the wildtype) responses of the abaxial leaf surface, compared tothe wild type (Table 1). As the mutant produces only;30% of wild-type levels of ascorbate (Conklin et al.,1997), the responses of stomatal density and index to CO2

of the abaxial surface may be regulated in part byascorbate concentrations. The second result is a lack ofstomatal density and index responses of the adaxialsurface. Epidermal cell densities, although slightly higherthan wild-type levels, are not significantly differentbetween ambient and elevated CO2-treated plants. Theseresults again suggest that both stomatal and epidermalnumbers of each leaf surface may be regulated indepen-dently. The vtc-1 mutant, lacking ascorbate, will exertless control over the detoxification of ROS, and H2O2 inparticular. Plants perceive accumulation of H2O2 as asignal of environmental change (Noctor and Foyer,1998). As a diffusible molecule, H2O2 is now implicatedin long distance, or systemic, signalling of severalresponse mechanisms including pathogen-induced sys-temic acquired resistance (Noctor and Foyer, 1998),drought, heavy metal toxicity, extremes of temperatureand atmospheric pollutants (May et al., 1998), and excesslight (Karpinski et al., 2000), which appear to beconcentration dependent (Foyer et al., 1997).

Research into ozone damage and acclimationresponses reveal that ROS are produced apoplasticallyby the pollutant causing the production of ROS by theplant (Kettunen et al., 1999), again suggesting that ROSconcentration may be utilized to differentiate betweenstresses and produce variable responses. It is also knownthat some ozone-treated plant species exhibit a changein stomatal density, generally increasing with increasingamounts of ozone. Minnocci et al. found an increase of37% in stomatal density with a leaf area decrease of 19%and stomatal aperture decrease of 63% in olive plantstreated with 100 ppb ozone (Minnocci et al., 1999), whilstPaakkonen et al. found significant increases in stomataldensity of two Betula species in response to increasedozone exposure (Paakkonen et al., 1997). The results ofthe vtc-1 mutant support the view that concentration-dependent control of ROS is the most likely effect onstomatal development in these experiments.

The aba-1 mutant response shows a significant increasein stomatal density and index of both leaf surfaces, withno change in epidermal cell numbers when mature leavesare supplied with elevated CO2. The overall stomataldensity (;400 mm�2) is almost twice that of the wild

type. This suggests that abscisic acid may exert aregulatory role over stomatal development in responseto CO2. Although in this set of experiments it was notpossible to include the parental wild-type, Landsberg,previous work suggests that Landsberg is largely non-responsive under CO2 enrichment, with respect tochanges in stomatal density (Woodward et al., 2001).The aba-1 mutant results, however, are of interest in thatABA and elevated CO2 concentration can have syner-gistic effects on stomatal closure in Arabidopsis, wherebyABA responses are enhanced (Leymarie et al., 1999).Differential roles of ABA in signalling stomatal apertureresponses to soil water potential and atmospheric watervapour pressure were found using ABA-deficient andABA-insensitive mutants (Assmann et al., 2000), suggest-ing that ABA control is subtle and specific to particularenvironmental cues.

Involvement of the sugar signalling pathway

The rate of photosynthesis is enhanced under elevatedCO2 due to direct effects on the rates of Rubiscocarboxylation and oxygenation. This leads to an increasein the rate of carbohydrate production. Plants grown atelevated CO2 often do not sustain this increase inphotosynthesis and frequently a down-regulation ofphotosynthetic gene expression reduces photosyntheticcapacity. Carbohydrate accumulation (Van der Kooijet al., 1999) and subsequent repression of photosyntheticgene expression (Cheng et al., 1998) is typically observedin Arabidopsis. Whether this regulation is due to sugar-mediated control of gene expression (Smeekens, 2000) ordue to induction of accelerated senescence (Ludewig andSonnewald, 2000) is unclear. Sugar signalling has recentlybeen identified as a major pathway involved in manyresponses including up- or down-regulation of photo-synthetic capacity (associated with changes in CO2 con-centration), and developmental responses, includingregulation of the cell-cycle and therefore, growth rate(Riou-Khamlichi et al., 2000). This developmentalresponse is known to be sensitive to CO2 in populationsof Dactylis glomerata (Kinsman et al., 1997). Sugar-induced pathways also interact with other signal trans-duction pathways including ethylene and PAL, as well asintegrating responses with ABA and other hormones(Smeekens, 2000). An involvement of sugar-mediatedsignalling in the systemic signalling of CO2 concentrationcannot be ruled out given the direct effect of CO2

on photosynthetic carbohydrate production. However,evidence from variegated leaves where achlorophyllousportions of leaves have functional stomata (Lafray et al.,1991) and much reduced carbohydrate content comparedto neighbouring photosynthetic regions (Madore, 1990)show normal reductions in stomatal density in responseto elevated CO2 (Beerling and Woodward, 1995). This

CO2 signalling in plants 189

would suggest that local sugar concentrations werenot influencing systemic control of stomatal develop-ment. Further, stomatal density is generally lower in theachlorophyllous regions of variegated leaves (Salisbury,1927; Virgin, 1957) analogous to the effect of increasedCO2 despite contrasting concentrations of carbo-hydrates that prevail in these conditions. A schematicthat summarizes our findings with these mutants ofArabidopsis is shown in Fig. 2.

Conclusion

Following the experimental investigations into long-termstomatal responses to CO2 it is clear that long-termchanges in stomatal numbers in the Arabidopsis ecotypeColumbia involve signal mechanisms from mature tonewly developing leaves. These mechanisms are utilized inresponses to both CO2 concentration and irradiance(Lake et al., 2001). The biochemical signal transductionpathways implicated in responses to CO2 suggest thatstomatal development is controlled independently fromepidermal cell development, both of which operate tooptimize stomatal spacing within the epidermis, and thatabaxial and adaxial leaf surfaces are also independentlycontrolled. Fatty acid metabolism is strongly implicatedin regulating developmental processes of both the abaxialand adaxial epidermis as evidenced by the inhibition ofsystemic CO2 signalling in the fad-4 mutant and thedisruption of stomatal responses to CO2 in the hic mutant(Gray et al., 2000). It is conceivable therefore, that fattyacids may play a crucial, even primary, role in theresponse pathways of stomatal regulation by CO2 thatmay involve the LOX-mediated signalling system. Afurther role in this process for ABA is suggested by a lackof normal wild-type responses of systemic CO2 signallingin the ABA-deficient (aba-1) mutant. It is intriguing thatABA, a hormone central to the biochemical functioning

of stomata, may also influence stomatal development.Ascorbate also appears to play an important role in thissignalling response albeit on the adaxial surface; its rolein the detoxification of ROS could suggest that theconcentration of ascorbate could modulate cellularresponses to these signalling molecules by altering theirconcentration in the apoplast. These results wouldsupport the recent evidence for the role of ROS insystemic signalling in plants. ROS, and H2O2 inparticular, and their control appear to be an importantcomponent of the long-distance signal-response to CO2

and ozone (Foyer et al., 1997). Hydroxy fatty acids areknown to elicit H2O2 production (Fauth et al., 1998),which is also implicated as a signal molecule in responsesto light (Karpinski et al., 1997, 2000) thereby providing apotential signalling link between systemic stomatalresponses to both light and CO2 (Lake et al., 2001) andto the involvement of fatty acid metabolism. A role forethylene in this signal transduction pathway is demon-strated by the inhibition of stomatal response to CO2

in the ethylene-insensitive ein-2 mutant, although thismutant is also known to be ABA-insensitive. This is inline with a previous suggestion for a direct effect ofethylene on stomatal development (Serna and Fenoll,1997). Further, the specificity of the ethylene response forthe abaxial surface provides two pathways for regulatingstomatal development independently on both surfaces of

Fig. 2. Diagram summarizing the signal mechanisms involved in long-term stomatal responses to CO2. (a) Signals from mature to new leavesare blocked in the fad-4 and aba-1 mutant. (b) Adaxial and abaxial leafsurfaces are independently controlled by involvement of ascorbate andethylene pathways shown by diminished and blocked responses of thevtc-1 and ein-2 mutants, respectively.

Fig. 3. Proposed model of signal-response mechanisms involved instomatal and epidermal cell developmental responses to pCO2. Blacklines indicate direct steps in the signal transduction pathways, red linesare potential routes by which pathways can interact, blue lines arehypothetical interactions with sugar-signalling pathways.

190 Lake et al.

the leaf, a phenomenon well known in the literature. Amodel that illustrates the results obtained from ourstudies of these signal transduction mutants is shown inFig. 3; this is clearly preliminary and much more workis required to elucidate the precise nature of these signaltransduction pathways. Many more mutants with lesionsin different pathways will need to be examined beforethe entire gambit of signalling pathways is exhausted andthe true nature of the systemic CO2 signalling pathwayis identified.

Finally, the question of how plants sense CO2 is onethat has not been addressed here thus far, probablybecause it is the most difficult to answer; however a recentreview has discussed this question (Assmann, 1999) andso this is only briefly mentioned here. No CO2 receptorshave yet been identified in any organism, yet it is appar-ent that many organisms have the ability to respond toCO2, including cyanobacteria (Sultemeyer et al., 1998),algae (Fukuzawa et al., 2001), insects (Keil, 1996),and mammals (Ofelia et al., 1999). The mechanism bywhich this occurs is still uncertain and whether a trueCO2-receptor exists or whether equilibrium of dissolvedCO2 with the bicarbonate pool causes pH-inducedchanges has yet to be established. However, electro-physiological data demonstrates that CO2 can have directeffects on ion channels located in the plasmamembrane(Brearley et al., 1997). Direct evidence for CO2 involve-ment in signal transduction pathways is at presenttenuous, however, a regulatory protein (CCM1) wasrecently identified from Chlamydomonas that is requiredfor the CO2-mediated induction of the carbon concentra-tion pathway (Fukuzawa et al., 2001), protein phos-phorylation is thought to be involved in this induction incyanobacteria (Sultemeyer et al., 1998) and CO2 activa-tion of HCO3

� uptake in mammals has been shown toinvolve the mitogen-activated protein kinases (MAPK)signal transduction pathway (Ofelia et al., 1999).

Acknowledgements

We would like to thank Dr Julie E Gray for critical reading ofthe manuscript and Dr P Conklin for donation of seeds. Theauthors are grateful to the BBSRC (50uP15690) and NERC(ROA13734) for financial support of this work.

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