the responses of guard and mesophyll cell photosynthesis to co2, o2, light, and water stress in a...

DOI: 10.1093/jxb/erg186

RESEARCH PAPER

The responses of guard and mesophyll cellphotosynthesis to CO2, O2, light, and water stress in arange of species are similar

Tracy Lawson, Kevin Oxborough, James I. L. Morison* and Neil R. Baker

Department of Biological Sciences, University of Essex, Colchester CO4 3SQ, UK

Received 20 December 2002; Accepted 7 April 2003

Abstract

High resolution chlorophyll a ¯uorescence imaging

was used to compare the photosynthetic ef®ciency of

PSII electron transport (estimated by Fq¢/Fm¢) in guard

cell chloroplasts and the underlying mesophyll in

intact leaves of six different species: Commelina

communis, Vicia faba, Amaranthus caudatus,

Polypodium vulgare, Nicotiana tabacum, and

Tradescantia albifora. While photosynthetic ef®ciency

varied between the species, the ef®ciencies of guard

cells and mesophyll cells were always closely

matched. As measurement light intensity was

increased, guard cells from the lower leaf surfaces of

C. communis and V. faba showed larger reductions

in photosynthetic ef®ciency than those from the

upper surfaces. In these two species, guard cell

photosynthetic ef®ciency responded similarly to that

of the mesophyll when either light intensity or CO2

concentration during either measurement or growth

was changed. In all six species, reducing the O2 con-

centration from 21% to 2% reduced guard cell photo-

synthetic ef®ciency, even for the C4 species A.

caudatus, although the mesophyll of the C4 species

did not show any O2 modulation of photosynthetic

ef®ciency. This suggests that Rubisco activity is sig-

ni®cant in the guard cells of these six species. When

C. communis plants were water-stressed, the guard

cell photosynthetic ef®ciency declined in parallel

with that of the mesophyll. It was concluded that

the photosynthetic ef®ciency in guard cells is

determined by the same factors that determine it in

the mesophyll.

Key words: Chlorophyll ¯uorescence, guard cell, mesophyll,

photosynthesis, stomata, water stress.

Introduction

The guard cells that form the stomatal pore control the ¯uxof CO2, H2O and other gases between the plant and theatmosphere and are regulated by both internal and externalfactors. Stomatal movements are due to the loss oraccumulation of ions, which require energy (Willmer andFricker, 1996). In the majority of species examined guardcells contain well-developed chloroplasts, unlike the otherepidermal cells from which they are formed. The role ofthese chloroplasts is still not clear, although they are notalways essential to stomatal function since achlorophyl-lous guard cells do open and close (in Paphiopedilum sp.,Nelson and Mayo, 1975). Guard cell chloroplasts havebeen proposed as signi®cant energy sources for H+

extrusion and ion transport (Wu and Assmann, 1993;Tominaga et al., 2001), and are involved in severaldifferent light transduction pathways (Zeiger et al., 2002).Although many studies have shown that guard cellscontain several of the main Calvin cycle enzymes(Shimazaki and Zeiger, 1985; Zemel and Gepstein, 1985;Gotow et al., 1988; Shimazaki et al., 1989), few havepresented conclusive evidence for signi®cant Calvin cycleactivity within these cells (Outlaw, 1989; Reckmann et al.,

* To whom correspondence should be addressed. Fax: +44 (0)1206 873416. E-mail: [email protected]: Ca, ambient CO2 concentration; F¢, chlorophyll ¯uorescence in the light-adapted state; Fm¢, chlorophyll ¯uorescence when PSII centres aremaximally closed in the light-adapted state; Fo¢, chlorophyll ¯uorescence when PSII centres are maximally open in the light-adapted state; Fq¢, differencebetween F¢ and Fm¢; Fq¢/Fm¢, ¯uorescence parameter that provides an estimate of the operating ef®ciency of PSII photochemistry; Fq¢/Fv¢, factor relating theoperating and maximum ef®ciencies of PSII photochemistry; Fv¢, variable chlorophyll ¯uorescence (Fm¢±Fo¢); Fv¢/Fm¢, ¯uorescence parameter that provides anestimate of the maximum ef®ciency of PSII photochemistry (when all PSII centres are open); PPFD, photosynthetic photon ¯ux density; Rubisco, ribulose1,5-bisphosphate carboxylase oxygenase; VPD, vapour pressure de®cit.

Journal of Experimental Botany, Vol. 54, No. 388, ã Society for Experimental Biology 2003; all rights reserved

Journal of Experimental Botany, Vol. 54, No. 388, pp. 1743±1752, July 2003

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1990), and there is continuing debate about the role andnature of guard cell photosynthesis (Zeiger et al., 2002).Chlorophyll ¯uorescence is a powerful, non-invasivetechnique to investigate photosynthetic metabolism inguard cells. The majority of chlorophyll ¯uorescencemeasurements from guard cells have been restricted toepidermal peels (Melis and Zeiger, 1982; Ogawa et al.,1982) or guard cell protoplasts (Goh et al., 1997, 1999,2001) or have used variegated leaves; they have mainlybeen restricted to ¯uorescence induction curves orresponses of the steady-state ¯uorescence signal (F¢).However, using high spatial resolution chlorophyll a¯uorescence imaging in intact green leaves ofCommelina communis, it has recently been shown thatguard cell quantum ef®ciency for PSII photochemistry(Fq¢/Fm¢=(Fm¢±F¢)/Fm¢, `photosynthetic ef®ciency') wasapproximately 70±80% of that of the mesophyll cells(Baker et al., 2001; Lawson et al., 2002) across a widerange of light intensities. It has also been shown thatphotosynthetic ef®ciency in both guard and mesophyllcells of C. communis was similarly altered by O2

concentration at low, but not at high CO2 concentration,indicating that photorespiration is a major sink for ATPand NADPH produced through electron transport in guardcells, and that Rubisco activity is signi®cant (Lawson et al.,2002).

Some of the previous disagreements over the role ofchloroplast photosynthetic activity in guard cells could bedue to differences in plant material, as a range of speciesare widely used in stomatal physiological studies fromvery different taxa with different stomatal anatomy andoften grown in different conditions. As Zeiger et al. (2002)have recently emphasized, guard cell chloroplasts showremarkable functional plasticity. The aims of this studywere (1) to compare guard and mesophyll cell photosyn-thetic ef®ciencies in six species previously used instomatal physiology, and (2) to investigate the responseto different growth and measurement conditions, inparticular light, CO2 and O2 concentrations, and waterstress. The choice of species was largely dictated bywhether the stomata were large enough to be imagedclearly, but included a fern (Polypodium vulgare) and a C4

species (Amaranthus caudatus) to compare with the C3

species usually used in stomatal physiology.

Materials and methods

Plant material

Seeds of Commelina communis L., Nicotiana tabacum L. and Viciafaba L. were sown in a peat and loam based compost (F2, LevingtonHorticulture Ltd, Ipswich, UK) in a controlled environment chamber(SGC066 Fitotron, Sanyo Gallenkamp, Leicester, UK). After 3weeks, seedlings were transplanted into 100 mm diameter pots andused 6±7 weeks after sowing in the case of C. communis andN. tabacum and 4±5 weeks in V. faba. Cuttings of the variegatedplant Tradescantia albi¯ora Kunth. were grown in the same compost

and environment chamber. The chamber air temperature wasmaintained at 18 °C at night and 22 °C through the day. Light wasprovided by halogen quartz iodide lamps (Neutralweiss, Germany)from 06.00±21.00 h, at a constant PPFD of 530 mmol m±2 s±1 at plantheight. Relative humidity was maintained at 70% through the dayand 65% at night. Amaranthus caudatus L. and Polypodium vulgareL. were grown from seed in the same compost and maintained in theglasshouse where supplementary lighting was provided by high-pressure sodium lamps and the temperature was maintained between18 °C and 30 °C. All plants were kept well watered using capillarymatting, except those in the drought stress experiment.

Growth treatments

Drought stress: Plants of C. communis were grown in the controlledenvironment chamber until about 6-weeks-old. Water was thenwithheld from half the plants for 12 d, while control plants were keptwell watered. Leaf water potential was measured using a pressurechamber (SKPM1400, Skye Instruments, Powys, UK). After 12 d,stressed plants were rewatered, resulting in full recovery of the waterpotential after 2 d.

Elevated CO2: C. communis and V. faba were grown in twocontrolled environment chambers as above, but CO2 concentrationwas maintained at either 360 or 700 mmol mol±1 using an injectionsystem operated by an infrared gas analyser (WMA-2, PP Systems).

Growth PPFD: Plants of C. communis and V. faba were grown in theabove controlled environment chambers, but half of the chamberwas shaded, using neutral density tissue paper to give PPFDs at theheight of the plants of approximately 640 and 260 mmol m±2 s±1.

The microscope imaging system

The optical part of the imaging microscope used in these experi-ments is essentially the same as that described previously(Oxborough and Baker, 1997) with the modi®cation of the lowerlight source and a purpose-designed microscope cuvette attached toan infrared gas analyser to control CO2, O2 and VPD as described byLawson et al. (2002). CO2 of known concentration was suppliedthrough the gas analyser system (CIRAS1; PP Systems, Hitchin,UK), and known O2 concentration was supplied from external gasbottles (BOC, Surrey, UK) attached to the air inlet on the gasanalyser. Unless otherwise stated, conditions in the microscopecuvette were 23±25°C with 21% O2, a Ca of 360 mmol mol±1 and aVPD of approximately 0.6 kPa. Chlorophyll ¯uorescence wasde®ned by a 680 nm bandpass ®lter (Coherent, Watford, UK). Fo¢and Fm¢ de®ne the minimal and maximal ¯uorescence levels fromleaves in the light, respectively. F¢ is the ¯uorescence level at anypoint between Fo¢ and Fm¢. For the construction of parameterizedimages, the speci®c term Fq¢ was recently introduced (Oxboroughand Baker, 1997; Oxborough et al., 2000) which denotes thedifference between Fm¢ and F¢ measured immediately beforeapplication of a saturating pulse to measure Fm¢. Under theseconditions, Fq¢/ Fm¢ equates to the operating quantum ef®ciency ofPSII photochemistry. Images of Fv¢/Fm¢ and Fq¢/Fv¢ were generatedfrom images of Fo, Fm and Fm¢ as described previously (Lawsonet al., 2002). There was no attempt to estimate rates of electrontransport from Fq¢/ Fm¢ because there are uncertainties concerningthe exact light absorption and contribution of PSI ¯uorescence forthe guard and mesophyll cell chloroplasts. All images were takenfrom the abaxial surface of leaves (except where stated) using a 403objective, which provided images of 3103205 mm with a pixel sizeof 534 nm2. Replicates are individual stomatal complexes on leavesof different plants. The mesophyll areas used for comparison werethose immediately adjacent to the guard cells. Chloroplasts withinguard cell pairs were isolated from images using the ends-in search

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and other editing tools described in Oxborough and Baker (1997)and Oxborough et al. (2000).

Statistical analysis

Mean values of chloroplast photosynthetic ef®ciencies (Fq¢/Fm¢)were calculated from the images, and differences between species,cell type (mesophyll or guard cell, abaxial or adaxial) or treatments(light, CO2 or O2 concentration) were compared using ANOVAswith mixed `between subjects' (e.g. species) and `within subjects'(e.g. cell type, CO2 or O2 concentration) designs as appropriate. Thedata in the regression in Fig. 6b were examined for the effect of anytreatments using analysis of covariance. All statistical analyses werecarried out with SPSS v. 10, or Systat v. 5.

Results

Fluorescence images and guard cell chloroplastappearance

Images of steady-state ¯uorescence (F¢) from guard cellsin the six species examined are shown in Fig. 1, anddemonstrate the variation in stomatal pore and guard cellsize in the different species and the differences inchloroplast number and orientation within individualguard cells. For example, in N. tabacum (Fig. 1d) thechloroplasts are located on the outer wall of the guard cells,

whereas in T. albi¯ora (Fig. 1e) chloroplasts appear to bedistributed evenly throughout the cells around the vacuole.Guard cells of the fern P. vulgare (Fig. 1c) contain manychloroplasts, as do the other epidermal cells, as previouslynoted (Willmer and Fricker, 1996). Despite these differ-ences, all the chlorophyll ¯uorescence parameters werereadily measured and hence photosynthetic electrontransport ef®ciency (estimated by Fq¢/Fm¢) could becalculated for both guard cells and underlying mesophyllcells in all of the species.

Photosynthetic ef®ciencies in guard and mesophyllcells in different species

There were signi®cant differences between species inFq¢/Fm¢ of guard and underlying mesophyll cells duringsteady-state photosynthesis under identical measurementconditions (species difference P <0.001; Fig. 2a). The fernP. vulgare exhibited the lowest ef®ciencies, with guardcell and mesophyll Fq¢/Fm¢ values of 0.24 and 0.31,respectively, while the maximum values of 0.62 and 0.65,respectively, were found in V. faba. The low values ofFq¢/Fm¢ observed in P. vulgare are notable, particularly asthe large number of chloroplasts present might have beenexpected to result in a high degree of self-shading, whichwould reduce the average incident-light intensity and,consequently, result in a higher photosynthetic ef®ciency.One explanation might be the higher light level duringgrowth reducing photosynthetic ef®ciency as these plantswere glasshouse-grown, but A. caudatus was grown in asimilar environment and yet showed higher ef®ciencies.For P. vulgare, N. tabacum and T. albi¯ora guard cellFq¢/Fm¢ values were 79%, 92% and 87% of the values ofthe adjacent mesophyll cells, respectively, although nosigni®cant differences were observed for A. caudatus,V. faba and C. communis (overall cell type differenceP <0.001; species3cell type interaction P=0.049), andthere was a close linear correlation between mesophyll andguard cell Fq¢/Fm¢ (Fig. 2b).

Effect of growth and measurement PPFD

Photosynthetic ef®ciency is dependent both on measure-ment PPFD and on growth conditions which affect thedevelopment of the photosynthetic apparatus, as exempli-®ed by the large differences observed for sun and shadeleaves (Pearcy, 1998). In species that are amphistomatous,stomatal guard cells provide an interesting system toexamine the effect of growth PPFD on photosyntheticbehaviour, as guard cells in the upper surface are exposedto much higher PPFD than those on the lower surface. InC. communis and V. faba, plants grown at moderate lightintensity (530 mmol m±2 s±1) guard cell Fq¢/Fm¢ in bothupper (adaxial) and lower (abaxial) leaf surfaces decreasedwith increasing incident PPFD (Fig. 3; P <0.001). The twospecies differed in the response of the photosyntheticef®ciency of the upper and lower guard cells to PPFD

Fig. 1. Steady-state chlorophyll ¯uorescence images (F¢) obtainedunder the microscope from intact leaves showing abaxial stomatal-guard cells with chloroplasts of (a) Amaranthus caudatus; (b)Commelina communis; (c) Polypodium vulgare; (d) Nicotianatabacum; (e) Tradescantia albi¯ora, and (f) Vicia faba.

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(P=0.003). In C. communis the decrease of Fq¢/Fm¢ wassubstantially larger in guard cells in the lower surface(Fig. 3a). In V. faba at the lowest measurement PPFD of 93mmol m±2 s±1 guard cells in the lower surface operated at ahigher ef®ciency than those in the upper surface (Fig. 3b).However, as PPFD increased there were large decreases inef®ciency in guard cells in both surfaces and no signi®cantdifferences were observed between the guard cells on thelower and upper surfaces (Fig. 3b). Fq¢/Fm¢ was lower inguard cells of V. faba than those from C. communis at thehigher PPFD levels, particularly in the upper surface.

The in¯uence of growth PPFD on the lower surfacestomata and any possible acclimation to light was studiedby growing C. communis and V. faba at PPFD of 260 and640 mmol m±2 s±1 and measuring photosynthetic ef®ciencyat PPFDs of 93 and 428 mmol m±2 s±1 (Fig. 4). WhileFq¢/Fm¢ declined in higher measurement PPFD in both cell

types in both species (P <0.001), overall the photosyntheticef®ciencies measured in V. faba were about 0.10 lowerthan those of C. communis (overall species differenceP=0.026), and there was a larger relative response ofFq¢/Fm¢ to measurement PPFD in V. faba (P=0.012). Therewas also a small (but signi®cant, P=0.018) differencebetween species in the response to growth PPFD, with C.communis plants grown at 640 mmol m±2 s±1 having ahigher Fq¢/Fm¢ for both guard and mesophyll cells,regardless of the measurement PPFD (Fig. 4a). In contrast,V. faba showed no differences in Fq¢/Fm¢ between plantsgrown under the two different light intensities (Fig. 4b).While there were no signi®cant differences between Fq¢/Fm¢ of guard and mesophyll cells in each combination ofgrowth and measurement PPFD treatments for C. com-munis, guard cells in V. faba had signi®cantly lower Fq¢/Fm¢ values (approximately 8%) than the mesophyll cells,for all growth and measurement PPFDs (overall celltype3species interaction P=0.018). Both the short-termresponses of Fq¢/Fm¢ to measurement PPFD and the longerterm acclimation to growth PPFD were very similar forboth guard and mesophyll cells (Fig. 4c).

Effect of CO2 and O2 concentration

The effects of CO2 and O2 concentration on thephotosynthetic ef®ciencies of the guard and mesophyllcells were determined for the six species (Table 1). As in

Fig. 2. (a) Comparison of guard cell (open bars) and mesophyll cell(®lled bars) photosynthetic ef®ciency (estimated by Fq¢/Fm¢) in intactleaves of six species. Measurements were taken after 10 minstabilization under the following conditions Ca 360 mmol mol±1, 21%O2, PPFD 114 mmol m±2 s±1, 24 °C and a VPD of 0.6 kPa. Data arethe means of three replicates 6SE. (b) Relationship between Fq¢/Fm¢of guard cells and mesophyll cells for individual stomata of sixspecies under the conditions detailed above. The broken linerepresents the y=x relationship. (Amar.=Amaranthus caudatus (®lleddiamonds); Comm.=Commelina communis (®lled squares);Polyp.=Polypodium vulgare (®lled triangles); Nicot.=Nicotianatabacum (open squares); Trades.=Tradescantia albi¯ora (opentriangles); and Vicia=Vicia faba (®lled circles).

Fig. 3. Effect of measurement PPFD on guard and mesophyll cellphotosynthetic ef®ciency (estimated by Fq¢/Fm¢) from the lower (openbars) and upper surface (®lled bars) in (a) Commelina communis and(b) Vicia faba. Data are the means of three replicates 6SE.

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the previous experiments, Fq¢/Fm¢ of the guard cells werelower than those of the adjacent mesophyll in some, but notall, species (signi®cant cell type3species interactionP=0.003). Overall, there was a more pronounced increaseof Fq¢/Fm¢ with an increase of [CO2] from 150 to 360 mmolmol±1 for the C3 species than for the C4 species Amaranthuscaudatus (overall CO23species interaction P=0.036; ifdata from A. caudatus are excluded there is no signi®cantinteraction, P=0.274). In mesophyll cells in the ®ve C3

species, decreases of [O2] from 21% to 2% decreasedFq¢/Fm¢ substantially at the lower [CO2] (7±26% reduc-tion), but not in higher [CO2] (there were no signi®cantspecies differences in response to CO2, P=0.232, but there

was a signi®cant overall CO23O2 interaction, P=0.036).The reduction in Fq¢/Fm¢ in low [O2] or low [CO2] wasalmost entirely due to a decrease in Fq¢/Fv¢, with littlechange being observed in Fv¢/Fm¢ (data not shown),indicating that the reduced availability of terminal electronacceptors for electron transport from PSII was primarilyresponsible for the decrease in photosynthetic ef®ciency. Inthe C4 species no effects of [O2] on Fq¢/Fm¢ of themesophyll at either [CO2] were observed. Clearly, in low[CO2] in the mesophyll of C3, but not C4 species, O2 isacting as a sink for the products of photosynthetic electrontransport due to Rubisco oxygenase activity and photo-respiratory metabolism. However, low [O2] decreased Fq¢/Fm¢ of the guard cells in all six species (range from 7±24%)at both CO2 concentrations (O2 effect P <0.001, noCO23O2 interaction, P=0.301), indicating that Rubisco isactive in the guard cells of the C4 species as well as of theC3 species.

Effect of growth CO2 concentration

The effects of growth [CO2] (360 or 700 mmol mol±1) onFq¢/Fm¢ was examined for C. communis and V. faba todetermine if photosynthetic acclimation resulted in differ-ent behaviour of guard cells compared with mesophyllcells (Fig. 5). Fq¢/Fm¢ of guard cells was slightly (2±9%)lower than that of mesophyll cells, except for V. fabagrown in high [CO2] where there was no difference(overall cell type effect, P=0.002). Fq¢/Fm¢ in both guardand mesophyll cells increased by approximately 10% withan increase in measurement [CO2] from 150 to 360 mmolmol±1, but not in higher [CO2]. The effect of growth [CO2]on Fq¢/Fm¢ of both guard and mesophyll cells differedbetween the two species (P=0.014). In C. communis,growth in high [CO2] substantially reduced Fq¢/Fm¢ of bothguard and mesophyll cells in all CO2 measurementconcentrations (Fig. 5a; P <0.001), whereas V. fabashowed no signi®cant effect of growth CO2 concentration(Fig. 5b). The decrease in Fq¢/Fm¢ in C. communis wasattributable to a decrease in Fq¢/Fv¢ since Fv¢/Fm¢ did notchange signi®cantly (data not shown); again this indicatesthat the decrease in photosynthetic ef®ciency is due to adecrease in the ability to use the products of electrontransport.

Effect of water stress on the relationship betweenguard and mesophyll cells

Close correlations were found between the values ofFq¢/Fm¢ of guard and adjacent mesophyll cells for differentspecies and in differing measurement and growth condi-tions (Figs 2c, 4c, 5c). To examine if such correlationsbetween the guard and mesophyll cell photosyntheticef®ciencies are conserved during water stress, C. communisplants were subjected to mild water stress by withholdingwater for 12 d. Leaf water potentials decreased toapproximately ±0.75 MPa in the drought-stressed plants,

Fig. 4. Effect of growth and measurement PPFD on guard andmesophyll cell photosynthetic ef®ciency (estimated by Fq¢/Fm¢) for (a)Commelina communis and (b) Vicia faba. Plants were grown at twoPPFD (260 and 640 mmol m±2 s±1) and measured at 93 and 428 mmolm±2 s±1. Data are the means of three replicates 6SE. (c) Relationshipbetween Fq¢/Fm¢ of guard cells and mesophyll cells for individualmeasurements on C. communis and V. faba at the differentmeasurement and growth PPFD. The broken line represents the y=xrelationship.



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while well-watered controls remained at c. ±0.05 MPathroughout. Stomatal aperture showed a steady decline aswater was withheld, but recovered within 48 h afterrewatering (data not shown). Both guard and mesophyllcell Fq¢/Fm¢ declined during the stress, but recovered alongwith stomatal aperture 48 h after rewatering (Fig. 6a). Thisdecrease in Fq¢/Fm¢ was again largely due to a decrease inFq¢/Fv¢ not in Fv¢/Fm¢ (data not shown), again indicatingthe limitation on photosynthetic ef®ciency being the abilityto use the products of electron transport. However, therewas a close linear relationship between guard andmesophyll cell Fq¢/Fm¢ as the values changed through thedrying cycle which was not distinguishable from that of thecontrol plants (Fig. 6b, pooled regression for all treatmentsr2=0.902). Increasing measurement Ca from 360 to700 mmol mol±1 resulted in a substantial increase inFq¢/Fm¢ of both guard and mesophyll cells in the water-stressed plants (mean increase at the end of the cycle wasfrom 0.23 to 0.37 for mesophyll cells), but only a smallincrease was observed in control plants (Fig. 6b).However, even with increased CO2 concentration Fq¢/Fm¢of drought-stressed plants did not increase to that of thecontrol plants. This suggests that as water stress increased,stomata adjusted to reduce water loss, and restricted CO2

diffusion into the leaf, resulting in a decrease in Fq¢/Fm¢through a reduction in the capacity for the consumptionof the products of electron transport. The relationshipbetween Fq¢/Fm¢ of the guard and mesophyll cells was notaffected by CO2 or water stress (although there is astatistically signi®cant difference of the intercept forthe water stress treatment (P <0.001) at 0.028 it isnegligible).

Discussion

Much of the previous work investigating guard cellphotosynthesis has been con®ned to species such asC. communis or V. faba due to the ease with which theepidermis can be removed from the leaf and the ability to

obtain uncontaminated guard cell protoplasts (Willmer andFricker, 1996). In this study, photosynthetic ef®ciency inguard cells during steady-state photosynthesis has beenmeasured in intact leaves from a number of species.Taxonomically, these species are very diverse, includingone fern and ®ve angiosperms, and the angiospermsinclude two monocots (C. communis and T. albi¯ora, bothin the Commelinaceae) and three dicots, each fromfamilies in different subclasses (V. faba: Fabaceae,subclass Rosidae; A. caudatus: Amaranthaceae, Caryo-phillidae; and N. tabacum: Solanaceae, Asteridae).Consequently, it is believed that these conclusions on theresponses of the photosynthetic ef®ciency of guard cells toenvironmental factors have considerable general applica-tion. In leaves of all species the values of photosyntheticef®ciency for guard cells were either indistinguishablefrom or only slightly lower (minimum of 79%) than thoseof the underlying, spongy mesophyll cells. In all speciesexamined the responses of guard and mesophyll photo-synthetic ef®ciency to changes in light, [CO2] and [O2]were similar, although there were demonstrable differ-ences between species in the actual values of guard cellsand mesophyll, and their responses to growing conditions(Figs 2±5; Table 1). Some of these differences may be dueto the plants being grown under different environmentalconditions, as it has been demonstrated that photosyntheticef®ciency can vary with the growth environment (Figs 4±6). However, the lower values of Fq¢/Fm¢ for T. albi¯orathan for four other species from the chambers is consistentwith previous work (Lawson et al., 2002). The loweref®ciencies observed in T. albi¯ora and P. vulgare are alsore¯ected in lower photosynthetic rates compared with theother species, indicating lower intrinsic light use ef®cien-cies for photosynthesis. These two species also tend tohave a greater number of chloroplasts in the guard cellsthan other species, which may result in increased lightabsorption and a reduced ef®ciency at any given incidentPPFD.

Table 1. Response of guard and mesophyll cell Fq¢/Fm¢ in intact leaves of six different species to reductions in O2 from 21% to2% at high (360 mmol mol±1) and low (150 mmol mol±1) CO2 concentration

PPFD was approximately 240 mmol m±2 s±1. Data are the means of three replicates, and the pooled SE appropriate for each column is shown.

Species 150 mmol mol±1 CO2 360 mmol mol±1 CO2

Guard cell Mesophyll Guard cell Mesophyll

21% O2 2% O2 21% O2 2% O2 21% O2 2% O2 21% O2 2% O2

Amaranthus caudatus 0.528 0.507 0.538 0.555 0.562 0.529 0.566 0.568Vicia faba 0.547 0.489 0.606 0.553 0.640 0.598 0.672 0.655Nicotiana tabacum 0.453 0.422 0.533 0.483 0.557 0.532 0.608 0.584Polypodium vulgare 0.139 0.108 0.219 0.181 0.244 0.219 0.309 0.324Tradescantia albi¯ora 0.263 0.200 0.340 0.253 0.387 0.330 0.443 0.420Commelina communis 0.537 0.495 0.544 0.508 0.587 0.570 0.592 0.586Pooled SE 0.019 0.017 0.025 0.025 0.020 0.018 0.028 0.028

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Effect of light

Some differences were observed in the values andresponses of photosynthetic ef®ciency between the guardcells on the adaxial and abaxial surfaces, and the way itwas affected by PPFD (Fig. 3). In V. faba a difference wasobserved only at low PPFD, agreeing with the report ofsimilar photosynthetic rates per unit chlorophyll foradaxial and abaxial guard cell protoplasts from the samespecies by Goh et al. (1997). In C. communis the differentlight responses of the guard cells in adaxial and abaxialsurfaces were similar to those seen in leaf photosyntheticresponses for sun and shade leaves, respectively (Pearcy,1998). In addition, photosynthetic ef®ciency of C. communis

guard and mesophyll cells was affected by the PPFDduring growth, while this was not the case in V. faba. Thecontrast between the two species may be due to the growthhabit. Leaves of C. communis are more horizontal,consequently the adaxial surface is better illuminatedthan in V. faba, and the abaxial surface only receives thelight that is transmitted though the leaf. On the other hand,leaves of V. faba are less rigid in their angle to the stems,and are frequently twisted, exposing both surfaces tosimilar, but reduced, PPFD. Such differences in photo-synthetic ef®ciency may lie behind the higher sensitivity ofabaxial stomata to light (Travis and Mans®eld, 1981), andmay be associated with differences in guard cell pigmentcontent (Lu et al., 1993; Goh et al., 1997).

Effect of changes in CO2 and O2

In the C3 species, the photosynthetic ef®ciencies of theguard and mesophyll cells were reduced at low CO2

Fig. 6. (a) Time-course of guard (squares) and mesophyll cell(triangles) photosynthetic ef®ciency (estimated by Fq¢/Fm¢) forCommelina communis for either drought-stressed plants (opensymbols) or well-watered plants (solid symbols). Arrow indicatestime of rewatering. Data are the means of three replicates 6SE.(b) Relationship between guard cell and mesophyll cells Fq¢/Fm¢ forwell-watered (closed symbols) and drought-stressed plants (opensymbols). Ambient CO2 concentration was maintained at 360 or 700mmol mol±1. The broken line represents the y=x relationship, and thesolid line a linear regression with the equation y=0.914x, r2=0.902.

Fig. 5. Effect of growth and measurement CO2 on guard andmesophyll cells for (a) Commelina communis and (b) Vicia faba.Plants were grown at CO2 concentrations of 360 and 700 mmol mol±1

and measured in 150, 360 and 700 mmol mol±1 CO2. Data are themeans of three replicates 6SE. (c) Relationship between Fq¢/Fm¢ ofguard cells and mesophyll cells for individual measurements on C.communis and V. faba at the different measurement and growth CO2

concentrations. The broken line represents the y=x relationship.



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concentration (Fig. 4; Table 1). Similar reductions wereobserved in Fq¢/Fm¢ of both the guard and the mesophyllcells in ®ve C3 species when the ambient O2 was decreasedfrom 21% to 2% at low CO2 concentrations (Table 1). Weargue, as others have done (Cardon and Berry, 1992), thatsuch effects of CO2 and O2 on photosynthesis indicate thatRubisco is a major sink for the products of photosyntheticelectron transport in guard cells, and con®rm and extendearlier evidence for this from T. albi¯ora (Lawson et al.,2002). The argument is strengthened by the lack ofresponse of Fq¢/Fm¢ to [O2] in the mesophyll inAmaranthus caudatus, a C4 species, which is as expectedas there is no Rubisco activity in mesophyll cells of C4

leaves. However, there was an O2 response in the guardcells (Table 1) which agrees with recent immunogoldlabelling studies on Amaranthus viridis which showedsubstantial amounts of Rubisco in guard cells, but nolabelling in mesophyll cells (Ueno, 2001). Consequently, itappears that although the mesophyll cells of the C4 leaveslack Rubisco and do not operate a carbon reduction cycleas expected, the guard cells do exhibit Rubisco and Calvincycle activity.

Furthermore, the guard cells in C. communis showed thesame reduction in Fq¢/Fm¢ that occurred in the mesophyllcells in response to growth in high [CO2] (Fig. 5). Thecurrent explanations for photosynthetic acclimation duringgrowth in high CO2 are either that increased carbohydratesupply is not matched by sink demand, thus inhibitingCalvin cycle activity either by limiting RuBP regenerationor by causing the down-regulation of Rubisco or that theamount of Rubisco is reduced due to a change in Nallocation (Drake et al., 1997). Therefore, the matchingreduction of photosynthetic ef®ciency in the guard andmesophyll cells in high CO2 also argues for comparableCalvin cycle activity in guard and mesophyll cells. SuchRubisco regulation in both guard and mesophyll cells maybe the mechanism behind the parallel acclimation ofstomatal conductance and mesophyll photosynthesis tohigh [CO2] sometimes observed (Morison, 1998;Assmann, 1999; Lodge et al., 2001).

Effect of water stress

During slowly imposed water stress, there were paralleldeclines in the photosynthetic ef®ciencies of the guard andmesophyll cells over a time-course of days (Fig. 6). Gohet al. (2001) have described declines in photosyntheticef®ciency in the guard and mesophyll cell protoplastsunder hypertonic osmotic stress, but this was accompaniedby declines in photochemical and non-photochemicalquenching. In their experiment there were differencesbetween the guard and mesophyll cells, but they imposedrather severe osmotic stress on cells without walls. Someof the decline in photosynthetic ef®ciency in this study wasdue to reductions in CO2 supply since doubling Ca

increased Fq¢/Fm¢ and reductions of Ca below ambient

reduced Fq¢/Fm¢ markedly, as shown in Fig. 5 (see alsoLawson et al., 2002). However, it is also likely that someof the decline in Fq¢/Fm¢ was due to other water stresseffects causing `impaired photosynthetic metabolism' (seereview by Lawlor, 2002) because increased Ca did notcompletely offset the decline in Fq¢/Fm¢. Previous workindicates that Ca, and by inference intercellular [CO2],would have to be very low to cause the declines observedhere (Lawson et al., 2002; Fig. 5b). While these resultscannot distinguish which of the possible mechanisms areinvolved in this photosynthetic inhibition, an importantresult is that both the guard and mesophyll cells aresimilarly affected. This suggests that if guard cellphotosynthetic electron transport or Calvin cycle activityis important to aperture maintenance, then there must be anadditional positive feedback of reduced photosynthesis inguard cells during water stress, contributing to reductionsin stomatal aperture.

Photosynthesis in guard cells

There is a long-standing controversy over the role andactivity of the Calvin cycle in guard cells (see recentreview by Zeiger et al., 2002). A possible explanation forthe discrepancy between results indicating substantialCalvin cycle activity and those concluding that there islittle activity is that photosynthetic regulation in guardcells re¯ects the pretreatment of leaves and the measure-ment conditions (Zeiger et al., 2002). For example, it hasbeen proposed that the accumulation of sucrose near theguard cells in the apoplastic phloem loader V. faba maysuppress Calvin cycle enzymes (Lu et al., 1997) so thatstudies of guard cell photosynthetic activities using intactleaves may give different results to those with peels.Secondly, Talbott and Zeiger (1996) observed changes inthe osmotic regulation of guard cells of V. faba during theday, with K+ being the main osmoticum early in the day,but replaced by sucrose later. Thirdly, the light regulationof stomatal function is complex: both blue and red lightstimulate photosynthesis and sucrose accumulation inguard cells (Tallman and Zeiger, 1988; Talbott andZeiger, 1993), but the relative proportion of red and bluelight appears to change the balance between the starch±sugar and the K+±malate osmotic mechanisms (Talbott andZeiger, 1993). The present work provides strong evidencefor Calvin cycle activity in guard cells of six species,including V. faba, during steady-state photosynthesis inintact leaves, using moderate to high ¯uence rates of bluelight when sucrose supply in the apoplast should have beensubstantial. Such moderate and high ¯uence rates will havedriven normal photosynthetic activity in the guard cells(Wu and Assmann, 1993) and this probably contributed tostomatal opening in addition to any low intensity, chloro-phyll-independent blue light response (Tallman andZeiger, 1988; Taylor and Assmann, 2001). Furthermore,these measurements of guard cell photosynthetic ef®ciency

1750 Lawson et al.


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agree well with those of Goh et al. (1999, 2001) usingchlorophyll ¯uorescence in the guard cell protoplasts.

An important result emerging from these studies is thatphotosynthetic ef®ciency of guard cells in intact leavesresponded quantitatively to light, CO2, O2 and water stressin a similar way to adjacent mesophyll cells. As high-lighted by Wong et al. (1979), there is often a closepositive correlation at the leaf scale between stomatalconductance and mesophyll CO2 assimilation rate across arange of environmental conditions. This close relationshiphas been attributed to the in¯uence of internal CO2

concentration (Raschke, 1976), but there have also beensuggestions that there is another signal transmitted fromthe mesophyll cells to the guard cells such that mesophyllphotosynthesis controls the degree of stomatal opening(Heath and Russell, 1954; Wong et al., 1979; Lee andBowling, 1995). However, the nature of any suchmessengers is not clear. Sucrose movement within thetranspiration stream has been suggested recently byOutlaw and colleagues, as this has been shown to be amajor source of organic carbon for the guard cells, and canalso exert an osmotic effect by accumulation in the cellapoplast (Lu et al., 1997; Outlaw and De Vlieghere-He,2001). Alternatively, photosynthetic metabolism in theguard cells may be behind the co-ordination of the stomataand the mesophyll (Farquhar and Wong, 1984; Jarvis andDavies, 1998). Figures 2c, 4c, 5c, and 6b show that a close,linear correlation between the guard cell and mesophyllphotosynthetic activity exists at the cell level. Thissuggests that the guard cell photosynthetic activity mayprovide the sensing mechanism linking stomatal move-ment to mesophyll photosynthetic rate.

Acknowledgement

This work was funded by the BBSRC (grant 84/P10409).

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the responses of guard and mesophyll cell photosynthesis to co2, o2, light, and water stress in a...

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