PHYSIOLOGIA PLANTARUM 116: 127–133. 2002 Copyright C Physiologia Plantarum 2002

Printed in Denmark – all rights reserved ISSN 0031-9317

Profiles of photosynthesis within red and green leaves ofQuintinia serrata

Kevin S. Goulda,*, Thomas C. Vogelmannb,1, Tao Hanb and Michael J. Clearwaterc

aPlant Sciences Group, School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, New ZealandbBotany Department, University of Wyoming, Laramie, WY 82071-3165, USAcHorticulture and Food Research Institute of New Zealand Ltd, Te Puke Research Centre, RD2 Te Puke, New Zealand1Present address: Botany and Agricultural Biochemistry, University of Vermont, Burlington, VT 05405-0086, USA*Corresponding author, e-mail: k.gould/auckland.ac.nz

Received 14 January 2002; revised 21 March 2002

We have measured photosynthesis at the cellular, tissue, andwhole leaf levels to understand the role of anthocyaninpigments on patterns of light utilization. Profiles of chloro-phyll fluorescence through sections of red and green leavesof Quintinia serrata showed that anthocyanins in the meso-phyll restricted absorption of green light to the uppermostpalisade mesophyll. The distribution was further restrictedwhen anthocyanins were also present in the upper epidermis.

Introduction

Anthocyanin production can modify the photosyntheticperformance of leaves. For most reported species,photosynthesis is lower in red leaves than in green leaves.For example, maximum quantum yields for photosyn-thetic oxygen evolution in Coleus were significantly lowerfor red-leaf varieties than for green-leaf varieties (Burgerand Edwards 1996), and species of Syzygium which pro-duce red juvenile leaves had lower photosynthetic capac-ities than related species with green juvenile leaves(Dodd et al. 1998). The dark-adapted efficiencies of PSIIcentres were lower for the red, flushing leaves of Coty-ledon orbiculata than for the mature, green leaves(Barker et al. 1997), and light-saturated rates of carbonassimilation in the rapidly expanding leaves of Brachys-tegia spiciformis were inversely correlated to red pigmentlevels (Tuohy and Choinski 1990, Choinski and Johnson1993). However, departures in the effects of antho-cyanins on photosynthesis have been noted. In Prunus,the net photosynthesis of red leaves was only marginallylower than those of green leaves (Marini 1986), and redmorphs of Begonia pavonina and in Triolena hursuta had

Abbreviations – PAR, photosynthetically active radiation; PS, photosystem.

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Mesophyll cells located beneath a cyanic light-filter assumedthe characteristic photosynthetic features of shade-adaptedcells. As a result, red leaves showed a 23% reduction inCO2 assimilation under light-saturating conditions, and alower threshold irradiance for light-saturation, relative tothose of green leaves. The photosynthetic characteristics ofred leaves are comparable to those of shade-acclimatedplants.

significantly higher light-saturated rates of carbon as-similation than the green morphs (Gould et al. 1995).

Differences in quantum efficiency of photosynthesisbetween red and green leaves have been attributed to theabsorption of quanta by anthocyanin pigments in thecell vacuole (Gould et al. 1995, Krol et al. 1995, Pietriniand Massacci 1998, Smillie and Hetherington 1999).Purified anthocyanin fractions absorb strongly in thegreen wavebands (Harborne 1967), and red leaves typic-ally absorb more green light than do green leaves (Elleret al. 1981, Gausman 1982, Burger and Edwards 1996,Woodall et al. 1998, Neill and Gould 1999). Green lightis a key driver of photosynthesis in the lower mesophylllayers (Sun et al. 1998). The absorption of green lightby anthocyanins has been postulated to protect chloro-plasts from photoinhibition by intercepting high-energyquanta that would otherwise be absorbed by chlorophyllb (Gould et al. 1995).

Photoprotective hypotheses for the function of antho-cyanins in leaves implicitly assume that the light energyabsorbed by the anthocyanins cannot be transferred to

the chloroplasts. This is, perhaps, a reasonable assump-tion given that anthocyanins most frequently reside inthe cell vacuole, and are therefore physically separatedfrom the photosynthetic machinery. Accordingly, antho-cyanins would function as simple light filters, impactingdirectly on photosynthesis by reducing the quantumfluence rate absorbed by chlorophyll. The overall effecton photosynthesis would depend on the concentrationand distribution of anthocyanins in the leaf; a red filterwould reduce the quantum efficiency of photosynthesismost effectively if it were optically dense, and locatedabove, rather than beneath, the chlorenchyma.

The impact of anthocyanins on the distribution andquality of light within a leaf needs to be tested empiric-ally at the cellular level. Measurements of whole-leafphotosynthesis record only the net outcome of possiblelocalized variations in the attenuation, transfer, or util-ization of light energy. In the absence of cellular infor-mation, we cannot exclude the possibility that some orall of the energy absorbed by anthocyanins is re-emittedas light that is useful for photosynthesis. Indeed, puri-fied anthocyanin solutions emit blue and green fluor-escence when excited by ultraviolet radiation (Cherepyet al. 1997, Drabent et al. 1999), and anthocyanin-richextracts have been reported to enhance the Hill reactionactivity of chloroplast suspensions from several species(Sharma and Banerji 1981, Dhawale et al. 1983). Theseeffects, if realised in planta, would partially offset thelosses associated with the absorption of light by cyanictissues.

Here, we compare the gradients of absorbed lightwithin red and green leaf laminae of Quintinia serrataA. Cunn. by measuring the chlorophyll fluorescence pro-files in leaf sections (Vogelmann and Han 2000). Photo-synthetic responses of the red and green leaves are de-scribed both at the whole-leaf level, and for individualcell layers within the leaves. Our data indicate thatanthocyanins can modify photosynthetic profiles by re-stricting the absorption of light to chloroplasts withinthe uppermost palisade mesophyll cells.

Materials and methods

Q.serrata plants approximately 0.5 m tall were obtainedin pots from a nursery, having been raised from seedcollected from the Waitakere Ranges, 25 km west ofAuckland, New Zealand. This species is a canopy treeindigenous to the North Island of New Zealand. It isexceptionally variable with respect to anthocyanin con-tent and distribution in leaf laminae; red and greenleaves are anatomically identical and can contain similarquantities of chlorophyll (Gould et al. 2000). Leaveswere preferentially selected from these plants to providea range of phenotypes ranging from entirely red to ex-clusively green. Measurements were taken only on fullyexpanded leaf laminae.

Light response curves for carbon assimilation were ob-tained for intact red and green leaves using an LI-6400open photosynthesis system (LI-COR Instruments, NE,

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USA) fitted with a red-blue LED light source and a CO2

mixer. Fully expanded leaves of a range of colours wereenclosed in the leaf chamber, and allowed to reach aconstant rate of photosynthesis at 500 mmol mª2 sª1

photosynthetically active radiation (PAR) before PARwas stepped up to saturation and back down to darknessover a 30-min period. The CO2 concentration, leaf tem-perature, and vapour pressure deficit at the leaf surfacevaried between 355 and 370 mmol molª1, 20 and 22æC,and 1 and 1.5 kPa, respectively. After measurements, allleaves were sectioned, and the presence and histologicallocation of anthocyanins noted under a compound mi-croscope. A 1-cm diameter disk was taken from eachleaf, and extracted in 1 ml of 1 M HCl: MeOH (1:4, v/v). Anthocyanin concentrations were estimated as A528values of these extracts using a Hewlett Packard HP8453spectrophotometer.

Cellular profiles of photosynthetic oxygen evolutionwere measured for thick (700 mm) transverse sectionsthrough red and green portions of a single leaf usingthe photoacoustic equipment and methods described byHan and Vogelmann (1999). The leaf sections wereplaced with a cut transverse face uppermost on moistfilter paper in a photoacoustic cell. A narrow linearbeam (50 mm wide, 6 mm long) of red (650 nm) light wasobtained using a 5 mW laser diode (model TOLD9421,Toshiba, Japan) and diode laser driver (model06DLD201, Melles Griot, Irvine, CA, USA), and modu-lated at 3 Hz with a mechanical chopper. The light wasfocused on the uppermost cell layers of each transversesection, providing an irradiance of 600 mmol mª2 sª1.The position of the light on the sample was viewed witha long-working distance microscope. Photoacoustic sig-nals were detected by a random-incidence and pressure-response microphone (model 2560, Larson Davis, Provo,UT, USA), routed to a second preamplifier (model5R560; Stanford Research Systems, Sunnyvale, CA,USA), then to two lock-in amplifiers (model 5R830DSP; Stanford Research Systems), and finally to a dual-channel, strip-chart recorder. The signals were measuredat 20æC under ambient CO2, both before and aftersuperimposition of a beam of non-modulated saturatinglight (670 nm) at 1500 mmol mª2 sª1 obtained from a10 mW laser diode (model TOLD9215, Toshiba, Japan).Signals for oxygen evolution were calculated as de-scribed by Han and Vogelmann (1999). The procedurewas repeated for consecutive 50-mm intervals across theleaf sections, by moving the photoacoustic chamber rela-tive to the light source using an X-Y stage controlledby two linear actuators and a programmable controller(models 850 A and 855C, Newport, Irvine, CA, USA).

Gradients of absorbed light in the Q.serrata leaveswere measured from chlorophyll fluorescence profiles oftransverse sections, as described by Vogelmann and Han(2000). Leaf sections, 1 mm thick ¿ 2 mm wide, weretaken from the proximal, central, and distal regions of8 leaves for which pigmentation patterns were uniformacross the lamina surface. They were mounted on an in-verted microscope such that they could be irradiated on

Fig. 1. Light-response curves for CO2assimilation in green (P) and red (S)leaves of Q.serrata. Error bars show .n Ω 4 per leaf type.

their adaxial surfaces with monochromatic blue (450nm), red (650 nm) and then green (550 nm) light. Thelight was obtained from a 75-W xenon arc lamp (modelA-1010B, Photon Technology International, MonmouthJunction, NJ, USA) and interference filters (Corion,Franklin, MA, USA). Irradiance of the actinic light wasadjusted to 2000 mmol mª2 sª1 using neutral densityfilters. Images of red chlorophyll fluorescence from thecut transverse face of the leaf were captured with a cryo-genically cooled charge-coupled device (CCD) camera(CH270 camera head, CF200A 16/40 camera electronicsunit, AT200 controller board, 35-mm shutter; all fromPhotometrics, Tucson, AZ, USA), using a narrow bandinterference filter (680 nm; Corion) as the barrier filter.A fourth image was taken with epi-illumination mono-chromatic blue light incident on the cut face of each leafsection in order to provide a measure of chlorophyll dis-tribution. Shutter times were 2 s for exposures withgreen light, and 1 s for exposures with red and blue lightand for a dark control. Average grey scale values bothacross and down the fluorescence images (thick profileanalysis) were quantified using Image Pro Plus (version2.0, Media Cybernetics, Silver Spring, MD, USA) witha spatial resolution of 1.8 mm pixelª1. Grey scale valuesfor each image, after subtracting those from the imageof the corresponding dark control, were normalized suchthat maximum fluorescence read 100%, and minimumfluorescence zero. The images were colour-indexed usingAdobe Photoshop 3.0.5. The histological distributionsof anthocyanins in the leaf sections were noted, and forthe three locations within each lamina, anthocyanin con-centrations were estimated as A528 from 1-cm diameterleaf discs extracted in 1 ml of 1 HCl:MeOH (1:4 v/v).

Results

Fully expanded leaf laminae of Q.serrata ranged in ap-pearance from entirely red, through blotchy, to entirelygreen. Anthocyanin levels (A528 values) varied 8-foldacross a sample of 16 leaves. All red regions held antho-cyanins in the palisade mesophyll tissue. Anthocyanins

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were also present, although less frequently and at lowerconcentrations, in the spongy mesophyll and epidermaltissues.

Rates of CO2 assimilation under saturating white lightwere on average 23% lower for red than for green Q.serrata leaves (Fig. 1), a statistically significant difference(; P 0.02). Among the red leaves, rates of light-saturated CO2 assimilation were greatest in those leaveswhich held the least anthocyanin. Saturation wasachieved with irradiances above 350 and 500 mmol mª2

sª1 in the red and green leaves, respectively. The ap-parent quantum yields were similar for red (j Ω 0.043)and green (j Ω 0.048) leaves under light-limiting con-ditions, and there were no significant differences instomatal conductance or rates of dark respiration (P0.05).

Measurement of oxygen evolution from 50 mm-wide

Fig. 2. Profiles for oxygen evolution capacity in transverse sectionsthrough green (P) and red (S) regions of a leaf. In the red region,anthocyanins were located in the palisade and spongy mesophyll.Relative positions of upper epidermis (UE), uppermost palisademesophyll (PM1), second palisade mesophyll layer (PM2), spongymesophyll (SM), and lower epidermis (LE) are indicated.

bands of cells in transverse sections through Q.serrataleaves yielded photoacoustic signals approximately 5-fold lower than those for spinach (Han et al., 1999). Asa result, the data for profiles of oxygen evolution ca-pacity were relatively noisy (Fig. 2). Nonetheless, differ-ences were detectable between the profiles for green andred leaves. When anthocyanins were present in the meso-phyll tissues, oxygen evolution was confined largely tothe palisade mesophyll (Fig. 2). The green leaves, by con-trast, yielded oxygen from both the palisade and spongymesophyll (Fig. 2).

Potential differences between the photosynthesis ofgreen and red leaves were more clearly indicated fromthe profiles of absorbed light revealed by chlorophyllfluorescence in transverse sections irradiated on their ad-axial surfaces (Fig. 3). Differences were most pro-nounced under green light (Fig. 3C,G), for which fluor-escence extended down into the lower spongy mesophyllin the green leaves but was restricted predominantly tothe uppermost palisade mesophyll layer in the red leaves.Similar differences, though smaller in magnitude, wereevident under red light (Fig. 3D,H).

Fig. 3. Light micrographs (A, E) and colour-indexed images of red chlorophyll fluorescence for transverse sections through a green (A-D)and a red (E-H) leaf of Q.serrata. Fluorescence images were captured with a CCD digital camera after irradiating the adaxial surfaces withmonochromatic blue (B, F), green (C, G) and red (D, H) light. Bars Ω 100 mm.

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Profiles of light absorption revealed by chlorophyllfluorescence for green Q.serrata leaves under monochro-matic blue, green and red light (Fig. 4A) were similar tothose reported previously for other species. Blue lightgave a relatively narrow fluorescence profile with maxi-mum fluorescence located in the uppermost palisademesophyll. The fluorescence profiles for red and greenlight were broader. A slightly greater proportion of thefluorescence from green light originated from the lowerleaf tissues than did that from red light; the spongymesophyll accounted for 11 ∫ 2% of the total fluor-escence under blue light, 32 ∫ 3% under green light, and29 ∫ 2% under red light (Fig. 4A). By contrast, in redleaves, the profiles for green and (to a lesser extent) redlight shifted significantly towards the uppermost photo-synthetic tissues (Fig. 4B). For those leaves, the spongymesophyll accounted for 9 ∫ 2% of the total fluorescenceunder blue light, 9 ∫ 2% under green light, and 22 ∫ 2%under red light (Fig. 4B). Variation in light profiles wasnot associated with differences in chloroplast distri-bution between red and green leaves, as revealed by epi-illumination (data not shown).

Fig. 4. Chlorophyll fluorescence profiles for transverse sectionsthrough the green (A) and red (B) leaves of Q.serrata shown in Fig.3. Adaxial surfaces of leaves were irradiated with monochromaticblue (H), green (g), and red (P) light. Relative positions of upperepidermis (UE), uppermost palisade mesophyll (PM1), second pali-sade mesophyll layer (PM2), spongy mesophyll (SM), and lower epi-dermis (LE) are indicated. Symbols identify the lines; actual datapoints are spaced 1.8 mm apart. Data are averages of three regionsper leaf. Error bars show .

Differences in anthocyanin concentration among thered leaves did not appreciably alter the fluorescence pro-files. However, the locations of red cells within the leavessignificantly affected both the positions of fluorescencemaxima, and the relative contributions of the palisadeand spongy mesophyll to the fluorescence profiles (Fig.5A-C). These positional effects were greatest undergreen light (Fig. 5B). Leaves for which anthocyaninswere present in both the epidermal and mesophyll tissuesproduced a narrow fluorescence profile, with maximumfluorescence located at the epidermis-palisade interface.When anthocyanins were present in the mesophyll butabsent from epidermal tissues, the fluorescence profileswere broader, with maximum fluorescence located lowerinto the palisade mesophyll.

Discussion

Anthocyanins appear to impact significantly on thephotosynthesis of Q.serrata leaves by restricting absorp-

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tion of green light, and to a lesser extent red light, tochloroplasts within the uppermost palisade mesophyllcells (Figs 3–5). The location of cyanic cells is critical.When present in the epidermal layers, light absorption isrestricted to an extremely narrow band at the epidermis-palisade boundary (Fig. 5). Most Q.serrata leaves, how-

Fig. 5. Chlorophyll fluorescence profiles for transverse sectionsthrough leaves of Q.serrata irradiated with monochromatic blue(A), green (B), and red (C) light. Anthocyanins were absent (H),were present in all cell layers (P), or were distributed irregularlythrough the palisade and spongy mesophyll (g). Relative positionsof upper epidermis (UE), uppermost palisade mesophyll (PM1),second palisade mesophyll layer (PM2), spongy mesophyll (SM),and lower epidermis (LE) are indicated. Symbols identify the lines;actual data points are spaced 1.8 mm apart. Each line is the averageof nine profiles on three leaves (P), or six profiles on two leaves (H,g). Error bars show .

ever, hold anthocyanins exclusively in the mesophyll(Neill and Gould 1999, Gould et al. 2000), which has theeffect of limiting photosynthesis within the uppermostpalisade mesophyll and reducing the overall contributionfrom the spongy mesophyll (Fig. 5). Gradients in theinternal light flux within the mesophyll are common fordorsiventral green leaves (Smith et al. 1997). The ab-sorption of quanta by anthocyanins in the upper leaftissues accentuates these gradients.

A cyanic light-filter would, in the short term, reducethe quantum efficiency of photosynthesis with respectto the total light absorbed, and increase the thresholdirradiance at which saturation of photosynthesis isachieved. However, prolonged exposure to the filteredlight could also lead to permanent alterations in physi-ology as a result of chloroplasts developing under, oracclimating to, the shaded conditions. Differences in thelight-response curves for photosynthesis between the redand green leaves of Q.serrata (Fig. 1) are consistent withthe long-term acclimation to light abatement by antho-cyanins. The lower thresholds for light saturation, andthe lower light-saturated rates of photosynthesis in redversus green leaves are typical of the differences betweenshade and sun species, and for sun species acclimatedto low- and high-light environments (Björkman 1981).Acclimation to shade is attributable to a host of struc-tural and biochemical modifications to the chloroplasts(Björkman 1981).

Shade-acclimation in red Q.serrata leaves was mostevident among cells of the spongy mesophyll. When sec-tions were irradiated on their cut faces, the spongymesophyll cells of red leaves gave weaker oxygen evolu-tion signals than did those of green leaves (Fig. 2). Thesignals were also substantially lower than those of thepalisade mesophyll in the same red leaves, even thoughboth tissues contained anthocyanin and had receivedidentical irradiance. Photosynthetic capacities of chloro-plasts in the spongy mesophyll were probably reduced inresponse to the long-term exposure to low levels of greenlight imposed by anthocyanins in the upper tissues.Chloroplasts in the lower tissues of red leaves are ex-treme examples of shade-adapted organelles (Outlaw1987, Nishio et al. 1993).

Our observation that the location of anthocyanins isan important determinant of where light is absorbedmay explain reported differences in the photosyntheticresponses of red leaves across other species. In red-leafedvarieties of Coleus, for which anthocyanins drasticallyreduce apparent quantum yields (by 76% under greenlight!), the anthocyanins reside exclusively in the upperepidermis (Burger and Edwards 1996). Smaller differ-ences in photosynthesis are apparent in species for whichanthocyanins are located in the palisade and spongymesophyll (Dodd et al. 1998, and the current study). Ina third group for which anthocyanins reside exclusivelyin the lower spongy mesophyll and/or the lower epider-mis (Lee et al. 1979, Lee 1986), quantum efficiencies arelower but maximum yields are greater in the red versusgreen leaves (Gould et al. 1995).

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A 23% reduction in CO2 assimilation of red versusgreen Q.serrata leaves would seem potentially disadvan-tageous for these slow-growing trees, which are typicallyexposed to full sunlight. Nonetheless, in their naturalforest environment, trees bearing entirely red leaves, ormixtures of red and green leaves, apparently thrivealongside those with only green leaves (Gould et al.2000). It is possible that anthocyanins in vegetative or-gans are simply the by-products of a saturated flavonoidmetabolism, which are shunted into the cell vacuole forergastic storage. However, the strong association be-tween anthocyanins and the chlorenchyma in leavesfrom disparate plant taxa (Lee and Collins 2001) sug-gests that the absorption of high-energy quanta byanthocyanins serves a useful function. The functionalimplications of anthocyanins in leaves require furtherstudies of plants in their natural environments.

Acknowledgements – We thank Candice Barclay for help with thephotosynthesis measurements, Dr Mary Neighbours for her kindhospitality during KSG’s visit to Wyoming, and Prof. David W. Leefor inspiring the project. The work was supported by a RoyalSociety Marsden grant UOA707 and NSF DBI-9724499.

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