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Are red leaf phenotypes more or less fit? The case of winter leaf reddening in Cistus creticus
Environmental and Experimental Botany 67 (2010) 509–514
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Are red leaf phenotypes more or less fit? The case of winter leaf reddening in Cistus creticus Constantinos Nikiforou, Konstantina Zeliou, Velissarios-Phaedon Kytridis, Alexandra Kyzeridou, Yiannis Manetas ∗ Laboratory of Plant Physiology, Section of Plant Biology, Department of Biology, University of Patras, GR-265 00 Patras, Greece
a r t i c l e
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Article history: Received 2 June 2009 Received in revised form 3 September 2009 Accepted 21 September 2009 Keywords: Anthocyanins Chlorophyll fluorescence Growth Photosynthetic performance Reproductive effort Reproductive success
a b s t r a c t Winter leaf reddening occurs in some plants as a response to the combination of cold temperatures and high light. Hence, a protective function against photoinhibition of photosynthesis has been suggested. However, it is unknown whether the leaf anthocyanic trait confers long term benefits to the plant and to that aim parameters related to fitness were measured. We took advantage of intra-species variation in the expression of the red leaf character displayed by the Mediterranean shrub Cistus creticus, a field of which during winter becomes a mosaic of green and red individuals under apparently similar environmental conditions. The red individuals are known to be more sensitive to winter photoinhibition of photosynthesis, hence anthocyanins might serve a compensatory function through light screening and/or detoxifying reactive oxygen species. If anthocyanins are indeed beneficial in this regard, this might be reflected in altered growth and reproductive output of red compared to green individuals. Both phenotypes displayed similar photosynthetic performance indices before and after the winter red period, yet reds suffered a considerable drop in this parameter concomitant with anthocyanin accumulation. This photosynthetic inferiority was irregularly linked to growth, since red plants produced fewer new leaves during the following spring, yet shoot relative growth rate was higher. Moreover both phenotypes displayed similar flower numbers, pollination success and seed yield, mass and germinability. As judged by the similar final reproductive output, vulnerability to the winter stress does not render the red phenotype less fit, nor anthocyanin accumulation render it more fit. Moreover, the photosynthetic inferiority of the red phenotype, although linked to slightly reduced leaf number, it was not limiting for reproductive effort and success. Regardless of function, winter leaf redness in C. creticus may indicate photosynthetically weak individuals. However, neither a fitness cost nor benefit of anthocyanins can inferred in this system. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Anthocyanins advertise the presence of flowers to pollinating animals. Yet, in some plants and under some circumstances, they are accumulated in leaves, masking the green chlorophyll color. We may distinguish between developmentally controlled redness, occurring either in young, developing or old, senescing leaves and environmentally induced in mature leaves under stress (Manetas, 2006; Archetti et al., 2009). Anthocyanins absorb visible radiation without being photosynthetic. Accordingly, they compete with photopigments for photon capture and as such, may entail a photosynthetic cost to the leaf due to lost photons under sub-saturating photosynthetic photon flux densities. Since the leaf anthocyanic
∗ Corresponding author. Tel.: +30 2610 997411x96; fax: +30 2610 997411. E-mail address: [email protected] (Y. Manetas). 0098-8472/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2009.09.005
trait has been preserved in most plants (especially angiosperms), it has been argued that a benefit is afforded to red leaves, compensating for the combined construction and photosynthetic costs. Many hypotheses have been proposed for a function of leaf anthocyanins, yet a consensus has not being reached. Ecological hypotheses link the anthocyanic trait to insect herbivory in one way or another. Leaf redness has been considered as a signal indicating a high defensive potential, provided that insects can perceive the red signal and avoid the bearer. Alternatively, it may be considered as a camouflage resulting from the masking of green leaf color which is usually perceived by folivorous insects (Stone, 1979; Archetti, 2000; Hamilton and Brown, 2001; Archetti and Brown, 2004; Archetti et al., 2009). In a third variant, the red leaf color is considered to undermine the insect camouflage, since a (usually green) folivorous insect is better seen by its predator when feeds on a red object (Lev-Yadun et al., 2004). There are sporadic reports supporting the ecological hypotheses for young or
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senescing red leaves (Furuta, 1986; Numata et al., 2004; Archetti and Leather, 2005; Karageorgou and Manetas, 2006). However, the significance of these hypotheses for the transient accumulation of anthocyanins during winter is apparently weakened, since folivorous insect activity is considerably less during this period. Among physiologists, the most prominent hypothesis is that of photoptotection against excess light, especially when other environmental stresses perturb the delicate balance between light capture and its use for CO2 assimilation. It is argued that when all the available biochemical and behavioral measures against photoinhibition are exhausted, anthocyanins may come to play by affording a light screen (reducing excitation pressure on photopigments) and/or detoxifying reactive oxygen species produced by chlorophyll over-excitation (Lee and Gould, 2002; Steyn et al., 2002; Manetas, 2006). The hypothesis is reasonable since those environmental stresses that increase the risk of photoinhibitory damage also induce leaf redness in some plants, but not in the majority of them (Lindoo and Caldwell, 1978; Christie et al., 1994; Hodges and Nozzolillo, 1996). However, recent experimental approaches gave conflicting results, some cases confirming (Feild et al., 2001; Hughes et al., 2005; Hughes and Smith, 2007a,b) and others rejecting the hypothesis (Burger and Edwards, 1996; Lee et al., 2003; Kyparissis et al., 2007; Esteban et al., 2008). Moreover, it has being argued that neither the optical properties of leaf anthocyanins are ideal for the sunscreen function, nor their intracellular or tissue location always appropriate for the anti-oxidative function (Kytridis and Manetas, 2006; Manetas, 2006). Regardless of function, one could argue that if anthocyanins are indeed beneficial, this could be manifested in enhanced vigor and fitness of the red phenotypes under field conditions. In this study, we approach for the first time the question of fitness in red- and green-leaf phenotypes, using the Mediterranean shrub Cistus creticus as a test system. The system is advantageous since some individuals of C. creticus become red during winter, while neighboring individuals, under apparently similar light exposure and on the same soil, remain green. Repeated observations with tagged individuals since 2002 indicated that the red character during winter was stable, i.e. the same individuals become red each year (Kytridis et al., 2008). However, this apparent advantage is confounded by the finding that the red phenotype of C. creticus exhibits also lower light-saturated PSII yields and pool sizes of photoprotective xanthophylls during winter and lower levels of leaf nitrogen throughout the year (Kytridis et al., 2008; Zeliou et al., 2009). Hence, any link between leaf redness and plant vigor should be seen within the context of these co-existing differences among the corresponding phenotypes. Assessment of vigor and fitness was made by measuring photosynthetic performance, growth and reproductive output. Photosynthesis plays a central role in energy and carbon acquisition and biomass accumulation and, indirectly, to fitness. Aspects of photosynthetic function can be assessed through in vivo chlorophyll fluorescence measurements which are accurate reliable, rapid and non-invasive (Strasser et al., 1995). They are based on the analysis of transients in chlorophyll fluorescence rise after closing all PSII reaction centers with sudden illumination. Since PSII is the target of various environmental stresses (Long et al., 1994), the assessment of its functionality is pivotal in the location of vulnerable individuals or populations. In this investigation, a newly proposed parameter, the so-called photosynthetic performance index (PI) is used. PI encompasses both structural and functional attributes of PSII, and it is much more sensitive as a stress indicator than the frequently used maximum PSII yield (as FV /FM, see Strasser et al., 2004). It has been shown to be positively correlated to CO2 assimilation rates (van Heerden et al., 2003), and proposed as an index for the assessment of tree quality (Hermans et al., 2003).
Although photosynthesis and growth are proxy (indirect) measures of plant fitness, the ultimate indicator is reproductive success, i.e. the ability of the individual to produce a satisfactory and viable seed yield. Reproductive parameters display species-specific phenotypic plasticity and are responsive to stress to a varying degree (Marshall et al., 1986; Sultan, 2001). In the present study, we use all the above indirect and direct criteria of fitness in the red/green leaf debate. 2. Materials and methods 2.1. Plant material and sampling area C. creticus L., Cistaceae is an evergreen Mediterranean shrub pioneering post fire sites. Our study site suffered a wild fire in 1989 and at present it is dominated mainly by C. creticus, C. salvifolius and regenerating evergreen sclerophyll and pine trees. The study area (38.14◦ N, 21.44 ◦ E, 250 m a.s.l.) covers the South facing foot of a hill. Twenty six apparently similar, fully exposed individuals (14 green, 12 red) were tagged during winter 2006, to monitor reproductive parameters in the subsequent spring and summer period. In 2008, 29 individuals (different from those used in 2006, 15 green, 14 red) were used to confirm previous observations on reproductive parameters and concurrently monitor shoot growth and leaf production during spring/early summer. In addition, photosynthetic performance and leaf anthocyanins were measured at the indicated sampling dates from December 2007 (i.e. 2 weeks before reddening) and up to August 2008, encompassing both the red and the green period of the year. For destructive measurements (i.e. photosynthetic performance and anthocyanins), the removed leaves were less than 2% of the current leaf population. Mean monthly temperatures for winter months of the 2 sampling years were10.3, 7.1 and 9.2 ◦ C for December 2005, January and February 2006 and 10.2, 10.5 and 10.7 ◦ C for December 2007, January and February 2008. Corresponding 10 years (1997–2006) means were 11.1, 9.5 and 9.9 ◦ C, respectively. Data were kindly given by the Regional Institute of Plant Protection, located 3 km from the sampling site. 2.2. Photosynthetic performance (PI) Six mature leaves from each plant were harvested at predrawn of each sampling date, put in air-tight plastic envelopes lined internally with moist filter paper and kept in the dark for 1 h until measurement. Sampling dates encompassed both the “green” and the “red” period of the year, as shown in the corresponding Fig. 1. Leaves begin to senesce by mid spring and completely drop by mid June. Such leaves were not sampled for PI, hence the May measurement was done on mature leaves of current year growth. Chlorophyll a fluorescence transients were induced by red light (peak wavelength at 650 nm, 3000 mol photons m−2 s−1 ) given by a bank of three lightemitting diodes and fluorescence was captured by a Hansatech (Handy-PEA, Hansatech Instruments Ltd., King’s Lynn, Norfolk, UK) analyzer with appropriate data acquisition rates. The photosynthetic performance index (PI) was calculated from fluorescence values at cardinal points of the fluorescence versus time kinetics as
PI =
1 − (F0 − FM ) 4(FK − F0 )/(FJ − F0 )
(F − F ) (FM − F0 ) M J ×
F0
×
(FJ − F0 )
,
where F0 , FK and FJ the fluorescence yields at 20 s, 300 s and 2 ms, respectively, while Fm is the final maximum fluorescence
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Fig. 1. Photosynthetic performance index (PI) in the green (䊉) and red () phenotype along seasons. Values are means ± SD from 15 green and 14 red shrubs (six leaves per shrub). Arrows indicate the start and the end of the “red” period. Asterisks denote statistically significant differences between green and red plants for the indicated sampling dates (p < 0.05, student’s t-test).
yield obtained after c.a. 600–800 ms (Strasser et al., 2004). PI is a combined measure of performance related to the amount of active PSII reaction centers per absorbed energy, the maximum energy flux reaching the PSII reaction centers per absorbed energy, and the probability that this energy will be conserved as redox energy and drive electron transport beyond QA (for details see Strasser et al., 2004). PI is a sensitive index of plant vitality and is positively correlated to CO2 assimilation rates (van Heerden et al., 2003), hence to productivity based on photosynthesis. 2.3. Estimation of anthocyanins Four leaves from every shrub were sampled and extracted by grinding in a mortar with methanol: concentrated HCl 98:2 v/v. After centrifugation at 5000 g for 10 min, the supernatant was scanned in a Shimadzu (UV-160A) double-beam spectrophotometer and anthocyanins estimated according to Lindoo and Caldwell (1978). 2.4. Growth Three shoots per tagged shrub were tagged at late winter and shoot length and numbers of young, mature and senescing leaves were counted at regular (2–4 weeks intervals) up to late June. Relative shoot elongation rate was computed from length differences between successive sampling dates, normalized over initial shoot length and divided by the number of intervening days. Due to the small leaf size and the short petiole, non-invasive area measuring devices could not be used. Therefore, estimation of relative leaf growth rate was not feasible. Moreover, during the measuring spring period, new leaf burst co-occurs with old leaf drop. Hence, shoots bear a mixture of new, developing leaves, developed mature leaves of the previous and the current growth season and old senescing leaves. 2.5. Reproductive parameters Reproductive effort was measured on all tagged plants as the number of flowers per plant. Although care was taken to tag appar-
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ently similar individuals, some size deviation was unavoidable. Hence, flower number was normalized for similar plant size. Since flowers appear on the surface of the plant and not in its interior, the basis of the normalization was the plant surface area. This was computed geometrically from height and mean projected radius, assuming that the shrubs attain a half-sphere form. Flowers of C. creticus are ephemeral, i.e. they burst at pre-dawn and senesce at late afternoon (Manetas and Petropoulou, 2000). Accordingly, the study site was visited daily at mid-day during the whole blossom period (i.e. late April to early June) and all flowers counted. All fruits (capsules) from all tagged plants were harvested after maturation at mid July, counted and opened to release seeds. Fifty seeds per plant were weighed to obtain the mean seed mass and the total seed number per plant was assessed from weighing its total seed harvest. Pollination success was computed as fruits per flowers and seeds per intact fruit. Reproductive success was computed as total seed number per plant, normalized for similar plant size. Seed germination tests were performed in Petri dishes lined with moist filter paper in growth chambers at a 12 h photoperiod (PAR = 30 mol m−2 s−1 , 250 A light meter, LI-COR, NE) and 20 ◦ C/15 ◦ C day/night temperatures. A seed was considered germinated when the radicle penetrated the seed coat. Germination tests were performed after breaking dormancy by treating the seeds at high temperature (80 ◦ C for 5 min) as suggested by Thanos and Georghiou (1988). Twenty five seeds from each tagged plant were put in each dish. 2.6. Statistics Tests for assessing significance of differences between red and green plants are given in the legends of Figures and Table. 3. Results Anthocyanin accumulation in the red phenotype started abruptly at late December and peaked at mid February. Anthocyanin levels were lowered thereafter, yet some redness remained up to late April (not shown). Photosynthetic performance index (PI) was almost similar in the two phenotypes before leaf reddening and 1 week after the onset of anthocyanin accumulation (Fig. 1). Thereafter, PI dropped considerably in the red phenotype and the established significant difference to the green phenotype lasted up to the end of the “red” period i.e. up to mid April. Similar photosynthetic performances were evident in the subsequent “green” period and the summer decrease in PI was equally strong in both phenotypes. Shoot elongation rates were significantly higher in the red phenotype (Fig. 2). Although initial leaf number on a unit shoot length basis was found to be similar at the beginning of growth measurements, the green phenotype displayed significantly higher leaf numbers in subsequent measurements (Fig. 3). This could be due to the higher elongation rates of red shoots (Fig. 2). However, after complete shedding of old leaves by late June, the resulting total number of new leaves was c.a. 20% more in the green phenotype, implying higher rates of new leaf production in the greens. The combination of slightly longer shoots with slightly fewer leaves resulted in a somewhat thinner canopy of the red phenotype. Flowering started at late April and followed a roughly bellshaped kinetics in both phenotypes up to early June (not shown). No differences were observed for the start, peak and concluding flowering dates and the reproductive effort, measured as the number of flowers per plant (normalized for similar canopy size), was similar in the two phenotypes (Table 1). Moreover, no differences were observed in the pollination success, expressed either as the ratio of fruits to flowers or seeds per fruit (Table 1). Finally, reproductive success, (as the normalized number of seeds per plant), seed mass and seed germination rates were also similar (Table 1).
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4. Discussion
Fig. 2. Relative shoot growth rate in the green and the red phenotype during the growing season. Values are means ± SE from 15 green (䊉) and 14 red () shrubs (tree shoots per shrub). Red shrubs displayed higher shoot growth rates (two-way ANOVA).
Fig. 3. . Leaf numbers per unit shoot length in the green (䊉) and red () phenotype during the growing season. Senescing leaves were not included. Values are means±SE from 15 green and 14 red shrubs (three shoots per shrub). Red shrubs displayed lower leaf numbers (two-way ANOVA).
Table 1 Reproductive parameters of the green and red phenotype for the years 2006 and 2008. Values are means ± SE from 14 green and 12 red plants (2006) and 15 green and 14 red plants (2008), respectively. ND, not determined. Sampled plants in the two sampling years were different. Differences in the various parameters between the green and the red phenotype within each sampling year are not statistically significant (student’s t-test). Flower numbers and seed yields per plant were normalized for similar canopy size. Green
Red
Year 2006
Flowers/plant Fruit/flower Seeds/fruit Seeds/plant Seed mass (mg) Germinability (%)
486 ± 8 0.51 ± 0.05 52 ± 3 12896 ± 1966 0.56 ± 0.02 ND
516 ± 7 0.48 ± 0.04 53 ± 4 13116 ± 2913 0.58 ± 0.03 ND
Year 2008
Flowers/plant Fruit/flower Seeds/fruit Seeds/plant Seed mass (mg) Germinability (%)
320 ± 4 0.48 ± 0.04 56 ± 4 8602 ± 1320 0.57 ± 0.02 55 ± 3.8
350 ± 4 0.55 ± 0.06 53 ± 3 10203 ± 1300 0.56 ± 0.02 55 ± 4
Although Mediterranean winter is rather mild, many native plants suffer a reduction in photosynthetic performance during this period (Garcia-Plazaola et al., 1999; Karavatas and Manetas, 1999). However, our results indicate that in C. creticus we can distinguish between a strong, green-leaf phenotype maintaining its photosynthetic capacity during winter and a weak, red-leaf phenotype vulnerable to cold stress (Fig. 1). This was not unexpected, as previous reports have shown that the red-leaf phenotype of C. creticus is also characterized by lower light-saturated PSII yields and lower pool sizes of the xanthophyll cycle components during winter and lower leaf nitrogen regardless of season (Kytridis et al., 2008; Zeliou et al., 2009). A link between xanthophyll cycle pool sizes and photoprotection (Demmig-Adams et al., 1996) and between nitrogen levels and photosynthetic capacity (Hikosaka, 2004) is well established. However, photosynthetic inferiority and vulnerability to winter stress of the red phenotype seems to be in apparent contrast to the assumed photoprotective function for leaf anthocyanins since, in this case, one would expect PI of red leaves to remain similar or even be higher than that of green leaves. Alternatively, the low PI value in reds may be a compromise of some (yet slight) protection afforded by anthocyanins, which can not fully alleviate the inherent inferior capability of the red phenotype to cope with winter stress. Regardless of anthocyanin function, one would reasonably expect that photosynthetic inferiority of the red phenotype could be reflected in both growth and reproduction. We may notice at this point that shoot elongation in both phenotypes is already active at late winter and new leaf production seems to be highly active at mid spring (Figs. 2 and 3), i.e. at the period when red phenotypes possess red mature leaves of low photosynthetic performance. Moreover, photosynthetic performance in the red phenotype is already low since mid winter, i.e. well ahead the start of new leaf growth (Fig. 1). Flowering and seed maturation immediately follows the red leaf period and may be supported by current photosynthesis, which is performed by green leaves of equal photosynthetic capacity in both phenotypes (Fig. 1). Concerning vegetative growth, our results are conflicting, since expectations for a lower growth rate in the red phenotype were only fulfilled when the number of new leaves was considered (Fig. 3). Yet, stem elongation rates were higher in the red leaf phenotype (Fig. 2). The resulting slightly longer intermodes may reduce self-shading and compensate for carbon loss due to fewer leaves. Although detailed carbon balance estimation was not the aim of this investigation and growth assessment did not include mass measurements, the results may allow the conclusion that growth in C. creticus is only slightly affected by reduced winter photosynthesis. In any case, during the subsequent flowering and fruiting period (late spring/early summer) both phenotypes displayed similar photosynthetic performance, yet red plants had less leaves (Fig. 3). In spite of that, reproductive effort, reproductive success and seed mass and germinability were similar, suggesting that reproductive output was independent of both previous and current photosynthesis. A redundant acquisition of resources, i.e. carbon and nitrogen assimilation rates in excess of that needed for normal growth and reproduction may be inferred for C. creticus (for a review on redundancy in resource acquisition in plants, see Thomas and Sadras, 2001). Alternatively, one may assume that maintenance of reproductive output has a priority and the reduction in leaf number is a way of re-directing resources to that aim. In any case, a protective function of winter leaf reddening against photoinhibition of photosynthesis is not substantiated on the basis of these results. If carbon acquisition is enough for growth and reproduction (even after a severe winter inactivation of photosynthesis and a subsequent reduction of photosynthetic area during spring and summer), the assumed protective function of anthocyanins may be extremely
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this character for locating physiologically weak, yet equally strong reproductive performers.
References
Fig. 4. A radar plot of physiological (PI, nitrogen, VAZ/Chl), growth (RSGR, LN) and reproductive (RE, PS, RS, SG, SM) parameters of C. creticus. Data compiled from this and a previous study (Kytridis et al., 2008). Values for the red phenotype (empty circles) were normalized over those of the green phenotype (filled circles). Note that physiological parameters refer to the “red” period of the year. Corresponding values for nitrogen levels and VAZ/Chl ratios are lower for the red phenotype all year round (not shown). PI, photosynthetic performance index; VAZ/Chl, (i.e. violaxanthin + antheraxanthin + zeaxanthin)/chlorophyll, denoting photoprotective potential; RSGR, relative shoot growth rate; LN, leaf number; RE, reproductive effort; PS, pollination success (as seeds/fruit); RS, reproductive success; SM, seed mass; SG, seed germinability. Growth parameters are means from the whole sampling period. Reproductive parameters are those of year 2008.
marginal. We may add, however, that winters in both sampling years were normal in respect to temperatures, only slightly deviating from the long-term means (see Section 2). We cannot exclude the possibility that during exceptionally severe winters the reproductive output of the red phenotype may be finally affected. The hypothesis of a signal against herbivores, originally proposed for red senescing leaves (Archetti, 2000; Hamilton and Brown, 2001; Archetti and Brown, 2004), is also weak for our case since insect herbivory is negligible in winter and no apparent loss of leaf mass to consumers was observed (unpublished observations). Moreover, this hypothesis predicts that the optical signal is afforded by individuals, with a high defensive commitment. Yet, in the case of C. creticus the signal may not be honest, as total defensive phenolic levels do not differ between the two phenotypes (Kytridis et al., 2008). We may note, however, that other classes of defensive compounds have not been measured in this plant. In addition, the apparent redundancy in resource acquisition would allow considerable leaf loss to consumers without appreciable effect in growth and reproduction. Similar arguments can be put against the camouflage hypothesis for red leaf color, which may be well applied to young leaves developing during the period of high insect herbivore pressure (Karageorgou and Manetas, 2006), but not for mature leaves becoming red during the winter period of low insect abundance. 5. Conclusion Combining information from this and previous work on the same plant (Kytridis et al., 2008; Zeliou et al., 2009) in a radar plot (Fig. 4), we notice that physiological inferiority of the red phenotype in terms of nitrogen levels, photoprotective potential and photosynthetic performance (i.e. vulnerability to stress), has a rather negligible effect on growth and reproductive success, hence on final fitness. Accordingly, a fitness benefit of winter leaf redness cannot be easily inferred. Although the adaptive significance of winter leaf redness is still obscure, the observer may be benefited from
Archetti, M., 2000. The origin of autumn colours by coevolution. J. Theor. Biol. 205, 625–630. Archetti, M., Brown, S.P., 2004. The coevolution theory of autumn colours. Proc. R. Soc. B-Biol. Sci. 271, 1219–1223. Archetti, M., Leather, S.R., 2005. A test of the coevolution theory of autumn colours: colour preference of Rhopalosiphum padi on Prunus padus. Oikos 110, 339–343. Archetti, M., Döring, T.F., Hagen, S.B., Hughes, N.M., Leather, S.R., Lee, D.W., Lev-Yadun, S., Manetas, Y., Ougham, H.J., Schaberg, P.G., Thomas, H., 2009. Unravelling the evolution of autumn colours: an interdisciplinary approach. Trends Ecol. Evol. 24, 166–173. Burger, J., Edwards, G.E., 1996. Photosynthetic efficiency, and photodamage by UV and visible radiation in red versus green leaf in Coleus varieties. Plant Cell Physiol. 37, 395–399. Christie, P.J., Alfenito, M.R., Walbot, V., 1994. Impact of low-temperature stress on general phenylpropanoid and anthocyanin pathways: enhancement of transcript abundance and anthocyanin pigmentation in maize seedlings. Planta 194, 541–549. Demmig-Adams, B., Gilmore, A.M., Adams, W.W., 1996. In vivo functions of carotenoids in higher plants. Faseb J. 10, 403–412. Esteban, R., Fernandez-Marın, B., Becerril, J.M., García-Plazaola, J.I., 2008. Photoprotective implications of leaf variegation in E. dens-canis L. and P. officinalis L. J. Plant Physiol. 165, 1255–1263. Feild, T.S., Lee, D.W., Holbrook, M., 2001. Why leaves turn red in autumn. The role of anthocyanins in senescing leaves of red-osier dogwood. Plant Physiol. 127, 566–574. Furuta, K., 1986. Host preference and population dynamics in an autumnal population of the maple aphid, Periphyllus californiensis Shinji (Homoptera, Aphididae). J. Appl. Entomol. 102, 93–100. Garcia-Plazaola, J.I., Artetxe, U., Dunabeitia, M.K., Becceril, J.M., 1999. Role of photoprotective systems of Holm-Oak (Quercus ilex) in the adaptation to winter conditions. J. Plant Physiol. 155, 625–630. Hamilton, W.D., Brown, S.P., 2001. Autumn tree colours as a handicap signal. Proc. R. Soc. B-Biol. Sci. 268, 1489–1493. Hermans, C., Smeyers, M., Rodriguez, R.M., Eyletters, M., Strasser, R.J., Delhaye, J.-P., 2003. Quality assessment of urban trees: a comparative study of physiological characterisation, airborne imaging and on site fluorescence monitoring by the OJIP-test. J. Plant Physiol. 160, 81–90. Hikosaka, K., 2004. Interspecific difference in the photosynthesis–nitrogen relationship: patterns, physiological causes and ecological importance. J. Plant Res. 117, 481–494. Hodges, D.M., Nozzolillo, C., 1996. Anthocyanin and anthocyanoplast content of cruciferous seedlings subjected to mineral nutrient deficiencies. J. Plant Physiol. 147, 749–754. Hughes, N.M., Neufeld, H.S., Burkey, K.O., 2005. Functional role of anthocyanins in high-light winter leaves of the evergreen herb Galax urceolata. New Phytol. 168, 575–587. Hughes, N.M., Smith, W.K., 2007a. Attenuation of incident light in Galax urceolata (Diapensiaceae): concerted influence of adaxial and abaxial anthocyanic layers on photoprotection. Am. J. Bot. 94, 784–790. Hughes, N.M., Smith, W.K., 2007b. Seasonal photosynthesis and anthocyanin production in ten broadleaf evergreen species. Funct. Plant Biol. 34, 1072–1079. Karageorgou, P., Manetas, Y., 2006. The importance of being red when young: anthocyanins and the protection of young leaves of Quercus coccifera from insect herbivory and excess light. Tree Physiol. 26, 613–621. Karavatas, S., Manetas, Y., 1999. Seasonal patterns of photosystem 2 photochemical efficiency in evergreen sclerophyll sand drought semi-deciduous shrubs under Mediterranean field conditions. Photosynthetica 36, 41–49. Kyparissis, A., Grammatikopoulos, G., Manetas, Y., 2007. Leaf morphological and physiological adjustments to the spectrally selective shade imposed by anthocyanins in Prunus cerasifera. Tree Physiol. 27, 849–857. Kytridis, V.-P., Manetas, Y., 2006. Mesophyll versus epidermal anthocyanins as potential in vivo antioxidants: evidence linking the putative antioxidant role to the proximity of oxy-radical source. J. Exp. Bot. 57, 2203–2210. Kytridis, V.-P., Karageorgou, P., Levizou, E., Manetas, Y., 2008. Intra-species variation in transient accumulation of leaf anthocyanins in Cistus creticus during winter: evidence that anthocyanins may compensate for an inherent photosynthetic and photoprotective inferiority of the red-leaf phenotype. J. Plant Physiol. 165, 952–959. Lee, D.W., Gould, K.S., 2002. Anthocyanins in leaves and other vegetative organs: an introduction. Adv. Bot. Res. 37, 1–16. Lee, D.W., O’Keefe, J., Holbrook, N.M., Feild, T.S., 2003. Pigment dynamics and autumn leaf senescence in a New England deciduous forest, eastern USA. Ecol. Res. 18, 677–694. Lev-Yadun, S., Dafni, A., Flaishman, M.A., Inbar, M., Izhaki, I., Katzir, G., Neeman, G., 2004. Plant coloration undermines herbivorous insect camouflage. BioEssays. 26, 1126–1130. Lindoo, S.J., Caldwell, M.M., 1978. Ultraviolet-B radiation induced inhibition of leaf expansion and promotion of anthocyanin production. Plant Physiol. 61, 278–282.
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Long, S.P., Humphries, S., Falkowski, P.G., 1994. Photoinhibition of photosynthesis in nature. Annu. Rev. Plant Physiol. Plant. Mol. Biol. 45, 633–662. Manetas, Y., Petropoulou, Y., 2000. Nectar amount, pollinator visit duration and pollination success in the Mediterranean shrub Cistus creticus. Ann. Bot. 86, 815–820. Manetas, Y., 2006. Why some leaves are anthocyanic and why most anthocyanic leaves are red? Flora 201, 163–177. Marshall, D.L., Levin, D.A., Fowler, N.L., 1986. Plasticity of yield components in response to stress in Sesbania macrocarpa and Sesbania vesicaria (Leguminosae). Am. Nat. 127, 508–521. Numata, S., Kachi, N., Okuda, T., Manokaran, N., 2004. Delayed greening, leaf expansion, and damage to sympatric Shorea species in a lowland rain forest. J. Plant Res. 117, 19–25. Steyn, W.J., Wand, S.J.E., Holcroft, D.M., Jacobs, G., 2002. Anthocyanins in vegetative tissues: a proposed unified function in photoprotection. New Phytol. 155, 349–361. Stone, B.C., 1979. Protective coloration of young leaves in certain Malaysian palms. Biotropica 11, 126. Strasser, R.J., Srivastava, A., Govindjee, 1995. Polyphasic chlorophyll a fluorescence transient in plants and cyanobacteria. Photochem. Photobiol. 61, 32–42.
Strasser, R.J., Tsimilli-Michael, M., Srivastava, A., 2004. Analysis of the chlorophyll a fluorescence transient. In: Papageorgiou, G.C., Govindjee (Eds.), Chlorophyll Fluorescence: A Signature of Photosynthesis. Springer, Dordrecht, The Netherlands, pp. 321–362. Thanos, C.A., Georghiou, K., 1988. Ecophysiology of fire stimulated seed germination in Cistus incanus spp creticus (L) Heywood and C. salvifolius L. Plant Cell Environ. 11, 841–849. Sultan, S.E., 2001. Phenotypic plasticity for fitness components in Polygonum species of contrasting ecological breadth. Ecology 82, 328–343. Thomas, H., Sadras, V.O., 2001. The capture and gratuitous disposal of resources by plants. Funct. Ecol. 15, 3–12. van Heerden, P.D.R., Tsimilli-Michael, M., Kruger, G.H.J., Strasser, R.J., 2003. Dark chilling effects on soybean genotypes during vegetative development: parallel studies of CO2 assimilation, chlorophyll a fluorescence kinetics O-J-I-P and nitrogen fixation. Physiol. Plant. 117, 476–491. Zeliou, K., Manetas, Y., Petropoulou, Y., 2009. Transient winter leaf reddening in Cistus creticus characterizes weak (stress-sensitive) individuals, yet anthocyanins cannot alleviate the adverse effects on photosynthesis. J. Exp. Bot. 60, 3031–3042.
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Are red leaf phenotypes more or less fit? The case of winter leaf reddening in Cistus creticus Constantinos Nikiforou, Konstantina Zeliou, Velissarios-Phaedon Kytridis, Alexandra Kyzeridou, Yiannis Manetas ∗ Laboratory of Plant Physiology, Section of Plant Biology, Department of Biology, University of Patras, GR-265 00 Patras, Greece
a r t i c l e
i n f o
Article history: Received 2 June 2009 Received in revised form 3 September 2009 Accepted 21 September 2009 Keywords: Anthocyanins Chlorophyll fluorescence Growth Photosynthetic performance Reproductive effort Reproductive success
a b s t r a c t Winter leaf reddening occurs in some plants as a response to the combination of cold temperatures and high light. Hence, a protective function against photoinhibition of photosynthesis has been suggested. However, it is unknown whether the leaf anthocyanic trait confers long term benefits to the plant and to that aim parameters related to fitness were measured. We took advantage of intra-species variation in the expression of the red leaf character displayed by the Mediterranean shrub Cistus creticus, a field of which during winter becomes a mosaic of green and red individuals under apparently similar environmental conditions. The red individuals are known to be more sensitive to winter photoinhibition of photosynthesis, hence anthocyanins might serve a compensatory function through light screening and/or detoxifying reactive oxygen species. If anthocyanins are indeed beneficial in this regard, this might be reflected in altered growth and reproductive output of red compared to green individuals. Both phenotypes displayed similar photosynthetic performance indices before and after the winter red period, yet reds suffered a considerable drop in this parameter concomitant with anthocyanin accumulation. This photosynthetic inferiority was irregularly linked to growth, since red plants produced fewer new leaves during the following spring, yet shoot relative growth rate was higher. Moreover both phenotypes displayed similar flower numbers, pollination success and seed yield, mass and germinability. As judged by the similar final reproductive output, vulnerability to the winter stress does not render the red phenotype less fit, nor anthocyanin accumulation render it more fit. Moreover, the photosynthetic inferiority of the red phenotype, although linked to slightly reduced leaf number, it was not limiting for reproductive effort and success. Regardless of function, winter leaf redness in C. creticus may indicate photosynthetically weak individuals. However, neither a fitness cost nor benefit of anthocyanins can inferred in this system. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Anthocyanins advertise the presence of flowers to pollinating animals. Yet, in some plants and under some circumstances, they are accumulated in leaves, masking the green chlorophyll color. We may distinguish between developmentally controlled redness, occurring either in young, developing or old, senescing leaves and environmentally induced in mature leaves under stress (Manetas, 2006; Archetti et al., 2009). Anthocyanins absorb visible radiation without being photosynthetic. Accordingly, they compete with photopigments for photon capture and as such, may entail a photosynthetic cost to the leaf due to lost photons under sub-saturating photosynthetic photon flux densities. Since the leaf anthocyanic
∗ Corresponding author. Tel.: +30 2610 997411x96; fax: +30 2610 997411. E-mail address: [email protected] (Y. Manetas). 0098-8472/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2009.09.005
trait has been preserved in most plants (especially angiosperms), it has been argued that a benefit is afforded to red leaves, compensating for the combined construction and photosynthetic costs. Many hypotheses have been proposed for a function of leaf anthocyanins, yet a consensus has not being reached. Ecological hypotheses link the anthocyanic trait to insect herbivory in one way or another. Leaf redness has been considered as a signal indicating a high defensive potential, provided that insects can perceive the red signal and avoid the bearer. Alternatively, it may be considered as a camouflage resulting from the masking of green leaf color which is usually perceived by folivorous insects (Stone, 1979; Archetti, 2000; Hamilton and Brown, 2001; Archetti and Brown, 2004; Archetti et al., 2009). In a third variant, the red leaf color is considered to undermine the insect camouflage, since a (usually green) folivorous insect is better seen by its predator when feeds on a red object (Lev-Yadun et al., 2004). There are sporadic reports supporting the ecological hypotheses for young or
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senescing red leaves (Furuta, 1986; Numata et al., 2004; Archetti and Leather, 2005; Karageorgou and Manetas, 2006). However, the significance of these hypotheses for the transient accumulation of anthocyanins during winter is apparently weakened, since folivorous insect activity is considerably less during this period. Among physiologists, the most prominent hypothesis is that of photoptotection against excess light, especially when other environmental stresses perturb the delicate balance between light capture and its use for CO2 assimilation. It is argued that when all the available biochemical and behavioral measures against photoinhibition are exhausted, anthocyanins may come to play by affording a light screen (reducing excitation pressure on photopigments) and/or detoxifying reactive oxygen species produced by chlorophyll over-excitation (Lee and Gould, 2002; Steyn et al., 2002; Manetas, 2006). The hypothesis is reasonable since those environmental stresses that increase the risk of photoinhibitory damage also induce leaf redness in some plants, but not in the majority of them (Lindoo and Caldwell, 1978; Christie et al., 1994; Hodges and Nozzolillo, 1996). However, recent experimental approaches gave conflicting results, some cases confirming (Feild et al., 2001; Hughes et al., 2005; Hughes and Smith, 2007a,b) and others rejecting the hypothesis (Burger and Edwards, 1996; Lee et al., 2003; Kyparissis et al., 2007; Esteban et al., 2008). Moreover, it has being argued that neither the optical properties of leaf anthocyanins are ideal for the sunscreen function, nor their intracellular or tissue location always appropriate for the anti-oxidative function (Kytridis and Manetas, 2006; Manetas, 2006). Regardless of function, one could argue that if anthocyanins are indeed beneficial, this could be manifested in enhanced vigor and fitness of the red phenotypes under field conditions. In this study, we approach for the first time the question of fitness in red- and green-leaf phenotypes, using the Mediterranean shrub Cistus creticus as a test system. The system is advantageous since some individuals of C. creticus become red during winter, while neighboring individuals, under apparently similar light exposure and on the same soil, remain green. Repeated observations with tagged individuals since 2002 indicated that the red character during winter was stable, i.e. the same individuals become red each year (Kytridis et al., 2008). However, this apparent advantage is confounded by the finding that the red phenotype of C. creticus exhibits also lower light-saturated PSII yields and pool sizes of photoprotective xanthophylls during winter and lower levels of leaf nitrogen throughout the year (Kytridis et al., 2008; Zeliou et al., 2009). Hence, any link between leaf redness and plant vigor should be seen within the context of these co-existing differences among the corresponding phenotypes. Assessment of vigor and fitness was made by measuring photosynthetic performance, growth and reproductive output. Photosynthesis plays a central role in energy and carbon acquisition and biomass accumulation and, indirectly, to fitness. Aspects of photosynthetic function can be assessed through in vivo chlorophyll fluorescence measurements which are accurate reliable, rapid and non-invasive (Strasser et al., 1995). They are based on the analysis of transients in chlorophyll fluorescence rise after closing all PSII reaction centers with sudden illumination. Since PSII is the target of various environmental stresses (Long et al., 1994), the assessment of its functionality is pivotal in the location of vulnerable individuals or populations. In this investigation, a newly proposed parameter, the so-called photosynthetic performance index (PI) is used. PI encompasses both structural and functional attributes of PSII, and it is much more sensitive as a stress indicator than the frequently used maximum PSII yield (as FV /FM, see Strasser et al., 2004). It has been shown to be positively correlated to CO2 assimilation rates (van Heerden et al., 2003), and proposed as an index for the assessment of tree quality (Hermans et al., 2003).
Although photosynthesis and growth are proxy (indirect) measures of plant fitness, the ultimate indicator is reproductive success, i.e. the ability of the individual to produce a satisfactory and viable seed yield. Reproductive parameters display species-specific phenotypic plasticity and are responsive to stress to a varying degree (Marshall et al., 1986; Sultan, 2001). In the present study, we use all the above indirect and direct criteria of fitness in the red/green leaf debate. 2. Materials and methods 2.1. Plant material and sampling area C. creticus L., Cistaceae is an evergreen Mediterranean shrub pioneering post fire sites. Our study site suffered a wild fire in 1989 and at present it is dominated mainly by C. creticus, C. salvifolius and regenerating evergreen sclerophyll and pine trees. The study area (38.14◦ N, 21.44 ◦ E, 250 m a.s.l.) covers the South facing foot of a hill. Twenty six apparently similar, fully exposed individuals (14 green, 12 red) were tagged during winter 2006, to monitor reproductive parameters in the subsequent spring and summer period. In 2008, 29 individuals (different from those used in 2006, 15 green, 14 red) were used to confirm previous observations on reproductive parameters and concurrently monitor shoot growth and leaf production during spring/early summer. In addition, photosynthetic performance and leaf anthocyanins were measured at the indicated sampling dates from December 2007 (i.e. 2 weeks before reddening) and up to August 2008, encompassing both the red and the green period of the year. For destructive measurements (i.e. photosynthetic performance and anthocyanins), the removed leaves were less than 2% of the current leaf population. Mean monthly temperatures for winter months of the 2 sampling years were10.3, 7.1 and 9.2 ◦ C for December 2005, January and February 2006 and 10.2, 10.5 and 10.7 ◦ C for December 2007, January and February 2008. Corresponding 10 years (1997–2006) means were 11.1, 9.5 and 9.9 ◦ C, respectively. Data were kindly given by the Regional Institute of Plant Protection, located 3 km from the sampling site. 2.2. Photosynthetic performance (PI) Six mature leaves from each plant were harvested at predrawn of each sampling date, put in air-tight plastic envelopes lined internally with moist filter paper and kept in the dark for 1 h until measurement. Sampling dates encompassed both the “green” and the “red” period of the year, as shown in the corresponding Fig. 1. Leaves begin to senesce by mid spring and completely drop by mid June. Such leaves were not sampled for PI, hence the May measurement was done on mature leaves of current year growth. Chlorophyll a fluorescence transients were induced by red light (peak wavelength at 650 nm, 3000 mol photons m−2 s−1 ) given by a bank of three lightemitting diodes and fluorescence was captured by a Hansatech (Handy-PEA, Hansatech Instruments Ltd., King’s Lynn, Norfolk, UK) analyzer with appropriate data acquisition rates. The photosynthetic performance index (PI) was calculated from fluorescence values at cardinal points of the fluorescence versus time kinetics as
PI =
1 − (F0 − FM ) 4(FK − F0 )/(FJ − F0 )
(F − F ) (FM − F0 ) M J ×
F0
×
(FJ − F0 )
,
where F0 , FK and FJ the fluorescence yields at 20 s, 300 s and 2 ms, respectively, while Fm is the final maximum fluorescence
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Fig. 1. Photosynthetic performance index (PI) in the green (䊉) and red () phenotype along seasons. Values are means ± SD from 15 green and 14 red shrubs (six leaves per shrub). Arrows indicate the start and the end of the “red” period. Asterisks denote statistically significant differences between green and red plants for the indicated sampling dates (p < 0.05, student’s t-test).
yield obtained after c.a. 600–800 ms (Strasser et al., 2004). PI is a combined measure of performance related to the amount of active PSII reaction centers per absorbed energy, the maximum energy flux reaching the PSII reaction centers per absorbed energy, and the probability that this energy will be conserved as redox energy and drive electron transport beyond QA (for details see Strasser et al., 2004). PI is a sensitive index of plant vitality and is positively correlated to CO2 assimilation rates (van Heerden et al., 2003), hence to productivity based on photosynthesis. 2.3. Estimation of anthocyanins Four leaves from every shrub were sampled and extracted by grinding in a mortar with methanol: concentrated HCl 98:2 v/v. After centrifugation at 5000 g for 10 min, the supernatant was scanned in a Shimadzu (UV-160A) double-beam spectrophotometer and anthocyanins estimated according to Lindoo and Caldwell (1978). 2.4. Growth Three shoots per tagged shrub were tagged at late winter and shoot length and numbers of young, mature and senescing leaves were counted at regular (2–4 weeks intervals) up to late June. Relative shoot elongation rate was computed from length differences between successive sampling dates, normalized over initial shoot length and divided by the number of intervening days. Due to the small leaf size and the short petiole, non-invasive area measuring devices could not be used. Therefore, estimation of relative leaf growth rate was not feasible. Moreover, during the measuring spring period, new leaf burst co-occurs with old leaf drop. Hence, shoots bear a mixture of new, developing leaves, developed mature leaves of the previous and the current growth season and old senescing leaves. 2.5. Reproductive parameters Reproductive effort was measured on all tagged plants as the number of flowers per plant. Although care was taken to tag appar-
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ently similar individuals, some size deviation was unavoidable. Hence, flower number was normalized for similar plant size. Since flowers appear on the surface of the plant and not in its interior, the basis of the normalization was the plant surface area. This was computed geometrically from height and mean projected radius, assuming that the shrubs attain a half-sphere form. Flowers of C. creticus are ephemeral, i.e. they burst at pre-dawn and senesce at late afternoon (Manetas and Petropoulou, 2000). Accordingly, the study site was visited daily at mid-day during the whole blossom period (i.e. late April to early June) and all flowers counted. All fruits (capsules) from all tagged plants were harvested after maturation at mid July, counted and opened to release seeds. Fifty seeds per plant were weighed to obtain the mean seed mass and the total seed number per plant was assessed from weighing its total seed harvest. Pollination success was computed as fruits per flowers and seeds per intact fruit. Reproductive success was computed as total seed number per plant, normalized for similar plant size. Seed germination tests were performed in Petri dishes lined with moist filter paper in growth chambers at a 12 h photoperiod (PAR = 30 mol m−2 s−1 , 250 A light meter, LI-COR, NE) and 20 ◦ C/15 ◦ C day/night temperatures. A seed was considered germinated when the radicle penetrated the seed coat. Germination tests were performed after breaking dormancy by treating the seeds at high temperature (80 ◦ C for 5 min) as suggested by Thanos and Georghiou (1988). Twenty five seeds from each tagged plant were put in each dish. 2.6. Statistics Tests for assessing significance of differences between red and green plants are given in the legends of Figures and Table. 3. Results Anthocyanin accumulation in the red phenotype started abruptly at late December and peaked at mid February. Anthocyanin levels were lowered thereafter, yet some redness remained up to late April (not shown). Photosynthetic performance index (PI) was almost similar in the two phenotypes before leaf reddening and 1 week after the onset of anthocyanin accumulation (Fig. 1). Thereafter, PI dropped considerably in the red phenotype and the established significant difference to the green phenotype lasted up to the end of the “red” period i.e. up to mid April. Similar photosynthetic performances were evident in the subsequent “green” period and the summer decrease in PI was equally strong in both phenotypes. Shoot elongation rates were significantly higher in the red phenotype (Fig. 2). Although initial leaf number on a unit shoot length basis was found to be similar at the beginning of growth measurements, the green phenotype displayed significantly higher leaf numbers in subsequent measurements (Fig. 3). This could be due to the higher elongation rates of red shoots (Fig. 2). However, after complete shedding of old leaves by late June, the resulting total number of new leaves was c.a. 20% more in the green phenotype, implying higher rates of new leaf production in the greens. The combination of slightly longer shoots with slightly fewer leaves resulted in a somewhat thinner canopy of the red phenotype. Flowering started at late April and followed a roughly bellshaped kinetics in both phenotypes up to early June (not shown). No differences were observed for the start, peak and concluding flowering dates and the reproductive effort, measured as the number of flowers per plant (normalized for similar canopy size), was similar in the two phenotypes (Table 1). Moreover, no differences were observed in the pollination success, expressed either as the ratio of fruits to flowers or seeds per fruit (Table 1). Finally, reproductive success, (as the normalized number of seeds per plant), seed mass and seed germination rates were also similar (Table 1).
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4. Discussion
Fig. 2. Relative shoot growth rate in the green and the red phenotype during the growing season. Values are means ± SE from 15 green (䊉) and 14 red () shrubs (tree shoots per shrub). Red shrubs displayed higher shoot growth rates (two-way ANOVA).
Fig. 3. . Leaf numbers per unit shoot length in the green (䊉) and red () phenotype during the growing season. Senescing leaves were not included. Values are means±SE from 15 green and 14 red shrubs (three shoots per shrub). Red shrubs displayed lower leaf numbers (two-way ANOVA).
Table 1 Reproductive parameters of the green and red phenotype for the years 2006 and 2008. Values are means ± SE from 14 green and 12 red plants (2006) and 15 green and 14 red plants (2008), respectively. ND, not determined. Sampled plants in the two sampling years were different. Differences in the various parameters between the green and the red phenotype within each sampling year are not statistically significant (student’s t-test). Flower numbers and seed yields per plant were normalized for similar canopy size. Green
Red
Year 2006
Flowers/plant Fruit/flower Seeds/fruit Seeds/plant Seed mass (mg) Germinability (%)
486 ± 8 0.51 ± 0.05 52 ± 3 12896 ± 1966 0.56 ± 0.02 ND
516 ± 7 0.48 ± 0.04 53 ± 4 13116 ± 2913 0.58 ± 0.03 ND
Year 2008
Flowers/plant Fruit/flower Seeds/fruit Seeds/plant Seed mass (mg) Germinability (%)
320 ± 4 0.48 ± 0.04 56 ± 4 8602 ± 1320 0.57 ± 0.02 55 ± 3.8
350 ± 4 0.55 ± 0.06 53 ± 3 10203 ± 1300 0.56 ± 0.02 55 ± 4
Although Mediterranean winter is rather mild, many native plants suffer a reduction in photosynthetic performance during this period (Garcia-Plazaola et al., 1999; Karavatas and Manetas, 1999). However, our results indicate that in C. creticus we can distinguish between a strong, green-leaf phenotype maintaining its photosynthetic capacity during winter and a weak, red-leaf phenotype vulnerable to cold stress (Fig. 1). This was not unexpected, as previous reports have shown that the red-leaf phenotype of C. creticus is also characterized by lower light-saturated PSII yields and lower pool sizes of the xanthophyll cycle components during winter and lower leaf nitrogen regardless of season (Kytridis et al., 2008; Zeliou et al., 2009). A link between xanthophyll cycle pool sizes and photoprotection (Demmig-Adams et al., 1996) and between nitrogen levels and photosynthetic capacity (Hikosaka, 2004) is well established. However, photosynthetic inferiority and vulnerability to winter stress of the red phenotype seems to be in apparent contrast to the assumed photoprotective function for leaf anthocyanins since, in this case, one would expect PI of red leaves to remain similar or even be higher than that of green leaves. Alternatively, the low PI value in reds may be a compromise of some (yet slight) protection afforded by anthocyanins, which can not fully alleviate the inherent inferior capability of the red phenotype to cope with winter stress. Regardless of anthocyanin function, one would reasonably expect that photosynthetic inferiority of the red phenotype could be reflected in both growth and reproduction. We may notice at this point that shoot elongation in both phenotypes is already active at late winter and new leaf production seems to be highly active at mid spring (Figs. 2 and 3), i.e. at the period when red phenotypes possess red mature leaves of low photosynthetic performance. Moreover, photosynthetic performance in the red phenotype is already low since mid winter, i.e. well ahead the start of new leaf growth (Fig. 1). Flowering and seed maturation immediately follows the red leaf period and may be supported by current photosynthesis, which is performed by green leaves of equal photosynthetic capacity in both phenotypes (Fig. 1). Concerning vegetative growth, our results are conflicting, since expectations for a lower growth rate in the red phenotype were only fulfilled when the number of new leaves was considered (Fig. 3). Yet, stem elongation rates were higher in the red leaf phenotype (Fig. 2). The resulting slightly longer intermodes may reduce self-shading and compensate for carbon loss due to fewer leaves. Although detailed carbon balance estimation was not the aim of this investigation and growth assessment did not include mass measurements, the results may allow the conclusion that growth in C. creticus is only slightly affected by reduced winter photosynthesis. In any case, during the subsequent flowering and fruiting period (late spring/early summer) both phenotypes displayed similar photosynthetic performance, yet red plants had less leaves (Fig. 3). In spite of that, reproductive effort, reproductive success and seed mass and germinability were similar, suggesting that reproductive output was independent of both previous and current photosynthesis. A redundant acquisition of resources, i.e. carbon and nitrogen assimilation rates in excess of that needed for normal growth and reproduction may be inferred for C. creticus (for a review on redundancy in resource acquisition in plants, see Thomas and Sadras, 2001). Alternatively, one may assume that maintenance of reproductive output has a priority and the reduction in leaf number is a way of re-directing resources to that aim. In any case, a protective function of winter leaf reddening against photoinhibition of photosynthesis is not substantiated on the basis of these results. If carbon acquisition is enough for growth and reproduction (even after a severe winter inactivation of photosynthesis and a subsequent reduction of photosynthetic area during spring and summer), the assumed protective function of anthocyanins may be extremely
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this character for locating physiologically weak, yet equally strong reproductive performers.
References
Fig. 4. A radar plot of physiological (PI, nitrogen, VAZ/Chl), growth (RSGR, LN) and reproductive (RE, PS, RS, SG, SM) parameters of C. creticus. Data compiled from this and a previous study (Kytridis et al., 2008). Values for the red phenotype (empty circles) were normalized over those of the green phenotype (filled circles). Note that physiological parameters refer to the “red” period of the year. Corresponding values for nitrogen levels and VAZ/Chl ratios are lower for the red phenotype all year round (not shown). PI, photosynthetic performance index; VAZ/Chl, (i.e. violaxanthin + antheraxanthin + zeaxanthin)/chlorophyll, denoting photoprotective potential; RSGR, relative shoot growth rate; LN, leaf number; RE, reproductive effort; PS, pollination success (as seeds/fruit); RS, reproductive success; SM, seed mass; SG, seed germinability. Growth parameters are means from the whole sampling period. Reproductive parameters are those of year 2008.
marginal. We may add, however, that winters in both sampling years were normal in respect to temperatures, only slightly deviating from the long-term means (see Section 2). We cannot exclude the possibility that during exceptionally severe winters the reproductive output of the red phenotype may be finally affected. The hypothesis of a signal against herbivores, originally proposed for red senescing leaves (Archetti, 2000; Hamilton and Brown, 2001; Archetti and Brown, 2004), is also weak for our case since insect herbivory is negligible in winter and no apparent loss of leaf mass to consumers was observed (unpublished observations). Moreover, this hypothesis predicts that the optical signal is afforded by individuals, with a high defensive commitment. Yet, in the case of C. creticus the signal may not be honest, as total defensive phenolic levels do not differ between the two phenotypes (Kytridis et al., 2008). We may note, however, that other classes of defensive compounds have not been measured in this plant. In addition, the apparent redundancy in resource acquisition would allow considerable leaf loss to consumers without appreciable effect in growth and reproduction. Similar arguments can be put against the camouflage hypothesis for red leaf color, which may be well applied to young leaves developing during the period of high insect herbivore pressure (Karageorgou and Manetas, 2006), but not for mature leaves becoming red during the winter period of low insect abundance. 5. Conclusion Combining information from this and previous work on the same plant (Kytridis et al., 2008; Zeliou et al., 2009) in a radar plot (Fig. 4), we notice that physiological inferiority of the red phenotype in terms of nitrogen levels, photoprotective potential and photosynthetic performance (i.e. vulnerability to stress), has a rather negligible effect on growth and reproductive success, hence on final fitness. Accordingly, a fitness benefit of winter leaf redness cannot be easily inferred. Although the adaptive significance of winter leaf redness is still obscure, the observer may be benefited from
Archetti, M., 2000. The origin of autumn colours by coevolution. J. Theor. Biol. 205, 625–630. Archetti, M., Brown, S.P., 2004. The coevolution theory of autumn colours. Proc. R. Soc. B-Biol. Sci. 271, 1219–1223. Archetti, M., Leather, S.R., 2005. A test of the coevolution theory of autumn colours: colour preference of Rhopalosiphum padi on Prunus padus. Oikos 110, 339–343. Archetti, M., Döring, T.F., Hagen, S.B., Hughes, N.M., Leather, S.R., Lee, D.W., Lev-Yadun, S., Manetas, Y., Ougham, H.J., Schaberg, P.G., Thomas, H., 2009. Unravelling the evolution of autumn colours: an interdisciplinary approach. Trends Ecol. Evol. 24, 166–173. Burger, J., Edwards, G.E., 1996. Photosynthetic efficiency, and photodamage by UV and visible radiation in red versus green leaf in Coleus varieties. Plant Cell Physiol. 37, 395–399. Christie, P.J., Alfenito, M.R., Walbot, V., 1994. Impact of low-temperature stress on general phenylpropanoid and anthocyanin pathways: enhancement of transcript abundance and anthocyanin pigmentation in maize seedlings. Planta 194, 541–549. Demmig-Adams, B., Gilmore, A.M., Adams, W.W., 1996. In vivo functions of carotenoids in higher plants. Faseb J. 10, 403–412. Esteban, R., Fernandez-Marın, B., Becerril, J.M., García-Plazaola, J.I., 2008. Photoprotective implications of leaf variegation in E. dens-canis L. and P. officinalis L. J. Plant Physiol. 165, 1255–1263. Feild, T.S., Lee, D.W., Holbrook, M., 2001. Why leaves turn red in autumn. The role of anthocyanins in senescing leaves of red-osier dogwood. Plant Physiol. 127, 566–574. Furuta, K., 1986. Host preference and population dynamics in an autumnal population of the maple aphid, Periphyllus californiensis Shinji (Homoptera, Aphididae). J. Appl. Entomol. 102, 93–100. Garcia-Plazaola, J.I., Artetxe, U., Dunabeitia, M.K., Becceril, J.M., 1999. Role of photoprotective systems of Holm-Oak (Quercus ilex) in the adaptation to winter conditions. J. Plant Physiol. 155, 625–630. Hamilton, W.D., Brown, S.P., 2001. Autumn tree colours as a handicap signal. Proc. R. Soc. B-Biol. Sci. 268, 1489–1493. Hermans, C., Smeyers, M., Rodriguez, R.M., Eyletters, M., Strasser, R.J., Delhaye, J.-P., 2003. Quality assessment of urban trees: a comparative study of physiological characterisation, airborne imaging and on site fluorescence monitoring by the OJIP-test. J. Plant Physiol. 160, 81–90. Hikosaka, K., 2004. Interspecific difference in the photosynthesis–nitrogen relationship: patterns, physiological causes and ecological importance. J. Plant Res. 117, 481–494. Hodges, D.M., Nozzolillo, C., 1996. Anthocyanin and anthocyanoplast content of cruciferous seedlings subjected to mineral nutrient deficiencies. J. Plant Physiol. 147, 749–754. Hughes, N.M., Neufeld, H.S., Burkey, K.O., 2005. Functional role of anthocyanins in high-light winter leaves of the evergreen herb Galax urceolata. New Phytol. 168, 575–587. Hughes, N.M., Smith, W.K., 2007a. Attenuation of incident light in Galax urceolata (Diapensiaceae): concerted influence of adaxial and abaxial anthocyanic layers on photoprotection. Am. J. Bot. 94, 784–790. Hughes, N.M., Smith, W.K., 2007b. Seasonal photosynthesis and anthocyanin production in ten broadleaf evergreen species. Funct. Plant Biol. 34, 1072–1079. Karageorgou, P., Manetas, Y., 2006. The importance of being red when young: anthocyanins and the protection of young leaves of Quercus coccifera from insect herbivory and excess light. Tree Physiol. 26, 613–621. Karavatas, S., Manetas, Y., 1999. Seasonal patterns of photosystem 2 photochemical efficiency in evergreen sclerophyll sand drought semi-deciduous shrubs under Mediterranean field conditions. Photosynthetica 36, 41–49. Kyparissis, A., Grammatikopoulos, G., Manetas, Y., 2007. Leaf morphological and physiological adjustments to the spectrally selective shade imposed by anthocyanins in Prunus cerasifera. Tree Physiol. 27, 849–857. Kytridis, V.-P., Manetas, Y., 2006. Mesophyll versus epidermal anthocyanins as potential in vivo antioxidants: evidence linking the putative antioxidant role to the proximity of oxy-radical source. J. Exp. Bot. 57, 2203–2210. Kytridis, V.-P., Karageorgou, P., Levizou, E., Manetas, Y., 2008. Intra-species variation in transient accumulation of leaf anthocyanins in Cistus creticus during winter: evidence that anthocyanins may compensate for an inherent photosynthetic and photoprotective inferiority of the red-leaf phenotype. J. Plant Physiol. 165, 952–959. Lee, D.W., Gould, K.S., 2002. Anthocyanins in leaves and other vegetative organs: an introduction. Adv. Bot. Res. 37, 1–16. Lee, D.W., O’Keefe, J., Holbrook, N.M., Feild, T.S., 2003. Pigment dynamics and autumn leaf senescence in a New England deciduous forest, eastern USA. Ecol. Res. 18, 677–694. Lev-Yadun, S., Dafni, A., Flaishman, M.A., Inbar, M., Izhaki, I., Katzir, G., Neeman, G., 2004. Plant coloration undermines herbivorous insect camouflage. BioEssays. 26, 1126–1130. Lindoo, S.J., Caldwell, M.M., 1978. Ultraviolet-B radiation induced inhibition of leaf expansion and promotion of anthocyanin production. Plant Physiol. 61, 278–282.
514
C. Nikiforou et al. / Environmental and Experimental Botany 67 (2010) 509–514
Long, S.P., Humphries, S., Falkowski, P.G., 1994. Photoinhibition of photosynthesis in nature. Annu. Rev. Plant Physiol. Plant. Mol. Biol. 45, 633–662. Manetas, Y., Petropoulou, Y., 2000. Nectar amount, pollinator visit duration and pollination success in the Mediterranean shrub Cistus creticus. Ann. Bot. 86, 815–820. Manetas, Y., 2006. Why some leaves are anthocyanic and why most anthocyanic leaves are red? Flora 201, 163–177. Marshall, D.L., Levin, D.A., Fowler, N.L., 1986. Plasticity of yield components in response to stress in Sesbania macrocarpa and Sesbania vesicaria (Leguminosae). Am. Nat. 127, 508–521. Numata, S., Kachi, N., Okuda, T., Manokaran, N., 2004. Delayed greening, leaf expansion, and damage to sympatric Shorea species in a lowland rain forest. J. Plant Res. 117, 19–25. Steyn, W.J., Wand, S.J.E., Holcroft, D.M., Jacobs, G., 2002. Anthocyanins in vegetative tissues: a proposed unified function in photoprotection. New Phytol. 155, 349–361. Stone, B.C., 1979. Protective coloration of young leaves in certain Malaysian palms. Biotropica 11, 126. Strasser, R.J., Srivastava, A., Govindjee, 1995. Polyphasic chlorophyll a fluorescence transient in plants and cyanobacteria. Photochem. Photobiol. 61, 32–42.
Strasser, R.J., Tsimilli-Michael, M., Srivastava, A., 2004. Analysis of the chlorophyll a fluorescence transient. In: Papageorgiou, G.C., Govindjee (Eds.), Chlorophyll Fluorescence: A Signature of Photosynthesis. Springer, Dordrecht, The Netherlands, pp. 321–362. Thanos, C.A., Georghiou, K., 1988. Ecophysiology of fire stimulated seed germination in Cistus incanus spp creticus (L) Heywood and C. salvifolius L. Plant Cell Environ. 11, 841–849. Sultan, S.E., 2001. Phenotypic plasticity for fitness components in Polygonum species of contrasting ecological breadth. Ecology 82, 328–343. Thomas, H., Sadras, V.O., 2001. The capture and gratuitous disposal of resources by plants. Funct. Ecol. 15, 3–12. van Heerden, P.D.R., Tsimilli-Michael, M., Kruger, G.H.J., Strasser, R.J., 2003. Dark chilling effects on soybean genotypes during vegetative development: parallel studies of CO2 assimilation, chlorophyll a fluorescence kinetics O-J-I-P and nitrogen fixation. Physiol. Plant. 117, 476–491. Zeliou, K., Manetas, Y., Petropoulou, Y., 2009. Transient winter leaf reddening in Cistus creticus characterizes weak (stress-sensitive) individuals, yet anthocyanins cannot alleviate the adverse effects on photosynthesis. J. Exp. Bot. 60, 3031–3042.