DOI: 10.1007/s11099-016-0235-5
PHOTOSYNTHETICA 55 (1): 31-40, 2017
Specific leaf area variations drive acclimation of Cistus salvifolius in different light environments G. PUGLIELLI+, L. VARONE, L. GRATANI, and R. CATONI Department of Environmental Biology, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy
Abstract Cistus salvifolius L. is the most widely spread Cistus species around the Mediterranean basin. It colonizes a wide range of habitats growing from sea level to 1,800 m a.s.l., on silicolous and calcicolous soils, in sun areas as well as in the understory of wooded areas. Nevertheless, this species has been mainly investigated in term of its responsiveness to drought. Our aim was to understand which leaf traits allow C. salvifolius to cope with low-light environments. We questioned if biochemical and physiological leaf trait variations in response to a reduced photosynthetic photon flux density were related to leaf morphological plasticity, expressed by variations of specific leaf area (SLA) and its anatomical components (leaf tissue density and thickness). C. salvifolius shrubs growing along the Latium coast (41°43ʹN,12°18ʹE, 14 m a.s.l., Italy) in the open and in the understory of a Pinus pinea forest, were selected and the relationships between anatomical, gas exchange, chlorophyll (Chl) fluorescence, and biochemical parameters with SLA and PPFD variations were tested. The obtained results suggested long-term acclimation of the selected shrubs to contrasting light environments. In high-light conditions, leaf nitrogen and Chl contents per leaf area unit, leaf thickness, and Chl a/b ratio increased, thus maximizing net photosynthesis, while in shade photosynthesis, it was downregulated by a significant reduction in the electron transport rate. Nevertheless, the increased pigment-protein complexes and the decreased Chl a/b in shade drove to an increased light-harvesting capacity (i.e. higher actual quantum efficiency of PSII). Moreover, the measured vitality index highlighted the photosynthetic acclimation of C. salvifolius to contrasting light environments. Overall, our results demonstrated the morphological, anatomical, and physiological acclimation of C. salvifolius to a reduced light environment. Additional key words: carotenoids; chlorophyll fluorescence; gas exchange; leaf absorptance; leaf nitrogen partitioning.
Introduction The reduction in light environment has phenotypic consequences on plant morphology and physiology (Price et al. 2003) and such acclimation process varies among species (Hallik et al. 2012). In particular, morphological adjustments are relatively slow and occur at the time scale of leaf growth (Yamashita et al. 2002). Specific leaf area (SLA) is one of the main morphological traits which changes in response to light variations (Gratani 2014). The
plasticity of SLA implies the morphogenetic control of leaves which tends to increase leaf area in the shade in order to intercept more light. Moreover, the increased total lamina thickness in sun leaves compared to shade leaves is mainly due to the greater palisade parenchyma, spongy parenchyma and epidermis thickness, suggesting that leaf internal structure plays an important role in light capture (Evans 1999). Alterations in light availability influence
——— Received 2 March 2016, accepted 20 April 2016, published as online-first 28 April 2016. +Corresponding author; e-mail:
[email protected] Abbreviations: C/N – ratio between soil organic carbon content and total soil nitrogen content; Ca – CO2 concentration in the leaf chamber; Cab – abaxial cuticle thickness; Cad – adaxial cuticle thickness; Car – carotenoids; Car/Chl – ratio between Car and Chl (a+b); Ce – apparent carboxylation efficiency; Chl – chlorophyll; Ci – intercellular CO2 concentration; DM – dry mass; E – transpiration rate; ETab – abaxial epidermis thickness; ETad – adaxial epidermis thickness; ETR – electron transport rate; fias – fraction of the mesophyll occupied by intercellular air spaces; FM – fresh mass; Fm – maximal fluorescence yield of the dark-adapted state; Fm' – maximal fluorescence yield of the light-adapted state; Fs – steady-state fluorescence yield; gs – stomatal conductance; LA – leaf area; LMA – leaf mass area; LT – total leaf thickness; LTD – leaf tissue density; Narea – nitrogen content per unit leaf area; Nleaf – leaf nitrogen content; NORG – organic nitrogen content per unit of leaf area; NP – nitrogen allocated to the pigment-protein complexes; NP% – NP as percentage of NORG; P – palisade parenchyma thickness; PN – net photosynthetic rate; R/FR – ratio between irradiance in the red and far red wavelengths; RD – respiration rate; RD/PN – ratio between RD and PN; Rfd – vitality index; S – spongy parenchyma thickness; SD – stomatal density; SLA – specific leaf area; SPA – stomatal pore area; SPL – stomatal pore length; SPW – stomatal pore width; Tl – leaf temperature; Tm – mean air temperature; Tmax – mean maximum air temperature; Tmin – mean minimum air temperature; WUEi – intrinsic water-use efficiency; α – absorptance; ΦPSII – effective quantum yield of PSII photochemistry; χ – chlorophyll content per unit of leaf area.
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leaf physiology (Hallik et al. 2012). In high-light environments, the amount of the outer antenna proteins is reduced (Niinemets 2007) resulting in an increased Chl a/b ratio (Niinemets 2010) and a decreased pigment-protein nitrogen (Evans and Poorter 2001). Sun leaves are generally characterized by higher nitrogen content per unit of leaf area which is correlated with an increased electron transport capability and carboxylation rate (Kull and Niinemets 1993). As a consequence, the photosynthetic capacity is higher in leaves under high-light conditions (Evans 1989). The higher photosynthetic rates of sun leaves are supported by a higher stomatal density and stomatal conductance to maximize CO2 absorption (Sack et al. 2006). Leaf trait adjustments to low light increase the capability of light absorption at the expense of the photosynthetic capability (Evans 1989). Nevertheless, the tolerance to one stress is typically reduced by other cooccurring stresses and tolerance to shade, for example, has been reported to compromise tolerance to other environmental limitations, such as drought (Valladares and Niinemets 2008). In a global climate change context, the trade-offs between shade tolerance and tolerance to other environmental factors point to important global change effects on species relative abundances mediated by species-specific differences in their tolerance to shade (Kursar 1998, Valladares and Niinemets 2008). Thus, it is important to understand if species-specific pattern of trait variations in response to light reduction can be referred as acclimation or just as passive responses to a reduced light availability (Puglielli et al. 2015). The species belonging to the genus Cistus are mainly distributed in the Mediterranean area (Southern Europe, North Africa, and Western Asia) (Fernandez-Muzuecos and Vargas 2010), including the Mediterranean Basin (Núñez-Olivera et al. 1996), where they colonize degraded areas (Attaguile et al. 2000). Among the species of the
genus, C. salvifolius L. is a diploid insect-pollinated shrub, and is outcrossing and highly self-incompatible (Bosch 1992). C. salviifolius is the most widely spread species of the genus Cistus around the Mediterranean basin. It has a circum-Mediterranean distribution, from Portugal and Morocco to Palestine and the eastern coast of the Black Sea, also occurring in the south of the Eurosiberian floristic region. It has also been recorded in Macaronesia (Madeira), where it is probably an introduced species (Short 1994). At least three intercontinental colonizations are responsible for its wide distribution, leading to little geographical isolation with high genetic diversity within populations, but no genetic differentiation between the different populations (Farley and Mc Neilly 2000, Fernández-Mazuecos and Vargas, 2010). The factors that caused the dispersion of C. salviifolius around the Mediterranean were mostly ecological, such as the climate and the soil (Papaefthimiou et al. 2014). It grows on silicolous, calcicolous, and sandy soils in a wide range of habitats (Guzmán et al. 2009) from sea level to 1,800 m and in the understory of wooded areas (Farley and McNeilly 2000). Moreover, C. salvifolius populations differ strongly in a leaf size, with a wide range in both leaf size and form (Warburg 1968). At the best of our knowledge, leaf trait variations of C. salvifolius in different light conditions has been poorly investigated (e.g. Zunzunegui et al. 2016), thus the main objective of this research was to analyze C. salvifolius response to light variations. In fact, the selected species has been mainly investigated in term of its responsiveness to drought (e.g. Harley et al. 1987, Zunzunegui et al. 2009, Grent et al. 2014). In particular, we questioned if biochemical and physiological leaf trait variations in response to a reduced PPFD can be related to leaf morphological plasticity, which was analyzed in term of variations in SLA and its anatomical components.
Materials and methods Study area and plant material: The study was carried out along the Latium coast (41°43ʹN,12°18ʹE, 14 m a.s.l., Italy) from the middle of April to the beginning of May 2015, since previous studies have demonstrated that longer-term foliage traits vs. light relationships are stable during most of the growing season (Niinemets et al. 2004, Hallik et al. 2012). Ten representative and comparable C. salviifolius shrubs (height = 0.50 ± 0.10 m) growing in the open (site A, sun plants), and ten shrubs growing in the understory of the Pinus pinea forest (site B, shade plants) were selected. The selected shrubs were of the same age (i.e. seven years old), thus avoiding that any difference in the selected leaf traits could be due to differences in the plant age. Site A was characterized by a pH ranging from 5.60 to 5.97 and a C/N ratio of 13.9 ± 2.1. In site B, the pH ranged from 6.34 to 6.53 and the C/N ratio was 12 ± 0.5. During the study period, sun and shade conditions were characterized by a PPFD [µmol(photon) m–2 s–1] of
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1,786 ± 110 and 98 ± 34, respectively. The ratio between irradiance in the red and far red wavelengths (R/FR) was 2.93 ± 1.1 and 0.93 ± 0.08, in sun and shade conditions, respectively. PPFD was measured by radiometers (Li-185B, LI-COR, USA) and R and FR by plant growth photometers (IL150, International Light, USA). The study area is characterized by a Mediterranean type of climate. The mean minimum air temperature (Tmin) of the coldest month (February) was 4.4 ± 2.9°C, the mean maximum air temperature (Tmax) of the hottest month (August) was 30.7 ± 2.4°C, and the mean air temperature (Tm) was 16.1 ± 6.1°C. Dry period was from the beginning of June to the end of August (65 mm of total rainfall for the period). Total annual rainfall was 884 mm, with most of it occurring in autumn and winter. During the study period, total rainfall was 1291 mm, Tm of 15.3 ± 5.1°C, Tmax (August) of 27.2 ± 1.2°C, and Tmin (January) of 5.5 ± 1.2°C (Data from the Metereological Station of Roma-
CISTUS SALVIFOLIUS ACCLIMATION TO SHADE
Capocotta, ARSIAL, Lazio Regional Agency for Development and Agricultural Innovation for the years 2004–2014). Morphological and anatomical leaf traits: Fully expanded leaves (n = 3 per each sun and shade shrub) from the external medium portion of the crown of the considered shrubs were collected in the middle of May. The following parameters were measured: leaf area (excluding petiole) (LA), obtained by the image analysis system (Delta-T Devices, UK), and dry mass (DM, mg), determined drying leaves at 80°C to constant mass. Specific leaf area (SLA) was calculated by the ratio between LA and DM. Leaf tissue density (LTD) was calculated by the ratio of leaf mass area (LMA = 1/SLA) and total leaf thickness (LT) (Wright and Westoby 2002). Leaf anatomy was analyzed by a light microscopy using an image analysis system (Axiovision, AC software). The following parameters were measured: LT, palisade (P) and spongy parenchyma thickness (S), adaxial and abaxial epidermis thickness (ETad and ETab), adaxial and abaxial cuticle thickness (Cad and Cab). All measurements were restricted to vein free areas, according to Chabot and Chabot (1977). Stomatal density (SD) and stomatal length were measured. SD was determined from nail varnish impressions of the abaxial lamina, according to Sack et al. (2003). The number of stomata were counted on separate impressions of the lamina, each measuring 0.5×1.0 cm. Stomatal length was measured on at least 30 randomly selected stomata, according to Mishra (1997). Stomatal pore length (SPL) and width (SPW) were measured on the same recorded digital images. Dimension of the stomata was used to calculate the equivalent area of the ellipsoid representing the stomatal pore area (SPA) by the following formula: (π × length × width)/4, according to Bartolini et al. (1997). The fraction of the mesophyll occupied by intercellular air spaces (fias) was calculated, according to Syvertsen et al. (1995) as fias= 1 – (Am/lW) where Am is the crosssectional area of the mesophyll cells, l the mesophyll thickness, and W the width of the measured section. Gas exchange and Chl fluorescence: Measurements of gas exchange were carried out using the infrared gas analyzers ADC LCPro+ (UK) equipped with a leaf chamber (PLC, Parkinson Leaf Chamber, ADC, UK). Measurements were made on three leaves per each sun and shade shrubs. Net photosynthetic rate (PN), PPFD, stomatal conductance (gs), transpiration rate (E), intercellular CO2 concentration (Ci), and leaf temperature (Tl) were measured. Measurements were carried out under natural conditions, on cloudless days at saturating PPFD [ 1,000 mol(photon) m ̶ 2 s ̶ 1 in sun conditions], in the morning from 8:00 a.m. to 12:00 a.m. (Reich et al. 1995). CO2 concentration in the leaf chamber (Ca) was set at 400 ppm, and relative air humidity of the incoming air ranged between 40 and 60%. Apparent carboxylation
efficiency (Ce) was also determined by the ratio between PN and Ci (Flexas et al. 2001). The intrinsic water-use efficiency (WUEi) was calculated as the ratio between PN and gs. On each sampling occasion, respiration rate (RD) measurements were carried out after the PN measurements (on the same leaves) as CO2 efflux, darkening leaf chamber by a black paper, according to Cai et al. (2005), for 30 min prior to each measurement, to avoid transient post-illumination bursts of CO2 (Atkin et al. 1998). The ratio RD/PN was calculated. Chl fluorescence measurements were carried out by portable modulated fluorometers (OS5p, Opti ̶ Sciences, USA) on the same leaves as gas-exchange measurements, for sun and shade shrubs. The effective quantum yield of PSII photochemistry (ΦPSII) was calculated on light-adapted leaves according to Genty et al. (1989) asPSII = (Fm' ̶ Fs)/Fm', where Fm' was the maximal fluorescence yield of the light-adapted state obtained with a light-saturating pulse [~8,000 µmol (photon) m ̶ 2 s ̶ 1] and Fs was the steady-state fluorescence yield of illuminated leaves [1,600 µmol(photon) m ̶ 2 s ̶ 1]. The electron transport rate (ETR) was calculated according to Krall and Edwards (1992) as ETR = PSII × PPFD × 0.5 × 0.84. Vitality index (Rfd) was also calculated according to Lichtenthaler and Rinderle (1988) by using the equation Rfd = (Fm – Fs)/Fs , where Fm is the maximal fluorescence yield of the dark-adapted state obtained on the same leaves dark-adapted for 30 min with leaf clips. Biochemical leaf traits: Leaf nitrogen content (Nleaf), Chl a, Chl b, and carotenoids (Car) contents were measured on fully expanded leaves (n = 3 per each sun and shade shrubs) from the external medium portion of the crown of the considered shrubs collected in the middle of May. Immediately after collection, leaf samples were kept cool in the dark and transported immediately to the laboratory. Chl and Car content were determined after grinding leaves in acetone [n = 10, 1.5 g of fresh mass (FM) each, per sun and shade shrubs, respectively, and per each occasion]. The homogenates were centrifuged in a refrigerated centrifuge (4237R. A.L.C., Italy). Absorbance of the supernatants was measured by a Jasco model 7800LCD (Japan) spectrophotometer at the wavelengths of 645, 663, and 440 nm for Chl a, Chl b, and Car, respectively. The Chl content was calculated according to MacLachlan and Zalik (1963) and Car according to Holm (1954). The Chl (a+b), Chl a/b, and the ratio of Car/Chl were calculated being related with the capacity of light absorption in PSI and PSII (Zunzunegui et al. 2016). The fraction of the photosynthetically active photon irradiance, which is absorbed by the leaf (absorptance, α) depending on the Chl content per unit leaf area (χ), was calculated according to Evans and Poorter (2001) as: α χ⁄ χ 76 . Nitrogen content was determined by drying leaf samples at 70°C (n = 3 samples, 0.5 g of DM each, per sun
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and shade shrubs, respectively, per each occasion) grounded into a fine powder. The Nleaf content was measured by the Kjeldahl method (Mendes et al. 2001). Nitrogen content per unit of leaf area (Narea) was also determined. The organic nitrogen content per unit of leaf area (NORG) was calculated according to Evans and Poorter 10 N ∗ / SLA. The average value of (2001) as N ∗ 3.3 of N [mmol(N) g1(DM)] proposed by Evans and Poorter (2001) for ten dicotyledonous C3 species with different growth forms (woody trees, shrubs, and herbaceous species) was used. The same N ∗ value for sun and shade shrubs was used since it is independent from the growth irradiance (Evans and Poorter 2001). The nitrogen allocated to the pigment-protein complexes (NP) was calculated according to Evans and Poorter (2001) as χη⁄10 where η is the mean nitrogen to Chl ratio for N
pigment-protein complexes [53.7 and 51.7 mol(N) mol(Chl) ̶ 1 for sun and shade shrubs, respectively]. The value of NP as percentage of NORG (NP%) was calculated. Statistics: Differences in the considered leaf traits between sun and shade shrubs were analysed by one-way analysis of variance (ANOVA) at p≤0.05. Multiple comparisons were analyzed by a Tukey's test. Pearson pair-wise correlation coefficients were used to characterise bivariate relationships between the traits. A multiple regression analysis by using LT as response variable and P, S, ETad, ETab, Cad, Cab as predictors was carried out in order to evaluate the main determinants of total leaf thickness. Statistical analyses were carried out using Statistica 10 (Statsoft, USA).
Results Morphological and anatomical leaf traits: SLA was 55% higher in shade than in sun leaves, while LTD was 5% lower in shade than that in sun leaves (Table 1). SLA was significantly correlated with PPFD, LA, LT, ETad, P, Table 1. Leaf morphological, anatomical, and stomatal parameters. Mean values ± SD are shown. Different letters indicate significant differences between sun and shade (one-way ANOVA, p≤0.05, n = 30). LA leaf area; DM leaf dry mass; SLA specific leaf area; LTD leaf tissue density; LT total leaf thickness; P palisade parenchyma thickness; S spongy parenchyma thickness; ETad adaxial epidermis thickness; ETab abaxial epidermis thickness; Cutad adaxial cuticle thickness; Cutab abaxial cuticle thickness; fias the fraction of the mesophyll occupied by intercellular air spaces; SD stomatal density; SPA stomatal pore area. Parameter
Sun
Shade
Morphological leaf traits LA [cm2] DM [g] SLA [cm2 g ̶ 1] LTD [mg cm ̶ 3]
2.77 ± 0.03a 0.49 ± 0.004a 106 ± 10a 430 ± 36a
6.99 ± 0.04b 0.92 ± 0.005b 164 ± 17b 409 ± 30a
Anatomical leaf traits LT [µm] P [µm] S [µm] ETad [µm] ETab [µm] Cad [µm] Cab [µm] fias [%]
213 ± 17a 96.2 ± 4a 59 ± 2.5a 23.2 ± 6.3a 17.3 ± 3.7a 1.3 ± 0.4a 0.9 ± 0.2a 22.4 ± 2.1a
150 ± 8b 88.7 ± 5b 53.4±3b 22.4 ± 2.7a 18.1 ± 2a 0.97 ± 0.1b 0.8 ± 0.2a 23.6 ± 2.4a
Stomatal parameters SD [n mm ̶ 2] SPA [µm2]
230.9 ± 17.4a 23.8 ± 4.4a
171.5 ± 13.1b 35.2 ± 7.2b
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L, and Cad. The same parameters were significantly correlated with PPFD (Table 2). LT was 42% higher in sun than in shade leaves. Cad, P and L were 34, 80, and 50% higher in sun than in shade leaves, respectively. ETab, ETad, and Cab did not show any significant difference between sun and shade leaves. SD was 35% higher in sun than that in shade leaves, while SPA was 49% higher in shade than in sun leaves. fias was 3% lower in sun than that in shade leaves. The multiple regression analysis showed a significant effect of P, S, ETad, and Cad in determining LT variations. Table 2. Pearson pair-wise correlation coefficients of specific leaf area (SLA) and photosynthetic photon flux density (PPFD) vs. morphological and anatomical leaf traits. Correlation coefficients are calculated for sun and shade shrubs pooled. *p
