L a G D a b c d e f a A R R A K A C L S 1 s d p w m n s a 0 d Environmental and Experimental Botany 71 (2011) 10–17 Contents lists available at ScienceDirect Environmental and Experimental Botany journa l homepage: www.e lsev ier .com/ locate /envexpbot eaf paraheliotropism in Styrax camporum confers increased light use efficiency nd advantageous photosynthetic responses rather than photoprotection ustavo Habermanna,∗, Patricia F.V. Ellsworthb, Juliana L. Cazotoc, Aline M. Feistlera, Leandro da Silvad, ario A. Donatti e,1, Silvia R. Machadof,1 Univ Estadual Paulista, Unesp, Departamento de Botânica, IB, Rio Claro-SP, 13506-900, Brazil University of Miami, Department of Biology, Coral Gables, FL, 33124, USA Universidade Estadual de Campinas, Unicamp, Departamento de BIOLOGIA Vegetal, IB, Campinas-SP, 13083-970, Brazil Instituto Agronômico de Campinas, Centro de Ecofisiologia e Biofísica, Campinas-SP, 13001-970, Brazil Univ Estadual Paulista, Unesp, Departamento de Física, IGCE, Rio Claro-SP, 13506-900, Brazil Univ Estadual Paulista, Unesp, Departamento de Botânica, IB, Botucatu-SP, 18618-000, Brazil r t i c l e i n f o rticle history: eceived 29 November 2009 eceived in revised form 11 October 2010 ccepted 13 October 2010 eywords: baxial and adaxial leaf surfaces errado ight curves tyracaceae a b s t r a c t Styrax caporum is a native shrub from the Brazilian savanna. Most of its leaves are diaheliotropic, whereas some are paraheliotropic, mainly at noon. A previous study of this species revealed higher stomatal conductance (gs) and transpiration rates (E) in para- compared to diaheliotropic leaves, and a rise in CO2 assimilation rates (A) with an increase of irradiance for paraheliotropic leaves. We hypothesized that this species exploits the paraheliotropism to enhance the light use efficiency, and that it is detected only if gas exchange is measured with light interception by both leaf surfaces. Gas exchange was measured with devices that enabled light interception on only one of the leaf surfaces and with devices that enabled light interception by both leaf surfaces. Water relations, relative reflected light intensity, leaf temperature (Tl), and leaf anatomical analyses were also performed. When both leaf surfaces were illuminated, a higher A, E, and gs were observed in para- compared to diaheliotropic leaves; however, A did not depend on gs, which did not influence CO2 accumulation in the stomatal cavity (Ci). When only the adaxial leaf surface was illuminated, a greater A was detected for para- than for diaheliotropic leaves only at 11:00 h; no differences in T were observed between leaf types. Light curves revealed that under non-saturating l light the adaxial side of paraheliotropic leaves had higher A than the abaxial side, but they showed similar values under saturating light. Although the abaxial leaf side was highly reflective, both surfaces presented the same response pattern for green light reflection, which can be explained by the compact spongy parenchyma observed in the leaves, increasing light use efficiency in terms of CO2 consumption for paraheliotropic leaves. We propose that paraheliotropism in S. camporum is not related to leaf heat tion. avoidance or photoprotec . Introduction Trees and shrubs growing in the cerrado, or the Brazilian avanna, which is characterized by wet (October–March) and ry (April–September) seasons, have to adjust their morpho- hysiological traits seasonally to successfully cope with the soil ater availability. The cerrado vegetation is a vertically structured osaic of grassland, scrubland, and dense woodland physiog- omies (Haridasan, 2008). The soils of these areas are deep, acidic, andy, contain low levels of organic matter and phosphorus, and re rich in aluminum (Haridasan, 2008). ∗ Corresponding author. Tel.: +55 19 3526 4210; fax: +55 19 3526 4201. E-mail address: ghaber@rc.unesp.br (G. Habermann). 1 CNPq fellowship. 098-8472/$ – see front matter © 2010 Elsevier B.V. All rights reserved. oi:10.1016/j.envexpbot.2010.10.012 © 2010 Elsevier B.V. All rights reserved. Concomitant to seasonal water deficit, the cerrado environment experiences a high irradiance load and elevated vapor pressure deficits (VPD). Under such conditions, paraheliotropic leaf move- ment is one of the strategies used by many plants. Diaheliotropic leaves, which are oriented at an angle perpendicular to incom- ing light, maximize light interception, while paraheliotropic leaves, which orient parallel to the light, minimize it (Koller, 1986, 1990; Bielenberg et al., 2003; Pastenes et al., 2005; Arena et al., 2008). In leguminous species, paraheliotropic leaf movements are rapidly induced by unfavorable conditions. In beans, leaf para- heliotropism is induced by water deficit (Pastenes et al., 2005), leaf heat and excess sunlight interception (Bielenberg et al., 2003). In the soybean, leaf paraheliotropism is induced with increasing irradiance (Jiang et al., 2006). For leguminous cerrado species, paraheliotropism may be a strategy to avoid excess sunlight inter- ception at noon (Caldas et al., 1997; Rodrigues and Machado, 2006), dx.doi.org/10.1016/j.envexpbot.2010.10.012 http://www.sciencedirect.com/science/journal/00988472 http://www.elsevier.com/locate/envexpbot mailto:ghaber@rc.unesp.br dx.doi.org/10.1016/j.envexpbot.2010.10.012 and Ex w E a a p 2 e s ( u ( b c o t s d w T l l l l s i g t 6 s p i t a S l s 2 2 P s m 4 t n o 2 a t r m r t p G. Habermann et al. / Environmental hich reinforces the photo-protective role of paraheliotropism. ven the leaf wilting movement of non-leguminous species, such s cotton plants, confers photo-protection and maintains carbon ssimilation (Zhang et al., 2010). Another leguminous species, Robinia pseudoacacia exhibits araheliotropic leaf movement at high irradiance levels (Liu et al., 007), reducing light interception and leaf temperature (Arena t al., 2008). However, paraheliotropic leaves of R. pseudoacacia how higher stomatal conductance (gs) and CO2 assimilation rates A), which are attributed to a higher photochemical performance of nrestrained paraheliotropic leaves, compared to restrained leaves Arena et al., 2008). However, the physiological role of paraheliotropism may not e universal. Styrax camporum, a non-leguminous species from the errado, possesses leaves that are always diaheliotropic as well as ther leaves that assume a paraheliotropic position from 10:00 h o 17:00 h (Habermann et al., 2008). Paraheliotropic leaves of this pecies have higher A and transpiration rates (E) compared to the iaheliotropic leaves, although this high E has a weak relationship ith the reduction in leaf temperature (Habermann et al., 2008). his study also demonstrated compact spongy parenchyma in both eaf types, and a rise in A with the increase of irradiance for parahe- iotropic leaves. Thus, this species might exploit the paraheliotropic eaf movement to enhance light use efficiency. In the present study, we hypothesized that, compared to diahe- iotropic leaves, the paraheliotropic leaves of S. camporum would how higher values of gas exchange rates if measured with light ntercepted by adaxial and abaxial leaf surfaces. Measurements of as exchange with devices that enabled artificial light intercep- ion only by adaxial or abaxial leaf surfaces (LI-6400 Irga with a 400-02B red/blue LED light source) and with devices that enabled unlight interception by both leaf sides (LI-6200 Irga with a trans- arent 6000-12 one liter chamber) were performed in April 2006, n Botucatu-SP and Corumbataí-SP, Brazil. The leaf water potential, he leaf temperature of para- and diaheliotropic leaves, and the rel- tive reflected light intensity of adaxial and abaxial leaf surfaces of . camporum were also measured. Anatomical and ultra-structural eaf analyses were performed as a framework for the functional tudies (see online supplementary information, Fig. S1). . Materials and methods .1. Site description This study was conducted using adult plants of S. camporum ohl. from cerrado fragments characterized by scattered trees and hrubs and a large proportion of grassland (“closed field”) in the unicipalities of Botucatu, São Paulo (SP) state, Brazil (22◦ 51′ S, 8◦ 26′ W) and Corumbataí, SP state, Brazil (22◦ 13′ S, 47◦ 37′ W). Five individual plants between 1.5 and 2-m tall from each of hese sites were used. Plants were completely leafy at the begin- ing of the fall season in April 2006, when the measurements were btained. .2. Leaf angle measurement In order to classify leaves as para- or diaheliotropic, the petiole ngle formed with the horizon was measured. A fine wire was posi- ioned between the petiole and a ruler with a water level, which epresented the horizon. The curvature radius formed by the wire irrored the petiole angle. Then, the angle defined by the wire was eproduced on paper and determined with a goniometer, similar to he method presented by Arena et al. (2008). Leaves showing petiole angle greater than 50◦ were classified as araheliotropic leaves, which were marked on each plant at noon perimental Botany 71 (2011) 10–17 11 on the day before the measurements were performed. Leaves dis- playing a petiole angle between 0◦ and 10◦ were considered as diaheliotropic. Both leaf types occurred on woody stems, and had mature fully expanded leaf blades. Very young and very old leaves were avoided. 2.3. Leaf gas exchange measurements Gas exchange was measured using an infrared gas analyzer (LI- 6200, LI-Cor, USA) with a 6000-12 one liter chamber, which is made of Lexan® and MargardTM transparent materials. These materials have a transmittance of 90% in the visible and near infra-red spectra, but bellow 450 nm it falls markedly to 60% at 400 nm (Li-Cor, 1990). Therefore, this leaf cuvette enables sunlight absorptance from 300 to 1100 nm (Li-Cor, 1990). This leaf cuvette has inserts which were used to fix leaf area to 6 cm2 (a predetermined size), allowing faster measurements and exposure of adaxial and abaxial leaf surfaces to direct sunlight or diffuse irradiance (soil reflection and scattered irradiance). Additionally, gas exchange was also measured with an infra-red gas analyzer (LI-6400, Li-Cor, USA) using a leaf cuvette that enables artificial red and blue LED light (6400-02B, Li-Cor, USA) interception by only one of the leaf surfaces. The 6400-02B red blue light source spectral output has one peak centered at about 670 nm and a secondary peak at about 465 nm (Li-Cor, 2004). Because paraheliotropic leaves were not flat on both sides of the midrib, and showed different inclination planes, it was not possible to measure just one of the leaf sides of the midrib without disturb- ing the leaf planes. Otherwise, it would not match the minimum area necessary to make measurements in gas exchange chambers. Thus, the leaf planes of paraheliotropic leaves became entirely flat when leaf cuvettes of both equipments were closed. However, the petiole angle was not disturbed. Diaheliotropic leaves were nat- urally completely flat, following the petiole angle, which was not disturbed as well. CO2 assimilation (A) and transpiration (E) rates, stomatal con- ductance (gs), and intercellular CO2 (Ci) were determined by the Irgas’ data analysis programs, which employ the Von Caemmerer and Farquhar (1981) general gas exchange equations for both equipments. The photosynthetic radiation use efficiency (PhRUE), was also calculated (See online supplementary data for more details about the method for calculating PhRUE, Table S1). Both leaf cuvettes had external quantum sensors, which were used to measure the incoming sunlight. In the case of the leaf cuvette that enabled light interception by both leaf sides, the quantum sensor measured ambient PPFD intercepted by leaves. The leaf cuvette that enabled artificial light interception by only one of the leaf sides was set to provide 1800 �mol photons m−2 s−1, as ambient PPFD varied from 1000 to 1600 �mol photons m−2 s−1. The leaf temperature (Tl) was obtained using a small thermocouple within the leaf cuvettes of both systems, according to Bielenberg et al. (2003). Curves of A, gs, and Ci as a function of the PPFD values estab- lished in the leaf cuvette that enables artificial light interception by only one of the leaf sides were also constructed to detect the sole responses of the adaxial and abaxial leaf surfaces of para- and diaheliotropic leaves. These curves were generated at a controlled leaf temperature (25 ± 1 ◦C). 2.4. Estimation of leaf reflectance An estimation of the relative intensity of reflected light from both leaf sides was performed using a fluorometer (Cary Eclipse, Varian, USA), which detects scattered light reflected within the same spectrum (�) of incident light. Five young one-year-old S. camporum potted plants were maintained under natural sunlight. As no previous differences were detected from the same leaf surface between para- or diaheliotropic leaves, one randomly selected leaf 1 and Ex f i c n 2 t s D 2 s ( ( d o f 2 o i 3 3 m f a o t e i ( p S ( c o b o 3 l p b T a l l i d s l 2 G. Habermann et al. / Environmental rom each of the five plants was detached and immediately inserted nto the equipment. The angle between the leaf and light beam was lose (but not exactly) to 45◦, so that reflected light intensity could ot saturate the detector. .5. Leaf water potential The leaf water potential at predawn (� pd) and midday (when he vapor pressure deficit, VPD, was maximum) (� md) were mea- ured by the pressure chamber method (Turner, 1981) using a IK-7000 (Daiki Kogyo, Japan) pressure chamber. .6. Data analysis Statistical analysis was carried out using two leaves randomly elected from five plants (replicates) within an area of five hectares ha) in Botucatu and 37 ha in Corumbataí. Gas exchange variables A, E, gs, and Ci), Tl, � pd, and � md were determined (mean and stan- ard deviation), and they were then subjected to one-way analysis f variance (comparisons between para- and diaheliotropic leaves), ollowed by the Tukey’s test (P < 0.05). .7. Light and electron microscopy See online supplementary data for more details about the meth- ds used for obtaining and analyzing light and electron microscopic mages (Fig. S1). . Results .1. Morpho-anatomical traits Leaves of S. camporum are almost all diaheliotropic, but some ature fully expanded leaves assume paraheliotropic position rom 10:00 h to 17:00 h. These mature completely expanded para- nd diaheliotropic leaves are observed on woody stems, and also n primary branches of adult plants (Fig. S1a). Young leaves of S. camporum presented the trichome indumen- um on both surfaces of the leaf blade (Fig. S1b), while mature fully xpanded leaves displayed a glabrous adaxial surface, regardless of ts heliotropic position (Fig. S1d). The mesophyll of both young (Fig. S1c) and mature Figs. S1d and e) leaves was differentiated into a unistratified alisade and a two- to three-layered spongy chlorenchyma. pongy parenchyma cells showed numerous wall ingrowths Figs. S1d and e), which developed into slit-like gas spaces. Such ells presented thin walls and a peripheral cytoplasm with numer- us chloroplasts (Fig. S1e) and a single central vacuole that could e translucent or dense if filled with phenolic substances. See nline supplementary data (Fig. S1) for details. .2. Gas exchange variables When the leaves were measured with light interception by both eaf sides, the CO2 assimilation rate (A) was significantly higher for ara- than for diaheliotropic leaves at 9:00 h, 11:00 h, and 14:00 h, ut A was similar between leaf types at the end of the day (Fig. 1a). he stomatal conductance (gs) and transpiration rates (E) followed lmost the same response pattern (Fig. 1c and e), with parahe- iotropic leaves demonstrating higher values than diaheliotropic eaves during most parts of the day. Paraheliotropic leaves exhib- ted a significant lower value of internal CO2 (Ci) compared to iaheliotropic leaves, but only at 9:00 h (Fig. 1g). Leaves measured with light interception by only the adaxial leaf urface demonstrated similar A values for para- and diaheliotropic eaves throughout the day, except at 11:00 h, when paraheliotropic perimental Botany 71 (2011) 10–17 leaves exhibited a higher A compared to diaheliotropic leaves (Fig. 1b). For gs and E, differences were detected only at 9:00 h and 14:00 h, respectively (Fig. 1d and f). Internal CO2 concentra- tions were similar in para- and diaheliotropic leaves, with the latter showing greater values at 14:00 h, although this increase was not significantly higher than the CO2 concentrations in paraheliotropic leaves (Fig. 1h). Variations in gs did not seem to influence the A of leaves mea- sured with sunlight interception by both leaf sides; daily results revealed a conserved low gs range with respective low carbon assimilation rates for diaheliotropic leaves, while most of the data for paraheliotropic leaves demonstrated a higher gs range with respective higher CO2 assimilation rates (Fig. 2a). In contrast, when leaves were measured with light interception by only the adax- ial leaf surface, increases in gs resulted in greater CO2 assimilation rates, regardless of the leaf type (Fig. 2b). The Ci values of leaves measured with sunlight interception by both leaf sides were not dependent on gs (Fig. 3a). However, when leaves were measured with light interception by only the adaxial leaf surface, Ci fluctuated between 200 and 300 �mol mol−1, and this effect was dependent on gs (Fig. 3b). Similarly, A was not influenced by Ci in either paraheliotropic or diaheliotropic leaves intercepting light by both leaf sides (Fig. 4a); but when measuring gas exchange with light interception by only the adaxial leaf side, increases in Ci resulted in greater carbon assimilation rates, regardless of the leaf type (Fig. 4b). Both surfaces of para- and diaheliotropic leaves displayed a sim- ilar (P < 0.05) A from 0 to 100 PPFD; but from 200 to 1800 �mol photons m−2 s−1, diaheliotropic leaves showed significantly higher (P < 0.05) A for the adaxial than for the abaxial side (Fig. 5a). The adaxial surface, compared to the abaxial surface of paraheliotropic leaves, showed a higher (P < 0.05) A within the range of 200 and 800 �mol photons m−2 s−1; however, from 1000 to 1800 PPFD, adaxial and abaxial sides of the paraheliotropic leaves exhibited a similar (P < 0.05) A (Fig. 5a). Regarding the gs/PPFD curves, adaxial and abaxial sides of both leaf types presented similar (P < 0.05) val- ues at each PPFD (Fig. 5b). For Ci, the abaxial side presented greater values than the adaxial side for only diaheliotropic leaves, consid- ering a range between 200 and 800 �mol photons m−2 s−1; from 1000 to 1800 PPFD, both surfaces of the two leaf types had similar (P < 0.05) Ci values (Fig. 5c). The photosynthetic radiation use efficiency (PhRUE) was higher in paraheliotropic than in diaheliotropic leaves at every time of day considered, when the leaves were measured with sunlight inter- ception by both leaf sides. However, when leaves were artificially illuminated only on the adaxial leaf surface, PhRUE was the same between para- and diaheliotropic leaves, except at 11:00 h, when paraheliotropic leaves presented increased PhRUE in relation to diaheliotropic leaves. See online supplementary data (Table S1) for details. 3.3. Leaf temperature The leaf temperature remained between 30 ◦C and 38 ◦C for leaves measured when light intercepted both leaf sides, and para- heliotropic leaves demonstrated lower values than diaheliotropic leaves only at 11:00 h and 16:00 h (Fig. 6a). When leaves were mea- sured with light interception by only the adaxial leaf side, the leaf temperature fluctuated between 25 ◦C and 32 ◦C, although no sig- nificant differences between leaf types were detected throughout the day (Fig. 6b). 3.4. Water relations The leaf water potentials at predawn (� pd) or midday (� md) were statistically similar among para- and diaheliotropic G. Habermann et al. / Environmental and Experimental Botany 71 (2011) 10–17 13 A ( µ m o l. m -2 .s -1 ) 0 2 4 6 8 10 12 14 16 18 Paraheliotropic Diaheliotropic gs ( m o l. m -2 .s -1 ) 0.0 0.1 0.2 0.3 0.4 0.5 0.6 E (m m o l. m -2 .s -1 ) 0 2 4 6 8 10 12 16:00h14:00h11:00h9:00h C i ( µm o l. m o l- 1 ) 180 200 220 240 260 280 300 16:00h14:00h11:00h9:00h ba dc fe hg * * * * * * * * * * * * * Light intercep�on by both leaf sides Light intercep�on only by the adaxial leaf side Fig. 1. Daily variations in CO2 assimilation rates (a and b), stomatal conductance (c and d), transpiration rates (e and f), and intercellular CO2 (g and h) for para- and d irect l olumn ( l ( 3 p ( l a e r iaheliotropic leaves of S. camporum (n = 5) in the field, measured using natural (d ight interception by only the adaxial leaf surface (b, d, f and h). Asterisks between c Vertical bars = SD). eaves measured in both locations (Botucatu and Corumbataí) Fig. 7). .5. Leaf reflectance Reflectance from both leaf sides showed a similar response attern, showing the peak of reflectance in the green spectrum 550 nm). The relative light intensity reflected from the abaxial eaf surface was significantly higher than light reflected from the daxial leaf surface within the visible light (400–700 nm). How- ver, considering wavelengths of 200–400 nm and 700–800 nm, eflectance was similar between both leaf surfaces (Fig. 8). and diffuse) irradiance interception by both leaf surfaces (a, c, e and g) as well as s indicate significant difference between para- and diaheliotropic leaves (P < 0.05). 4. Discussion 4.1. Water relations Plants from Botucatu and Corumbataí displayed a similar � w, indicating no differences in night rehydration (� pd) or in water uptake capacity under the highest VPD (� md) (Fig. 7); these results assure comparable physiological conditions between both popula- tions. It is unlikely that these plants were experiencing a soil water deficit, as the relative water content of both leaf types remained around 80% (data not shown). Moreover, S. camporum adult plants evaluated in the field during the dry season (July to September) 14 G. Habermann et al. / Environmental and Experimental Botany 71 (2011) 10–17 A ( µ m o l. m -2 .s -1 ) 0 2 4 6 8 10 12 14 16 18 Paraheliotropic ( ) Diaheliotropic ( ) y = -109.01x 2 + 77.44x - 0.45 R 2 = 0.77 y = -148.88x 2 +78.10x -1.14 R 2 = 0.58 gs (mol.m -2.s -1) 0.70.60.50.40.30.20.10.0 A ( µ m o l. m -2 .s -1 ) 0 2 4 6 8 10 12 14 16 y = -42.09x 2 + 46.37x + 3.08 R 2 = 0.86 y = -31.33x 2 + 39.09x + 3.59 R 2 = 0.81 a b Fig. 2. Individual daily readings (replicates) for CO2 assimilation rates in relation to t i b ( s g 4 i 7 c p t b n l c s i m n l A l t t o t c C i ( µm ol .m ol -1 ) 100 150 200 250 300 350 400 gs (mol.m -2 .s -1) 0.70.60.50.40.30.20.10.0 C i ( µm ol .m ol -1 ) 100 150 200 250 300 350 Paraheliotropic ( ) Diaheliotropic ( ) y = -624.46x2 + 657.62x + 113.82 R2 = 0.91 y = -221.59x2 + 386.1x + 161.54 R2 = 0.87 a b Fig. 3. Individual daily readings (replicates) of intercellular CO2 in relation to the stomatal conductance for para- and diaheliotropic leaves of S. camporum (n = 5) in he stomatal conductance for para- and diaheliotropic leaves of S. camporum (n = 5) n the field, measured using natural (direct and diffuse) irradiance interception by oth leaf surfaces (a) as well as light interception by only the adaxial leaf surface b). Only regression equations with R2 > 0.5 are shown. howed mean values of -0.5 MPa � pd and −1.6 MPa � md with low s compared to the wet season (January to March) (data not shown). .2. Leaf temperature Forseth and Teramura (1986) estimated that naturally orient- ng leaves of Pueraria lobata would maintain temperatures up to ◦C lower than horizontally restrained leaves. However, when S. amporum leaves were illuminated on both sides, the low leaf tem- erature (Tl) observed in paraheliotropic leaves at 16:00 h seemed o have a weak relationship with E or gs, which were similar etween the leaf types (Fig. 1c and e). Habermann et al. (2008) oted that S. camporum paraheliotropic leaves illuminated on both eaf sides had lower Tl and higher E, gs, and A during part of the day ompared to diaheliotropic leaves, but neither E nor gs was respon- ible for the low Tl of paraheliotropic leaves, and there was no ndication that the low Tl explained their high A. When leaves were easured with light interception only on the adaxial leaf sides, o differences in Tl were noted between para- and diaheliotropic eaves (Fig. 6b), even when evaluated for an entire year (March, pril, June, August, and October) (data not shown). Tl for the vertical eaflets of P. pubescens at midday was only 1.5 ◦C lower compared o horizontal leaflets (Caldas et al., 1997). For Arena et al. (2008), he reduction in Tl caused by paraheliotropism may be relevant nly if the air temperature reaches values higher than the optimum emperature for photosynthesis. Therefore, paraheliotropism in S. amporum does not seem to be related to leaf heat avoidance. More- the field, measured using natural (direct and diffuse) irradiance interception by both leaf surfaces (a) as well as light interception by only the adaxial leaf surface (b). Only regression equations with R2 > 0.5 are shown. over, cerrado species seem to be well adapted to high temperatures (Franco et al., 2007; Simon et al., 2009). 4.3. Leaf gas exchange and light interception S. camporum paraheliotropic leaves that intercepted light on both leaf sides displayed significantly higher A, E, and gs than dia- heliotropic leaves during most times of the day (Fig. 1a, c and e). This high A for paraheliotropic leaves could not be explained by the higher gs of para- compared to diaheliotropic leaves (Fig. 1c). When leaves were illuminated on both leaf sides, it was clear that A did not depend on gs (Fig. 2a). Separate groups of data demon- strate the greater response ranges for A and gs in paraheliotropic compared to diaheliotropic leaves (Fig. 2a). For both leaf types, the increased stomatal aperture did not result in enhanced intercellu- lar CO2 (Fig. 3a), the variation of which clearly did not affect the carbon assimilation rates (Fig. 4a). Furthermore, in nature, sunlight interception by the abaxial leaf side certainly does not explain the greater gs found in para- compared to diaheliotropic leaves (Fig. 1c), because adaxial and abaxial sides of both leaf types when illumi- nated by the artificial red/blue light demonstrated the same values of gs at each PPFD of the gs/PPFD curves (Fig. 5b). Leaves measured with light interception by only the adaxial leaf surface showed significantly higher A in para- than in diahe- liotropic leaves only at 11:00 h (Fig. 1b). However, leaves displayed daily Ci values that were clearly influenced by the opening of the stomatal pores (Fig. 3b). Consequently, higher carbon assimilation G. Habermann et al. / Environmental and Experimental Botany 71 (2011) 10–17 15 Ci (µmol.mol -1 ) 400350300250200150100 A ( µ m o l. m -2 .s -1 ) 0 2 4 6 8 10 12 14 16 Paraheliotropic (____) Diaheliotropic (__ __) y = 0.066x - 3.75 R2 = 0.723 y = 0.073x - 6.70 R2 = 0.695 A ( µ m o l. m -2 .s -1 ) 0 2 4 6 8 10 12 14 16 18 a b Fig. 4. Individual daily readings (replicates) of CO2 assimilation in relation to inter- cellular CO2 for para- and dia- heliotropic leaves of S. camporum (n = 5) in the field, m s r r ( i n ( s b i p t i e t r c i r w r 2 l u r s s A ( µ m o l. m -2 .s -1 ) -4 -2 0 2 4 6 8 10 12 14 16 Paraheliotropic adaxial Paraheliotropic abaxial Diaheliotropic adaxial Diaheliotropic abaxial gs ( m o l. m -2 .s -1 ) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 PPFD (µmol.m -2 .s -1) 2000180016001400120010008006004002000 C i ( µ m o l. m o l- 1 ) 150 200 250 300 350 400 450 a b c Fig. 5. Mean values (n = 5 plants) for the CO2 assimilation rates (a), stomatal con- easured using natural (direct and diffuse) irradiance interception by both leaf urfaces (a) as well as light interception by only the adaxial leaf surface (b). Only egression equations with R2 > 0.5 are shown. ates followed from increased Ci values, regardless of the leaf type Fig. 4b). The adaxial leaf surface of S. camporum seems to be specialized n direct light interception and absorptance, because it had a sig- ificantly lower light reflectance compared to the abaxial leaf side Fig. 8). For Helianthus annus leaves, direct light reflectance was only lightly higher in the abaxial compared to the adaxial leaf surface, ut diffuse light reflectance was significantly higher in the abax- al compared to the adaxial side (Gorton et al., 2010). The stellate ubescence, which covers the abaxial leaf surface and is absent on he adaxial side of S. camporum leaves (Habermann et al., 2008) s, then, responsible for this increased light reflectance. In fact, the ffective quantum yield of photosystem II (� PSII) and the electron ransport rate (ETR) for both leaf sides of one-year-old S. campo- um plants were similar between leaf types, but higher in adaxial ompared to abaxial sides (data not shown). However, when carbon assimilation was measured with light nterception by only one of the leaf sides using the artificial ed/blue light, which precisely emits the photosynthetic active aveband, it seems that the vertical leaf position of S. campo- um enables increased light use efficiency. When PPFD varied from 00 to 1800 �mol photons m−2 s−1, the adaxial sides of diahe- iotropic leaves displayed maximum CO2 assimilation rates (Amax nder saturating light, but non-saturating CO2, for photosynthesis esponses) that were significantly higher than those of their abaxial ides (Fig. 5a). Interestingly, non-saturating light for photosynthe- is responses (200-800 �mol photons m−2 s−1) did not induce CO2 ductance (b), and intercellular CO2 (c) in response to the photosynthetic photon flux density (PPFD) in para- and diaheliotropic leaves of S. camporum (n = 5) measured using light interception by only adaxial or abaxial leaf sides. (Vertical bars = SD). consumption on the abaxial sides of diaheliotropic leaves, which exhibited significantly higher Ci than those of their adaxial surfaces. When using more than 1000 �mol photons m−2 s−1 (saturating light), Ci was similar between both leaf sides of the two leaf types (Fig. 5c), but the adaxial leaf sides of diaheliotropic leaves main- tained a higher A compared to their abaxial sides (Fig. 5a). This suggests that increased light interception by the abaxial sides of diaheliotropic leaves does not improve their light use efficiency; therefore, some innate low Amax values are present for the abax- ial sides of diaheliotropic leaves. Adaxial surfaces of para- and diaheliotropic leaves presented the same (P < 0.05) A as the PPFD varied from 200 to 1800 �mol photons m−2 s−1 (Fig. 5a). Leaves illuminated only on the adaxial side, which did not render any con- spicuous differences in A between the two leaf types (Fig. 1 b, d and f) were measured using a PPFD of 1800 �mol photons m−2 s−1. Moreover, adaxial and abaxial sides of paraheliotropic leaves dis- played a distinct Amax when PPFD was below 800 �mol photons m−2 s−1, but similar values of Amax when PPFD was between 800 and 1800 �mol photons m−2 s−1 (Fig. 5a), indicating that parahe- liotropic leaves somehow integrate the photosynthetic capacities of both leaf sides under elevated irradiances. In fact, the leaves of Olea europaea, when intercepting light on both sides, showed higher apparent quantum yield [mol (CO2 assimilated) mol−1 (incident 16 G. Habermann et al. / Environmental and Experimental Botany 71 (2011) 10–17 16:00h14:00h11:00h9:00h L e a f te m p e ra tu re ( o C ) 22 24 26 28 30 32 34 36 38 L e a f te m p e ra tu re ( o C ) 22 24 26 28 30 32 34 36 38 40 Paraheliotropic Diaheliotropic * * a b Fig. 6. Daily variations in leaf temperature for para- and diaheliotropic leaves of S. camporum (n = 5) in the field, measured using natural (direct and diffuse) irradiance i l p q l ( u r b F h C d p b 800700600500400300200 0 100 200 300 400 500 600 Adaxial Leaf Surface Abaxial Leaf Surface R e la ti v e R e fl e c te d I n te n s it y ( a .u .) Wavelenght (nm) Fig. 8. Relative reflected light intensity within the 200–800 nm wavebands for nterception by both leaf surfaces (a) as well as light interception by only the adaxial eaf surfaces (b). Asterisks between columns indicate significant difference between ara- and diaheliotropic leaves (P < 0.05). (Vertical bars = SD). uanta of PPFD)] and greater A compared to cases in which their eaves intercepted light only on one leaf side under the same PPFD Proietti and Palliotti, 1997). Similarly, leaves of Eucalyptus mac- lata and E. pauciflora, which had been horizontally or vertically estrained, exhibited higher A values when equally illuminated on oth leaf sides compared to when abaxial or adaxial illumination ig. 7. Predawn (� pd) and midday (� md) leaf water potential in para- and dia- eliotropic leaves of S. camporum (n = 5) in the field, in Botucatu-SP, and in orumbataí-SP, southeastern Brazil. For each evaluation time (predawn and mid- ay), the same letters indicate the lack of statistical significance (P < 0.05) among ara- and diaheliotropic leaves of plants from Botucatu and Corumbataí. (Vertical ars = SD). leaves irradiated on the adaxial and abaxial leaf surfaces of S. camporum. Each curve represents data from leaves detached from five replicates (plants). (a.u. = arbitrary unit). alone was applied (Evans and Jakobsen, 1993). These results sup- port the integration of the photosynthetic capacities of adaxial and abaxial leaf sides, which may have occurred in S. camporum (Fig. 1a). We have not measured the apparent quantum yield, but when leaves were illuminated on both surfaces, the photosynthetic radi- ation use efficiency (PhRUE) was higher in para- compared to diaheliotropic leaves throughout the day, and when the same range of PPFD intercepted adaxial leaf surfaces only, para- and dia- heliotropic leaves showed similar PhRUE at 9:00 h, 14:00 h, and 16:00 h (See online supplementary data for details; Table S1). Thus, we propose that the leaves of S. camporum photosynthetically ben- efit from paraheliotropism, although there is no evidence that this great PhRUE increases plant ecological performance, for instance, by increasing plant biomass. The vertical leaf position in S. camporum enables increased pho- tosynthetic efficiency in terms of CO2 consumption, but not in terms of light absorptance, especially when considering the contri- bution of the abaxial leaf surface, which is highly reflective. In fact, for some epiphytic fern leaves, the higher A values observed for the leaf side mostly exposed to direct sunlight was attributable to a greater CO2 consumption (Martin et al., 2009). Notwithstanding, the reflectance pattern of the adaxial side is very similar to the abax- ial leaf side, with both sides exhibiting great reflectance within the green spectrum (525–575 nm) (Fig. 8). This indicates that, although pubescence promotes light reflectance, the abaxial leaf surface cer- tainly absorbs a small amount of light within the red spectrum, which is greater in direct sunlight than in scattered light. For H. annus leaves, there was almost the same direct light absorptance between the adaxial and abaxial leaf surfaces, but when diffuse light absorptance was measured, the abaxial side showed a slightly lower value (Gorton et al., 2010). Therefore, as supported by A/PPFD curves (Fig. 5a) abaxial leaf surfaces of S. camporum may not have light leaf absorptance significantly decreased under elevated irra- diances. S. camporum leaves have compact spongy parenchyma and amplified palisade parenchyma (See online supplementary data for details; Figs. S1d, e and f), suggesting that these cells have high chlorophyll content. Features such as stomata limited to the abaxial surface, developed palisade parenchyma, and compactly arranged spongy parenchyma are constant in leaves of cerrado woody species (Bieras and Sajo, 2009). Indeed, compact leaf tissues have previously been suggested to enable greater light interception and high water use efficiency (Chaves et al., 2002). and Ex i u a m t M c l n r P f m s s e 4 c f q ( F 2 m 2 h p i o t o t t e l a t f o ( i A d l C a D R A t R t B Von Caemmerer, S., Farquhar, G.D., 1981. Some relationships between the biochem- G. Habermann et al. / Environmental One could still argue that different devices, rather than light nterception, determined the differences in gas exchange, since nat- ral sunlight was used for double sided illumination measurements nd artificial red/blue light was used for single sided illumination easurements. However, regardless of the device/system used, he range of PPFD values reaching leaf surfaces was very similar. oreover, results of A, gs, and Ci obtained from PPFD curves indi- ated that there are distinct responses in para- and diaheliotropic eaves when considering each leaf surface under saturating and on-saturating red/blue light (Fig. 5). Finally, it is essential to emphasize that it was not the leaf age, ather than the leaf position, that determined our observations. araheliotropic leaves that were assessed were distinguishable rom diaheliotropic leaves. These paraheliotropic leaves had ature fully expanded blades, and they were localized on woody tems and primary branches of adult plants. Additionally, for some tems, paraheliotropic and diaheliotropic leaves were adjacent to ach other (See online supplementary data for details; Fig. S1a). .4. Ecophysiological significance of heliotropism for S. amporum It has been accepted that paraheliotropism is a mechanism or reducing transpiration rates, irradiance interception, conse- uently minimizing leaf heat and the potential for photoinhibition Ehleringer and Forseth, 1980; Forseth and Ehleringer, 1982; orseth and Teramura, 1986; Bielenberg et al., 2003; Pastenes et al., 005; Liu et al., 2007; Arena et al., 2008), although paraheliotropism ay also prevent optimum CO2 assimilation rates (Pastenes et al., 005). These traditional explanations for the significance of leaf eliotropism have been based on leguminous species, including P. ubescens from the cerrado (Caldas et al., 1997). Nonetheless, our data strongly suggest that, although the abax- al leaf surface is highly reflective, it may absorb a small amount f natural direct light within the red spectrum, which photosyn- hetically increases the efficiency of paraheliotropic leaves in terms f CO2 consumption, because adaxial and abaxial leaf sides seem o integrate their respective carbon assimilation rates. However, hese observations are detected only if measured with devices that nable light interception by both leaf surfaces. Moreover, parahe- iotropism in S. camporum does not seem to be related to leaf heat voidance (Fig. 6), neither does it minimize the potential for pho- oinhibition (data not shown). Therefore, in contrast to the results or leguminous species, the paraheliotropism of only some leaves f S. camporum, which do not even show pulvinus in their petioles Machado, 1991), could have novel significance for plant ecophys- ology. cknowledgements We thank Dr. Luis Alberti for Photoshop assistance; Fundação e Amparo à Pesquisa do Estado de São Paulo (Fapesp) for fel- owships to Patricia F. V. Ellsworth (Proc. 06/01125-8), Juliana L. azoto (Proc. 06/01180-9), and Aline M. Feistler (Proc. 09/04007-4); nd the Brazilian National Council for Scientific and Technological evelopment - CNPq (Proc. 405679/86) for financial support and for esearch Productivity Fellowships to Silvia R. Machado and Dario . Donatti. We extend our appreciation to Dr. João D. Rodrigues for he LI-6400 lent from Unesp, and Dr. Eduardo C. Machado and Dr. afael V. 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Zhang, Y.-L., Zhang, H.-Z., Du, M.-W., Li, W., Luo, H.-H., Chow, W.-S., Zhang, W.-F., 2010. Leaf wilting movement can protect water-stressed cotton (Gossypium hir- sutum L.) plants against photoinhibition of photosynthesis and maintain carbon assimilation in the field. J. Plant Biol. 53, 52–60. http://dx.doi.org/10.1016/j.envexpbot.2010.10.012 Leaf paraheliotropism in Styrax camporum confers increased light use efficiency and advantageous photosynthetic responses ... Introduction Materials and methods Site description Leaf angle measurement Leaf gas exchange measurements Estimation of leaf reflectance Leaf water potential Data analysis Light and electron microscopy Results Morpho-anatomical traits Gas exchange variables Leaf temperature Water relations Leaf reflectance Discussion Water relations Leaf temperature Leaf gas exchange and light interception Ecophysiological significance of heliotropism for S. camporum Acknowledgements Supplementary data Supplementary data