Plant Biology ISSN 1435-8603
RESEARCH PAPER
Paraheliotropism in Robinia pseudoacacia L.: an efficient strategy to optimise photosynthetic performance under natural environmental conditions C. Arena, L. Vitale & A. Virzo De Santo Dipartimento di Biologia Strutturale e Funzionale, Universita` degli Studi di Napoli Federico II, Napoli, Italy
Keywords Heat stress; light stress; net photosynthesis; paraheliotropism; photoprotection; Robinia pseudoacacia. Correspondence C. Arena, Dipartimento di Biologia Strutturale e Funzionale, Universita` degli Studi di Napoli Federico II, Complesso di Monte Sant’Angelo, via Cinthia 80126, Napoli, Italy. E-mail:
[email protected] Editor R. Leegood Received: 1 June 2007; Accepted: 20 August 2007 doi:10.1111/j.1438-8677.2008.00032.x
ABSTRACT We assessed the contribution of leaf movements to PSII photoprotection against high light and temperature in Robinia pseudoacacia. Gas exchange and chlorophyll a fluorescence measurements were performed during the day at 10:00, 12:00, 15:00 and 18:00 hours on leaves where paraheliotropic movements were restrained (restrained leaves, RL) and on control unrestrained leaves (UL). RL showed a strong decrease of net photosynthesis (An), stomatal conductance (gsH2O), quantum yield of electron transport (FPSII), percentage of photosynthesis inhibited by O2 (IPO) and photochemical quenching (qP) in the course of the day, whereas, a significant increase in Ci ⁄ Ca and NPQ was observed. Contrary to RL, UL had higher photosynthetic performance that was maintained at elevated levels throughout the day. In the late afternoon, An, gsH2O, FPSII and qP of RL showed a tendency to recovery, as compared to 15:00 hours, even if the values remained lower than those measured at 10:00 hours and in UL. In addition, contrary to UL, no recovery was found in Fv ⁄ Fm at the end of the study period in RL. Data presented suggest that in R. pseudoacacia, leaf movements, by reducing light interception, represent an efficient, fast and reversible strategy to overcome environmental stresses such as high light and temperature. Moreover, paraheliotropism was able to protect photosystems, avoiding photoinhibitory damage, leading to a carbon gain for the plant.
INTRODUCTION In natural environments, plants receive an amount of light that varies significantly during the course of the day, and photosynthetic processes respond rapidly to these changes. Generally, on a clear sunny day, saturation of photosynthesis occurs at photon flux densities (PPFD) lower than those reached under full sunlight, so the amount of light energy absorbed by the photosynthetic apparatus may often be in excess of that used in photochemistry. This unbalance may induce photoinhibition that limit CO2 fixation (Powles 1984). However, not only the excess light but also the increase in leaf temperature can lead to photoinhibition (Berry & Bjorkman 1980; Larcher 2000; Werner et al. 2002; Bombelli & Gratani 2003). In Mediterranean-type ecosystems, during summer, these constraints act simultaneously, enhancing
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the potential for photodamage. Photosynthetic efficiency of Mediterranean species is often down-regulated during the hottest hours of the day, allowing plants to cope with stress conditions; however, it is not the only strategy against photodamage. Other mechanisms involve both non-photochemical (i.e. thermal dissipation) and photochemical processes (such as photorespiration, nitrogen assimilation, Mehler–ascorbate peroxidase pathway, chlororespiration) able to decrease the excitation pressure at photosystem II (PSII) reaction centres (Demming-Adams et al. 1996; Horton et al. 1996; Kozaki & Takeba 1996; Niyogi 2000). Particularly at high irradiances, photorespiration and the Mehler–ascorbate peroxidase pathway, by consuming up to 20% and 30%, respectively, of reductive power of the electron transport chain can be considered the main safety valves for photosynthesis (Niyogi 2000; Ort & Baker 2002). Other
Plant Biology 10 (2008) 194–201 ª 2008 German Botanical Society and The Royal Botanical Society of the Netherlands
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photoprotective mechanisms consist of chlorophyll concentration changes (Giardi et al. 1996; Murchie & Horton 1997), and chloroplast and leaf movements (Ludlow & Bjorkman 1984; Haupt 1990). Leaf movement, such as paraheliotropism (light-avoiding), represents an efficient strategy to reduce light interception, and is able to limit excitation pressure to reaction centres and decrease the potential for photoinhibition (Ludlow & Bjorkman 1984; Pastenes et al. 2005; Jiang et al. 2006). Moreover, it can also be considered an effective mean to reduce leaf temperature and ameliorate plant resistance to drought conditions by limiting transpirational water loss (Forseth & Ehleringer 1982; Gamon & Pearcy 1989). It has been demonstrated that paraheliotropism is capable of protecting against photoinhibition in not only water-stressed plants but also well-watered plants. Leaf movements have largely been studied in alfalfa (Reed 1987), bean (Bielenberg et al. 2003; Pastenes et al. 2005), soybean (Jiang et al. 2006) and wild grape (Gamon & Pearcy 1989, 1990). In this study attention was focused on paraheliotropic movement of Robinia pseudoacacia L. (black locust) leaves and effects on their photosynthetic performance. Black locust is a native species of North America that was introduced into Europe where, at present, it constitutes one of the most invasive exotic plants. In southern Italy, it has colonised disturbed areas and poses the greatest threat to native woods. Some authors have characterised the photosynthetic activity of black locust in response to temperature, irradiance and CO2 concentration (Mebrahtu & Hanover 1991; Mebrahtu et al. 1991, 1993) but no study has been performed on paraheliotropic movement of R. pseudoacacia leaves as a photoprotective strategy. Thus, our goal was to evaluate the contribution of paraheliotropism resulting in PSII photoprotection as an effective means to cope with high irradiance and high temperature under natural environmental conditions. MATERIALS AND METHODS
Paraheliotropism in Robinia pseudoacacia
Gas exchange and Chlorophyll a fluorescence measurements
Gas exchanges parameters were measured on attached fully-expanded leaves, 20 days old, with a portable gas exchange system (HCM-1000; Walz, Effeltrich, Germany). The gas exchange parameters, i.e. net CO2 assimilation (An), water stomatal conductance (gsH2O) and intercellular CO2 concentration (Ci) were calculated by software in the HCM-1000 according to von Caemmerer & Farquhar (1981). The ratio of intercellular to ambient CO2 concentration (Ci ⁄ Ca) was also determined. Chlorophyll a fluorescence measurements were performed simultaneously to gas exchange with a portable pulse amplitude modulated fluorometer (MINI-PAM; Walz, Effeltrich, Germany) equipped with fibre optics of 2 mm. On dark-adapted leaves, the basal fluorescence signal (Fo) was obtained with a measuring light of 0.12 lmol photonsÆm)2Æs)1 at a frequency of 0.6 kHz, while the maximal fluorescence in the dark-adapted state (Fm) was measured with a 1-s saturating pulse at 10,000 lmol photonsÆm)2Æs)1. Under illumination, the steady-state fluorescence (Ft) was obtained by setting the measuring light to frequency of 20 kHz; maximal fluorescence in the light-adapted state (F¢m) was measured with a 1-s saturating pulse of 10,000 lmol photonsÆm)2Æs)1. The quantum yield of PSII linear electron transport (FPSII) was calculated according to Genty et al. (1989) as FPSII = (F¢m ) Ft ⁄ F¢m). Photochemical quenching (qP) was calculated as qP = (Fm¢ ) Ft) ⁄ (Fm¢ ) Fo¢) according to Van Kooten & Snel (1990). Non-photochemical quenching (NPQ) was expressed as NPQ = (Fm ) Fm¢) ⁄ Fm¢ (Bilger & Schreiber1986). Minimal fluorescence under light exposure (Fo¢) was indirectly determined as Fo¢ = Fo ⁄ [(Fv ⁄ Fm) + (Fo ⁄ Fm¢)] (Oxborough & Baker 1997). Although the formula used to calculate F¢o may sometimes be critical under photoinhibitory conditions (Maxwell & Johnson 2000), it has been successfully used for Chlorophyll a fluorescence measurements in plants exposed either to low temperatures (D’Ambrosio et al. 2006) or high temperatures (Sinsawat et al. 2004).
Plants and growth conditions
Young plants of R. pseudoacacia L., 60 to 70-cm tall, and the underlying soil were collected from Vesuvius National Park (NA, Italy) at the beginning of April 2005 and transplanted in 15-l pots. The plants were placed outdoors in the Botanical Garden of Naples University on a terrace exposed to east–south–west from April to June. During this period, plants were well watered every day and fertilised weekly with nutrient solution (Gesal Tecno-Pro, N:P:K 20:20:20 + micronutrients; Reckitt & Colman, Milan, Italy). R. pseudoacacia is able to perform heliotropic movements, orienting its leaflets in the morning about perpendicularly and at midday obliquely to the Sun’s direct rays. The leaf orientation returns to the horizontal plane in the late afternoon.
Leaf movements measurements
Fifteen sun leaves, representative of the population of unshaded leaves were chosen randomly for the measurements at each selected measuring time. In order to evaluate the range of leaf heliotropic movements, an analysis of leaf movements was performed through diurnal measurements of leaf orientation in the morning (10:00 hours), at midday (12:00 hours) and in the afternoon (15:00 and 18:00 hours). Leaf inclination (a) was obtained by measuring the angle of the adaxial leaflet surface formed with its rachis. A fine wire was positioned between the leaflet petiole and rachis, taking care to not alter the leaf position and light exposure; the curvature radius of the fine wire mirrored precisely the leaflet angle. The angle described by fine wire inclination
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was then reproduced on paper and measured with a goniometer. Experimental design
Chlorophyll a fluorescence and gas exchange measurements were done at the end of June 2005 under clear-sky conditions on three consecutive days with natural sunlight, temperature and relative humidity. During each day, measurements were done at 10:00, 12:00, 15:00 and 18:00 hours on R. pseudoacacia attached unrestrained (UL) and restrained (RL) leaves. RL were blocked by fixing the rachis on both sides with narrow adhesive bands to prevent leaf and leaflets movements. The adhesive band covered only the length of the rachis and the petioles of leaflets, thus avoiding any possible influence of the sticky tape on leaflet temperature and gas exchange (Fig. 1). For measurements, UL and RL were selected from three different plants, at the same height from ground and with the same orientation to incident sunlight, taking care not to alter leaf position during measurements. In order to determine the IPO coefficient, gas exchange at each time was first measured under ambient
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O2 (photorespiratory condition) and subsequently at low O2 (non-photorespiratory condition). The non-photorespiratory condition was obtained by passing compressed air (370 lmolÆmol)1 CO2 and 2% O2) through a humidifier before entering the gas exchange system, to achieve values of relative air humidity comparable to those measured under photorespiratory conditions. The maximal PSII photochemical efficiency (Fv ⁄ Fm) was determined on leaves kept in the dark from 20:00 hours of the day before measurements until 9:00 hours of the following day to ensure complete reoxidation of PSII reaction centres. Leaves were dark-adapted using a thin aluminium sheet positioned around leaf in such a way as to allow free air circulation. The acquired Fo and Fm values were used for calculation of fluorescence parameters. Successively, leaves were exposed to natural daylight and gas exchange followed by chl a fluorescence measurements at different hours of the day on the same leaf area for both UL and RL groups. Data acquisition was done when a steady-state of CO2 net assimilation was achieved. In the late afternoon (18:30–19:00 hours), the maximal PSII photochemical efficiency was again measured on 30-min dark-adapted UL and RL leaves to assess possible differences induced by lack of paraheliotropic movements. Data analyses
The statistical analysis of the data was performed by oneway anova followed by Student–Newman–Keuls test (sigma-stat 1.0), based on a significance level of P < 0.05. Data are means ± SE (n = 8). RESULTS B
Gas exchange measurements
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Fig. 1. Unrestrained leaf with position of leaflets in the morning (A) and at noon (B). Restrained leaves (C) with leaflets maintained during the whole day in the same position as in the morning, by fixing their petioles to the rachis with narrow adhesive bands put on both the adaxial and abaxial rachis surface ( ).
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At 10:00 and 18:00 hours, no differences in PPFD and leaf temperatures were found between UL and RL (Fig. 2A and B). At 12:00 and 15:00 hours, incident light on RL reached 1600 and 1400 lmol photonsÆm)2Æs)1, respectively, and leaf temperatures were 37 and 39 C. In UL, PPFD and leaf temperature were lower than those for RL (Fig. 2A and B). Net photosynthesis (An) measured at 10:00 hours was not significantly different between UL and RL (Fig. 3A), when, in both groups, the angle that leaflets formed with the rachis was 80. After 10:00 hours, An decreased for both leaf groups (P < 0.01), showing a stronger decline in RL than in UL, where the leaflet angle was reduced from 80 to 50. At 15:00 hours in RL, An decline was 59% as compared to initial values, while in UL, it was only 10% and leaflet angle was about 60. In the late afternoon, An slightly increased in RL but remained significantly lower compared to UL. As in photosynthesis, also gsH2O showed a progressive reduction in both leaf groups; however, in UL, gsH2O was always higher than in RL (Fig. 3B); in RL the lowest value of gsH2O was found
Plant Biology 10 (2008) 194–201 ª 2008 German Botanical Society and The Royal Botanical Society of the Netherlands
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at 15:00 hours. At 18:00 hours, when PPFD declined to 400 lmol photonsÆm)2Æs)1, a 38% recovery in gsH2O was observed. Conversely, in UL, gsH2O continued to diminish despite a PPFD reduction from 1000 to 400 lmol photonsÆm)2Æs)1. The trend in intercellular CO2 concentration around mesophyll cells, measured as the ratio of intercellular to ambient CO2 concentration (Ci ⁄ Ca) was similar between RL and UL at 10:00 hours, but became different (P < 0.01) during the course of the day (Fig. 3C). In particular, in UL at midday and at 15:00 hours, when leaf temperatures reached 40 C (Fig. 2B), the ratio of Ci ⁄ Ca significantly increased (P < 0.05) as compared to 10:00 hours, and in RL it remained still high after 15:00 hours, when both leaf temperature and PPFD declined; in contrast, in UL, the Ci ⁄ Ca remained almost constant throughout the course of the day. Photorespiration, expressed as percentage of photosynthesis inhibited by O2 (IPO), was statistically different (P < 0.01) in RL and UL (Fig. 3D). In UL, IPO increased with rising temperature and irradiance and reached the highest value, nearly 30%, at 15:00 hours, followed by a decline at 18:00 hours. Conversely, in the RL, IPO
The quantum yield of PSII linear electron transport (FPSII) and photochemical quenching coefficient (qP) were always lower (P < 0.001) in RL as compared to UL, except at 10:00 hours (Fig. 4A and B). The minimum values of these parameters were detected at 15:00 hours, and a tendency to recovery was observed in the late afternoon, even if the values remained lower than those measured at 10:00 hours. Unlike RL, in UL FPSII as well as qP did not decrease at midday and 15:00 hours compared to 10:00 hours. At 18:00 hours, when temperature and PPFD on the leaf surface declined, a significant increase in both parameters was detected (Fig. 4A and B). The non-photochemical quenching coefficient (NPQ) was significantly higher (P < 0.001) in RL as compared to UL throughout the day, except at 10:00 hours (Fig. 4C). In UL, unlike RL, no difference in NPQ was detected from 10:00 to 15:00 hours as compared to the initial value. At 18:00 hours, NPQ declined strongly (P < 0.01) in both RL and UL, even if it remained significantly higher (P < 0.05) in RL. In the morning, when sunlight was still weak and leaves of both plant groups were almost in a horizontal position, no difference was detected between UL and RL in Fv ⁄ Fm values (Fig. 4). Conversely, in the evening, a significant decrease (P < 0.05) of Fv ⁄ Fm was found in RL as compared to UL, which showed a Fv ⁄ Fm ratio similar to that measured in the morning (Fig. 5). DISCUSSION Gas exchange measurements
Paraheliotropic movements allowed unrestrained leaves (UL), compared to restrained leaves (RL), to reduce both radiation load on the leaf surface and leaf temperature (Fig. 2A and B). These results are consistent with data reported in the literature for other species (Forseth & Teramura 1986; Gamon & Pearcy 1989; Pastenes et al. 2005). It is well known that combined conditions of high irradiance and temperature, similar to those found in the present study, negatively affect photosynthesis by increasing the potential for photoinhibition (Foyer & Noctor 1999; Horton 2000). Changes in leaf angle allowed UL to maintain photosynthetic rates (An) at midday as well as at 15:00 hours near to those recorded in the morning (Fig. 3A); conversely, RL experienced irradiance (1400– 1600 lmol photonsÆm)2Æs)1) and leaf temperatures (37.5– 39 C) higher than optimum for photosynthesis in this species (Mebrahtu et al. 1991, 1993) (Fig. 3A and B) and showed a depression of CO2 uptake. These results support the hypothesis that leaf movements in UL allow a compromise between light interception and stress avoidance in order to maximise carbon gain. A slight but significant reduction of An was observed at 15:00 hours, in parallel
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with the highest incident light and leaf temperature. As no change in Ci ⁄ Ca was observed throughout the day (Fig. 3C), the An decline was not attributable to a decrease in stomatal conductance (Fig. 3B), but rather to a rise in photorespiration activity associated with an increase of irradiance and leaf temperature, as suggested by the highest IPO values (Fig. 3D). However, it should be emphasised that stomatal closure, reducing water loss by transpiration, could have contributed to the rise in leaf temperature that, in turn, affected the photorespiration rate. At 18:00 hours, the decline in CO2 fixation represents a physiological response of the photosynthetic apparatus to decreasing light. In RL, the minimum value for photosynthesis was reached at 15:00 hours, when gsH2O was lowest and Ci ⁄ Ca highest (Fig. 3A–C); however, stomatal closure was not responsible for photosynthetic depression, and An inhibition was likely the consequence of photochemical and ⁄ or biochemical limitations due to high-leaf temperatures. This hypothesis is based on the fact that CO2 fixation and photorespiratory rates decreased simultaneously (Fig. 3D), suggesting that photorespiration was not the cause of the An decline. Many studies have reported a heat-induced An decrease even in non-photorespiratory conditions, indicating the involvement of factors other than photorespiration in the An inhibition. Heat may induce a direct impairment of Rubisco or inactivation of Rubisco activase and, conse-
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quently, a decline in photosynthesis (Feller et al. 1998; Law & Crafts-Brandner 1999; Crafts-Brandner & Law 2000; Salvucci et al. 2001). According to Feller et al. (1998), deactivation of Rubisco and Rubisco activase occurs at temperatures similar to those experienced by RL of R. pseudoacacia in the course of the central hours of the day. Our results are inconsistent with those reported by Pastenes et al. (2005) and Bielenberg et al. (2003), who studied the role of movement in bean leaves. Pastenes et al. (2005) found a higher An in watered-restrained as compared to watered-unrestrained leaves and suggested that paraheliotropism required a cost in term of photosynthesis, even if the decrease in PPFD and temperature confers on the plant the capacity to protect against photoinhibition. On the other hand, Bielenberg et al. (2003) suggested that the potential loss of carbon assimilation associated with lower PPFD may be counteracted by a reduction in temperature and transpiration that leads to a substantial increase in water use efficiency. However, it should be noted that in the above experiments, bean leaves experienced leaf temperatures close to optimum for photosynthesis (Bielenberg et al. 2003), whereas in R. pseudoacacia, the temperature increased to values higher than the optimum for this species, reaching 40 C, a temperature known to inhibit photosynthesis in most plants. In addition, as in R. pseudoacacia, saturation of An occurs at irradiance values ranging from 600 to 800 lmol
Plant Biology 10 (2008) 194–201 ª 2008 German Botanical Society and The Royal Botanical Society of the Netherlands
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photonsÆm)2Æs)1 (data not shown), the progressive angle closure in response to an increase of both light and temperature conditions does not affect net CO2 gas exchange. The benefits of leaf angle changes have been reported also by others authors. Gamon & Pearcy (1990) found positive effects of leaf movements on carbon fixation in wild grape plants, but only at leaf temperatures over 45 C; whereas, Jiang et al. (2006) suggested that leaf orientation in cooperation with photorespiration and thermal dissipation successfully protect young soybean leaves against high irradiance in the field. In black locust plants, the leaf movement was advantageous also at lower temperatures, constituting the main photoprotective strategy against high light and temperature that allowed the leaves to maintain an elevated carbon gain throughout the day at no cost to the plant.
In RL, the simultaneous decreases of FPSII and qP at 12:00 and 15:00 hours (Fig. 4A and B) indicate that leaf exposure to high light and temperature strongly reduces photochemistry, enhancing the possibility of photoinhibitory damage to PSII (Niyogi 2000; Ort & Baker 2002). Consistent with the hypothesis of PSII damage is the drop at 15:00 hours of qP below the threshold of 0.5 indicated as the limit value for the onset of photoinhibition (Chow 1994). In contrast, UL did not experience the harmful effect of elevated light and temperature on photosystems, as evidenced by high FPSII and qP during the day (Fig. 4A and B). In the late afternoon, with the return of light and temperature to values not detrimental for photosynthesis, leaflets orientation in UL reverted to the horizontal position and FPSII and qP reached the highest values of the day, indicating the absence of photodamage to reaction centres; in RL, FPSII and qP improved, compared to 15:00, but the values remained lower than at 10:00 (Fig. 4A and B). The limited capacity to recover in RL likely suggests the occurrence of photoinhibitory damage induced by prolonged exposure to high irradiance and temperature in the more critical hours of the day (Fig. 4A and B). These results are not consistent with the findings of Pastenes et al. (2005) who observed full restoration of photochemistry in Phaseolus vulgaris L. leaves in the late afternoon. The lack of recovery in R. pseudoacacia leaves was probably due to the more severe stress conditions experienced by black locust as compared to P. vulgaris. Our data show that changes in leaf orientation clearly alleviate high light and temperature stress on PSII, enabling reaction centres to efficiently balance light capture and utilization, thus minimizing photoinhibitory damage risks. The leading role of paraheliotropism to afford photoprotection was confirmed by the full recovery of Fv ⁄ Fm in the late afternoon, compared to values measured in the morning (Fig. 5). It is well known that the Fv ⁄ Fm ratio is a good indicator of photoinhibition
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(Bjo¨rkman & Demmig 1987; Filella et al. 1998), so its complete restoration indicates the absence of irreversible damage at PSII ascribed to over-reduction of the electron transport chain (Pastenes et al. 2005). In contrast in RL, the absence of Fv ⁄ Fm recovery indicated that PSII reaction centres were likely damaged by high light and leaf temperature. The strong increase in NPQ found in RL during the hottest hours of the day indicates that thermal dissipation, when photochemical pathways are reduced and paraheliotropic movements disabled, becomes the most important dissipative process in consuming the excess of excitation energy (Fig. 4C). Our data confirm the fundamental role of paraheliotropism in R. pseudoacacia, where it represents an effective, reversible and rapid mean to offset the effects of excess irradiance and temperature on the photosynthetic apparatus, allowing maintenance of PSII efficiency and high-carbon gain throughout the day. This strategy could have contributed significantly to the wide diffusion of such species in Mediterranean-type ecosystems, where, during summer, plants are exposed to a combination of high light, temperature and vapour pressure deficit. REFERENCES Berry J., Bjorkman O. (1980) Photosynthetic response and adaptation to temperature in higher plants. Annual Review of Plant Physiology, 31, 491–543. Bielenberg D.G., Miller J.D., Berg V.S. (2003) Paraheliotropism in two Phaseolus species: combined effects of photon flux density and pulvinus temperature, and consequences for leaf gas exchange. Environmental and Experimental Botany, 49, 95–105. Bilger W., Schreiber U. (1986) Energy-dependent quenching of dark-level chlorophyll fluorescence in intact leaves. Photosynthesis Research, 10, 303–308. Bjo¨rkman O., Demmig B. (1987) Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77K among vascular plants of diverse origins. Planta, 170, 489–504. Bombelli A., Gratani L. (2003) Interspecific differences of leaf gas exchange and water relations of three evergreen Mediterranean shrub specie. Photosynthetica, 41, 619–625. von Caemmerer S., Farquhar G.D. (1981) Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta, 153, 376–387. Chow W.S. (1994) Photoprotection and photoinhibitory damage. In: Bittar E.E., Barber J. (Eds), Advances in Molecular and Cell Biology, Vol. 10. Elsevier, The Netherlands: 151–196. Crafts-Brandner S.J., Law R.D. (2000) Effect of heat stress on the inhibition and recovery of the ribulose-1,5-bisphosphate carboxylase ⁄ oxygenase activation state. Planta, 212, 67–74. D’Ambrosio N., Arena C., Virzo De Santo A. (2006) Temperature response of photosynthesis, excitation energy dissipation and alternative electron sinks to carbon assimilation in Beta vulgaris L. Environment and Experimental Botany, 55, 248–257.
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