Leaf Orientation, Photorespiration and Xanthophyll Cycle Protect Young Soybean Leaves Against High Irradiance in ﬁeld

Environmental and Experimental Botany 55 (2006) 87–96

Leaf orientation, photorespiration and xanthophyll cycle protect young soybean leaves against high irradiance in ﬁeld

Chuang-Dao Jiang a, Hui-Yuan Gao b,∗,QiZoub, Gao-Ming Jiang a, Ling-Hao Li a

a Laboratory of Quantitative Vegetation Ecology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, PR China b Department of Plant Science, Shandong Agricultural University, Taian 271018, PR China

Accepted 6 October 2004

Abstract

In order to fully understand the adaptive strategies of young leaves in performing photosynthesis under high irradiance, leaf orientation, chloroplast pigments, gas exchange, as well as chlorophyll a fluorescence kinetics were explored in soybean plants. The chlorophyll content and photosynthesis in young leaves were much lower than that in fully expanded leaves. Both young and fully expanded leaves exhibited down-regulation of the maximum quantum yield (FV/FM) at noon in their natural position, no more serious down-regulation being observed in young leaves. However, when restraining leaf movement and vertically exposing −2 −1 the leaves to 1200 ␮mol m s irradiance, more pronounced down-regulation of FV/FM was observed in young leaves; and the actual photosystem II (PS II) efficiency (ФPSII) drastically decreased with the significant enhancement of non-photochemical −2 −1 quenching (NPQ) and ‘High energy’ quenching (qE) in young leaves. Under irradiance of 1200 ␮mol m s , photorespiration (Pr) in young leaves measured by gas exchange were obviously lower, whereas the ratio of photorespiration/gross photosynthetic rate (Pr/Pg) were higher than that in fully expanded leaves. Compared with fully expanded leaves, young leaves exhibited higher xanthophyll pool and a much higher level of de-epoxidation components when exposure to high irradiance. During leaf development, the petiole angle gradually increased all the way. Especially, the midrib angle decreased with the increasing of irradiance in young leaves; however, no distinct changes were observed in mature leaves. The changes of leaf orientation greatly reduced the irradiance on young leaf surface under natural positions. In this study, we suggested that the co-operation of leaf angle, photorespiration and thermal dissipation depending on xanthophyll cycle could successfully prevent young leaves against high irradiance in field. © 2004 Elsevier B.V. All rights reserved.

Keywords: Photosynthetic rate; Chlorophyll a ﬂuorescence; Photorespiration; Xanthophyll cycle; Leaf orientation; Soybean

Abbreviations: A, antheraxanthin; Chl, chlorophyll; F0, minimal fluorescence in dark-adapted state; FM, maximum fluorescence in dark- adapted state; FV, maximum variable fluorescence in dark-adapted state (=FM−F0); FV/FM, maximum quantum yield of photosystem II; FS, F F steady-state fluorescence under irradiance; M, maximum fluorescence in ligh-adapted state; V, maximum variable fluorescence in light-adapted F − F state (= M 0); PFD, photon flux density; Pn, net photosynthetic rate; Pg, gross photosynthetic rate; Pr, photorespiration; PSII, photosystem II; ФPSII, the actual PSII efficiency under irradiance; NPQ, non-photochemical quenching; qE, the fast relaxing component of non-photochemical quenching; V, violaxanthin; Z, zeaxanthin ∗ Corresponding author. Tel.: +86 538 8241341; fax: +86 538 8249608. E-mail address: [email protected] (H.-Y. Gao).

1. Introduction zeaxanthin (Z) and antheraxanthin (A) (Bjorkman¨ and Demmig, 1987). All organisms that exhibit qE have a During leaf development, the newly initiating leaves xanthophyll cycle (Muller¨ et al., 2001). For the last few are often exposed to full sunlight at the topmost canopy, years, many investigators have paid special attentions indicating that those young leaves have to endure ex- to the role of xanthophyll cycle under conditions of tremely high irradiance. However, young leaves have cold-temperature stress (Verhoeven et al., 1999), water lower photosynthesis activity per unit area compared stress (Munne-Bosch´ and Alegre, 2000), and nutrition with fully developed leaves (Krause et al., 1995; Choin- deficiency (Verhoeven et al., 1997; Jiang et al., 2001; ski et al., 2003). These will inevitably result in more Jiang et al., 2002). Just recently, some people have fur- excessive excited energy in young leaves. It is well ther explored the characteristics of xanthophyll cycle known that too much light can lead to largely increased pigments and excited energy dissipation in senescence production of damaging reactive oxygen as byproducts leaves (Munne-Bosch´ et al., 2001; Lu et al., 2001; Lu of photosynthesis, during which photosynthetic rate is et al., 2003). Specifically, a few physiologists argued depressed (Osmond, 1994; Muller¨ et al., 2001). In ex- that xanthophyll cycle pigments and excited energy dis- treme cases, reactive oxygen can cause pigment bleach- sipation were enhanced in young leaves so that the ing and death. Such is the case well known to anyone photosynthetic apparatus could be protected (Krause who tries to move a houseplant outdoors into full sun- et al., 1995; Yoo et al., 2003). However, such ques- light (Osmond, 1994; Muller¨ et al., 2001). Therefore, it tion is still in debate (Ogren,¨ 1991; Krause et al., 1995; is a great challenge for young leaves to be subjected to Bertamini and Nedunchezhian, EEB1511BIB312003). strong irradiance. Nevertheless, plants have developed In all these studies, we noticed that detached and almost a number of strategies to balance the captured light fully expanded leaves were chosen to explore xantho- energy, thereby protecting photosynthetic apparatus phyll cycle. Under field conditions, can these mecha- against photodamage (Anderson et al., 1997). nisms successfully protect young leaves against high Photorespiration provides an effective electron sink irradiance at the early development stages? when CO2 assimilation is low (Kozaki and Takeba, Some authors also noticed that leaves, especially 1996). It is well documented that photorespiration pro- some leguminous plants, can change their orientation tects leaves against high irradiance through not only by inclining upwards and downwards under higher acting as a sink for reducing equivalents but also pre- irradiance, thereby minimizing the interception of venting over-reduction of the electron carriers between irradiance for avoiding photodestruction (Gamon and PS II and PS I (Kozaki and Takeba, 1996; Osmond Pearcy, 1989; Ogren¨ and Evan, 1992; Bjorkman¨ and and Grace, 1995). However, there were few studies Demmig-Adams, 1995; James and Bell, 2000; Feng focusing on changes of photorespiration during leaf et al., 2002). We wonder whether or not leaf orienta- expansion. tions plays a more important role in newly initiating Thermal dissipation of excess irradiance measured leaves than that in fully developed ones when legumi- as non-photochemical quenching (NPQ) is believed to nous plants are subjected to strong irradiance. be of paramount importance in the protection of the The objective of this study is to explore how young photosynthetic apparatus against the deleterious effects soybean leaves cope with high irradiance under field of excess light. It has been known for many years that at conditions, and whether the co-operation of leaf ori- least two main components of NPQ can be resolved by entation, photorespiration and xanthophyll cycle could analyzing its dark relaxation kinetics, the rapidly relax- effectively protect young leaves against strong sunlight ing component (qE) and the slowly reversible compo- in field. nent (often termed qI). qE, the rapidly relaxing component of NPQ is considered to be an important photopro- 2. Materials and methods tective mechanism to cope with excessive irradiance (Bjorkman¨ and Demmig, 1987; Muller¨ et al., 2001). 2.1. Plant materials The value of qE is always associated with pH gradient across the thylakoid membrane (Briantais et al., 1980) Soybean (Glycine max L.) plants were grown in ten and the formation of the xanthophyll cycle pigments, plastic pots (22 cm in diameter and 30 cm in height) at C.-D. Jiang et al. / Environmental and Experimental Botany 55 (2006) 87–96 89 the beginning of May. The plants grown in pots were ech, UK). The maximum quantum yield of photo- placed in field subjected to natural solar radiation, with system II (FV/FM) was determined in dark-adapted a daily maximum photosynthetic photon flux density (15 min) samples. After the initial Chl fluorescence −2 −1 (PPFD) of above 1600 ␮mol m s , and the maxi- yield (F0) was determined in low modulated mea- mum air temperature was about 33 ◦C. The soybean suring light, a 0.7-s pulse of saturating white light plants were thinned to one plant per pot 2 weeks after (>3000 ␮mol m−2 s−1) was applied to obtain the max- sowing. Nutrients and water were supplied sufficiently imum Chl fluorescence yield (FM) and the FV/FM throughout, to avoid potential nutrients and drought (FV, the variable Chl fluorescence yield, is defined as stresses. After growing for 5 weeks, newly expanding FM−F0). The steady-state fluorescence level (FS) and F leaves with an area of about 33% of fully expanded the maximum Chl fluorescence level ( M) during expo- leaves (33% A), near fully expanded leaves with an sure to illumination were also measured, respectively. area of about 78% of fully expanded leaves (78% A) The fluorescence transient was induced by continu- and fully expanded leaves (100% A) were studied in ous light (1200 ␮mol m−2 s−1) for 2 h. While the pho- the experiments. Generally, three leaves (33% A, 78% tochemical fluorescence-quenching coefficient (qP) A and 100% A) of each plant were used in the differ- and non-photochemical quenching (NPQ) were dis- ent measurements, and at least three replications were criminated by applying saturating pulses after every made. 30 min of the continuous light treatment. In the experiments carried out in open field, all The actual PS II efficiency (ФPSII) was calcu- F − F leaves were kept in their natural positions. While in lated as ( M FS)/ M (Genty et al., 1989), and non- F − the experiments performed in the laboratory, attached photochemical quenching (NPQ) as FM/ M 1(Bilger leaves were restrained in horizontal positions using and Bjorkman,¨ 1990). NPQ was resolved into fast flexible wire and vertically illuminated by a man-made relaxing (qE) and slowly relaxing (qI) components by lamp (Philips HPLR 400 W). Between the lamp and extrapolation in semi-logarithmic plots of the maxi- leaves, a water bath with flowing water was used to mum fluorescence yield versus time as described by keep temperature in steady. Johnson et al. (1993).

2.2. Measurement of photosynthetic rate 2.4. Pigment analysis

Photosynthetic rate–Photosynthetic photon flux The leaf chlorophyll and carotenoid were extracted density (Pn–PFD) response curves were made at leaf with 80% acetone, with the extracts being analyzed chamber temperature of 30 ◦C, and at 350 ␮mol mol−1 with a UV-120 system (Shimadzu, Japan) accord- CO2 with a portable photosynthetic system (CIRAS- ing to Arnon (1949). Then the carotenoid compo- 1, PP systems, UK). PFD was fixed every 10 min in nents of xanthophyll cycle were determined, according a sequence of 2000, 1600, 1200, 800, 600, 400, 300, to the method developed by Thayer and Bjorkman¨ −2 −1 200, 150, 100, 0 ␮mol m s . Light intensity, CO2 (1990). After dark adaptation for 12 h, leaves were concentration and leaf chamber temperature were con- all horizontally exposed to a strong irradiance of trolled by automatic control device of the CIRAS-1 1200 ␮mol m−2 s−1 for 0, 1, 2 h, respectively. After- photosynthetic system. Photosynthetic rate measured wards, they were quickly frozen in liquid nitrogen. −1 at two O2 concentrations (21% O2 + 350 ␮mol mol Latter, leaf samples were extracted with 85% acetone. −1 CO2 and 2% O2 + 350 ␮mol mol CO2) under Content of the pigments were estimated by applying 1200 ␮mol m−2 s−1 PFD was used to calculate pho- the conversion factors for peak area to nmol as deter- torespiration. mined for this solvent mixture by Thayer and Bjorkman¨ (1990). 2.3. Measurement of chlorophyll fluorescence parameters 2.5. Measurement of leaf orientation

In vivo chlorophyll fluorescence was measured A petiole angle is the angle of branch to which leaves using a pulse-modulated fluorimeter (FMS-2, Hansat- are attached and a midrib angle is defined as the devia- 90 C.-D. Jiang et al. / Environmental and Experimental Botany 55 (2006) 87–96 tion of the midrib from vertical. Both leaf angles were and 3.59 ± 0.11 in 100% A, respectively. Such a result investigated using a clinometer (PM-5/360 PC, Suunto indicates that the increase magnitude of Chl a was much Co., Finland). The midrib angle was measured at both higher than that of Chl b during leaf development. sunny and cloudy days to exclude the rhythmical movement. 3.2. Changes of photosynthesis and photorespiration 2.6. Statistical analysis Fig. 2 shows the photosynthesis–photosynthetic Data of measurements were inputted into Microsoft photon flux density (Pn–PFD) response curves dur- Excel 2000 spreadsheet, and each value of mean and ing leaf development. The maximum assimilation S.E. in the figures represents 3–6 replications of mea- rates under saturation photon flux density were − − − − surements. And data were subjected to analysis of 5.1 ± 0.43 ␮mol m 2 s 1, 10.2 ± 0.5 ␮mol m 2 s 1 − − variance using SPSS (10.0 for Windows). The least and 16.7 ± 0.46 ␮mol m 2 s 1 in 33% A, 78% A, significant differences between the means were esti- 100% A leaves, respectively. Obviously, young leaves mated at 95% confidence level. Plots and fit curves were exhibited lower CO2 assimilation capacity than that of performed using SigmaPlot 2000 and Microsoft Excel fully expanded ones. Young leaves also showed lower 2000. Unless otherwise indicated, significant differ- saturation light of photosynthetic rate (SLP) (Fig. 2). In ences among different leaf types are given at P < 0.05. parallel with the lower CO2 assimilation capacity, the photorespiration (Pr) in young leaves was also lower (Fig. 3), whereas the ratio of photorespiration/gross 3. Results photosynthetic rate (Pr/Pg) in young leaves was higher than that in fully expanded leaves (Fig. 3). 3.1. Changes in chlorophyll content 3.3. Changes of the maximum PS II quantum yield Chlorophyll (Chl) contents per unit area in young under high irradiance leaves (33 and 78% expanded) were significantly lower than that in fully expanded ones (Fig. 1). Chl a/Chl b The maximum PS II quantum yield (FV/FM) after ratios were 2.20 ± 0.07 in 33% A, 3.06 ± 0.12 in 78% A full dark-adaptation were 0.78 ± 0.01, 0.81 ± 0.01

Fig. 1. Changes of chlorophyll pigments during the development of soybean leaves. The newly expanding leaves with an areas of about Fig. 2. Typical light response curves of different expanding soybean 33% of fully expanded leaves (33% A), near fully expanded leaves leaves measured in ambient CO2 (about 350 ␮mol/mol) at leaf cham- ◦ with an areas of about 78% of fully expanded leaves (78% A) and ber temperature of 30 C. (᭹), () and () represent fully expanded fully expanded leaves (100% A) were studied in the experiment. leaves (100% A), almost fully expanded leaves (78% A) and just ini- Values are means ± S.E., n =3. tiated leaves (33% A), respectively. Valuesare means ± S.E., n = 3–5. C.-D. Jiang et al. / Environmental and Experimental Botany 55 (2006) 87–96 91

Fig. 3. Changes of photorespiration (Pr) and the ratio of photorespiration to gross photosynthetic rate (Pr/Pg) during expansion of soybean leaves under 1200 ␮mol m−2 s−1 irradiance measured at leaf chamber temperature of 30 ◦C. Values are means ± S.E., n =4. and 0.84 ± 0.01 in 33% A, 78% A and 100% A leaves, respectively (Fig. 4A). When leaf movements were restrained and vertically subjecting leaves to 1200 ␮mol m−2 s−1 irradiance, young leaves were more susceptible to strong light than mature ones, as indicated by the more pronounced decrease in FV/FM ratios in young leaves; when the light was turned off, the values of FV/FM were almost completely restored Fig. 4. (A) Changes of the maximum efficiency of PS II photo- in 1 h in the three leaf types (Fig. 4A). However, there chemistry (FV/FM) in different expanding soybean leaves under −2 −1 were no appreciable differences in FV/FM (P > 0.05) 1200 ␮mol m s irradiance and the dark recovery courses measured in ambient CO2 (about 350 ␮mol/mol) at room temperature at noon when all leaves in their natural positions ◦ were exposed to full sun light in field condition (25–30 C). Attached leaves were kept vertically to the irradiance. (B) Changes of the maximal efficiency of PS II photochemistry (Fig. 4B). (FV/FM) in different expanding soybean leaves in field. Attached leaves were all kept in natural positions and measured in ambient CO2 (about 350 ␮mol/mol). Values are means ± S.E., n = 4–6. 3.4. Regulation of PS II photochemistry under high irradiance

As shown in Fig. 5A, a general decline in ФPSII was observed upon exposure to high irradiance, but the fully expanded leaves remained substantially higher ФPSII Xanthophyll cycle pigments, which are closely cor- than others. In young leaves, a significant decrease related with energy dissipation, were also analyzed. On of ФPSII together with a marked increase in non- chlorophyll basis, the xanthophyll cycle pool size was photochemical quenching (NPQ) occurred (Fig. 5B). significantly higher in young leaves than that in fully Meanwhile, NPQ in young leaves was dramatically expanded leaves (Fig. 6A). And the de-epoxidation higher compared with mature ones (Fig. 5B). To esti- components of the xanthophyll cycle pigments were mate the contribution of qE, the dark relaxation kinetics much more enhanced in young leaves than that in of fluorescence in the three leaf types were analyzed. fully expanded leaves when vertically exposed to ␮ −2 −1 Clearly, qE was significantly greater in young leaves 1200 mol m s irradiance for 1 and 2 h, respec- than that in mature ones (Fig. 5C). tively (Fig. 6B and C). 92 C.-D. Jiang et al. / Environmental and Experimental Botany 55 (2006) 87–96

Fig. 6. (A) Changes of xanthophyll cycle pigment pool size, (B) the de-epoxidation components per Chl, and (C) the de-epoxidation level at various developmental stages of soybean leaves in dark and vertically exposed to 1200 ␮mol m−2 s−1 irradiance for 1 and 2 h, respectively. Values are means ± S.E., n =4.

Fig. 5. Changes of (A) actual photosystem II efficiency (ФPSII), (B) non-photochemical quenching (NPQ) and (C) ‘high-energy’ fluores- 3.5. Changes in leaf angle and irradiance on leaf cence quenching (qE) in different expanding soybean leaves under surface −2 −1 1200 ␮mol m s irradiance measured in ambient CO2 (about ◦ 350 ␮mol/mol) at room temperature (25–30 C). Attached leaves Fig. 7 showed that petiole angles were smaller in ± were kept vertically to the irradiance. Values are means S.E., young leaves than that in mature ones. At dawn or n = 3–4. sunset, the midrib angles of all the three leaf types were almost the same. However, the angles in young leaves were significantly reduced at noon on clear days (Fig. 8A). Noticeably, no appreciable diurnal variation C.-D. Jiang et al. / Environmental and Experimental Botany 55 (2006) 87–96 93

Fig. 9. Typical daily changes of irradiance on leaf surface at various developmental stages of soybean leaves on clear day in field. Values Fig. 7. Changes of petiole angle during various developmental stages are means ± S.E., n =6. of soybean leaves in field. Values are means ± S.E., n =6. (P > 0.05) was noted on cloudy day (Fig. 8B). Obvi- ously, it is the light rather than rhythmical movement that induced the change of midrib angle. We also noted that the changes of leaf orientation under high irradiance in young leaves were steeper than that in fully expanded ones (Figs. 7 and 8). Among the three leaf types, mature leaves received the greatest amount of daily irradiance on leaf surface, and had a large peak of irradiance at noon (Fig. 9). Along with the development of soybean leaves, it was the changes in leaf angle that resulted in the increase of intercepted photon flux density.

4. Discussion

4.1. Development of photosynthetic apparatus and changes of leaf orientation

The increase of chlorophyll content and Chl a/Chl b ratio with the process of leaf expansion indicated a gradual development of photosynthetic apparatus (Fig. 1), which was supported by the increase of photosynthetic rate with the leaf expansion (Fig. 2), so that more excited energy would be utilized in CO2 assimilation rather than dissipated with the process of leaf expansion. It was reported that FV/FM could represent original Fig. 8. Changes of midrib angle in different expanding soybean activity of PS II (Hulsebosch et al., 1996). However, leaves on clear day (A) and cloudy day (B) in field. Values are high FV/FM was observed among the three leaf types means ± S.E., n =6. (Fig. 4A) indicating that young leaves had almost the 94 C.-D. Jiang et al. / Environmental and Experimental Botany 55 (2006) 87–96 same activity of primary charge separation as that in energy was dissipated in young leaves (Fig. 5B), which mature leaves. It is thus deduced that the activity of PS was also supported by the fact that young leaves had II might not be the limiting step of the photosynthesis larger qE, one of the major components of NPQ, than in young leaves. the mature leaves under the same irradiance (Fig. 5C). Young leaves had a more vertical leaf orientation Some authors reported that qE is promoted by the for- than mature leaves (Figs. 7 and 8) indicating that the mation of the xanthophyll pigments zeaxanthin (Z) and mature leaves can effectively intercept more irradiance antheraxanthin (A) (Demmig-Adams, 1990; Gilmore, than young leaves under filed conditions. Considering 1997). We noticed that much more de-epoxidation that with the development of photosynthetic apparatus components were produced in young leaves than that and increase of CO2 assimilation capacity, more and in fully expanded ones when exposed to irradiance more irradiance capture is needed to promote photo- (Fig. 6B and C), which was in accordance with the synthesis, the change of leaf angle is an adaptation to change of qE. Additionally, it is believed that Z might intercept irradiance during leaf expansion. act as an important antioxidant, which can directly 1 deactivate O2 and also quench the excited triplet state 4.2. Development of photoprotective mechanisms of chlorophyll in the thylakoid membrane under high during leaf expansion irradiance (Havaux and Niyogi, 1999; Havaux et al., 2000). The higher content of xanthophyll cycle pig- It has been widely known that leaves developed ments per Chl and the higher de-epoxidation state of several regulation mechanisms to protect themselves xanthophylls [(A + Z)/(V+A+Z)]% in young leaves against high irradiance, thus can efficiently balance the might be a strengthened acclimation to cope with light capture and utilization (Osmond, 1994). Data in excess irradiance during the initial stages of leaf expan- this study revealed that young leaves that did not have sion to excess irradiance (Fig. 6B and C). The reversible accomplished photosynthetic apparatus ad already had changes of FV/FM in young leaves revealed that photo- efficient mechanisms to consume excited energy cap- protective mechanisms in young leaves worked well to tured by light harvesting complexes (Figs. 3 and 5) avoid photodestruction to the photosynthetic apparatus except carbon assimilation. under excessive irradiance (Fig. 4A and B). Foyer and Noctor (2000) reported that photorespiration acts as a route for energy consumption in C3 4.3. Changes of leaf orientation alleviate high plant; Kozaki and Takeba (1996) also demonstrated light stress in young leaves that photorespiration plays a key role in the protection of leaves against over reduction and uses energy when Data in the present study demonstrated that young CO2 assimilation is restricted. The pronounced higher leaves have timely developed photoprotective mecha- ratio of Pr/Pg in young leaves (Fig. 3) implies that rel- nisms, such as: photorespiration, xanthophylls cycle, to ative more excited energy captured by light harvesting cope with high irradiance. Significant down-regulation complexes was allocated to photorespiration in young in FV/FM in young leaves when vertically exposed to leaves. Increased allocation of excited energy to pho- high irradiance reflected that young leaves were more torespiration can effectively maintain linear electron susceptible to high irradiance (Fig. 4A). Additionally, transport and utilization of excited energy by allowing when FV/FM measured during daily courses were plot- metabolism to continue using the products of photo- ted as a function of irradiance (Fig. 10), it was also synthetic electron transport (Osmond and Grace, 1995; noticed that young leaves were more susceptible to high Foyer and Noctor, 2000), mitigating deleterious effects, irradiance than mature ones in field. such as photodestruction during the initial stages of leaf Theoretically, horizontal leaves receive more irradi- expansion. ance on leaf surface than steep ones (Ogren¨ and Evan, The lower ФPSII in young leaves under controlled 1992; James and Bell, 2000). Obviously, the changes conditions indicates that less light energy was utilized in petiole angle and midrib angle kept young leaves in photochemical reaction than that in mature ones in more vertical positions than fully expanded leaves (Fig. 5A) so that much more excessive energy was pro- (Figs. 7 and 8), enabling the young leaves to reduce duced in young leaves. In fact, much more excessive light intensity on the leaf surface (Fig. 9). Therefore, C.-D. Jiang et al. / Environmental and Experimental Botany 55 (2006) 87–96 95

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