Environmental and Experimental Botany 65 (2009) 177–182 Contents lists available at ScienceDirect Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot Effects of shade treatments on the photosynthetic capacity, chlorophyll fluorescence, and chlorophyll content of Tetrastigma hemsleyanum Diels et Gilg Yajuan Dai a , Zonggen Shen b , Ying Liu a , Lanlan Wang a , David Hannaway c , Hongfei Lu a,∗ a b c College of Chemistry and Life Science, Zhejiang Normal University, Jinhua, Zhejiang 321004, China Department of Biological and Food Engineering, Changshu Institute of Technology, Changshu, Jiangsu 215500, China Department of Crop and Soil Science, Oregon State University, Corvallis, OR 97331-3002, United States a r t i c l e i n f o Article history: Received 22 April 2008 Received in revised form 2 October 2008 Accepted 7 December 2008 Keywords: Tetrastigma hemsleyanum Light intensity Photosynthesis Chlorophyll fluorescence Leaf morphology Shade tolerance a b s t r a c t Tetrastigma hemsleyanum Diels et Gilg was grown under full sunlight and moderate and high levels of shade for one month to evaluate its photosynthetic and chlorophyll fluorescence response to different light conditions. The results showed that T. hemsleyanum attained greatest leaf size and Pn when cultivated with 67% shade. Leaves of seedlings grown with 90% shade were the smallest. Leaf color of plants grown under full sunlight and 50% shade was yellowish-green. The Pn value increased rapidly as PPFD increased to 200 ␮mol m−2 s−1 and then increased slowly to a maximum, followed by a slow decrease as PPFD was increased to 1000 ␮mol m−2 s−1 . Pn was highest for the 67% shade treatment and the LSP for this shade treatment was 600 ␮mol m−2 s−1 . Full sunlight and 50% shade treatments resulted in significant reduction of ETR and qP and increased NPQ. Chl a, Chl b and total chlorophyll content increased and Chl a/b values decreased with increased shading. Results showed that light intensity greater than that of 50% shade depressed photosynthetic activity and T. hemsleyanum growth. Irradiance less than that of 75% shade limited carbon assimilation and led to decreased plant growth. Approximately 67% shade is suggested to be the optimum light irradiance condition for T. hemsleyanum cultivation. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Tetrastigma hemsleyanum Diels et Gilg, belonging to the family Vitaceae, is a herbaceous perennial species native to China. It is distributed in the eastern, central, southern and southwestern provinces of China and is best suited to shady and moist hillsides and valleys. The entire herb and its root tubers possess anti-inflammatory, analgesic, and antipyretic properties. It also is used in Chinese folk medicine for dispelling phlegm and improving blood circulation. It is used for the treatment of high fever, infantile febrile convulsion, pneumonia, asthma, hepatitis, rheumatism, menstrual disorders, sore throat, and scrofula (Liu et al., 2002). In addition, it recently has been reported to work well in improving the immune system and for anti-cancer properties (Feng et al., 2006; Xu et al., 2006; Ding et al., 2005). Thus, in recent years, it has become an important species in China for its Abbreviations: Pn, net photosynthetic rate; PPFD, photosynthetic photon flux density; Yield, effective quantum yield of photochemical energy conversion; ETR, relative rate of electron transport through PSII; qP, photochemical quenching; NPQ, nonphotochemical quenching; E, transpiration rate; WUE, water use efficiency; LSP, light saturation point; Chl, chlorophyll; Chl a, chlorophyll a; Chl b, chlorophyll b. ∗ Corresponding author. Fax: +86 579 82282531. E-mail addresses: luhongfei0164@sina.com, luhongfei63@yahoo.com.cn (H. Lu). 0098-8472/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2008.12.008 medicinal properties and economic value. However, due to human overexploitation coupled with its specific environmental growth requirements for cultivation, T. hemsleyanum has become an endangered species. Climate, soil nutrients, and water have long been understood to be primary factors influencing agricultural productivity (Boyer, 1982; Fischer and Turner, 1978; Novoa and Loomis, 1981). It is relatively easy to control water and nutrient supplies through irrigation and fertilization. In contrast, light intensity (one of the most important plant growth requirements) is more difficult to control (Wang et al., 2007). Through the process of photosynthesis light energy is used to produce ATP and NADPH in the light reaction and subsequently, in the light-independent reaction, carbon is fixed into carbohydrates and oxygen is produced. Under high irradiance, however, the photosynthetic apparatus absorbs excessive light energy, resulting in the inactivation or impairment of the chlorophyllcontaining reaction centers of the chloroplasts (Bertaminia et al., 2006). As a consequence, photosynthetic activity is depressed by photoinhibition (Osmond, 1994). In contrast, under low irradiance, insufficient ATP is produced to allow for carbon fixation and carbohydrate biosynthesis. This leads to reduced plant growth. Although T. hemsleyanum has been reported to be a shade-preferring plant (based on its primary occurrence in the shaded understory) (Xu et al., 2006), no studies have determined the optimum light intensity for its growth. 178 Y. Dai et al. / Environmental and Experimental Botany 65 (2009) 177–182 Fm′ are the fluorescence at steady-state photosynthesis and maximum fluorescence in the light, respectively. qP was calculated as (Fm − Fm′ )/(Fm′ − F0 ). NPQ quenching was calculated as (Fm − Fm′ )/Fm′ (Genty et al., 1989). The relative rate of electron transport through PSII (ETR) was calculated as Yield × PPFDa × 0.5 (Krall and Edwards, 1992), where PPFDa is the absorbed light (␮mol photons m−2 s−1 ) by leaf (measured using an integrating sphere). 2.4. Chl contents Fig. 1. Curves of diurnal variation of full sunlight during September 2007 in Jinhua. The objective of the present study was to determine the optimum light intensity for the growth of T. hemsleyanum by quantifying the effects of different shade treatments on chlorophyll content, chlorophyll fluorescence, and photosynthetic capacity. Following the final measurements described above, leaves were collected for determination of chlorophyll content (Chl a, Chl b, Chl a + b). Chlorophyll pigments were extracted by grinding leaves in 80% acetone in the dark at room temperature and were expressed as mg/gfm from the equations of Porra (2002). 2.5. Data analysis 2. Materials and methods 2.1. Plants and growth conditions All experiments were conducted in a completely randomized block design replicated three times. Significance at P ≤ 0.05 was assessed by ANOVA using SAS version 9.0 (SAS Institute, Cary, NC, USA). T. hemsleyanum plants (with root tubers) were collected from the North Mountain area of Jinhua City and planted in the Zhejiang Normal University campus greenhouse in September 2006. Plantlets were obtained by layering and planting in pots containing a mixture of peat, sand, and humic soil (1:2:1). Plants were grown under five shade treatments (0, 50, 67, 75, and 90% of natural incident irradiance) with 3 replications. Diurnal variation of September full sunlight in Jinhua, measured with a TES-1332 Digital Lux Meter (TES, Taiwan), is displayed in Fig. 1. Shading was accomplished by using one or two layers of commercial black cloth shade for 30 days beginning on September 1, 2007. Daily maximum air temperatures were between 36 and 40 ◦ C. Irrigation was provided manually to saturation at 18:00 h each day. Light conditions had significant effects on T. hemsleyanum leaf morphology. Leaves grown under 67% (Fig. 2c) and 75% (Fig. 2d) shade were larger than leaves from other treatments. Leaves from the 90% shade treatment (Fig. 2e) were the smallest. Leaf color of plants grown under 67% (Fig. 2c) and 75% shade (Fig. 2d) were dark green, while those grown under full sunlight (Fig. 2a) and 50% shade (Fig. 2b) were yellowish-green. 2.2. Photosynthetic parameters 3.2. Photosynthesis Photosynthetic photon flux density (PPFD) response curves were developed using a GFS-3000 portable photosynthesis system (WALZ, Effeltrich, Germany). The parameters were measured on fully expanded leaves from 09:00 to 17:00 h on a clear, cloudless day. The air cuvette temperature and the air CO2 concentration were maintained at 25 ◦ C and 750 ␮L L−1 , respectively. PPFD was increased from 0 to 1000 ␮mol m−2 s−1 (0, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 ␮mol m−2 s−1 ). Water use efficiency (WUE) was calculated as Pn /E (␮mol CO2 ␮mol−1 H2 O) (Galmés et al., 2007). Assimilation was recorded at each of the 10 light levels following a 10 min acclimation period. Five replications were used for each plant. Regardless of shading treatment, the Pn value increased rapidly as PPFD increased to 200 ␮mol m−2 s−1 and then increased slowly to a maximum, followed by a slow decrease as PPFD was increased to 1000 ␮mol m−2 s−1 (Fig. 3a). The light compensation points (LCP) in full sunlight and 90% shade treatment plants were a little lower than the ones in 50%, 67%, and 75% shade treatment plants (Fig. 3a). The light saturation points (LSP) in 0% and 50% shade treatments were lower than for other treatments (Fig. 3a). Both Pn and maximum Pn varied significantly (P < 0.05) with light intensity treatments. The Pn value increased with increased shading; the highest Pn was observed in the 67% shade treatment plants and the LSP for this shade treatment was 600 ␮mol m−2 s−1 . Lower light intensities reduced the Pn value. The Pn –PPFD curves for natural light intensity and 90% shade treatments were almost coincident beyond 200 ␮mol m−2 s−1 . Transpiration rate (E)–PPFD curves of the five light intensity treatments are shown in Fig. 3b; E values varied significantly (P < 0.05) with light intensity treatments. The E values of full sunlight and 50% shade treatment plants were lower and E–PPFD curves appeared to be a marked single-peak curve. The E values of full sunlight treatment plants (when PPFD was >200 ␮mol m−2 s−1 ) were always lower than that from the 50% shade treatment plants. The E values from the other three treatments increased as PPFD increased. The average E in 90% shaded plants was always the highest with increasing PPFD. The average E value of 67% shade plants was lower than that of 75% shade plants. When PPFD was increased above 800 ␮mol m−2 s−1 , however, the E value was higher than that of the 75% shade treatment plants. 2.3. Chl fluorescence Chl fluorescence was measured with a MINIPAM (pulseamplitude modulation) fluorometer (WALZ, Effeltrich, Germany). Fluorescence measurements were taken simultaneously with gas exchange measurements since the fiber optic bundle of the fluorometer was fitted with a gas-tight seal within the gas exchange cuvette. Leaves were light-adapted for approximately 10 min prior to measurements of the effective quantum yield of photochemical energy conversion (Yield), photochemical (qP) and nonphotochemical (NPQ) quenching of chl fluorescence. Measurements were obtained over a range of PAR values between 0 and 1455 ␮mol m−2 s−1 . The relative effective quantum yield of photochemical energy conversion at steady-state photosynthesis was calculated as Yield = (Fm′ − Fs)/Fm′ , where Fs and 3. Results 3.1. Leaf morphology Y. Dai et al. / Environmental and Experimental Botany 65 (2009) 177–182 179 Fig. 2. Randomly chosen T. hemsleyanum leaves of plants from the various shade treatments. (a) Full sunlight. (b) 50% shade. (c) 67% shade. (d) 75% shade. (e) 90% shade. (Bar = 1 cm.) Water use efficiency (WUE)–PPFD curves are shown in Fig. 3c. The WUE–PPFD curves of full sunlight and 50% shade treatment plants display a marked double-peak appearance. WUE in 90% shade treatment plants was lowest. WUE in 67% shade treatment plants was highest when PPFD was 500–600 ␮mol m−2 s−1 and decreased slowly as PPFD increased above 600 ␮mol m−2 s−1 . 3.3. Chl fluorescence Full sunlight and 50% shade treatment resulted in a significant (P < 0.05) reduction in the apparent electron transport rate (ETR) and photochemical quenching (qP), and an increase in nonphotochemical quenching (NPQ) (Fig. 4a–c). ETR increased rapidly as PPFD increased for the 67, 75, and 90% shade treatment plants but decreased as PPFD was increased above 544 ␮mol m−2 s−1 . The ETR value for leaves of 67% shade treatment plants was highest while the value was smallest for plants grown under full sunlight. Quenching coefficients plotted as a function of PAR showed a steady decline in qP and a clear increase in NPQ with increasing irradiance. The qP value in 67% shade treatment plants was the highest, while values for natural intensity treatment plants were lowest. The NPQ for 67% shade treatment plants was lower than for 75% shade treatment plants until PAR increased above 544 ␮mol m−2 s−1 . The NPQ value for natural intensity treatment plants was always the highest with increasing irradiance. 3.4. Chl contents Chl content was affected significantly (P < 0.05) by the different light intensity treatments (Fig. 5). The full sunlight and 50% shade treatments resulted in significant reductions in Chl a, Chl b and total chlorophyll content and an increase in the Chl a:b ratio. The highest Chl a, Chl b and total chlorophyll content and the lowest Chl a:b ratio were observed in the 90% shade treatment plants. Chl a, Chl b and total chlorophyll content and Chl a:b ratios in 67% shade treatment plants were 0.79, 0.72, 0.77 and 1.08 times the values for 90% shade treatment plants, and 0.98, 0.97, 0.98 and 1.01 times the values observed in 75% shade treatment plants. Fig. 3. (a) Photosynthesis (Pn )-irradiance (PAR) response curves from leaves of T. hemsleyanum grown under full sunlight (), 50% shade (), 67% shade (), 75% shade (), 90% shade (*). (b) The transpiration rate (E)-irradiance (PAR) response curves from leaves grown under full sunlight (), 50% shade (), 67% shade (), 75% shade (), 90% shade (*). (c) Water use efficiency (WUE)-irradiance (PAR) response curves from leaves grown under full sunlight (), 50% shade (), 67% shade (), 75% shade (), 90% shade (*). Values are means ± S.E. 4. Discussion Leaf chl content is well established as a common reference system when physiological reactions are quantified (Wittmann et al., 2001). Decreases in chl b content have been suggested to be an 180 Y. Dai et al. / Environmental and Experimental Botany 65 (2009) 177–182 Fig. 4. (a) Apparent electron transport rate (ETR)-irradiance (PAR) response curves from leaves of T. hemsleyanum grown under full sunlight (), 50% shade (), 67% shade (), 75% shade (), 90% shade (*). (b) Photochemical quenching (qP)-irradiance (PAR) response curves from leaves grown under full sunlight (), 50% shade (), 67% shade (), 75% shade (), 90% shade (*). (c) Nonphotochemical quenching (NPQ)irradiance (PAR) response curves from leaves grown under full sunlight (), 50% shade (), 67% shade (), 75% shade (), 90% shade (*).Values are means ± S.E. indication of chlorophyll destruction by excess irradiance (Jason et al., 2004). Our results showed significant (P < 0.05) decreases in chl content (chl a, chl b, and chl a + b) (Fig. 5) and leaf yellowing (Fig. 2) in 0% and 50% shaded conditions suggesting that a light intensity of greater than that of 50% shade conditions may seriously impair or totally inactivate the photosynthetic system. Plants grown under shaded conditions are known to optimize their effectiveness of light absorption by increasing pigment density per unit leaf area (Wittmann et al., 2001). The reductions we observed in chl a/b ratio in leaves of 67, 75 and 90% shaded plants were due primarily to significant (P < 0.05) increases in chl b content (Fig. 5) and are most likely due to changes in the organization of both lightharvesting and electron transport components (Schiefthaler et al., 1997). The marked increase in leaf chl content in the 90% shaded condition demonstrated the plant’s ability to maximize the lightharvesting capacity in low-light growth conditions (Kura-Hotta et al., 1987; Lei et al., 1996). Leaf chl content also is one of the most important factors in determining the photosynthesis rate (Mao et al., 2007) and dry matter production (Ghosh et al., 2004). Naidu et al. (1984) suggested that reduced rates of photosynthesis may be due to reduced levels of Chl, particularly Chl a which is a more directly involved in determining photosynthetic activity (Sestak, 1996). The lower Chl a contents we observed in 0% and 50% shaded plants may at least partially explain the lower photosynthetic rates found in these two treatments plants leaves. Leaf size (Fig. 2) was smallest when plants were grown under 90% shade. This result confirms the report by Gordon et al. (1994) in which leaf size decreased under low-light conditions in Posidonia sinuosa plants. This adjustment reduces the respiratory demand of the shoot to help compensate for the greatly decreased the photosynthetic capacity of the leaves (Campbell and Miller, 2002). The 90% shade and full sunlight treatments decreased the light compensation points. Lower compensation irradiance in 90% shade plants was used to judge the shade adaptation (Rena et al., 1994). The LCPs in full sunlight plants in our experiment differed from previous reports (Lambers et al., 1998) that suggested that plants have acclimated to a high light environment demonstrated increased respiration and LCP. This would imply that full sunlight will be intolerant for T. hemsleyanum growth. Our results showed that full sunlight and 50% shade treatments reduced the light saturation points (LSPs) in T. hemsleyanum plants (Fig. 3a). This suggests that these treatments provided light in excess of what could be used for photosynthesis and photoinhibition was increased (Galmés et al., 2007). The T. hemsleyanum plants in this test had their highest Pn when grow under the light irradiance of 67% shade treatment. The WUE of this treatment plants declined when PPFD increased after 600 ␮mol m−2 s−1 because the net CO2 assimilation became light saturated, while transpiration constantly increased with increasing PPFD (Schapendonk et al., 1997). Although, the E values of 90% shade-grown plants, and their chl content (especially Chl b content) were the highest among the five treatments plants, their Pn and WUE values were no higher than the 0% shaded treatments. This suggests that although this shade-tolerant plant could adapt to growing under a light intensity of 90% shade, such low light intensity would still decrease its growth. The lower E values of 0% shade and 50% shade plantlets suggests that they adapted to high light irradiance and used stomatal closure to decrease water loss. WUE of these two treatments plants increased again after 700 and 600 ␮mol m−2 s−1 which suggests that stomatal closure at high irradiance reduced transpiration more than photosynthesis. Chl fluorescence continues to be a mainstay in studies of photosynthetic regulation and plant responses to the environment due to its sensitivity, convenience, and nonintrusive characteristics (Rascher et al., 2000; Schreiber et al., 1995). The ETR value represents the relative quantity of electrons passing through PSII during steady-state photosynthesis (Tezara et al., 2003). Exposure to the high irradiance conditions of the 0% and 50% shade treatments resulted in a greatly reduced ETR value (Fig. 4a). Reductions in ETR may be due to the loss of chlorophyll via reduction in the efficiency of excitation capture, most likely from photoinhibition (Flowers et al., 2007). In the 67% shade treatments, ETR was saturated at 500–600 ␮mol m−2 s−1 ; almost coincident with photosynthesis light saturation values. The qP is an indication of the proportion of PSII reaction centers that are open (Maxwell and Johnson, 2000). High qP is advantageous for the separation of electric charge in the reaction center, and is beneficial to electron transport and PSII yield (Guo et al., 2006; Mao et al., 2007). In this experiment, the differences in the values of qP showed that this species has significant differences in the activities of electron transport in PSII when plants are grown under varied shade treatments. The separation of electric charge in the reaction center, the ability to transport electrons, and the quantum yield of PSII were enhanced in 67% shade treatment Y. Dai et al. / Environmental and Experimental Botany 65 (2009) 177–182 181 Fig. 5. Comparison of chlorophyll (Chl) content in leaves of T. hemsleyanum grown under full sunlight, 50% shade, 67% shade, 75% shade, 90% shade. Values are means ± S.E. (Different letters mark significant differences, P < 0.05.) plants and weakened in the 0% and 50% shaded treatments. The NPQ can represent the energy which cannot be utilized to transport photosynthetic electrons being dissipated harmlessly as heat energy from PSII antennae (Muller et al., 2001; Vasil’ev et al., 1998; Veres et al., 2006). The lower NPQ in 67% and 75% shaded plants indicates that these plants effectively reduced the irradiance heat and efficiently utilized the energy absorbed by antenna pigments in PSII (Guo et al., 2006). The higher NPQ in full sunlight and 50% shade treatment plants showed the energy absorbed in the physiological range of irradiances was much higher than photochemical utilization by which will cause inhibition of photosynthetic capacity (Vasil’ev et al., 1998). The low ETR and qP values, and the high NPQ value combined with unhealthy leaf morphology (smaller leaf size and yellow leaf color), and the low Pn of the plants grown under full sunlight and 50% shade suggested that excess light energy damaged plants (Melis, 1999). This is thought to be due to the formation of destructive oxidative molecules (such as singlet oxygen radicals), resulting in damage to the photosynthetic apparatus via photoinhibition (Krause, 1988; Aro et al., 1993). These results indicate that a light intensity greater than that of 50% shade will be excessive irradiance and result in damage to the photosynthetic apparatus of T. hemsleyanum. On the other hand, the low Pn and WUE value and the smallest leaf size of plants in the 90% shade treatment plants indicated that the light intensity of 90% shade led to a decline in plant growth. 5. Conclusions Light irradiance levels significantly affected the growth of T. hemsleyanum. Leaf appearance was best and Pn values were greatest when plants were grown under 67% shade. When light intensity exceeded that of 50% shade, photosynthetic activity was depressed likely due to photoinhibition. Light intensity reductions greater than that of 75% shade, however, resulted in insufficient irradiation to maintain Pn , influencing carbon balance and consequently leading to a decline in plant growth. Thus, approximately 67% shade is concluded to be the optimum light irradiance condition for T. hemsleyanum cultivation. In view of the low light acclimation capacity of this plant, wild T. hemsleyanum is not expected to occur in the shaded understory where the light intensity is greater than 50% of ambient values. Light intensity of natural habitat areas with less than that of 25% ambient light will decrease the productivity of T. hemsleyanum. For agricultural purposes, in order to obtain high yields of T. hemsleyanum, we recommend trying to achieve approximately 33% ambient light with a 67% shade net. Acknowledgements This study was partially supported by the Emerging Talents scheme of Zhejiang Province and Research Learning and Innovation Experiment Foundation of Zhejiang Normal University. The authors thank Mr. L.H. Zhang, Ms. X.X. Lin, and Ms. Y.F. Chen for their help in chl content measurements. The authors also thank Mr. B. Jiang and Ms. L.Z. Mao for their assistance in reviewing the English manuscript. References Aro, E.M., Virgin, I., Andersson, B., 1993. Photoinhibition of Photosystem II. 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