Abstract
Photosynthetic physiology of Dendrobium nobile, Dendrobium pendulum, Dendrobium chrysotoxum, and Dendrobium densiflorum was studied. A bimodal diurnal variation of the net photosynthetic rate (Pn) was observed in the four Dendrobium species with the first peak [5.09 to 6.06 μmol (CO2) per m−2·s−1] ≈1100 hr and the second peak [3.83 to 4.58 μmol (CO2) per m−2·s−1] at 1500 hr. No CO2 fixation was observed at night. For all four Dendrobium species, the light compensation point (LCP) was 5 to 10 μmol·m−2·s−1, light saturation point (LSP) ranged from 800 to 1000 μmol·m−2·s−1, apparent quantum yield (AQY) was 0.02, and CO2 compensation points (CCP) and saturation point (CSP) were 60 to 85 μmol·mol−1 and 800 to 1000 μmol·mol−1, respectively. Carboxylation efficiency (CE) values ranged from 0.011 to 0.020. The optimum temperature for photosynthesis was between 26 and 30 °C. The measurement of Pn seasonal variation indicated that July to August had the higher Pn for Dendrobium species. Additionally, the chlorophyll a/b (Chl a/b) ratios of the leaves were 2.77 to 2.89. Measurement of key enzymes in the photosynthetic pathway indicated relatively high Ribulose-1,5-bisphosphate carboxylase (RuBPCase) and glycolate oxidase (GO) activities but very low phosphoenolpyruvate carboxylase (PEPCase) activities. It suggested that these four Dendrobium species are typical semishade C3 plants.
The genus Dendrobium is one of the largest genera in Orchidaceae; there are ≈1500 species around the world. In China there are 74 species and two varieties (belonging to nine sections) and mainly distributed in the mountain ranges of southern and western China (Tsi et al., 1999). Most Dendrobium orchids are endangered species and are overexploited as a result of their ornamental and medicinal values, e.g., Dendrobium. nobile, D. pendulum, D. chrysotoxum, and D. densiflorum (Chen and Tsi, 1997). These four species all blossom in spring and can be cultivated as ornamental potted plants or used for extraction of polysaccharides and alkaloids from the stems. At present, Dendrobium is listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora. There have been considerable efforts in large-scale commercial cultivation of medicinal Dendrobium orchids. However, research on the physiology of wild Dendrobium species has been scanty, and only a few wild Dendrobium species was investigated (Chou et al., 2001; Su and Zhang, 2003a, 2003b; Zhu et al., 2013a, 2013b).
Generally, orchids can be divided into thick-leaved orchids and thin-leaved orchids according to their leaf thickness. The thick-leaved orchids belong to CAM plants, e.g., Phalaenopsis (Endo and Ikusima, 1989), Dendrobium Phalaenopsis (He and Woon, 2008), and Cattleya (Stancato et al., 2002); and the thin-leaved orchids are C3 plants, e.g., Dendrobiums (Zhu et al., 2013a, 2013b). There are fairly extensive studies on the photosynthetic physiology of thin-leaved Oriental Cymbidium (Pan et al., 1997; Pan and Ye, 2006). The photosynthetic characteristics of thin-leaved Oncidium have also been shown to be a C3 orchid (He et al., 2011; Li et al., 2002). No C4 plant was found in Orchidaceae. In our previous studies, photosynthetic characteristics of wild Dendrobium (D. williamsoii, D. longicornu, D. chrysanthum, and D. dixanthum) in China were reported to be semishade C3 orchids There is no conclusive evidence to indicate that the photosynthetic pathway of wild Dendrobium species endemic to China belong to the nobile type that blossom in spring, and they are different from the CAM orchid Dendrobium, which blossom in fall (Khoo et al., 1997). More works are needed to understand the ecophysiological and photosynthetic characteristics of the wild Chinese Dendrobium orchids.
The objective of the present investigation is to carry out a systemic study of the photosynthetic characteristics of these four well-known wild Dendrobium species (D. nobile, D. pendulum, D. chrysotoxum, and D. densiflorum) in China. This study provides useful information for the conservation and rational use of these four endangered orchids.
Materials and Methods
Plant materials.
Four wild Chinese Dendrobium species (D. nobile and D. pendulum in Sect. Eugenanthe; D. chrysotoxum and D. densiflorum in Sect. Callista) were collected from Yunnan Province in China and cultivated in pots using sawdust as a substrate in a greenhouse kept with 60% to 70% of shade, which provided a maximum midday light radiation intensity was ≈1250 ± 100 μmol·m−2·s−1. The shaded greenhouse was 28 ± 2 °C in the day and 25 ± 3 °C at night, and relative humidity (RH) was between 65% and 80%. Plants were fertilized with one-third Hoagland nutrient solution weekly. The fourth mature leaf from the apex was selected for testing of photosynthetic characteristics in all experiments. All determinations were replicated three times.
Leaf structure.
The anatomical structure of the leaves was examined under an optical microscope (DM 6000B; Leica, Germany) and a scanning electron microscope (S-3500N; Hitachi, Japan). Preparation of the sample for the scanning electron microscope was as described in the Cytology Laboratory, Institute of Botany, the Chinese Academy of Sciences (1974).
Measurement of photosynthetic characteristics.
Various parameters including Pn, stomatal conductance, intercellular CO2 concentration, transpiration rate (Tr), photosynthetically active radiation (PAR), and air temperature were measured simultaneously by an LI−6400 portable photosynthetic system (LI-COR).
Light-response curve.
Measurement was conducted between 0930 hr and 1130 hr. Using the automatic measurement function of the light response curve of the LI-6400 photosynthesis system, the built-in red and blue light sources (6400−02B) were set at a series of PAR gradients within the range of 0 to 1200 μmol·m−2·s−1, and the leaf Pn corresponding to each gradient was measured. The corresponding curve was plotted with the paired values of Pn and PAR, and the LCP, LSP, and related parameters were obtained. Linear regression was performed on the paired values of PAR and Pn below 200 μmol·m−2·s−1, and the initial slope of the response curve Pn − PAR was the AQY of photosynthesis. For photosynthesis measurement, the CO2 concentration was set at 380 ± 10 μmol·mol−1, leaf chamber temperature at 25 ± 0.5 °C, and RH at 70 ± 15%.
CO2-response curve.
The CO2 concentration was regulated using the injection function (6400-01) of the LI-6400 portable photosynthesis system. The PAR was maintained at 800 ± 10 μmol·m−2·s−1, temperature at 25 ± 0.5 °C, and RH at 70 ± 15%. The CO2 concentration gradients were set within the range of 0 to 1500 μmol·mol−1 and the Pn corresponding to each gradient was measured. The corresponding curve was plotted with the paired values of Pn and PAR, and the CCP, CSP, and related parameters were acquired. Linear regression was performed on the paired values of CO2 concentration and Pn below 200 μmol·m−2·s−1, and the initial slope was the CE of RuBPCase. The Pn at the CO2 light saturation point was the regenerating rate of RuBP.
Temperature response curve.
The Pn value of each temperature gradient within the range of 18 to 34 °C was measured from low to high temperature using the leaf chamber temperature adjustment function (6400-13) of the LI-6400 portable photosynthesis system. During the measurements, PAR was set at 800 ± 10 μmol·m−2·s−1, CO2 concentration at 380 ± 10 μmol·mol−1, and RH at 70 ± 15%.
Measurement of Pn diurnal variation and seasonal variation.
For measurement of diurnal variation, the Pn and the related parameters were measured from 0700 hr to 1900 hr during 3 sunny days in the middle of May. The Pn seasonal variations were measured under natural conditions at 1000 hr to 1100 hr on 3 sunny days in the middle of each month from January to October.
Measurement of chlorophyll content.
Chlorophyll measurement was done according to methods described by Wintermans and De Mots (1965).
Enzyme extraction and activity determination.
Leaves of each Dendrobium species were illuminated for 2 h before enzyme extract. The sliced leaf tissues (0.2 g) were grounded in 1.5 mL of pre-cooled 100 mm Tris-HCL buffer solution (containing 10 mm MgCl2, 5 mm mercaptoethanol, 1 mm EDTA, 12.5% glycerol, and 1% PVP, pH7.4) with N2 in a semimicro Waring blender for 3 min. The homogenates were filtered through four layers cheesecloth and then centrifuged at 15,000 rpm for 20 min at 4 °C. The supernatant was used for testing enzyme activity of RuBPCase (EC 4.1.1.39), PEPCase (EC 4.1.1.31), and GO (EC 1.1.3.1) (Zhang, 1990).
Determination of protein content.
The protein content in the enzyme extracts was determined by Coomassie brilliant blue staining, and the standard curve was plotted using bovine serum albumin.
Results
Anatomical structure of Dendrobium species leaves.
Dendrobium showed bifacial anatomy with thin leaves ≈800 ± 50 μm (Fig. 1E–H), suggesting these species were thin-leaved orchids compared with thick-leaved CAM orchids such as Phalaenopsis amabilis, which have a leaf thickness of ≈3000 μm (Endo and Ikusima, 1989). The adaxial epidermis cells of the leaves were covered by cuticle with no stomata. These cells were larger and square in shape and formed a single layer of cells ≈20 μm thick, which arranged in an orderly rectangular pattern. The abaxial epidermis cells were irregular in shape and sizes with stomatal intensities ranging from 110 to 130 mm−2. The stomata were elliptical and the guard cell length and width were ≈30 μm and 20 μm, respectively. All stomata were slightly sunken into the leaf epidermis and were covered by a waxy stomatal cover (Fig. 1A–D). The mesophyll tissue was well developed and differentiated into palisade and spongy tissues (Fig. 1E–H). The palisade tissue was composed of one to two layers of orderly arranged cylindrical cells with thickness of 60 μm. The cells in the outer layer were densely arranged, deeply stained, and idioblast-shaped, whereas the cells in the inner layer were lightly stained and contained abundant chloroplasts. The spongy tissue cells were loosely arranged with a layer thickness of 40 μm and also contained many chloroplasts. The vascular bundles were arranged in a ring and the parenchyma cells in the bundle sheath were relatively small and distinctively arranged, containing no chloroplasts. No Kranz leaf anatomy was observed. Therefore, the anatomical structure of these four Dendrobium species indicated they were typical semishade C3 plants.
Electron microscope images of the abaxial surface (A–D) and cross-section (E–H) of leaves of the four Dendrobium species. (A, E) Dendrobium nobile; (B, F) Dendrobium pendulum; (C, G) Dendrobium chrysotoxum; (D, H) Dendrobium densiflorum.
Citation: HortScience horts 49, 8; 10.21273/HORTSCI.49.8.1023
Electron microscope images of the abaxial surface (A–D) and cross-section (E–H) of leaves of the four Dendrobium species. (A, E) Dendrobium nobile; (B, F) Dendrobium pendulum; (C, G) Dendrobium chrysotoxum; (D, H) Dendrobium densiflorum.
Citation: HortScience horts 49, 8; 10.21273/HORTSCI.49.8.1023
Electron microscope images of the abaxial surface (A–D) and cross-section (E–H) of leaves of the four Dendrobium species. (A, E) Dendrobium nobile; (B, F) Dendrobium pendulum; (C, G) Dendrobium chrysotoxum; (D, H) Dendrobium densiflorum.
Citation: HortScience horts 49, 8; 10.21273/HORTSCI.49.8.1023
Light intensity, CO2, and temperature response curves of the Dendrobium leaves.
The four species exhibited similar Pn–PAR curves (Fig. 2A). Within 0 to 300 μmol·m−2·s−1, Pn increased linearly, but the increased rates became slow when PAR ranged from 300 to 600 μmol·m−2·s−1; the Pn reached its peak at PAR of 800 μmol·m−2·s−1 and began to decrease with the increase of PAR. The maximum of Pn, LCP, and LSP for the four Dendrobium species was 5 to 6 μmol (CO2) per m−2·s−1, 5 to 8 μmol·m−2·s−1, and 800 to 1000 μmol·m−2·s−1, respectively (Fig. 2A; Table 1). Maximum photosynthetic rate reflected leaf photosynthetic efficiency, and D. chrysotoxum had the higher photosynthetic efficiency [5.89 μmol (CO2) per m−2·s−1] and the highest demand for light, whereas D. nobile had the lowest photosynthetic potential [4.38 μmol (CO2) per m−2·s−1]. Generally, the four Dendrobium species have relatively low LSPs and LCPs, which reflect their shade habit. In addition, AQY is a key index for the light use efficiency of leaves that reflects the ability of leaves to use dim light. The AQYs of the four Dendrobium species were ≈0.02 and no significant differences were observed among the four Dendrobium species, which indicated that they had relatively low light use efficiency but relatively high requirements for the light quantum for photosynthesis.
Light intensity (A), CO2 concentration (B), and temperature (C) response curves of the leaves of the four Dendrobium species.
Citation: HortScience horts 49, 8; 10.21273/HORTSCI.49.8.1023
Light intensity (A), CO2 concentration (B), and temperature (C) response curves of the leaves of the four Dendrobium species.
Citation: HortScience horts 49, 8; 10.21273/HORTSCI.49.8.1023
Light intensity (A), CO2 concentration (B), and temperature (C) response curves of the leaves of the four Dendrobium species.
Citation: HortScience horts 49, 8; 10.21273/HORTSCI.49.8.1023
Characteristics of light and CO2 responses of the leaves of the four Dendrobium species.
The photosynthetic rates of the four Dendrobium species showed almost identical responses to CO2 concentration (Fig. 2B; Table 1) and their CCPs were between 60 and 90 μmol·mol−1. For CO2 in the range of 0 to 700 μmol·mol−1, the photosynthetic rates of the four species increased rapidly with increasing CO2 concentration. The Pn peaked at ≈9 to 10 μmol (CO2) per m−2·s−1 in CO2 concentrations of 800 μmol·mol−1, indicating that the CO2 saturation points of the Dendrobium species were in the range of 800 to 1000 μmol·mol−1. The CEs of the four Dendrobium species were ≈0.011 to 0.020, showing their ability to low use of CO2. High CO2 concentration significantly increased the photosynthetic rates of these species. Temperature was the main influencing factor for the photosynthetic rate of plants. The Pn for the four Dendrobium species increased linearly with a rise in temperature between 18 and 26 °C and peaked at 28 to 30 °C. The maximum Pn of the four Dendrobium species were between 4.89 and 5.31 μmol (CO2) per m−2·s−1 (Fig. 2C). The Pn decreased when temperature was over 30 °C, indicating that the optimum temperatures for Dendrobium photosynthesis were in the range of 26 to 30 °C.
Pn diurnal and annual variation of the four Dendrobium species.
The diurnal variation in the photosynthetic rates of the four Dendrobium species showed the same bimodal curve (Fig. 3). There was an rapid increase of Pn from 700 h, reaching a peak of 5.09 to 6.06 μmol (CO2) per m−2·s−1 ≈1100 hr but then decreased thereafter, and dropped to a minimum at 1300 hr to 1400 hr (Fig. 3A). From 1100 hr to 1300 hr, the environmental PAR and temperature rose continuously and exceeded the optimum conditions for Dendrobium photosynthesis. The high PAR and temperature exerted stress on the plant resulting in an increase in stomatal resistance, a decrease in CO2 absorption, and an increased of Tr (Fig. 3B–D), which, in turn, caused “midday depression.” The second Pn peak [3.83 to 4.58 μmol (CO2) per m−2·s−1] began at 1500 hr to 1600 hr, after which Pn rapidly decreased. Furthermore, it showed that the four Dendrobium species showed no CO2 absorption at night at our preliminary experiment (unpublished data). Therefore, these four species were C3 rather than CAM plants.
Diurnal variation of net photosynthetic rate (Pn) (A), stomatal conductance (gS) (B), transpiration rate (Tr) (C), and environmental photosynthetically active radiation (PAR) (D) of the leaves of the four Dendrobium species.
Citation: HortScience horts 49, 8; 10.21273/HORTSCI.49.8.1023
Diurnal variation of net photosynthetic rate (Pn) (A), stomatal conductance (gS) (B), transpiration rate (Tr) (C), and environmental photosynthetically active radiation (PAR) (D) of the leaves of the four Dendrobium species.
Citation: HortScience horts 49, 8; 10.21273/HORTSCI.49.8.1023
Diurnal variation of net photosynthetic rate (Pn) (A), stomatal conductance (gS) (B), transpiration rate (Tr) (C), and environmental photosynthetically active radiation (PAR) (D) of the leaves of the four Dendrobium species.
Citation: HortScience horts 49, 8; 10.21273/HORTSCI.49.8.1023
The Pn of the four Dendrobium species had relatively similar seasonal variation patterns (Fig. 4) with the highest value in June [the peak values of D. nobile, D. pendulum, D. chrysotoxum, and D. densiflorum were 5.87, 5.51, 6.27, and 6.22 μmol (CO2) per m−2·s−1, respectively]. Measurement of seasonal photosynthesis was carried out from January to October and the photosynthesis rates of these Dendrobium species were higher during May to August, which may be correlated with temperature, light intensity, and leaf maturity. The Pn during March and April was relatively low, which may be the result of the low temperature or immature leaves during this period.
Seasonal variation of net photosynthetic rate (Pn) of the four Dendrobium species.
Citation: HortScience horts 49, 8; 10.21273/HORTSCI.49.8.1023
Seasonal variation of net photosynthetic rate (Pn) of the four Dendrobium species.
Citation: HortScience horts 49, 8; 10.21273/HORTSCI.49.8.1023
Seasonal variation of net photosynthetic rate (Pn) of the four Dendrobium species.
Citation: HortScience horts 49, 8; 10.21273/HORTSCI.49.8.1023
Variation of chlorophyll content and activities of key photosynthetic enzymes.
The Chl a/b ratios of the four Dendrobium species were varied from 2.77 to 2.89 (Table 2). Measurement of enzyme activities (Table 3) indicated that PEPCase activity in the four Dendrobium species were very low, all within a range of 1.2 to 1.3 nmol (CO2)/mg (protein)/min.
Variation of chlorophyll content in leaves of four Dendrobium species.
Variation of the activities of key photosynthetic enzymes in leaves of four Dendrobium species.
Discussion
The photosynthesis of tropical orchids has been studied and documented (Hew and Yong, 1997). Generally thin-leaved orchids are C3 plants, whereas thick-leaved orchids are CAM plant. To date, no C4 orchid has been reported (Pan et al., 1997; Pan and Ye, 2006; Ye et al., 1993). The four Dendrobium species have bifacial leaf structure and are thin-leaved orchids. No Kranz structure that displayed the characteristics of C3 plants in the vascular bundle sheath cells was observed (Fig. 1E–H). Furthermore, the photosynthetic characteristics, especially the high CCP and CSP (Table 1) and low PEPCase activity (Table 3), demonstrated that these studied species were C3 plants and not C4 or CAM orchids. Our results differ from that of Su and Zhang (2003a, 2003b) who stated that D. nobile and D. officinale have a facultative CAM photosynthetic pathway (transformation of photosynthesis pathway in the CAM pathway and C3 pathway) but were similar to our previous results that the four Dendrobium species (D. williamsonii, D. longicornu, D. chrysanthum, and D. dixanthum) were C3 plants (Zhu et al., 2013a, 2013b). The Pn diurnal variation of the leaves of the four Dendrobium species showed bimodal curves with a noticeable “midday depression” (Fig. 3A). That phenomenon is a known light defense mechanism that protects the plant’s photosynthetic apparatus from damage resulting from high PAR and temperature (Edwards and Walker, 1985). The LSPs of the four Dendrobium species were 40% to 50% (Table 1) of maximum light intensity (1800 to 2200 μmol·m−2·s−1), indicating that the Dendrobium species exhibited characteristics of shade plants. The optimum temperature of photosynthesis for these Dendrobium plants ranged from 26 to 30 °C (Fig. 2C), indicating they should be suitable for growing in the subtropical and tropical area. These results are consistent with the optimum temperature range reported for the photosynthesis of D. chrysanthum and D. dixanthum (26 to 30 °C; Zhu et al., 2013b). The CO2 response curves showed that the photosynthetic rates improved with increasing CO2 concentration, and the photosynthetic rates in CSPs were nearly twice (Fig. 2B; Table 1) as great as that of atmospheric CO 2 concentration (370 μmol·mol−1). Meanwhile, the values of Chl a/b ratio (2.77 to 2.89) of these plants (Table 2) also showed the characteristic of C3 plants (Hew and Yong, 1997). Moreover, the Pn seasonal variation of the four Dendrobium species showed that the most vigorous growth was exhibited in May to August with the peak values of D. nobile, D. pendulum, D. chrysotoxum, and D. densiflorum being 5.80, 5.28, 6.14, and 6.06 μmol (CO2) per m−2·s−1, respectively (Fig. 4). Finally, PEPCase is a key enzyme for C4 plants, and its activity has been measured at over 100 nmol (CO2)/mg (protein)/min in the leaves of C4 sugarcane plant (Ye et al., 1993). In our present study, PEPCase is usually very low [1.2 to 1.3 nmol (CO2)/mg (protein)/min], similar to C3 plants (Table 3). Those results are consistent with previous findings (Edwards and Walker, 1985) as well as our own previous research on Cymbidium species (Ye et al., 1993) and Dedrobium species (Zhu et al., 2013a, 2013b). Their relatively high RuBPCase and GO activities further supported that they were typical C3 plants with high photorespiration rates, results also similar to our previous studies (Zhu et al., 2013a, 2013b).
This study revealed for the four Dendrobium species were C3 plants, which would help in improving the cultivation techniques for their ex situ conservation as well as increase sustainable production. The correlation between the photosynthetic rate and environmental factors in the leaves of the four Dendrobium species indicated that CO2 concentration, air temperature, and light intensity were very important for the growth of those plants. To clarify systematically the rule of Dendrobium blooming in spring photosynthesis pathway, it is necessary to continue to investigate the photosynthetic characteristics of more Chinese Dendrobium species that blossom in spring.
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