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Phalaenopsis efficiently acclimate to highlight environment through orchid mycorrhization
Scientia Horticulturae 179 (2014) 184–190
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Phalaenopsis efficiently acclimate to highlight environment through orchid mycorrhization Ming-Chih Lee a , Doris C.N. Chang a , Chun-Wei Wu a , Yin-Tung Wang b , Yu-Sen Chang a,∗ a b
Department of Horticulture and Landscape Architecture, National Taiwan University, Taipei 10617, Taiwan Adjunct Professor and Graduate Faculty, Texas A&M University, College Station, TX 77843, USA
a r t i c l e
i n f o
Article history: Received 1 May 2014 Received in revised form 12 September 2014 Accepted 16 September 2014 Keywords: Photosynthetic performance Radiation use efficiency Photoacclimation Photoprotection
a b s t r a c t Phalaenopsis (Phalaenopsis amabilis ‘KC1410’) grow slowly and experience photoinhibition during the daytime. Thus, this study used orchid mycorrhizal (OM) fungus to promote orchid growth hypothetically via increasing the photoacclimation capacity of Phalaenopsis in response to high light (HL). By combining chlorophyll fluorometer and spectral reflectance techniques, the influences of OM colonization on the growth potentials and the photosynthetic performance of HL-grown Phalaenopsis were investigated. OM symbiosis significantly promoted the growth of HL-grown orchids, such as leaf span, fresh weight, and dry weight. Consistent with these observations, OM symbiosis obtained higher value of normalized difference vegetation index (NDVI) and enhanced CO2 uptake rate. This could be attributed to an increase in photosynthetic performance in OM plants. Notably, maximum quantum efficiency of PSII photochemistry (Fv/Fm) was not altered by OM symbiosis, however, the light-adapted fluorescence measurements of PSII operating efficiency (ФPSII) and electron transport rate (ETR) were increased. Nevertheless, OM orchids had higher photochemical quenching (qP) and lower non-photochemical quenching (NPQ). This indicates a relative higher photosynthetic performance and radiation-use efficiency (RUE) in OM plants. The photochemical reflectance index (PRI) as a photoprotection indicator was also enhanced. Based on these results, we proposed that OM symbiosis increases both the photosynthetic performance and the RUE in the HL-acclimated Phalaenopsis, resulting in an advantage to the OM plants to survive the HL stress. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Orchidaceae is one of the most advanced plant families, which establish a symbiotic relationship with fungi, thus allowing them to adapt to a wide range of habitats. The main group of fungus inhabiting orchid roots is Basidiomycetes (Currah et al., 1997; Dearnaley, 2007; Rasmussen, 2002) and largely in the genus Rhizoctonia (Bougoure et al., 2005; Waterman and Bidartondo, 2008) which is well known for enhancing orchid growth (Takahashi et al., 2007). Mycorrhizal relations in orchid plants are known to facilitate the nutrient uptake of plant and, subsequently, resulting in
Abbreviations: OM, orchid mycorrhizal; HL, high light; chl, chlorophyll; NDVI, normalized difference vegetation index; Fv/Fm, maximum quantum efficiency of PSII photochemistry; ФPSII, PSII operating efficiency; ETR, electron transport rate; qP, photochemical quenching; NPQ, non-photochemical quenching; RUE, radiationuse efficiency; PRI, photochemical reflectance index; V, violoxanthin; Z, zeaxanthin; XC, xanthophyll cycle. ∗ Corresponding author. Tel.: +886 2 33664856; fax: +886 2 33664855. E-mail addresses: [email protected], [email protected] (Y.-S. Chang). http://dx.doi.org/10.1016/j.scienta.2014.09.033 0304-4238/© 2014 Elsevier B.V. All rights reserved.
growth stimulation. During seed germination and seedling establishment, the fungus translocates vitamins, amino acids and sugars to the orchid (Arditti et al., 1990). Stable isotope labeling study also revealed that adult orchid Goodyera repens passes 14 CO2 to the mycobiont and mycorrhizal fungi continued to provide carbon, nitrogen, phosphorus, and water to adult photosynthetic plants (Cameron et al., 2006, 2007). Previous studies performed on species of Orchidaceae, such as Anoectochilus, Haemaria, Phalaenopsis (P.), and Spiranthes magnicamporum have reported that OM symbiosis results in taller plants, with higher number of leaves, greater biomass, higher chlorophyll (chl) content, and improved reproductive fitness, i.e. flowering quantity and quality (Anderson, 1991; Chang, 2008; Chang and Chou, 2007; Wu et al., 2011). Phalaenopsis is an epiphytic orchid genus with obligate crassulacean acid metabolism (CAM) that exhibits disadvantages related to biomass productivity and accumulates organic acids at night in plant cells (McWilliams, 1970). Its saturating photosynthetic photon flux (PPF) is approximately 130–180 mol m−2 s−1 (Lee and Guo, 2012; Lootens and Heursel, 1998; Ota et al., 1991), indicating that Phalaenopsis is adapted to low light (LL) and shading is
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necessary for its optimal growth. Currently, Phalaenopsis plants are commercially grown at a PPF around 280–380 mol m−2 s−1 (Chen and Wang, 1996). During the day, light intensity higher than 200 mol m−2 s−1 PPF frequently causes photoinhibition (Horton and Ruban, 2004; Baker, 2008) on Phalaenopsis plants (Lee and Guo, 2012). The excessive light energy absorbed by leaves has been recognized as a physiological stress, causing photooxidative damage to the thylakoid membrane (Barber and Andersson, 1992; Horton and Ruban, 2004; Huner et al., 1998). Plants cope with irradiance (high light, HL) by several major mechanisms/strategies: photoacclimation, photoprotection, and photorepair. Photoacclimation is a process where the plant modulates and optimizes its photosynthetic performance according to the light environment it encounters. It is indicated by a decrease of chl content, chl to carbon ratio, photosynthetic to nonphotosynthetic ratios, size of photosystem II (PSII) antenna, and size and/or the number of the reaction centers (Ragni et al., 2008). Photoprotection of HL is a mechanism utilized by plants to prevent or minimize light energy from inducing photodamage via the generation of high levels of reactive oxygen species. The photoprotection capacity can be characterized by monitoring the energy flow used to drive photochemistry, being emitted as chlorophyll fluorescence (ChlF) and as heat (Murchie and Niyogi, 2011). Among them, NPQ, the latter route, is the major component of photoprotection (Murchie and Niyogi, 2011). Photorepair of plants deals with the disassembly of PSII complex and degradation and de novo biosynthesis of damaged D1 protein. The turnover of D1 protein, which is one of core subunits of PSII, is involved in photoinhibition of PSII caused by increasing irradiances (Aro et al., 1993). This can be characterized as sustained lowering of the quantum yield of PSII (Murchie and Niyogi, 2011). Plant chlorophyll (chl) molecules absorb light energy to drive photosynthesis (photochemistry), while excess energy can be dissipated as heat or re-emitted as ChlF. Among ChlF measurements the ФPSII indicates the portion of the absorbed energy used in photochemistry (Maxwell and Johnson, 2000). Large portions of the absorbed light are used to produce photoassimilates, which is referred to qP, or are dissipated as heat, which is referred to NPQ (Ralph and Gademann, 2005). When photosynthetic energy input exceeds its utilization, the plant acclimates by increasing the fraction of absorbed energy that is dissipated as heat in a process known as NPQ (Bilger and BjÖrkman, 1990; Maxwell and Johnson, 2000). NPQ competes to qP within a certain range of light irradiance, for instance, an increase of qP will result in a decrease in NPQ. Pulse-amplitude-modulated (PAM) fluorometer is a device used to provide quantitative information about PSII by measuring ChlF, and a better understanding of the early events of photosynthetic change influenced by the HL and mycorrhization. PAM fluorometer is a suitable monitoring tool because it is rapid and non-destructive, providing in-depth, quantitative physiological information about photosynthetic organisms (Ralph and Gademann, 2005). This information may be used to identify changes in photosynthetic status before morphological changes are observed. In combination with PAM fluorometer and the reflectance spectrometer, several indices related to photosynthesis, nutrition, biomass, and water status can be measured and calculated. NDVI is one of the most widely used spectral reflectance indices that relates to the difference between near infrared and red wavelength reflectance (Marti et al., 2007). It also has been reported as a sensitive foliar indicator of chl and nitrogen concentration, biomass, leaf area, and canopy structure (Gamon et al., 1995). The photosynthetic efficiency also can be indirectly assessed using the PRI derived from narrow-band reflectance ˜ at 531 and 570 nm (Fiella et al., 1996; Penuelas et al., 1995). The benefits of stimulated growth of Phalaenopsis due to mycorrhizal associations have been previously established (Chang, 2008; Wu et al., 2011). Since OM symbiosis has been shown to exchange
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nutrients with the plant, root colonization by the fungus increases the sink strength. We hypothesize that OM colonization would enhance the photosynthetic performance in leaves of Phalaenopsis to yield higher level of photoassimilates, thereby enhancing plant growth. The objectives of this study were to evaluate the OM effects on the growth of HL-grown orchid, and to elucidate the photochemical mechanisms involved in photoacclimation to photoinhibitory irradiance. Thereafter, an optimal strategy of light-intensity management that increases economic benefits for Phalaenopsis producers is proposed. 2. Materials and methods 2.1. Biological material and growth conditions Five-month-old micropropagated Phalaenopsis amabilis ‘KC1410’ plantlets was chosen as the model plants to study the HL-acclimation because of its slow growth and capability of establishing a symbiotic relationships with orchid mycorrhizae. Plants were purchased from King-Car Biotechnology Industrial Company (YiLan, Taiwan). OM and non-mycorrhizal (NM) plants were cultivated in 12-cm diameter pots filled with the processed bark of Pinus radiata (Pacific Wide Co., Cristchurch, New Zealand), then cultivated in a greenhouse (40% of full sun created by black shade cloth) with a pad and fan cooling system for 6 months. The maximum PPF measured at noon on a sunny day with HOBO data loggers (Onset, Massachusetts, USA) was 230–870 mol m−2 s−1 (due to cloudiness) about 40% of the prevailing solar radiation. Three days before experiment termination, seven plants in each treatment were transferred to shade environment with maximum PPF (less than 100 mol m−2 s−1 ) for the experiment of recovery of Fv/Fm. The photoperiod varied according to season and the air temperature was controlled at 28/22 ◦ C (day/night). All cultivates were watered twice a week. The fertilizer solution, made with distilled water and a 20N–8.7P–16.6 K (Peters Professional® 20-20-20, Scotts, Marysville, OH, USA) water-soluble fertilizer at 0.5 g L−1 was provided once every two weeks and allowed to drain. 2.2. Inoculum production and mycorrhization The OM fungal inoculums were prepared using OM isolates Ceratobasidium sp. (AG-A, binucleates) according to Chang and Chou (2007). This fungal isolates were previously identified as nonpathogenic fungi by inoculation test on seedlings of mung bean, cucumber, and rice. Fungal isolates were sub-cultured monthly on potato dextrose agar (PDA, Difco Labortories, Detroit, MI, USA) and maintained at room conditions. Sphagnum peat was homogeneously mixed with 1× potato-dextrose broth and adjusted to about 25% moisture content. After autoclaving, four myceliaclumps, each of 10(L) × 10(W) × 2(H) mm2 in size, were placed on top of the peat medium in a laminar flow hood and cultured for 2 weeks for compact growth at room temperature. For mycorrhizal treatment, 1 g of inocula consisting of peat and fungus mycelium was placed directly beneath the orchid roots, then the root sphere was refilled with pine bark. The NM control was prepared similar to the mycorrhizal treatment, except the myco-inocula was replaced with the autoclaved peat without fungal colonization. There were seven plants in NM and OM treatment, separately, and totally two experimental repeats were conducted in this study. 2.3. Biomass analysis All cultivates were harvested after 6 months of OM inoculation. Plant biomass was measured as leaf span (LS), fresh weight (FW), dry weight (DW), and shoot (S, including stem and leaves) and root (R) ratio (S/R, calculated using dry weights). Leaf span distance was
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measured from the end of one leaf to the longest opposite leaf tip according to Newton and Runkle (2009). Fresh shoots and roots were dried at 70 ◦ C for 120 h and weighed. Water content (WC) in whole plant was calculated using the formula (FW − DW)/FW. 2.4. Measurements
Table 1 Mycorrhizal effects on the growth characteristics, biomass, and dry matter partitioning of Phalaenopsis plantlets grown under high light for 6 months. All values are means ± standard error (n = 7). Different letters between treatments are significantly different at p ≤ 0.05 by Fisher’s LSD. NM: non-mycorrhizal control; OM: orchid mycorrhizal treatment; FW: fresh weight; DW: dry weight. Water content: (FW − DW)/FW. Growth characteristics
In order to estimate the photosynthetic performance, chl fluorometer, spectrometer, and gas exchange detector were used in the experiment. CO2 uptake rate was measured from 10 p.m. to 12 a.m. with a portable open flow gas exchange system LI-6400 (LI-COR, Lincoln, NE, USA). Measuring the photosynthesis on CAM Phalaenopsis is difficult because most of CO2 is assimilated at night. Thus, we measured the ChlF of photochemical reaction center of PSII instead of the determination of leaf photosynthesis. The ChlF parameters were measured at room temperature using a continuous monitoring device: MONITORING-PAM Multi-Channel Chlorophyll Fluorometer (Heinz Walz GmbH, Effeltrich, Germany) (Porcar-Castell et al., 2008). Fluorescence was measured from the upper surface of the 1st acropetal fully expended leaf. The fluorescence measurement was made on the 30 min dark-adapted plants during Phase III of CAM photosynthesis (2 p.m.). Saturating pulse analysis detected and calculated fluorescence parameters of leaves automatically, such as the fluorescence emission signal, minimum fluorescence signal (Fo), variable fluorescence signal (Fv), the maximum fluorescence signal (Fm), Fv/Fm, ETR, ФPSII, qP, and the NPQ. To monitor whether the mycorrhization regulates the RUE, we measured ChlF parameters qP and NPQ (Bilger and BjÖrkman, 1990), respectively, to study the PSII acclimation to the HL environment. The qP and NPQ were measured using rapid light curve (RLC) from 30 min dark-adapted NM and OM plants leaf. The measuring light (0.9 mol m−2 s−1 at Fo and Ft (steady yield of fluorescence in the light), and 9 mol m−2 s−1 at Fm), actinic light (1500 mol m−2 s−1 ), and saturating pulse (4000 mol m−2 s−1 ) were emitted by blue LED (light-emitting diode) with peak wavelength at 455 nm (full width at half maximum is 18 nm) and delivered to the leaf being measured through the MONI-head. The duration of the saturating pulse was 0.8 s. The detection and recording were performed using the RLC, of which exposed the leaf to nine incremental levels (0, 125, 190, 285, 420, 625, 820, 1150, and 1500 mol m−2 s−1 PPF) of irradiance, each with a 30 s interval. Measurements were recorded with WinControl-3 software (Heinz Walz GmbH, Inc., Effeltrich, Germany). Line-chart was drawn using data obtained from five of nine irradiance incremental levels (125, 190, 420, 820, and 1500 mol m−2 s−1 ). Leaf canopy spectrum of Phalaenopsis were measured using a portable spectrometer HandySpec Field (tec5 AG, Oberursel, Germany) fitted with a robust measurement probe and an internal halogen light source (400–2200 nm). This portable instrument measures spectral difference ranging from 305 to 2200 nm, and was calibrated with a white panel before use. The center of a leaf was selected for spectral measurement and each measurement was the mean of three consecutive individual full-range spectra scans. The equations (R, the reflectance) of spectral indices used to access the growth quality and photosynthesis are (1) NDVI computed as (R800 − R600)/(R800 + R600) (Rouse et al., 1974) and (2) PRI com˜ puted as (R531 − R570)/(R531 + R570) (Penuelas et al., 1995).
Leaf span (cm) FW (g) DW (g) DW ratio of S/R Water content (%) NDVI
Mycorrhizal treatment NM
OM
19.9 ± 0.6b 89.0 ± 2.3b 7.3 ± 0.2b 0.60 ± 0.03b 91.78 ± 0.21b 0.628 ± 0.026b
23.2 ± 0.6a 114.8 ± 5.9a 8.6 ± 0.4a 0.80 ± 0.06a 92.49 ± 0.12a 0.674 ± 0.027a
The level of confidence was 5% (P < 0.05). All data are presented as means ± standard error. 3. Results 3.1. OM symbiosis effects on plant morphology and growth Mycorrhizal Phalaenopsis grown under HL was enhanced by OM colonization (Table 1). OM plants had longer leaves that resulted in greater leaf span compared to NM plants. Also, OM plants had greater FW (an average increase of 27%) and DW (an average increase of 22%). Through the analysis of dry matter allocation, it is clearly known that OM plants had higher S/R ratio (0.8) than NM plants (0.6). OM plants exhibited higher water content (92.52%) compared to NM plants, which indicated that the former were growing more rapidly. NDVI of the OM leaves was significantly higher than the NM leaves, supporting the other observations of enhanced growth (LS, FW, DW, S/R, and WC) and demonstrably larger plants (Fig. 1). In order to determine the mechanism of this enhanced growth, we measured CO2 uptake rate for the evaluation of photosynthetic capacity. Leaf gas exchange results indicated that the OM leaves achieved higher rate of CO2 uptake than the NM plants at the night time (Fig. 2). 3.2. Light stress-recovery: dark adapted After six months cultivation under HL, the NM and OM plants underwent a photosynthetic photoinhibition, and the stress can be alleviated by exposure to LL (LL recovery) (Fig. 3). The value of Fv/Fm was similar in both OM and NM plants, approximately equal to 0.78,
2.5. Statistical analysis The effects of OM on shoot and root biomass, CO2 uptake rate, ChlF, and spectral reflectance indices were tested on seven plants for each treatment. Data were assessed by analysis of variance using Fisher’s LSD (CoStat ver. 6.4, Contact CoHort Software, USA).
Fig. 1. Phalaenopsis amabilis ‘KC1410’ plants grown under high-light environment with (OM) and without Ceratobasidium sp. (NM) inoculation. Scale bar represents 10 cm.
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Fig. 2. Carbon dioxide (CO2 ) uptake rate measured in the late evening (10 p.m.) 6 months after high-light cultivation of non-mycorrhizal (NM) and mycorrhizal (OM) orchids (Phalaenopsis amabilis ‘KC1410’). *** p ≤ 0.001.
indicating that the symbiotic cultivation did not significantly affect the maximum photochemical efficiency. After 3 days of LL recovery, both groups increased in Fv/Fm (≈0.81), while the OM leaves were more affected by the treatment, showing an increase of ≈3.8%. 3.3. Photosynthetic performance under actinic light Following the increase of actinic light intensity (refer to PPF), ФPSII decreased markedly in the NM and OM plants (Fig. 4). Both plant groups had similar ФPSII values when PPF <200 mol m−2 s−1 . Once light intensity increased above 200 mol m−2 s−1 PPF, ФPSII gradually increased and maintained significant difference between the NM and the OM plants in the range of 400–1200 mol m−2 s−1 PPF, and the most different values occurred around 820 mol m−2 s−1 PPF. RLC results showed that ETR appeared to be light sensitive and gradually increased with irradiance increase. In the actinic light intensity range from 0 to 500 mol m−2 s−1 PPF, the light-response curves of ETR for OM and NM orchids mostly overlapped. Once the PPF exceeded 500 mol m−2 s−1 , the ETR curve of the OM plants started separating from that of the NM plants, and reached the maximum difference around 820 mol m−2 s−1 PPF (Fig. 5).
Fig. 3. The maximum quantum yield of PSII photochemistry (Fv/Fm) measured with PAM fluorometer on dark-adapted samples of mycorrhizal (OM) and non-mycorrhizal (NM) Phalaenopsis during light-recovery experiment. Results are means ± standard error (n = 7). Black bar refers to high-light (HL: 230–870 mol m−2 s−1 ) culture, and white bar refers to low-light (LL: max. PPF under 100 mol m−2 s−1 ) culture. Different lower case letters indicate differences between mycorrhizal treatments and different capital letters represents differences between HL–LL-recovery treatments. Means followed by different letter(s) are significantly different at P ≤ 0.05 by Fisher’s LSD.
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Fig. 4. Analysis of light dependent changes in PSII operating efficiency (ФPSII) in an intact leaf of Phalaenopsis amabilis ‘KC1410’ with (OM) or without (NM) mycorrhization grown under high-light environment. The maximum difference of ФPSII determined at 820 PPF during response curve are indicated as black bar. Data are means ± standard error of seven plants. Vertical error bars represent the standard errors. ** p ≤ 0.01.
3.4. Radiation-use efficiency (RUE) under actinic light The photochemical quenching response, which was composed of qP and NPQ, revealed adverse patterns in both plant groups under given actinic light (Fig. 6), i.e., qP decreased and NPQ increased with PPF increase. Both qP and NPQ had no apparent difference between the treatments before 200 mol m−2 s−1 PPF, but exhibited evident difference when providing actinic light >420 mol m−2 s−1 PPF. Although qP decreased drastically and reached minimum at the end of RLC, the NPQ raised to a relative constant level over the excessive light interval in both plant groups. In the range of 420–820 mol m−2 s−1 PPF, the OM plants had significant higher qP and lower NPQ than the NM plants. 3.5. Photochemical reflectance index (PRI) and photoprotection To further investigate the capacity of photoprotection in leaves being exposed to HL, we measured the photochemical reflectance in Phalaenopsis (Fig. 7). The HL-acclimated leaf of the OM plant exhibited positive value of PRI, whereas the NM plants yielded negative value.
Fig. 5. Analysis of light dependent changes in electron transport rate (ETR) in an intact leaf of Phalaenopsis amabilis ‘KC1410’ with (OM) or without (NM) mycorrhization grown under high-light environment. The maximum difference of ETR determined at 820 PPF during response curve are indicated as black bar. Data are means ± standard error of seven plants. Vertical error bars represent the standard errors. ** p ≤ 0.01.
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Fig. 6. Analysis of light dependent changes in photochemical quenching (qP) and non-photochemical quenching (NPQ) in an intact leaf of Phalaenopsis amabilis ‘KC1410’ with (OM) or without (NM) mycorrhization grown under high-light environment. Data are means ± standard error of seven plants. Vertical error bars represent the standard errors.
4. Discussion 4.1. OM symbiosis stimulates growth of Phalaenopsis Plant yield is the result of its interaction to various environmental factors, e.g. light, air, water, temperature, soil, etc. The present study determined the effects of OM colonization on the yield of the HL-grown Phalaenopsis to reveal the photoacclimation strategy conferred by OM. Phalaenopsis inoculated with fungus Ceratobasidium exhibited stimulated growth similar to another orchid species inoculated with Rhizoctonia fungi (Chang, 2007). OM colonization had a positive impact on plant size and appearance of Phalaenopsis, manifested in greater leaf length (Fig. 1) and greener foliage (see NDVI in Table 1). The observed increase in biomass may imply the establishment of a beneficial symbiosis between fungus and Phalaenopsis. Noteworthy, the OM plants had the larger shoot biomass and thus resulted in high S/R (=0.80), which would indicate a mycorrhiza-mediated mechanism of photoassimilates allocation between shoots and roots. Based on the concept that photoassimilates-allocation changes alters the S/R, we suggested that the NM plants with S/R = 0.60 is in an allocation-balanced
Fig. 7. Assessing photoprotection capacity with evaluation of photochemical reflectance index (PRI) in an intact leaf of Phalaenopsis amabilis ‘KC1410’ with (OM) or without (NM) mycorrhization grown under high-light environment. Data are means ± standard error of seven plants. Vertical bars represent the standard errors. *** p ≤ 0.001.
status. On the contrary, OM plants is in an allocation-promoted status that a larger ratio of photoassimilates are allocated to the shoot part. It has been reported that mycorrhizal colonization may increase the sink strength of roots leading to stimulated photosynthesis and more photoassimilates for growth (Louche-Tessandier et al., 1999). Javot et al. (2007) also observed an increased shoot mass during the process of mycorrhization in Medicago truncatula plants. Higher WC in the OM leaves suggested an active cell growth to increase the shoot mass. This result agreed with the report of Yoder et al. (2000) that mycorrhization seems to enhance water uptake. Irradiance of 200–320 mol m−2 s−1 PPF is considered to promote growth and CO2 uptake of Phalaenopsis, but it also causes photodamage and photoinhibition (Guo et al., 2012; Wang, 1997). High PPF in excess of 320 mol m−2 s−1 PPF for 12 h would cause photoinhibition and reduce CO2 fixation in Phalaenopsis leaves (Guo et al., 2012). We cannot make inferences regarding CO2 uptake because we did not cultivate our plants under low irradiance conditions. However, if we assume that CO2 uptake was reduced, this carbon loss could be compensated by OM colonization because OM stimulated the HL-grown Phalaenopsis to uptake more CO2 and to enhance photochemical efficiency in comparison to the NM plants. The larger biomass of OM plants conceivably led to higher leaf area available for CO2 assimilation. We hypothesized that OM colonization would lead to increased demand for photosynthetic activity (Louche-Tessandier et al., 1999), and this carbon sink strength of symbiosis would stimulate the rate of photosynthesis (Harris et al., 1985). The general assumption is that OM symbiosis affects the whole plant photosynthetic performance, improving plant growth by increasing FW and DW. By using the spectral reflectance index NDVI, which is commonly applied to estimate chl content and plant growth potential, the significant difference in NDVI between the NM and the OM plants confirmed the observed results of greater vegetative biomass in OM Phalaenopsis. 4.2. Chlorophyll fluorescence (ChlF) and photosynthesis Acclimation to the light environment is a key ecophysiological feature for the growth and development of plants. Plant leaves generally exhibited a certain range of plasticity in relation to acclimation to the light environment (Valladares and Niinemets, 2008). Lin and Hsu (2004) improved photosynthetic production of Phalaenopsis by increasing the light exposure of lower leaves and indicated a photosynthetic plasticity in its leaves to adjust to irradiance. In the present study, Phalaenopsis exposed to HL for a long period decreased Fv/Fm below 0.80 (typical of photoinhibition). The Fv/Fm values of leaves in HL–LL recovery experiment were all >0.8 (typical of healthy leaves), which indicates a HL plasiticity, low efficiency of PSII photochemistry, and a photoinhibition on leaves of Phalaenopsis (Fig. 3). Similar results were reported by Naramoto et al. (2006) with shade-developed leaves of Fagus crenata plantlets exposed to HL. These phenomena may have been related to photochemistry changes of PSII during the transition from LL to HL, leading to decrease in Fv/Fm triggered by photoprotection mechanisms (Huner et al., 1993). Although HL resulted in similar Fv/Fm in both NM and OM plants, the higher recovery rate of Fv/Fm in OM plants after low PPF treatment, indicated that OM colonization could confer higher repair capacity on Phalaenopsis. This photorepair mechanism was also considered as one of the photoprotection mechanisms (Adir et al., 2003; Ragni et al., 2008). Photochemical efficiency ФPSII of plant leaves are sensitive to HL. When the leaf was illuminated, the decrease in the ФPSII occurred on the similar level in the OM and NM plants. In the HL environment, down regulation of ФPSII played a major role, as indicated by the drop of qP and the drastic development of NPQ (Fig. 6).
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In the present study, ChlF measurements showed that the OM plants lost less PSII photochemical efficiency than the NM plants and yielded higher ФPSII. Since ФPSII is highly correlated with the rate of carbon assimilation (Genty et al., 1989; Rolfe and Scholes, 1995), greater biomass was produced (Table 1). In addition, Fig. 4 shows that OM colonization significantly stimulated PSII photochemical efficiency of Phalaenopsis in the light intensity range of 400–1200 mol m−2 s−1 PPF that often cause photoinhibition, and consequently contributed a greater ability of photoacclimation to plants exposed to HL. Significant stable electron flow (ETR) in the OM plants also indicated a higher ФPSII in mycorrhizal Phalaenopsis (Fig. 4). It is consistent with the report of Tsimilli-Michael et al. (2000), where mycorrhizal Medicago exhibited increases in ETR. The increase of the various parameters obtained from the reflectance spectra, such as NDVI, and from the fluorescence measurements including ФPSII, ETR, and qP, all point to a raise in the photosynthetic performance of OM colonized orchids.
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PRI is an index of instantaneous photosynthetic light use efficiency (LUE) that can provide non-destructive assessment to ˜ determine the changes in leaf photosynthetic LUE (Penuelas et al., 1997; Nichol et al., 2006; Letts et al., 2008) and in xanthophyll cycle (Sims and Gamon, 2002; Nichol et al., 2006). In addition, PRI is significantly correlated to ФPSII and NPQ, and indicative the xanthophyll cycle-mediated thermal energy dissipation. It is well known that both PRI and RUE decreased along with the increase of PPF (Barton and North, 2001). Under HL stress, the OM plants sustained higher PRI (positive values) than the NM plants (negative values). We thus suggest that the OM plants are able to long-term acclimate to HL stress and that this acclimation is dependent of the XC (Fig. 6). In addition, high capacity of XC also responds to the elevated photosynthetic performance, e.g. ФPSII, ETR, and qP, of OM plants under HL environment. When plant leaves perceive excess light, its PRI value will drop below zero. Additionally, this indicates higher RUE in the OM plants when exposed to HL, which is consistent with the changes of qP and NPQ (Fig. 7).
4.3. Radiation-use efficiency and photoprotection 5. Conclusion Acclimatization of certain plants to change from LL to HL irradiation depends largely on its ability to minimize the magnitude and duration of photoinhibition of photosynthesis (Naidu and DeLucia, 1998; Krause et al., 2001; Guo et al., 2006). In the present study, the changes in growth potential, CO2 uptake rate, and photosynthetic performance in leaves of mycorrhizal Phalaenopsis appeared to be associated with the changes in RUE and protective mechanisms against excess light. Hence, photosynthetic energy balance and the partitioning of excitation energy between qP and NPQ processes were addressed. NPQ is an efficient photoprotective mechanism in chloroplasts to dissipate the excess energy absorbed by chl (Ruban et al., 2012). Its induction depends on the balance between PSII activity and ATP synthesis rate. According to Latowski et al. (2011), the conversion processes of violoxanthin (V) to zeaxanthin (Z) have been termed as xanthophyll cycle (XC), and Z-facilitated thermal dissipation of excess energy can cause the induction of NPQ. When light is not excessive, Z will convert back to V, thereby returning to an efficient utilization of light energy in photosynthesis. In our study, OM plants have less conversion of V to Z and, hence, contributed to a lower NPQ than the control (Fig. 6). The sustained lower NPQ and higher qP indicated that the dissipation mechanism was nearly sufficient to balance the requirements for the photoprotection and alleviated the RUE. On the other hand, the relative higher NPQ observed in the NM plants over the photoinhibition period indicated a lower capacity of photoprotection. The results indicated that the OM plants utilized light energy efficiently under strong actinic light that is close to the cultivation PPF in greenhouse, which reduced the loss of light energy and eventually resulted in lower NPQ. Thus, we suggested a greater and higher RUE on the OM plants upon photoinhibition. It is consistent with the observation in PRI variation (Fig. 7) on HL-grown Phalaenopsis, in which higher PRI resulted in higher RUE. The lower NPQ, which may reflect a higher RUE and photoprotection, also resulted in an up-regulation of PSII activity. It indicated that the OM plants with higher photochemical efficiency utilized photosynthetic energy to assimilate more carbon instead of to dissipate them as heat under HL stress (Fig. 6). In other words, PSII acclimation of OM plants involves modulation of the fractions of absorbed irradiance that is utilized in photochemistry more than that dissipated as heat. Thus, we assume that OM effects involve the long-term acclimation of PSII (Öquist and Huner, 2003) and lead to slow reversible acclimation in the PSII energy partitioning, which the photosynthetic protections occurred following the initiation of photoinhibition (Baker, 2008; Horton and Ruban, 2004; Lee and Guo, 2012). From our results, it is evident that the OM plants have considerable potential for HL acclimation.
In summary, mycorrhizal Phalaenopsis efficiently acclimate to HL environment and obtain more photoassimilates than NM plants by promotion of RUE and photoprotection capacity. Thus, OM inoculation could be applied for economic benefit in commercial production of Phalaenopsis. Acknowledgments This work was supported by the Research Grant NSC 100-2313B-002-004 from Taiwan’s National Science Council. I would like to thank Mr. Ting-Yi Chou for critical review of the manuscript. References Adir, N., Zer, H., Shochat, S., Ohad, I., 2003. Photoinhibition—a historical perspective. Photosynth. Res. 76, 343–370. Anderson, A.B., 1991. Symbiotic and asymbiotic germination and growth of Spiranthes magnicamporum (Orchidaceae). Lindleyana 6, 183–186. Arditti, J., Ernst, R., Yam, T.W., Glabe, C., 1990. The contributions of orchid mycorrhizal fungi to seed germination: a speculative review. Lindleyana 5, 249–255. Aro, E.M., McCaffery, S., Anderson, J., 1993. Photoinhibition and D1 protein degradation in peas acclimated to different growth irradiances. Plant Physiol. 103, 835–843. Baker, N.R., 2008. Chlorophyll fluorescence: a probe of photosynthesis in vivo. Ann. Rev. Plant Biol. 59, 89–113. Barber, J., Andersson, B., 1992. Too much of a good thing: light can be bad for photosynthesis. Trends Biochem. Sci. 12, 61–66. Barton, C.V.M., North, P.R.J., 2001. Remote sensing of canopy light use efficiency using the photochemical reflectance index model and sensitivity analysis. Remote Sens. Environ. 78, 264–273. Bilger, W., BjÖrkman, O., 1990. Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of Hedera canariensis. Photosynth. Res. 25, 173–185. Bougoure, J.J., Bougoure, D.S., Cairney, J.W.G., Dearnaley, J.D.W., 2005. ITS-RFLP and sequence analysis of endophytes from Acianthus, Caladenia and Pterostylis (Orchidaceae) in southeastern Queensland. Mycol. Res. 109, 452–460. Cameron, D.D., Johnson, I., Leake, J.R., Read, D.J., 2007. Mycorrhizal acquisition of inorganic phosphorus by the green-leaved terrestrial orchid Goodyera repens. Ann. Bot. 99, 831–834. Cameron, D.D., Leake, J.R., Read, D.J., 2006. Mutualistic mycorrhiza in orchids: evidence from plant-fungus carbon and nitrogen transfers in the green-leaved terrestrial orchid Goodyera repens. New Phytol. 171, 405–416. Chang, D.C.N., 2007. The screening of orchid mycorrhizal fungi (OMF) and their applications. In: Chen, W.H., Chen, H.H. (Eds.), Orchid Biotechnology. World Scientific, Toh Tuck Link, Singapore, pp. 77–98. Chang, D.C.N., 2008. Research and application of orchid mycorrhiza in Taiwan. Acta Hortic. 766, 299–305. Chang, D.C.N., Chou, L.C., 2007. Growth responses, enzyme activities, and component changes as influenced by Rhizoctonia orchid mycorrhiza on Anoectochilus formosanus Hayata. Bot. Study 48, 446–451. Chen, W.H., Wang, Y.T., 1996. Phalaenopsis orchid culture. Taiwan Sugar 43, 11–16. Currah, R.S., Zelmer, C.D., Hambleton, S., Richardson, K.A., 1997. Fungi from orchid mycorrhizas. In: Arditti, J., Pridgeon, A.M. (Eds.), Orchid Biology: Review and
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Phalaenopsis efficiently acclimate to highlight environment through orchid mycorrhization Ming-Chih Lee a , Doris C.N. Chang a , Chun-Wei Wu a , Yin-Tung Wang b , Yu-Sen Chang a,∗ a b
Department of Horticulture and Landscape Architecture, National Taiwan University, Taipei 10617, Taiwan Adjunct Professor and Graduate Faculty, Texas A&M University, College Station, TX 77843, USA
a r t i c l e
i n f o
Article history: Received 1 May 2014 Received in revised form 12 September 2014 Accepted 16 September 2014 Keywords: Photosynthetic performance Radiation use efficiency Photoacclimation Photoprotection
a b s t r a c t Phalaenopsis (Phalaenopsis amabilis ‘KC1410’) grow slowly and experience photoinhibition during the daytime. Thus, this study used orchid mycorrhizal (OM) fungus to promote orchid growth hypothetically via increasing the photoacclimation capacity of Phalaenopsis in response to high light (HL). By combining chlorophyll fluorometer and spectral reflectance techniques, the influences of OM colonization on the growth potentials and the photosynthetic performance of HL-grown Phalaenopsis were investigated. OM symbiosis significantly promoted the growth of HL-grown orchids, such as leaf span, fresh weight, and dry weight. Consistent with these observations, OM symbiosis obtained higher value of normalized difference vegetation index (NDVI) and enhanced CO2 uptake rate. This could be attributed to an increase in photosynthetic performance in OM plants. Notably, maximum quantum efficiency of PSII photochemistry (Fv/Fm) was not altered by OM symbiosis, however, the light-adapted fluorescence measurements of PSII operating efficiency (ФPSII) and electron transport rate (ETR) were increased. Nevertheless, OM orchids had higher photochemical quenching (qP) and lower non-photochemical quenching (NPQ). This indicates a relative higher photosynthetic performance and radiation-use efficiency (RUE) in OM plants. The photochemical reflectance index (PRI) as a photoprotection indicator was also enhanced. Based on these results, we proposed that OM symbiosis increases both the photosynthetic performance and the RUE in the HL-acclimated Phalaenopsis, resulting in an advantage to the OM plants to survive the HL stress. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Orchidaceae is one of the most advanced plant families, which establish a symbiotic relationship with fungi, thus allowing them to adapt to a wide range of habitats. The main group of fungus inhabiting orchid roots is Basidiomycetes (Currah et al., 1997; Dearnaley, 2007; Rasmussen, 2002) and largely in the genus Rhizoctonia (Bougoure et al., 2005; Waterman and Bidartondo, 2008) which is well known for enhancing orchid growth (Takahashi et al., 2007). Mycorrhizal relations in orchid plants are known to facilitate the nutrient uptake of plant and, subsequently, resulting in
Abbreviations: OM, orchid mycorrhizal; HL, high light; chl, chlorophyll; NDVI, normalized difference vegetation index; Fv/Fm, maximum quantum efficiency of PSII photochemistry; ФPSII, PSII operating efficiency; ETR, electron transport rate; qP, photochemical quenching; NPQ, non-photochemical quenching; RUE, radiationuse efficiency; PRI, photochemical reflectance index; V, violoxanthin; Z, zeaxanthin; XC, xanthophyll cycle. ∗ Corresponding author. Tel.: +886 2 33664856; fax: +886 2 33664855. E-mail addresses: [email protected], [email protected] (Y.-S. Chang). http://dx.doi.org/10.1016/j.scienta.2014.09.033 0304-4238/© 2014 Elsevier B.V. All rights reserved.
growth stimulation. During seed germination and seedling establishment, the fungus translocates vitamins, amino acids and sugars to the orchid (Arditti et al., 1990). Stable isotope labeling study also revealed that adult orchid Goodyera repens passes 14 CO2 to the mycobiont and mycorrhizal fungi continued to provide carbon, nitrogen, phosphorus, and water to adult photosynthetic plants (Cameron et al., 2006, 2007). Previous studies performed on species of Orchidaceae, such as Anoectochilus, Haemaria, Phalaenopsis (P.), and Spiranthes magnicamporum have reported that OM symbiosis results in taller plants, with higher number of leaves, greater biomass, higher chlorophyll (chl) content, and improved reproductive fitness, i.e. flowering quantity and quality (Anderson, 1991; Chang, 2008; Chang and Chou, 2007; Wu et al., 2011). Phalaenopsis is an epiphytic orchid genus with obligate crassulacean acid metabolism (CAM) that exhibits disadvantages related to biomass productivity and accumulates organic acids at night in plant cells (McWilliams, 1970). Its saturating photosynthetic photon flux (PPF) is approximately 130–180 mol m−2 s−1 (Lee and Guo, 2012; Lootens and Heursel, 1998; Ota et al., 1991), indicating that Phalaenopsis is adapted to low light (LL) and shading is
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necessary for its optimal growth. Currently, Phalaenopsis plants are commercially grown at a PPF around 280–380 mol m−2 s−1 (Chen and Wang, 1996). During the day, light intensity higher than 200 mol m−2 s−1 PPF frequently causes photoinhibition (Horton and Ruban, 2004; Baker, 2008) on Phalaenopsis plants (Lee and Guo, 2012). The excessive light energy absorbed by leaves has been recognized as a physiological stress, causing photooxidative damage to the thylakoid membrane (Barber and Andersson, 1992; Horton and Ruban, 2004; Huner et al., 1998). Plants cope with irradiance (high light, HL) by several major mechanisms/strategies: photoacclimation, photoprotection, and photorepair. Photoacclimation is a process where the plant modulates and optimizes its photosynthetic performance according to the light environment it encounters. It is indicated by a decrease of chl content, chl to carbon ratio, photosynthetic to nonphotosynthetic ratios, size of photosystem II (PSII) antenna, and size and/or the number of the reaction centers (Ragni et al., 2008). Photoprotection of HL is a mechanism utilized by plants to prevent or minimize light energy from inducing photodamage via the generation of high levels of reactive oxygen species. The photoprotection capacity can be characterized by monitoring the energy flow used to drive photochemistry, being emitted as chlorophyll fluorescence (ChlF) and as heat (Murchie and Niyogi, 2011). Among them, NPQ, the latter route, is the major component of photoprotection (Murchie and Niyogi, 2011). Photorepair of plants deals with the disassembly of PSII complex and degradation and de novo biosynthesis of damaged D1 protein. The turnover of D1 protein, which is one of core subunits of PSII, is involved in photoinhibition of PSII caused by increasing irradiances (Aro et al., 1993). This can be characterized as sustained lowering of the quantum yield of PSII (Murchie and Niyogi, 2011). Plant chlorophyll (chl) molecules absorb light energy to drive photosynthesis (photochemistry), while excess energy can be dissipated as heat or re-emitted as ChlF. Among ChlF measurements the ФPSII indicates the portion of the absorbed energy used in photochemistry (Maxwell and Johnson, 2000). Large portions of the absorbed light are used to produce photoassimilates, which is referred to qP, or are dissipated as heat, which is referred to NPQ (Ralph and Gademann, 2005). When photosynthetic energy input exceeds its utilization, the plant acclimates by increasing the fraction of absorbed energy that is dissipated as heat in a process known as NPQ (Bilger and BjÖrkman, 1990; Maxwell and Johnson, 2000). NPQ competes to qP within a certain range of light irradiance, for instance, an increase of qP will result in a decrease in NPQ. Pulse-amplitude-modulated (PAM) fluorometer is a device used to provide quantitative information about PSII by measuring ChlF, and a better understanding of the early events of photosynthetic change influenced by the HL and mycorrhization. PAM fluorometer is a suitable monitoring tool because it is rapid and non-destructive, providing in-depth, quantitative physiological information about photosynthetic organisms (Ralph and Gademann, 2005). This information may be used to identify changes in photosynthetic status before morphological changes are observed. In combination with PAM fluorometer and the reflectance spectrometer, several indices related to photosynthesis, nutrition, biomass, and water status can be measured and calculated. NDVI is one of the most widely used spectral reflectance indices that relates to the difference between near infrared and red wavelength reflectance (Marti et al., 2007). It also has been reported as a sensitive foliar indicator of chl and nitrogen concentration, biomass, leaf area, and canopy structure (Gamon et al., 1995). The photosynthetic efficiency also can be indirectly assessed using the PRI derived from narrow-band reflectance ˜ at 531 and 570 nm (Fiella et al., 1996; Penuelas et al., 1995). The benefits of stimulated growth of Phalaenopsis due to mycorrhizal associations have been previously established (Chang, 2008; Wu et al., 2011). Since OM symbiosis has been shown to exchange
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nutrients with the plant, root colonization by the fungus increases the sink strength. We hypothesize that OM colonization would enhance the photosynthetic performance in leaves of Phalaenopsis to yield higher level of photoassimilates, thereby enhancing plant growth. The objectives of this study were to evaluate the OM effects on the growth of HL-grown orchid, and to elucidate the photochemical mechanisms involved in photoacclimation to photoinhibitory irradiance. Thereafter, an optimal strategy of light-intensity management that increases economic benefits for Phalaenopsis producers is proposed. 2. Materials and methods 2.1. Biological material and growth conditions Five-month-old micropropagated Phalaenopsis amabilis ‘KC1410’ plantlets was chosen as the model plants to study the HL-acclimation because of its slow growth and capability of establishing a symbiotic relationships with orchid mycorrhizae. Plants were purchased from King-Car Biotechnology Industrial Company (YiLan, Taiwan). OM and non-mycorrhizal (NM) plants were cultivated in 12-cm diameter pots filled with the processed bark of Pinus radiata (Pacific Wide Co., Cristchurch, New Zealand), then cultivated in a greenhouse (40% of full sun created by black shade cloth) with a pad and fan cooling system for 6 months. The maximum PPF measured at noon on a sunny day with HOBO data loggers (Onset, Massachusetts, USA) was 230–870 mol m−2 s−1 (due to cloudiness) about 40% of the prevailing solar radiation. Three days before experiment termination, seven plants in each treatment were transferred to shade environment with maximum PPF (less than 100 mol m−2 s−1 ) for the experiment of recovery of Fv/Fm. The photoperiod varied according to season and the air temperature was controlled at 28/22 ◦ C (day/night). All cultivates were watered twice a week. The fertilizer solution, made with distilled water and a 20N–8.7P–16.6 K (Peters Professional® 20-20-20, Scotts, Marysville, OH, USA) water-soluble fertilizer at 0.5 g L−1 was provided once every two weeks and allowed to drain. 2.2. Inoculum production and mycorrhization The OM fungal inoculums were prepared using OM isolates Ceratobasidium sp. (AG-A, binucleates) according to Chang and Chou (2007). This fungal isolates were previously identified as nonpathogenic fungi by inoculation test on seedlings of mung bean, cucumber, and rice. Fungal isolates were sub-cultured monthly on potato dextrose agar (PDA, Difco Labortories, Detroit, MI, USA) and maintained at room conditions. Sphagnum peat was homogeneously mixed with 1× potato-dextrose broth and adjusted to about 25% moisture content. After autoclaving, four myceliaclumps, each of 10(L) × 10(W) × 2(H) mm2 in size, were placed on top of the peat medium in a laminar flow hood and cultured for 2 weeks for compact growth at room temperature. For mycorrhizal treatment, 1 g of inocula consisting of peat and fungus mycelium was placed directly beneath the orchid roots, then the root sphere was refilled with pine bark. The NM control was prepared similar to the mycorrhizal treatment, except the myco-inocula was replaced with the autoclaved peat without fungal colonization. There were seven plants in NM and OM treatment, separately, and totally two experimental repeats were conducted in this study. 2.3. Biomass analysis All cultivates were harvested after 6 months of OM inoculation. Plant biomass was measured as leaf span (LS), fresh weight (FW), dry weight (DW), and shoot (S, including stem and leaves) and root (R) ratio (S/R, calculated using dry weights). Leaf span distance was
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measured from the end of one leaf to the longest opposite leaf tip according to Newton and Runkle (2009). Fresh shoots and roots were dried at 70 ◦ C for 120 h and weighed. Water content (WC) in whole plant was calculated using the formula (FW − DW)/FW. 2.4. Measurements
Table 1 Mycorrhizal effects on the growth characteristics, biomass, and dry matter partitioning of Phalaenopsis plantlets grown under high light for 6 months. All values are means ± standard error (n = 7). Different letters between treatments are significantly different at p ≤ 0.05 by Fisher’s LSD. NM: non-mycorrhizal control; OM: orchid mycorrhizal treatment; FW: fresh weight; DW: dry weight. Water content: (FW − DW)/FW. Growth characteristics
In order to estimate the photosynthetic performance, chl fluorometer, spectrometer, and gas exchange detector were used in the experiment. CO2 uptake rate was measured from 10 p.m. to 12 a.m. with a portable open flow gas exchange system LI-6400 (LI-COR, Lincoln, NE, USA). Measuring the photosynthesis on CAM Phalaenopsis is difficult because most of CO2 is assimilated at night. Thus, we measured the ChlF of photochemical reaction center of PSII instead of the determination of leaf photosynthesis. The ChlF parameters were measured at room temperature using a continuous monitoring device: MONITORING-PAM Multi-Channel Chlorophyll Fluorometer (Heinz Walz GmbH, Effeltrich, Germany) (Porcar-Castell et al., 2008). Fluorescence was measured from the upper surface of the 1st acropetal fully expended leaf. The fluorescence measurement was made on the 30 min dark-adapted plants during Phase III of CAM photosynthesis (2 p.m.). Saturating pulse analysis detected and calculated fluorescence parameters of leaves automatically, such as the fluorescence emission signal, minimum fluorescence signal (Fo), variable fluorescence signal (Fv), the maximum fluorescence signal (Fm), Fv/Fm, ETR, ФPSII, qP, and the NPQ. To monitor whether the mycorrhization regulates the RUE, we measured ChlF parameters qP and NPQ (Bilger and BjÖrkman, 1990), respectively, to study the PSII acclimation to the HL environment. The qP and NPQ were measured using rapid light curve (RLC) from 30 min dark-adapted NM and OM plants leaf. The measuring light (0.9 mol m−2 s−1 at Fo and Ft (steady yield of fluorescence in the light), and 9 mol m−2 s−1 at Fm), actinic light (1500 mol m−2 s−1 ), and saturating pulse (4000 mol m−2 s−1 ) were emitted by blue LED (light-emitting diode) with peak wavelength at 455 nm (full width at half maximum is 18 nm) and delivered to the leaf being measured through the MONI-head. The duration of the saturating pulse was 0.8 s. The detection and recording were performed using the RLC, of which exposed the leaf to nine incremental levels (0, 125, 190, 285, 420, 625, 820, 1150, and 1500 mol m−2 s−1 PPF) of irradiance, each with a 30 s interval. Measurements were recorded with WinControl-3 software (Heinz Walz GmbH, Inc., Effeltrich, Germany). Line-chart was drawn using data obtained from five of nine irradiance incremental levels (125, 190, 420, 820, and 1500 mol m−2 s−1 ). Leaf canopy spectrum of Phalaenopsis were measured using a portable spectrometer HandySpec Field (tec5 AG, Oberursel, Germany) fitted with a robust measurement probe and an internal halogen light source (400–2200 nm). This portable instrument measures spectral difference ranging from 305 to 2200 nm, and was calibrated with a white panel before use. The center of a leaf was selected for spectral measurement and each measurement was the mean of three consecutive individual full-range spectra scans. The equations (R, the reflectance) of spectral indices used to access the growth quality and photosynthesis are (1) NDVI computed as (R800 − R600)/(R800 + R600) (Rouse et al., 1974) and (2) PRI com˜ puted as (R531 − R570)/(R531 + R570) (Penuelas et al., 1995).
Leaf span (cm) FW (g) DW (g) DW ratio of S/R Water content (%) NDVI
Mycorrhizal treatment NM
OM
19.9 ± 0.6b 89.0 ± 2.3b 7.3 ± 0.2b 0.60 ± 0.03b 91.78 ± 0.21b 0.628 ± 0.026b
23.2 ± 0.6a 114.8 ± 5.9a 8.6 ± 0.4a 0.80 ± 0.06a 92.49 ± 0.12a 0.674 ± 0.027a
The level of confidence was 5% (P < 0.05). All data are presented as means ± standard error. 3. Results 3.1. OM symbiosis effects on plant morphology and growth Mycorrhizal Phalaenopsis grown under HL was enhanced by OM colonization (Table 1). OM plants had longer leaves that resulted in greater leaf span compared to NM plants. Also, OM plants had greater FW (an average increase of 27%) and DW (an average increase of 22%). Through the analysis of dry matter allocation, it is clearly known that OM plants had higher S/R ratio (0.8) than NM plants (0.6). OM plants exhibited higher water content (92.52%) compared to NM plants, which indicated that the former were growing more rapidly. NDVI of the OM leaves was significantly higher than the NM leaves, supporting the other observations of enhanced growth (LS, FW, DW, S/R, and WC) and demonstrably larger plants (Fig. 1). In order to determine the mechanism of this enhanced growth, we measured CO2 uptake rate for the evaluation of photosynthetic capacity. Leaf gas exchange results indicated that the OM leaves achieved higher rate of CO2 uptake than the NM plants at the night time (Fig. 2). 3.2. Light stress-recovery: dark adapted After six months cultivation under HL, the NM and OM plants underwent a photosynthetic photoinhibition, and the stress can be alleviated by exposure to LL (LL recovery) (Fig. 3). The value of Fv/Fm was similar in both OM and NM plants, approximately equal to 0.78,
2.5. Statistical analysis The effects of OM on shoot and root biomass, CO2 uptake rate, ChlF, and spectral reflectance indices were tested on seven plants for each treatment. Data were assessed by analysis of variance using Fisher’s LSD (CoStat ver. 6.4, Contact CoHort Software, USA).
Fig. 1. Phalaenopsis amabilis ‘KC1410’ plants grown under high-light environment with (OM) and without Ceratobasidium sp. (NM) inoculation. Scale bar represents 10 cm.
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Fig. 2. Carbon dioxide (CO2 ) uptake rate measured in the late evening (10 p.m.) 6 months after high-light cultivation of non-mycorrhizal (NM) and mycorrhizal (OM) orchids (Phalaenopsis amabilis ‘KC1410’). *** p ≤ 0.001.
indicating that the symbiotic cultivation did not significantly affect the maximum photochemical efficiency. After 3 days of LL recovery, both groups increased in Fv/Fm (≈0.81), while the OM leaves were more affected by the treatment, showing an increase of ≈3.8%. 3.3. Photosynthetic performance under actinic light Following the increase of actinic light intensity (refer to PPF), ФPSII decreased markedly in the NM and OM plants (Fig. 4). Both plant groups had similar ФPSII values when PPF <200 mol m−2 s−1 . Once light intensity increased above 200 mol m−2 s−1 PPF, ФPSII gradually increased and maintained significant difference between the NM and the OM plants in the range of 400–1200 mol m−2 s−1 PPF, and the most different values occurred around 820 mol m−2 s−1 PPF. RLC results showed that ETR appeared to be light sensitive and gradually increased with irradiance increase. In the actinic light intensity range from 0 to 500 mol m−2 s−1 PPF, the light-response curves of ETR for OM and NM orchids mostly overlapped. Once the PPF exceeded 500 mol m−2 s−1 , the ETR curve of the OM plants started separating from that of the NM plants, and reached the maximum difference around 820 mol m−2 s−1 PPF (Fig. 5).
Fig. 3. The maximum quantum yield of PSII photochemistry (Fv/Fm) measured with PAM fluorometer on dark-adapted samples of mycorrhizal (OM) and non-mycorrhizal (NM) Phalaenopsis during light-recovery experiment. Results are means ± standard error (n = 7). Black bar refers to high-light (HL: 230–870 mol m−2 s−1 ) culture, and white bar refers to low-light (LL: max. PPF under 100 mol m−2 s−1 ) culture. Different lower case letters indicate differences between mycorrhizal treatments and different capital letters represents differences between HL–LL-recovery treatments. Means followed by different letter(s) are significantly different at P ≤ 0.05 by Fisher’s LSD.
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Fig. 4. Analysis of light dependent changes in PSII operating efficiency (ФPSII) in an intact leaf of Phalaenopsis amabilis ‘KC1410’ with (OM) or without (NM) mycorrhization grown under high-light environment. The maximum difference of ФPSII determined at 820 PPF during response curve are indicated as black bar. Data are means ± standard error of seven plants. Vertical error bars represent the standard errors. ** p ≤ 0.01.
3.4. Radiation-use efficiency (RUE) under actinic light The photochemical quenching response, which was composed of qP and NPQ, revealed adverse patterns in both plant groups under given actinic light (Fig. 6), i.e., qP decreased and NPQ increased with PPF increase. Both qP and NPQ had no apparent difference between the treatments before 200 mol m−2 s−1 PPF, but exhibited evident difference when providing actinic light >420 mol m−2 s−1 PPF. Although qP decreased drastically and reached minimum at the end of RLC, the NPQ raised to a relative constant level over the excessive light interval in both plant groups. In the range of 420–820 mol m−2 s−1 PPF, the OM plants had significant higher qP and lower NPQ than the NM plants. 3.5. Photochemical reflectance index (PRI) and photoprotection To further investigate the capacity of photoprotection in leaves being exposed to HL, we measured the photochemical reflectance in Phalaenopsis (Fig. 7). The HL-acclimated leaf of the OM plant exhibited positive value of PRI, whereas the NM plants yielded negative value.
Fig. 5. Analysis of light dependent changes in electron transport rate (ETR) in an intact leaf of Phalaenopsis amabilis ‘KC1410’ with (OM) or without (NM) mycorrhization grown under high-light environment. The maximum difference of ETR determined at 820 PPF during response curve are indicated as black bar. Data are means ± standard error of seven plants. Vertical error bars represent the standard errors. ** p ≤ 0.01.
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Fig. 6. Analysis of light dependent changes in photochemical quenching (qP) and non-photochemical quenching (NPQ) in an intact leaf of Phalaenopsis amabilis ‘KC1410’ with (OM) or without (NM) mycorrhization grown under high-light environment. Data are means ± standard error of seven plants. Vertical error bars represent the standard errors.
4. Discussion 4.1. OM symbiosis stimulates growth of Phalaenopsis Plant yield is the result of its interaction to various environmental factors, e.g. light, air, water, temperature, soil, etc. The present study determined the effects of OM colonization on the yield of the HL-grown Phalaenopsis to reveal the photoacclimation strategy conferred by OM. Phalaenopsis inoculated with fungus Ceratobasidium exhibited stimulated growth similar to another orchid species inoculated with Rhizoctonia fungi (Chang, 2007). OM colonization had a positive impact on plant size and appearance of Phalaenopsis, manifested in greater leaf length (Fig. 1) and greener foliage (see NDVI in Table 1). The observed increase in biomass may imply the establishment of a beneficial symbiosis between fungus and Phalaenopsis. Noteworthy, the OM plants had the larger shoot biomass and thus resulted in high S/R (=0.80), which would indicate a mycorrhiza-mediated mechanism of photoassimilates allocation between shoots and roots. Based on the concept that photoassimilates-allocation changes alters the S/R, we suggested that the NM plants with S/R = 0.60 is in an allocation-balanced
Fig. 7. Assessing photoprotection capacity with evaluation of photochemical reflectance index (PRI) in an intact leaf of Phalaenopsis amabilis ‘KC1410’ with (OM) or without (NM) mycorrhization grown under high-light environment. Data are means ± standard error of seven plants. Vertical bars represent the standard errors. *** p ≤ 0.001.
status. On the contrary, OM plants is in an allocation-promoted status that a larger ratio of photoassimilates are allocated to the shoot part. It has been reported that mycorrhizal colonization may increase the sink strength of roots leading to stimulated photosynthesis and more photoassimilates for growth (Louche-Tessandier et al., 1999). Javot et al. (2007) also observed an increased shoot mass during the process of mycorrhization in Medicago truncatula plants. Higher WC in the OM leaves suggested an active cell growth to increase the shoot mass. This result agreed with the report of Yoder et al. (2000) that mycorrhization seems to enhance water uptake. Irradiance of 200–320 mol m−2 s−1 PPF is considered to promote growth and CO2 uptake of Phalaenopsis, but it also causes photodamage and photoinhibition (Guo et al., 2012; Wang, 1997). High PPF in excess of 320 mol m−2 s−1 PPF for 12 h would cause photoinhibition and reduce CO2 fixation in Phalaenopsis leaves (Guo et al., 2012). We cannot make inferences regarding CO2 uptake because we did not cultivate our plants under low irradiance conditions. However, if we assume that CO2 uptake was reduced, this carbon loss could be compensated by OM colonization because OM stimulated the HL-grown Phalaenopsis to uptake more CO2 and to enhance photochemical efficiency in comparison to the NM plants. The larger biomass of OM plants conceivably led to higher leaf area available for CO2 assimilation. We hypothesized that OM colonization would lead to increased demand for photosynthetic activity (Louche-Tessandier et al., 1999), and this carbon sink strength of symbiosis would stimulate the rate of photosynthesis (Harris et al., 1985). The general assumption is that OM symbiosis affects the whole plant photosynthetic performance, improving plant growth by increasing FW and DW. By using the spectral reflectance index NDVI, which is commonly applied to estimate chl content and plant growth potential, the significant difference in NDVI between the NM and the OM plants confirmed the observed results of greater vegetative biomass in OM Phalaenopsis. 4.2. Chlorophyll fluorescence (ChlF) and photosynthesis Acclimation to the light environment is a key ecophysiological feature for the growth and development of plants. Plant leaves generally exhibited a certain range of plasticity in relation to acclimation to the light environment (Valladares and Niinemets, 2008). Lin and Hsu (2004) improved photosynthetic production of Phalaenopsis by increasing the light exposure of lower leaves and indicated a photosynthetic plasticity in its leaves to adjust to irradiance. In the present study, Phalaenopsis exposed to HL for a long period decreased Fv/Fm below 0.80 (typical of photoinhibition). The Fv/Fm values of leaves in HL–LL recovery experiment were all >0.8 (typical of healthy leaves), which indicates a HL plasiticity, low efficiency of PSII photochemistry, and a photoinhibition on leaves of Phalaenopsis (Fig. 3). Similar results were reported by Naramoto et al. (2006) with shade-developed leaves of Fagus crenata plantlets exposed to HL. These phenomena may have been related to photochemistry changes of PSII during the transition from LL to HL, leading to decrease in Fv/Fm triggered by photoprotection mechanisms (Huner et al., 1993). Although HL resulted in similar Fv/Fm in both NM and OM plants, the higher recovery rate of Fv/Fm in OM plants after low PPF treatment, indicated that OM colonization could confer higher repair capacity on Phalaenopsis. This photorepair mechanism was also considered as one of the photoprotection mechanisms (Adir et al., 2003; Ragni et al., 2008). Photochemical efficiency ФPSII of plant leaves are sensitive to HL. When the leaf was illuminated, the decrease in the ФPSII occurred on the similar level in the OM and NM plants. In the HL environment, down regulation of ФPSII played a major role, as indicated by the drop of qP and the drastic development of NPQ (Fig. 6).
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In the present study, ChlF measurements showed that the OM plants lost less PSII photochemical efficiency than the NM plants and yielded higher ФPSII. Since ФPSII is highly correlated with the rate of carbon assimilation (Genty et al., 1989; Rolfe and Scholes, 1995), greater biomass was produced (Table 1). In addition, Fig. 4 shows that OM colonization significantly stimulated PSII photochemical efficiency of Phalaenopsis in the light intensity range of 400–1200 mol m−2 s−1 PPF that often cause photoinhibition, and consequently contributed a greater ability of photoacclimation to plants exposed to HL. Significant stable electron flow (ETR) in the OM plants also indicated a higher ФPSII in mycorrhizal Phalaenopsis (Fig. 4). It is consistent with the report of Tsimilli-Michael et al. (2000), where mycorrhizal Medicago exhibited increases in ETR. The increase of the various parameters obtained from the reflectance spectra, such as NDVI, and from the fluorescence measurements including ФPSII, ETR, and qP, all point to a raise in the photosynthetic performance of OM colonized orchids.
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PRI is an index of instantaneous photosynthetic light use efficiency (LUE) that can provide non-destructive assessment to ˜ determine the changes in leaf photosynthetic LUE (Penuelas et al., 1997; Nichol et al., 2006; Letts et al., 2008) and in xanthophyll cycle (Sims and Gamon, 2002; Nichol et al., 2006). In addition, PRI is significantly correlated to ФPSII and NPQ, and indicative the xanthophyll cycle-mediated thermal energy dissipation. It is well known that both PRI and RUE decreased along with the increase of PPF (Barton and North, 2001). Under HL stress, the OM plants sustained higher PRI (positive values) than the NM plants (negative values). We thus suggest that the OM plants are able to long-term acclimate to HL stress and that this acclimation is dependent of the XC (Fig. 6). In addition, high capacity of XC also responds to the elevated photosynthetic performance, e.g. ФPSII, ETR, and qP, of OM plants under HL environment. When plant leaves perceive excess light, its PRI value will drop below zero. Additionally, this indicates higher RUE in the OM plants when exposed to HL, which is consistent with the changes of qP and NPQ (Fig. 7).
4.3. Radiation-use efficiency and photoprotection 5. Conclusion Acclimatization of certain plants to change from LL to HL irradiation depends largely on its ability to minimize the magnitude and duration of photoinhibition of photosynthesis (Naidu and DeLucia, 1998; Krause et al., 2001; Guo et al., 2006). In the present study, the changes in growth potential, CO2 uptake rate, and photosynthetic performance in leaves of mycorrhizal Phalaenopsis appeared to be associated with the changes in RUE and protective mechanisms against excess light. Hence, photosynthetic energy balance and the partitioning of excitation energy between qP and NPQ processes were addressed. NPQ is an efficient photoprotective mechanism in chloroplasts to dissipate the excess energy absorbed by chl (Ruban et al., 2012). Its induction depends on the balance between PSII activity and ATP synthesis rate. According to Latowski et al. (2011), the conversion processes of violoxanthin (V) to zeaxanthin (Z) have been termed as xanthophyll cycle (XC), and Z-facilitated thermal dissipation of excess energy can cause the induction of NPQ. When light is not excessive, Z will convert back to V, thereby returning to an efficient utilization of light energy in photosynthesis. In our study, OM plants have less conversion of V to Z and, hence, contributed to a lower NPQ than the control (Fig. 6). The sustained lower NPQ and higher qP indicated that the dissipation mechanism was nearly sufficient to balance the requirements for the photoprotection and alleviated the RUE. On the other hand, the relative higher NPQ observed in the NM plants over the photoinhibition period indicated a lower capacity of photoprotection. The results indicated that the OM plants utilized light energy efficiently under strong actinic light that is close to the cultivation PPF in greenhouse, which reduced the loss of light energy and eventually resulted in lower NPQ. Thus, we suggested a greater and higher RUE on the OM plants upon photoinhibition. It is consistent with the observation in PRI variation (Fig. 7) on HL-grown Phalaenopsis, in which higher PRI resulted in higher RUE. The lower NPQ, which may reflect a higher RUE and photoprotection, also resulted in an up-regulation of PSII activity. It indicated that the OM plants with higher photochemical efficiency utilized photosynthetic energy to assimilate more carbon instead of to dissipate them as heat under HL stress (Fig. 6). In other words, PSII acclimation of OM plants involves modulation of the fractions of absorbed irradiance that is utilized in photochemistry more than that dissipated as heat. Thus, we assume that OM effects involve the long-term acclimation of PSII (Öquist and Huner, 2003) and lead to slow reversible acclimation in the PSII energy partitioning, which the photosynthetic protections occurred following the initiation of photoinhibition (Baker, 2008; Horton and Ruban, 2004; Lee and Guo, 2012). From our results, it is evident that the OM plants have considerable potential for HL acclimation.
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