Photosynthetic Light Utilization Efficiency , Water Relations and Leaf Growth of C 3 and CAM Tropical Orchids under Natural Conditions

Native orchid species of Singapore in their natural conditions experience stress from high irradiance, high temperatures and periods of extended low rainfall, which impact orchid plant physiology and lead to reduced growth and productivity. In this study, it was found that there was a reduction in photochemical efficiency of photosystem II (PSII) in 6 native orchid species under high light (HL) and Bulbophyllum membranaceum under low light (LL). There was chronic photoinhibition in these 6 orchid species over a period of 3 months after transplanting onto the tree trunks without watering and fertilization, especially in Coelogynes mayeriana and Bulbophyllum membranaceum under both HL and LL. This chronic photoinhibition caused by sustained period of water deficit in their natural conditions was later reversed by natural re-watering conditions from higher rainfall. These results indicate that water deficit has a greater impact on photosynthetic light utilization efficiency than excess light. The present study also showed that after natural rewatering, relative water content (RWC) of leaves and pseudobulbs generally increased. During the natural re-watering, total leaf area also gradually increased and reached maximum expansion after 7 weeks under both HL and LL, with some exceptions due to leaf abscission or decline in total leaf area, possibly a strategy for water conservation.

S. Tay et al.

Introduction
Singapore has a tropical rainforest climate with no distinctive seasons, temperatures ranging from 22˚C to 35˚C, high relative humidity averages ranging from 60% to 100%, and annual rainfall about 2300 mm, with November to January being the wettest months.Considerable efforts have been made in the past decade to re-introduce native orchid species back into Singapore as part of conservation [1]- [3].Many of these re-introduced species are epiphytes adapted to relatively dry habitats, native to low-elevation forests, and are dependent upon their microclimate.Crucial to these epiphytic orchids is the requirement for water economy where high evapotranspiration combined with scarce water supply challenges plant water balance [4].Epiphytic orchids grow well under optimal environmental conditions and propagate naturally.However, when they experience stress from high irradiance, high temperatures and periods of extended low rainfall, these factors significantly impact their physiology and lead to reduced growth and productivity.
High irradiance and high temperature cause a reduction in photosynthetic quantum yield, loss in leaf chlorophyll, and a greater susceptibility to photoinhibition in orchids [5]- [7].The reduction in photosynthesis can be attributed to dynamic photoinhibition, photooxidation, photobleaching of chlorophyll and chloroplast damage leading to deletrious effects of the leaf photosynthetic apparatus [7]- [9].Furthermore, orchid leaves exposed to drastic increase in photon flux densities experienced a drop in CO 2 gas exchange and leaf chlorosis [10].
Epiphytic orchids experiencing stress from water deficit show significant decrease in relative water content (RWC) in leaves and pseudobulbs, reduction in photosynthetic light utilization, and greater susceptibility to photoinhibition caused by high irradiance [11].In other plants, periodic drought stress also leads to a closure of stomata [12], through the reduction of shoot water potential directly or reduced turgor [13].As a result, CO 2 depletes in the intercellular spaces and at the chloroplast level, limiting photosynthesis.Plants grown under low irradiance may also experience a reduction in the photosynthetic rate, resulting in a decline in growth rate [5] [14].Therefore it is necessary to strike a good balance in maximising photon energy utilization of leaves to increase photosynthetic activity whilst minimising photoinhibition [6].
Orchids are also able to tolerate and acclimatise to stressful conditions, dynamically achieved by the physiological and morphological adaptations.In some orchid species, carbon fixation through Crassulacean acid metabolism [15] provides a means of water conservation during times of water deficit, while preventing loss of carbon [16]- [19].Thick-leaved orchids, such as Bulbophyllum vaginatum, Dendrobium leonis and Phalaenopsis cornu-cervi, have features that are characteristic of plants that fix carbon primarily through Crassulacean acid metoblism [15], while thin-leaved orchids such as Coelogynes rochussenii, Coelogynes mayeriana and Bulbophyllum membranaceum are plants that fix carbon primarily through C 3 pathway [20]- [22].
There is little information about the photosynthetic light utilization efficiency and water relations of tropical orchid species in their natural environments, especially with regards to their responses to varying environmental conditions and the stress that comes with it.The information would be useful for improving the approach towards conservation of orchid species in their natural environments.Therefore, to complement the conservation efforts in Singapore, this study aims to better understand the photosynthetic light utilization efficiency, water relations and leaf growth of six selected C3 and CAM native tropical orchids under natural conditions.The results benefit sustainable conservation of these orchid species under natural conditions.

Plant Cultivation under Natural Conditions
Mature plants of Coelogynes rochussenii, Coelogynes mayeriana, Bulbophyllum vaginatum, Bulbophyllum membranaceum, Dendrobium leonis and Phalaenopsis cornu-cervi planted by the National Parks Board (NParks) Singapore in National Institute of Eduction (NIE), Singapore, were used in this study.These epiphytic orchid plants were planted on the trunks of rain trees, Albizia saman (syn.Samanea saman), under natural high light (HL) and low light (LL) conditions on 12 January 2015.LL was achieved by natural shading provided by the trees and positioning the plants out of direct sunlight.These orchids were neither watered nor fertilized after planting, and depended upon rainfall as the only source of water.The maximal PPFD ranged from 90 to 200 μmol•m −2 •s −1 for LL and from 400 to 1300 μmol•m −2 •s −1 for HL, with an ambient day temperature of about 30˚C to 35˚C during the photoperiod.6 plants of each species for each light condition were planted on the trees.

Measurement of Photosynthetic Photon Flux Density (PPFD)
PPFD was measured using a photosynthetically available radiation quantum sensor and reading unit (Skye Instruments Ltd, Llandrindod, UK).The stabilized PPFD, within a range of 0 to 1999 μmol•m −2 •s −1 , was measured from 6 different positions above the leaves for HL and LL respectively, and an average was calculated from the 6 values measured.

Measurement of Chl Fluorescence Fv/Fm Ratio
Diurnal changes of F v /F m ratios were taken every two hours from 0800 to 1600 h with the Plant Efficiency Analyser, PEA (Hansatech Instruments Ltd, England) on sunny and cloudy days, while weekly F v /F m ratios were taken at the time corresponding to the maximal midday PPFD.6 samples were taken from each species under each light condition.The leaves were pre-darkened with clips for 15 min prior to measurements.Dark adapted leaves were placed under the light pipe to measure F o , initial chlorophyll (Chl) fluorescence during the very early onset of illumination with excitation light.F m , maximum Chl fluorescence was determined by 0.8 seconds of saturated pulse (>6000 μmol•m −2 •s −1 ).The variable fluorescence yield F v , was determined by F m − F o .The efficiency of excitation energy captured by open PSII reaction centres in dark adapted leaves was estimated by the fluorescence F v /F m ratio.

Measurement of Chl Reading by Hand Held Chlorophyll Meter (SPAD-502)
Leaf Chl content was read by using a hand-held chlorophyll meter (SPAD-502, Minolta, Japan), which measures the absorbances of the leaf at wavelengths 645 nm and 663 nm.Using these two absorbances, the meter calculates a numerical SPAD value which is proportional to the amount of Chl present in the leaf, and expresses in terms of SPAD units.6 samples were taken from each species under each light condition.

Measurement of Midday RWC
Square cuts (1 cm by 1 cm) were made out of the leaves and 5 mm thick slices were made out of the pseudobulbs.These sample were first weighed with an analytical balance to determine fresh weight (FW), followed by immersion in water in the dark for 3 hours (for pseudobulb of C. mayeriana and leaf of B. membranaceum) or 24 hours (for the rest of the samples) prior to measurement of their saturated weight (SW).The samples were then dried in the oven at 80˚C for at least 72 hours to obtain their dry weight.RWC was expressed as RWC = (FW − DW)/(SW − DW) × 100%.10 samples each for leaves and for pseudobulbs were obtained from each species.

Measurement of Total Leaf Area
Young, budding leaves from 6 plants of each orchid species, cultivated under the respective light conditions (HL or LL), were selected and tracked over 16 weeks.The individual leaf area was measured every week by capturing the image of selected leaves against a measured scale and then using Image J software to calculate the leaf area from the image according the method outlined by Schneider et al. [23].The total leaf area was calculated as a sum of the individual leaf areas.Total leaf area is one of the parameters for leaf growth.

Statistical Analysis
One-way ANOVA was used to test for significant differences among different growth irradiances, using Tukey's multiple comparison tests to discriminate the means (IBM SPSS Statistics for Macintosh, Version 22.0, 2013).

Photosynthetic Light Utilization Efficiency
Diurnal PPFD was much higher for the plants grown under HL than LL on a sunny day at 1000 h, 1200 h and 1400 h (Figure 1(a) and Figure 1(b)) and the diurnal F v /F m ratio at these timings were also correspondingly lower in HL than LL (Figure 1 and B. membranaceum grown under LL did not recover to the same levels as at 0800 h, suggesting that these plants were suffering from chronic photoinhibition.
However, there was evidence of recovery from this chronic photoinhibition as shown in the difference in F v /F m ratios at midday, between 1 and 3 months of growing under HL and LL.Significant increase in the midday F v /F m ratios from 1 to 3 months of cultivation were observed in all species under both HL (Figure 2(a)) and LL (Figure 2(b)) except for C. mayeriana under LL (One-way ANOVA, p < 0.05, n = 6).Between species, B. vaginatum and B. membranaceum after 1 month under HL show significantly lower F v /F m ratios as compared with the other species, but recovered to levels comparable to D. leonis and P. cornu-cervi after 3 months under HL (Tukey's multiple comparison test, p < 0.05, n = 6).No significant difference in F v /F m ratio was found between species after 1 month and 3 months under LL.Further evidence of recovery from this chronic photoinhibition was provided in the data collected over 16 weeks for the changes to midday maximal PPFD and F v /F m ratio.Over the 16 weeks of cultivation under HL, midday maximal PPFD exceeded 800 μmol•m −2 •s −1 only in weeks 2 to 7 and week 13 (Figure 3(a)) while little fluctuation in midday PPFD (<200 μmol•m −2 •s −1 ) was seen in LL (Figure 3(b)).Based on rainfall data of the micro-climate of the area where the orchids were cultivated, weeks 2 to 7 coincided with a period of low rainfall (Figure 3(a)), while the drop in midday maximal PPFD (< 800 μmol•m −2 •s −1 ) from week 8 onwards, with the exception of week 13, coincided with an increase in weekly total rainfall.Corresponding with the fluctuations in midday maximal PPFD under HL and the weekly total rainfall, the F v /F m ratio decreased from weeks 2 to 8 for all 6 orchid species, under both HL (Figure 3(c , C. mayeriana and D. leonis.Therefore, the period of higher weekly total rainfall from week 8 onwards provided re-watering conditions, and this corresponded with plants showing recovery from chronic photoinhibition, where the F v /F m ratio that was decreasing below 0.700 in the first 8 weeks started to increase back to levels above 0.700 after week 8.
In contrast to the changes in F v /F m ratio over the 16 weeks of cultivation, difference between Chl content under HL and LL were lesser, with the exception of B. membranaceum which showed a higher Chl content under

Water Relations
To further understand the significance of the natural re-watering conditions provided by the higher weekly total rainfall from week 8 onwards, the water status of the plants was examined.RWC of leaves (Figure 5

Leaf Growth
The difference in leaf growth under HL and LL was also examined through the changes in total leaf area for all 6 species.Total leaf area generally increased for the first 7 weeks for all 6 orchid species, with the exception of D. leonis cultivated under HL (Figure 6(e)), where there was a sharp drop in total leaf area during weeks 5 to 7. The sharp drop was due to a loss of the selected leaves, and this could be further attributed to the sustained low rainfall period coupled with HL from weeks 2 to 9. Total leaf area continued to be sustained at around the same level from week 7 onwards for the rest of the plants, with the exception of C. rochussenii (Figure 6

Discussion
Some 70% of the native orchid species of Singapore are mostly vascular epiphytes [1].Vascular epiphytes are dependent upon their micro-climate in photosynthetic light utilization and water economy [4].In a review by Ort [24], it was reported that light intensities often exceed photosynthetic capacity in plants, while stomatal conductance limits CO 2 uptake into leaves, rendering even moderate irradiances in excess of photosynthetic capacity.Plants grown under high irradiance may lead to photoinhibition of the leaves, and this will result in a reduction in growth and productivity.Both C 3 and CAM orchids have been reported to show stress from exposure to high irradiances under natural conditions, such as decrease in photosynthetic light utilization, photoinhibition, photooxidative damage, and leaf chlorosis [5] [10] [11] [25]- [29].Our present study reports a reduction in the efficiency of open PSII reaction centres of all the 6 orchid species under exposure to HL and C 3 orchid B. membranaceum under exposure to LL, towards midday (Figure 1), indicating photoinhibition.This photoinhibition is particularly more severe in B. vaginatum and B. membranaceum grown under HL.All plants under HL were also not able to fully recover from the photoinhibition over the first month (Figure 2(a), open bars), indicating chronic photoinhibition.
However, photoinhibition of PSII in vivo provides photoprotection and is an acclimation strategy for plants under stress from excessive irradiances [30].Orchids have protective stategies to overcome the stress from high irradiances and to help balance maximising photon energy utilization while minimising photoinhibition [6].Previous studies reported higher tolerance to high PPFD and faster recovery from photoinbition in C 3 Oncidium orchid as compared to CAM Phalaenopsis orchid [29], greater photoprotection in leaves of C 3 orchid C. rochussenii under HL compared to LL [28], and recovery from photoinhibition in C 3 orchid Gramatophyllum speciosum in HL [27].The results of our study seem to contradict these cases because the diurnal F v /F m ratios decline sharply towards 1200 h in all the orchids exposed to HL (Figure 1(c)) as compared to LL (Figure 1(d)).All 6 species also showed chronic photoinhibition after 1 month under HL, but recovered after 3 months (Figure 2).However, in all previous studies, water deficit was not severe and this could be the reason why the C 3 orchids could better utilize photosynthetic light energy as compared to the CAM orchids.
Ort [24] argued that at high irradiances, water status take physiological precedence over maximizing photosynthesis.It has been reported that in thin-leaved, C 3 epiphytic orchid Catasetum viridiflavum, and other epiphytes, re-watered plants show recovery from photoinhibition despite high PPFD, indicating that water deficit has a greater impact on photoinhibition than high light [31] [32].Furthermore, strong photoinhibition and reduced photosynthetic capacity was observed in epiphytic CAM orchid hybrid, Cattleya forbesii Lindl.× Laelia tenebrosa Rolfe., following sustained water deficit for 42 days [11].It has also been reported that under water deficit stress, C. rochussenii under HL had a reduced photosynthetic light utilization than plants under LL, and water deficit stress took precedence over light stress in reducing the Chl reading of C. rochussenii.[28].Drought stress coupled with high light occurs when water supply is insufficient to sustain plant growth [27] [33].Our present study showed that a sustained period of water deficit caused a decline in photosynthetic light utilization in all 6 orchid species, followed by a period of natural re-watering conditions that enabled recovery from this chronic photoinhibition (Figure 3).The changes in photosynthetic light utilization were less likely to be attributed to changes in Chl content since there was little difference in Chl content between HL and LL (Figure 4), with the exception of B. membranaceum which showed a higher Chl content under LL than HL.Therefore, the results suggest that water deficit has a greater impact on photosynthetic light utilization efficiency than HL, and that B. vaginatum and B. membranaceum thrive better in LL, well-watered conditions.
Water deficit that drives closure of stomata result in reduction in CO 2 uptake and subsequent down-regulation of photosynthetic efficiency [34], which is alleviated in epiphytic orchids by carbon fixation through CAM, while achieving water economy [4] [35] [36].Epiphytic orchids have also been reported to lose water from the leaf slowly, yet maintain high values of RWC [37].However, another study reported a reduction in RWC and osmotic potential during a period of water shortage in some tropical epiphytes [38] [39].Moreover, studies had shown that pseudobulbs helped to reduce the loss of leaf water content during periods of water deficit stress by storing water in the pseudobulbs and then supplying this water to the leaves, thereby providing orchids with the ability to survive [11] [40]- [43].A previous study had also reported that C. rochussenii grown under LL had a significantly higher leaf RWC than those under HL [28].Our present study showed that after a period of natural re-watering, RWC of leaves and pseudobulbs increased significantly in all 6 orchid species, with the exception of the pseudobulbs of B. vaginatum and B. membranaceum under HL and C. mayeriana under LL (Figure 5).
Large pseudobulbs in C 3 orchids C. rochussenii and C. mayeriana could have stored water and helped maintain leaf water content so as to alleviate photoinhibition.The small size and thin leaves of B. membranaceum could have disadvantaged it in its ability to store water in the pseudobulbs to overcome periods of water deficit and for the leaves to overcome the stress from high irradiance, which explains the severity of photoinhibition.On the other hand, in both D. leonis and P. cornu-cervi, having fleshy leaves and the ability to fix carbon through CAM could be significant strategies in response to photoinhibition.Therefore, both of these species are more adaptable to both HL and LL conditions, and could have a wider range of tolerable irradiance.
Water deficits lead to reduction in leaf area, leaf abscission, and dieback of individual plant parts.However, reduction in leaf area and leaf abscission strongly reduced water loss, and epiphytic orchids that encounter shortages of water may stop growing when water is in short supply, only to resume growth when water supply is restored [31] [44].Our study showed a general increase in total leaf area for the 6 species for the first 7 weeks under HL and LL and maintained onwards as they had reached maximum leaf expansion.However, the drop in total leaf area in some species, such as D. leonis, was due to leaf abscission and decline in total leaf area.This could be a strategy for water conservation through reducing the total number of leaves, and therefore number of stomata, as well as reducing the surface area, leading to lesser water loss by evapotranspiration.

Conclusion
The results suggest that water deficit has a greater impact on photosynthetic light utilization efficiency than excess light.Over a period of water deficit, 6 orchid species experienced a decrease in F v /F m ratio and RWC of leaves and pseudobulbs, which could both be reversed by natural re-watering conditions from higher rainfall.Over 16 weeks, total leaf area increased and leaves reached maximum expansion after 7 weeks under both HL and LL, with some exceptions due to leaf abscission or decline in total leaf area, possibly a strategy for water conservation.Future work would be required to fully understand the adaptation strategies in response to stress from HL and water deficit that may occur in the natural environment.This will aid in improving the approach of re-introduction of native orchid species in Singapore.
(c) and Figure 1(d)), with the biggest difference in B. vaginatum and B. membranaceum.This suggests a reduction in the efficiency of open PSII reaction centres of the orchids under exposure

Figure 1 .
Figure 1.Diurnal changes of PPFD and F v /F m ratio on sunny days for C. rochussenii (), C. mayeriana (), B. vaginatum (), B. membranaceum (), D. leonis () and P. cornu-cervi () grown under HL and LL. to HL (PPFDs of 400 to 1300 μmol•m −2 •s −1 ) towards midday, particularly more severe in B. vaginatum and B. membranaceum, among the 6 orchid species examined.A reduction in the efficiency of open PSII reaction centres was also shown in B. membranaceum under exposure to LL (with a maximal PPFD of 200 μmol•m −2 •s −1 ) during midday.F v /F m ratio at 1600 h of all plants grown under HL except C. mayeriana,and B. membranaceum grown under LL did not recover to the same levels as at 0800 h, suggesting that these plants were suffering from chronic photoinhibition.However, there was evidence of recovery from this chronic photoinhibition as shown in the difference in F v /F m ratios at midday, between 1 and 3 months of growing under HL and LL.Significant increase in the midday F v /F m ratios from 1 to 3 months of cultivation were observed in all species under both HL (Figure2(a)) and LL (Figure2(b)) except for C. mayeriana under LL (One-way ANOVA, p < 0.05, n = 6).Between species, B. vaginatum and B. membranaceum after 1 month under HL show significantly lower F v /F m ratios as compared with the other species, but recovered to levels comparable to D. leonis and P. cornu-cervi after 3 months under HL (Tukey's multiple comparison test, p < 0.05, n = 6).No significant difference in F v /F m ratio was found between species after 1 month and 3 months under LL.Further evidence of recovery from this chronic photoinhibition was provided in the data collected over 16 weeks for the changes to midday maximal PPFD and F v /F m ratio.Over the 16 weeks of cultivation under HL, midday maximal PPFD exceeded 800 μmol•m −2 •s −1 only in weeks 2 to 7 and week 13 (Figure3(a)) while little fluctuation in midday PPFD (<200 μmol•m −2 •s −1 ) was seen in LL (Figure3(b)).Based on rainfall data of the micro-climate of the area where the orchids were cultivated, weeks 2 to 7 coincided with a period of low rainfall (Figure3(a)), while the drop in midday maximal PPFD (< 800 μmol•m −2 •s −1 ) from week 8 onwards, with the exception of week 13, coincided with an increase in weekly total rainfall.Corresponding with the fluctuations in midday maximal PPFD under HL and the weekly total rainfall, the F v /F m ratio decreased from weeks 2 to 8 for all 6 orchid species, under both HL (Figure3(c) and Figure 3(e)) and LL (Figure 3(d) and Figure 3(f)), and started increasing back to original levels or higher thereafter.The decrease in F v /F m ratio was greater in HL than LL, for B. vaginatum, B. membranaceum and P. cornu-cervi, C. rochussenii, C. mayeriana and D. leonis.Therefore, the period of higher weekly total rainfall from week 8 onwards provided re-watering conditions, and this corresponded with plants showing recovery from chronic photoinhibition, where the F v /F m ratio that was decreasing below 0.700 in the first 8 weeks started to increase back to levels above 0.700 after week 8.In contrast to the changes in F v /F m ratio over the 16 weeks of cultivation, difference between Chl content under HL and LL were lesser, with the exception of B. membranaceum which showed a higher Chl content under

) and Figure 3 (
e)) and LL (Figure 3(d) and Figure 3(f)), and started increasing back to original levels or higher thereafter.The decrease in F v /F m ratio was greater in HL than LL, for B. vaginatum, B. membranaceum and P. cornu-cervi, C. rochussenii

Figure 3 .
Figure 3. Changes of midday PPFD and F v /F m ratios of different orchid species over 16 weeks under HL and LL.Rainfall data adapted with permission from Meteorological Services Singapore (Historical Daily Records, 2015).
(a) and Figure 5(b)) and pseudobulbs (Figure 5(c) and Figure 5(d)) increased significantly in all 6 orchid species, between 1 to 4 months of cultivation under HL and LL, with the exception of the pseudobulbs of B. vaginatum and B. membranaceum under HL (One-way ANOVA, p < 0.05, n = 10).Between species, leaves of B. vaginatum, B. membranaceum, D. leonis and P. cornu-cervi after 1 month under HL and LL had significantly lower RWC compared to C. rochussenii and C. mayeriana (Tukey's multiple comparison test, p < 0.05, n = 10).However, after 4 months, RWC in the leaves of P. cornu-cervi under HL and LL and B. membranaceum under LL increased back to levels comparable with C. rochussenii and C. mayeriana.After 1 and 4 months under HL and LL, pseudobulbs of C. rochussenii, B. vaginatum and B. membranaceum remained significantly lower than that of C. mayeriana.
(a)) and B. membranaceum (Figure 6(d)) cultivated under LL, and C. mayeriana (Figure 6(b)) and P. cornu-cervi (Figure 6(f)) cultivated under HL.For the plants with total leaf area remaining unchanged from week 7 onwards, it could be attributed to the leaves reaching their maximum expanded area.

Figure 4 .
Figure 4. Changes of Chl reading of different orchid species over 16 weeks under HL and LL.

Figure 6 .
Figure 6.Total leaf area of different orchid species over 16 weeks under HL () and LL ().