C4 Equivalent Decarboxylation Competence in Tropical Orchids

Tropical xero-epiphytic orchids undergo a photosynthetic mechanism called ‘Crassulacean Acid Metabolism’, a modification of Calvin–Benson cycle. This anabolic process aids in the long-term retention of organically fixed CO2, imprinting them as active sequestrators and fixers of gaseous CO2. The malate decarboxylation competence of enzymes—phosphoenolpyruvate carboxykinase (PEPCK), Nicotinamide Adenine Dinucleotide malic enzyme (NAD-ME) and Nicotinamide Adenine Dinucleotide Phosphate malic enzyme (NADP-ME) in xero-epiphytic orchids that concentrate CO2 by employing RuBisCO to embark on a carboxylation route is defined in this paper. Physiological parameters like photosynthetic rates, and quantum yield, accompanied by histomorphometry were analysed. Our study indicated PEPCK as the dominant decarboxylating enzyme in tropical orchids. The efficiency of PEPCK was reinforced by two additional malic enzymes, which are dependent on cofactors, namely Nicotinamide Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Phosphate. These results indicated the need to appraise the potential of tropical orchids as strategic plant contenders for CO2 triggered greenhouse effect mitigator.


Introduction
Crop production, forestry, and resource use account for roughly 30% of worldwide Green House Gas (GHG) emissions (Panchasara et al. 2021; "United States Environmental Protection Agency," 2020). Increasing food production to meet consumption trends and nutritional needs and a reduction in GHG emissions are imperative if sustainable food security and eco-climate targets are to be met. However, since crop production is both a victim and perpetrator of GHG emissions, this has proved challenging.
Agricultural GHG contribution can be reduced by (a) Improving soil Carbon sequestration and reducing deforestation, (b) Improving N use efficiency, and (c) Water use efficiency of crops (Verge et al. 2007). Carbon sequestration or long-term carbon storage is primarily facilitated by photosynthesis. The enzyme Rubisco Nidhi S. Mukundan, Sunaina Banerjee and Shruti Kumar have equally contributed to the present manuscript. supports the key carbon fixing step called the Calvin-Benson cycle. Plants that use only the Calvin cycle for their carbon assimilation are called C3 plants. But the unspecific nature of Rubisco resulted in an uneconomical process called photorespiration which made it rather inefficient. This led to the C3 pathway's evolutionary anatomical and biochemical modification, giving rise to C4 (Hatch and Slack pathway) plants and CAM (Crassulacean acid metabolism pathway) plants.
Fluctuating CO 2 concentration resulted in the molecular evolution of Rubisco within these photosynthetic modifications (Hermida-carrera et al. 2020). C4 plants evolved to overcome the limitations of Rubisco, which are exacerbated in high temperatures, thus bypassing photorespiration. Selective factors for CAM photosynthesis are low daytime CO 2 and reduced ambient CO 2 concentration in aquatic plants. It is an adaptation to arid environment and proves advantageous in habitats with excess rainfall (Gamisch et al. 2021). The forest canopies and buffer precincts of Western Ghats, a 'Biodiversity Hotspot', are rich in diverse and persistent epiphytic and terrestrial orchids.
Epiphytic hydraulics have been shown to affect the forest microclimate evapotranspiration rates by employing various distinctive characteristics. Epiphytes are touted to be models for studying the hydraulic behaviour of plants to help contribute to global sustainable water management (Miller et al. 2021). The orchid family is one of few flowering plant lineages that thrive in epiphytic and terrestrial forms. In the late 1950s, studies on atmospheric CO 2 fixation by gas exchange in aerial roots (ARs) of orchids were demonstrated (Hew and Yong 2003). The carbon isotopic ratios indicated that the leaves and aerial roots in orchids rely on Crassulacean acid metabolism (CAM) (Zotz et al. 1997;Aschan and Pfanz 2003) for gaseous CO 2 fixation.
The degree of CAM expression is influenced by various environmental and endogenous factors, such as temperature, irradiance, moisture, growth stage and leaf age (Ota et al. 1991;Ehleringer 1994;Luttge 2004;Dayer et al. 2017;Lambers et al. 2019;Yadav et al. 2020). The CAM photosynthetic pathway in these plants is segregated into four phases based on time (Osmond et al. 1999). Nocturnal stomatal opening results in CO 2 uptake, followed by PEPC fixation and vacuolar storage as organic acid constitutes phase 1. The highly sensitive phase II involves the continued open phase of stomata in the early light for CO 2 uptake. After the remobilisation of the organic acid for decarboxylation and fixation, the more stable phase III presents assimilated carbon behind closed stomata. The transient Phase IV reopens the stomata in late light to replenish CO 2 , followed by direct assimilation due to depleted organic acid stock. Phase II and IV are transient due to the shift in the enzyme for CO 2 uptake. Whilst phase II is PEPC dominated, phase IV is Rubisco dominated C3 photosynthesis (Maxwell et al. 1999).
The characteristic traits that discriminate C4 (Hatch 1987) and CAM plants from C3 species are: (a) high optimal temperature and extraordinary irradiance saturation for maximum leaf photosynthetic rates, (b) reduced O 2 fixation and CO 2 evolution under high temperature and irradiance (photorespiration) (c) truncated CO 2 compensation point, (d) low mesophyll resistance to CO 2 diffusion coupled with high stomatal resistance leading to high water use efficiency, (e) higher internal exposed mesophyll surface area per cell volume and (f) ability to recycle respiratory CO 2 by illuminated leaves (Ota et al. 1991;Ehleringer 1994;Luttge 2004). These findings laid the foundation for a series of subsequent physiological, anatomical and biochemical studies that assisted in the elucidation of the C4 and CAM photosynthetic pathways. In an anthropologically altered milieu, it is predicted that C4 plants might lose their competitive benefit over C3 plants (Sage and Sage 2009;Silvera et al. 2009), unlike CAM plants.
Surprisingly CAM group of species shares similar features expressed by their C4 counterparts except for temporal and spatial dissimilarity in the localisation of substrates. Therefore, identifying tolerant CAM species with optimal photosynthetic efficiency would complement mitigating greenhouse effects in micro-ecological niches. With this view, orchid species in the Western Ghats were scrutinised for their photosynthetic efficiencies, as their ability to reduce CO 2 is less studied. There are no studies in Orchidaceae to date, which reported the decarboxylation efficiency of enzymes, especially Nicotinamide Adenine Dinucleotide Malic Enzyme (NAD-ME) Nicotinamide Adenine Dinucleotide Phosphate Malic Enzyme (NADP-ME) or Phosphoenolpyruvate Carboxykinase (PEPCK). These enzymes play a primary role in decarboxylating malate to pyruvate by releasing CO 2 for re-fixation by RuBisCO. Their decarboxylation efficiency is vital to computing the carbon assimilating ability in plants. This is crucial to assess their micro-niche climate re-tuning ability. Histomorphological studies were carried out on aerial roots, leaves and pseudo bulb tissues of the selected plants. We measured the photosynthetic efficiency (A), quantum yield (ФCO 2 ), and RuBisCO activity in intact leaf tissues of all the plants using the photosynthesis measuring system. The study interrelated the parameters above to identify photosynthetically efficient and tolerant tropical orchid candidates that can mitigate CO 2 -based greenhouse effects in a micro-ecological niche.

Plant Material, Collection, and Maintenance
In the present study, we identified the efficiencies of the three critical malate decarboxylation enzymes in eight common tropical orchids. These are Acampe praemorsa, Bulbophyllum neilgherrense, Dendrobium Sonia (white variety), Pholidota pallida, Rhynochostylis retusa, Sarcanthus pauciflorus, Vanda spathulata, Vanilla planifolia and three monocot control species representing C3, C4 and CAM photosynthetic types like Oryza sativa, Spathoglottis plicata, Saccharum officinarum, and Aloe vera respectively. The phenology of orchids (vegetative and reproductive cycles and their abundance with seasonal changes) was studied every three months in a year at four sites in central Western Ghats regions of Karnataka, India. These were Agumbe (13.502712°N, 75.0903191°E), Hebri (13.458111°N, 74.995251°E), Horanadu (13. 162679°N, 75.203887°E) and Sringeri (13.419814°N, 75.256738°E). Dendrobium Sonia and Vanilla planifolia were obtained commercially from an Indian Nursery (Fig. 1A). The species identification was done with the help of a taxonomist, Dr A. Radhakrishna Rao, and also by referring to various texts on regional floras (Abraham and Vatsala 1981;Bhat 2003). The inclusion of orchid species for this study was based on the following criteria: (a) high tolerance to seasonal changes, (b) high abundance throughout the year, with the predominant vegetative phase in natural conditions, (c) superior acclimatisation ability to greenhouse conditions and (d) species with CAM morphological characters. We excluded saprophytic orchid species. Four control plants, such as Oryza sativa, Spathoglottis plicata, Saccharum officinarum, and Aloe vera, along with the eight test orchids (Table 1), that served as sources of tissue samples were acclimatised to the greenhouse at Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, India (Fig. 1B). Orchids maintained in pots were well aerated and percolated with charcoal, stones, sand, and soil in 1:1:1:1 proportion. The orchids were grown in the greenhouse for three months to monitor their adaptability before the harvest of tissues for the experiment. The samples and readings were taken at 30 °C at 11am for every reading.
Sample Processing for Enzyme Analysis 500 mg of tissue was harvested from illuminated fresh young leaves, tender pseudo bulbs, and aerial roots of the plants. Enzyme extraction was carried out at a temperature of 4 °C in a pre-chilled mortar and pestle. Complete maceration of the tissue sample was ensured before grinding in an appropriate volume of extraction buffer (1 ml). The components of extraction buffer included 50 mM HEPES-KOH (pH 7.3), 2.5 mM MgCl 2 , 2.5 mM MnCl 2 , 1 mM Na 4 -EDTA, 20% (v/v) Glycerol and 10 mM 2-Mercaptoethanol (Ueno et al. 2005). The homogenate was filtered and centrifuged at 9000×g for 10 min at 4 °C, and then the supernatant was collected. The crude extract was stored in ice until assays were performed. The aliquots were used for three separate analyses. The analyses performed were a) activity detection of three malate decarboxylation enzymes, such as NADP-ME, NAD-ME, PEPCK, b) total chlorophyll content and c) total protein content. The substrates and the chemicals were purchased from Sigma-Aldrich, USA.

Malate Oxidative Decarboxylation Efficiency
The enzyme activity was detected in Quartz cuvettes based on the change in the absorbance using UV-1800 UV-VIS Spectrophotometer (Shimadzu Scientific Instruments). The reaction was performed at 28 °C in a total volume of 1 ml of assay buffer in which 10 µl of the crude extract was analysed. The principle of detection of the enzyme activity was the formation of NAD (P) H or the depletion of NADH which was monitored at 340 nm. The stability of each buffer was analysed before the addition of enzymes. Malic enzymes (NADP-ME and NAD-ME) were assayed in decarboxylase direction coupled to the reduction of cofactor NADP + and NAD + respectively. The assay medium for NADP-ME comprised 50 mM Tris-HCl (pH 8.2), 1 mM Na 4 -EDTA, 20 mM MgCl 2 , 0.5 mM NADP + and 5 mM Na-malate (Ku et al. 1991). The composition of the assay mix for NAD-ME was slightly modified from the buffer proposed elsewhere (Chen et al. 1993;Muhaidat et al. 2007). NAD-ME buffer mix comprised 25 mM HEPES-KOH (pH 7.2), 0.75 mM Na 4 -EDTA, 20 mM KCl, 2.5 mM NAD + , 75 µM coenzyme A, 8 mM MnCl 2 , 5 mM Na-malate. The PEPCK was assayed in the carboxylation direction. In a two-reaction system, the formation of oxaloacetic acid (OAA) from phosphoenolpyruvate (PEP) by PEPCK was monitored by the conversion of OAA to malate by the enzyme malic dehydrogenase (MDH). This reaction oxidises NADH. Therefore, the decrease in the absorbance at 340 nm was detected. The PEPCK assay buffer components include 100 mM HEPES-KOH (pH 7.0), 1 mM ADP,100 mM KCl, 4% (v/v) 2-Mercaptoethanol, 90 mM NaHCO 3 , 5 mM PEP, 4 mM MgCl 2 , 10 µM MnCl 2 , 0.14 mM NADH and 6 units of Porcine Heart MDH (Sigma Aldrich, USA). The production of 1 µmol of NAD (P) H or depletion of 1 µmol of NADH per hour at 28 °C is defined as the 1 unit of enzyme activity, which was later expressed both in µmol.mg total chlorophyll −1 h −1 and also as total protein in µmol. mg total protein −1 h −1 .

Estimation of Total Chlorophyll and Total Protein Contents
A crude extract was aliquoted and was re-extracted with an equal volume of 100% acetone, and the absorbance was read using UV-VIS Spectrophotometer. Chlorophyll a showed maximum absorbance at 662 nm, and chlorophyll b showed maximum absorbance at 645 nm. Three concordant readings were taken for each wavelength and values of chlorophyll a and b were calculated based on the following equations: Chlorophyll a [Chl a = 11.75 A 662 -2.350 A 645 ]; Chlorophyll b [Chl b = 18.61 A 645 -3.960 A 662 ] (Lichtenthaler 1987). Total chlorophyll content was the additive value of Chlorophyll a and Chlorophyll b, and the expression unit was µg chlorophyll/g tissue of the plant. Total protein concentrations of the crude enzyme extracts were determined in a 96-well plate assay protocol by spectrophotometer using Bradford's Reagent (Sigma-Aldrich, USA).

Histomorphometry and Calculations for Assessing Photosynthetic Efficiency
Three replicates of young photosynthetic tissues were excised from each plant and were incubated in Farmers' fixative (3:1 ratio of 100% ethanol to glacial acetic acid). Transverse section of leaves, pseudo bulbs and aerial roots was chosen for freehand sectioning. Sections were stained with 1% safranin O and also with 1% toluidine blue. Sections were viewed using Motic BA400 microscope. Images were captured and measured using Motic Images Plus 2.0. In leaves, a reference frame of 100,000 µm 2 was set enclosing a vascular bundle. Due to their broadleaf in Rhynochostylis retusa and Vanilla planifolia, a reference area measured was 150,000 µm 2 . The entire cross-sectional area of the pseudo bulb and aerial root was measured. The parameters analysed are defined below: 1. Total Photosynthetic Carbon Reductive (PCR) Tissue: PCR tissue stands for the area of the bundle sheath (BS Area ), which encircles a vascular bundle (VB). PCR holds a circular dimension denoted as factor 'X'. Area of bundle sheath was calculated, by measuring the radius of the vascular bundle (R) inclusive of the BS. Then the radius of the vascular bundle excluding BS is measured (r). The area of BS is calculated using a formula: 2. Total Photosynthetic Carbon Assimilative (PCA) Tissue: The area of PCA stands for the zone occupied by mesophyll tissue in the case of leaves/zone occupied by the cortical region in the case of stem and pseudo bulb exclusive of the vascular bundle within the reference frame. PCA is calculated using the formula = (A reference − πR 2 ).

BS to VB ratio estimates the Photosynthetic Carbon
Reductive (PCR) Tissue which is calculated by the formula (πR 2 − πr 2 )/πR 2 . In case of multiple vascular bundles (for example in aerial roots and pseudo bulbs), the total number of vascular bundles were counted and were subjected to the formula mentioned above. 4. The ratio of the PCA/Area of Reference (RA), is labelled as (PCA/RA = Y). The ratio of the PCR/Area of Reference (RA), is marked as (PCR/RA). The ratio of Y to PCR gives a factor that normalises every tissue (leaf, aerial root, and pseudo bulb) under scrutiny, irrespective of their stage or different dimensions.

Statistical Analysis
To compare and understand the degree of dissimilarity between the quantitative variables, we performed a one-way ANOVA, and the variables were statistically evaluated using post hoc test viz., Bonferroni's Multiple Comparison test. The mean ± standard deviation & post hoc tests were computed using Graph Pad Prism version 5.01, using the triplicate readings obtained from three independent enzyme isolations and three different cross sections. We also performed a Principal Component Analysis (PCA) using R programme to correlate the activity of three decarboxylating enzymes and to identify the most significant anatomical variable.

Physiological Response Curves
The mobile photosynthesis measuring system used for the present study was LICOR (LI-6400XT Portable Photosynthesis System), USA. The Infrared Gas Analyser (IRGA) was connected first to the console with chemical cylinders in a complete bypass mode. The instrument was set to be in Light control settings and was allowed to warm up for a minimum of 15 min. The reference value set was 380 μmol mol −1 CO 2 , after which the stabilisation and matching of CO 2 were performed.

Light Response Curve
A 6400-02B LED light source was used with a constant CO 2 reference set at ambient at 380 μmol CO 2 mol −1 and flow fixed at 500 μmol s −1 . Temperature control set was at 35ºC for constant leaf temperature. Insertion of the leaf into the chamber was done first after matching CO 2 values, and the leaf area was finally adjusted. The stomatal ratio was kept constant at 0.5. Measurements were taken using a light response panel as follows: 2000, 1500, 1000, 600, 200, 50, 0, 10, 100, 400, 600, 1000, 1500 and 2000 μmol m −2 s −1 . At each set value, the leaf was allowed to adjust for 10 min. The values recorded were, net photosynthetic rate (A), intercellular CO 2 concentration (Ci) and transpiration rate (T), and graphs plotted were for photosynthetic rate and quantum yield. Triplicate readings taken from individuals of each species were used for computing the mean ± standard error.

CO 2 Response Curves
The 6400-02B LED light source was used and set at 650 μmol m −2 s −1 with a constant CO 2 reference set above ambient at 420 μmol CO 2 mol −1 and flow fixed at 300 μmol s −1 . The temperature set was at 35ºC. After matching CO 2 values, leaf sample was inserted into the chamber. The leaf area and stomatal ratio were kept constant. Measurements were taken using a CO 2 response panel of 400, 300, 200, 100, 50, 0, 100, 200, 300, 400 μmol CO 2 mol −1 . At each set value, the leaf was allowed to adjust for 5 min.
The values recorded were the net photosynthetic rate (A), intercellular CO 2 concentration (Ci) and A-Ci curve, as well as photosynthetic efficiency (PE) against time, were plotted. Measurements were taken from four individuals of each species in triplicates.

Real-Time Monitoring of Photosynthetic Competence in CAM Plants
To assess the real-time photosynthetic competence in orchids, we used the Farquhar approach (Farquhar et al. 1980) that spectacled a biochemical growth model which computes photosynthesis as a function of demand and obtainability of CO 2. The advantage of this model is that photosynthesis is regulated not only by radiation and transpiration but also by air humidity, leaf temperature, CO 2 availability and leaf nitrogen content. For a precise performance, driving variables need to be given to the simulation at least once per hour. In this model, the calculation of photosynthesis [A] is computed as mole carbon per leaf area per second. 'R' is the RuBisCO (photosynthetic enzyme) or carboxylation limited rate of assimilation, which is a function of light, leaf nitrogen and temperature and soil moisture. Photosynthesis, as per this model, works as a function of internal CO 2 concentrations and is calculated accordingly, Eq.

and
(1) Fig. 2 A Malate decarboxylating activity based on total protein content. The enzyme activity of malate decarboxylating enzymes a PEP-CK, b NADP-ME and c NAD-ME, reported in µmol.mg protein hr.Values depicted indicate the mean ± standard deviation. The relative enzyme activity was statistically validated using oneway ANOVA with and the significance between groups was verified using Bonferroni's multiple comparison tests. The abbreviated letters stand for the parts of the plants selected for enzyme evaluations (L leaf, AR aerial root, PB pseudo bulb). d Principal Component Analysis plot depicting the enzyme dominance based on total protein content. B Malate decarboxylating activity based on total Chlorophyll content. The enzyme activity of malate decarboxylating enzymes a PEP-CK, b NADP-ME and c NAD-ME, reported in µmol.mg chlorophyll hr.Values depicted indicate the mean ± standard deviation. The relative enzyme activity was statistically validated using one-way ANOVA with and the significance between groups was verified using Bonferroni's multiple comparison tests. The abbreviated letters stand for the parts of the plants selected for enzyme evaluations (L Leaf., AR Aerial root, PB Pseudo bulb). d Principal Component Analysis plot depicting the enzyme dominance based on total chlorophyll content ◂ V max is the function of the maximum activity of RuBisCO. C c is the chloroplast CO 2 concentration. Γ* is the CO 2 compensation point in the light in the absence of mitochondrial respiration. K c is the Michaelis-Menten constant of RuBisCO for CO 2 , O is the oxygen concentration in the atmosphere, and K o is the Michaelis-Menten constant of RuBisCO for O 2 .

Profiles of Decarboxylation Enzymes Depicting the CO 2 Reduction Efficiency of Tropical Orchids
The enzymes NAD-ME, NADP-ME and PEPCK were seen in the C3, C4, and CAM control plants. The C3 control plant Oryza sativa and Spathoglottis plicata have a higher PEPCK activity than NADP-ME and NAD-ME ( Fig. 2A). The results indicated that the C3 plant used more PEPCK than NADP-ME and NAD-ME for decarboxylation of malate to pyruvate. In C4 control, Saccharum officinarum, NADP-ME was a dominant enzyme assisted by decarboxylation enzymes PEPCK and NAD-ME. Aloe vera, the CAM control, displayed predominant PEPCK activity closely followed by NAD-ME.
In chlorophyll-based enzyme activity calculations, highest PEPCK, NADP-ME, and NAD-ME activity were observed for homogenates of Pholidota pseudo bulbs and Vanilla pseudo bulb and leaf (Fig. 2B).
In total protein content-based enzyme activity, Pholidota leaf showed the highest NADP-ME activity, whereas Bulbophyllum pseudo bulb showed higher PEPCK activity. NAD-ME activity was highest in Bulbophyllum leaf and pseudo bulb. Only three tissues found in the eight orchids studied: Bulbophyllum leaf, Dendrobium aerial roots and Vanda spathulata aerial roots showed NAD-ME predominance. The remaining homogenates, when assayed, showed high levels of PEPCK independently or in combination with NADP/NAD-ME.
PEPCK dominance was observed in Rhynchostylis aerial root, Dendrobium leaf, Bulbophyllum pseudobulb, and the aerial root of Rhynchostylis and Vanilla. The PEPCK activity detected in leaf homogenates of Pholidota and Vanilla displayed a similar trend as that of CAM control Aloe. Pseudobulb homogenates of Pholidota and Bulbophyllum exhibited a closer resemblance to Saccharum officinarum (Table 2).
NADP-ME activity in leaf homogenates of Pholidota was on par with Saccharum officinarum. Homogenate of the elongated aerial root of Vanilla showed higher (2) R = V max for C4 plants.

Variables in Quantitative Histomorphometry that Aided in the Classification of Photosynthetic Subtypes
The ratio 'Y/PCR' could segregate CAM test species closer to C4 and C3 test plants, thus eliminating confounding anatomical dissimilarities (Fig. 4). The leaf thickness assessment and comparison (Fig. 4A) between the plant groups revealed that Sarcanthus has the highest midrib thickness, followed by Acampe and Vanda. All other species were closer to the CAM control plant Aloe vera.
All the test plants, irrespective of the leaf (Fig. 3A), aerial root ( Fig. 3B) or the pseudo bulb (Fig. 3C), comprise a tight wadding of cells to diminish the intercellular air spaces. The mesophyll region of the leaf tissue and cortical area of the pseudo bulb and aerial root were packed with large vacuoles for storing the C4 acid (malic acid or oxaloacetic acid). Bundle sheath cells encircling the vascular bundles (Fig. 3D) were highly condensed, indicating their water use efficiency is relatively more efficient.
The Y/PCR ratio was the highest for Oryza, followed by Saccharum. The PCA plot could precisely segregate plants with similar histomorphometric features of C3, C4 and CAM control groups (Fig. 4A-F

Evaluating the Photosynthetic Efficiency of CAM Tropical Orchids Using Light-Responsive Curve Plots
In our study, the typical CAM Aloe vera showed a light saturation point well below Oryza sativa [ Fig. 5A(a)]. It is evident from Fig. 5A(a) that the orchids exhibited negative photosynthetic rates. Of these adverse photosynthetic rates cluster, only the C3 orchid control Spathoglottis plicata displayed a positive photosynthetic rate [ Fig. 5A(b)], resembling the C3 and C4 control plants. Unusual behaviour was observed for Pholidota pallida, which stood out from the ordinary test plant clusters. Pholidota pallida demonstrated a negative photosynthetic rate.
The C4 test plant Saccharum officinarum exhibited high photosynthetic rates in our study. It is clear from Fig. 5B that in C4 type plants, light saturation point is higher than the C3 and the light compensation point for C4 is lower than the C3 plants. Both of these characteristic features relate to the ability of C4 plants to provide an increased amount of CO 2 availability to the Calvin-Benson cycle. We surveyed quantum yield in all eight orchid species and four controls of distinct photosynthetic types (Fig. 5B, C) and calculated the lightlimited quantum yield from CO 2 assimilation. We attempted to eliminate photo inhibitory conditions. The cause of low quantum yield is attributed to photorespiration and alternative electron sinks. Several physiological processes divert reducing power from carbon reduction, which accounts for evident variations in ΦCO 2 in the orchid population.

Assessing the Photosynthetic Efficiency of CAM Tropical Orchids Using Quantum Yield
Photosynthetic efficiency is defined as the segment of light energy converted to chemical energy during photosynthesis and is regarded as the rate of net photosynthesis at a particular time point. It is also represented as the net photosynthetic rate against time (Fig. 5A). The linear region of each light response curve varied considerably amongst the species. Thus, the current study determined the range of photosynthetically active radiation (PAR) valid for ΦCO2 measurements by individually evaluating each light response curve. The data are represented in Fig. 5A(a). We plotted PE in the present study using 'A' against time, as depicted by [ Fig. 5A(b)]. Highest PE was observed in Saccharum officinarum, followed by Oryza sativa and Spathoglottis plicata.
Saturation of CO 2 and light beyond 30 min gave a positive value for PE for the C3 plant, reaching a value almost close to C4. However, this was not observed in the CAM cluster. Comparative quantum yield values were represented in a bar diagram (Fig. 5C), where the values were tabulated and displayed as an inset. From the quantum yield statistics, it is Pseudobulbs are clustered and appears to be smooth and 4 angled. It bears long, broad leaves that are tough. Inflorescence bears creamy white flowers arranged in 2 or 3 rows

Rhynchostylis retusa
Leaf & aerial root Exotically blooming orchid. The inflorescence is a pendant raceme, consisting of more than 100 pink-spotted white flowers. The plant has a short, stout, creeping stem carrying up to 12, curved, fleshy, deeply channelled, keeled, retuse apically leaves and blooms on an axillary pendant to 60 cm long, racemose, densely flowered, cylindrical inflorescence that occurs in the winter (December) and early spring (June)

Sarcanthus pauciflorus
Leaf This species is a slender, pendulous orchid with linear, straight and falcately curved leaves that is narrowed at the base. Bears inflorescence (racemes) shorter than the plant body. Flowers are yellow with red margin and are found growing on tree trunks Spathoglottis plicata Leaf They are evergreen plants with crowded pseudobulbs just below the surface of the ground. The 3-lobed labellum projects forward with an extended mid-lobe which allows insects to land. Both sepals and petals are broad and the sepals are hairy on the outside. The inflorescence arises amongst sheaths from a recently matured pseudobulb

Vanda spathulata
Leaf & aerial root The species has a monopodial growth habit with highly variable leaves according to habitat. They have flat, strap-leaves to circumvent dry seasons. They are miniature plants which turn out to be quite massive with very large, rambling aerial roots

Vanilla planifolia
Leaf, pseudobulb & aerial root Grows as a vine, climbing up an existing tree (also called a tutor), pole, or other support. It can be grown in a wood (on trees), in a plantation (on trees or poles), or in a "shader", in increasing orders of productivity evident that Saccharum officinarum (C4) is photosynthetically efficient. Quantum yield is a confounding factor for photosynthetic competence, and from the C3 control, the C4 control displayed almost a three-fold increment in quantum yield (Fig. 5B). Spathoglottis plicata also showed a similar quantum yield value to C3 Oryza sativa. Spathoglottis plicata was superior in photosynthesis and was distinct from other CAM types. Saccharum officinarum, Oryza sativa and Spathoglottis plicata and the clustering of CAM displayed similar trends in net photosynthetic rates (Fig. 5D), relative quantum yield (Fig. 5 C) and photosynthetic efficiency (PE) [ Fig. 5A(b)]. Since the trend is the same in all the three calculations, we overruled underestimation/ overestimation of ΦCO 2 .

Gauging Net Carbon Assimilation Rate of CAM Tropical Orchids Using A-Ci Curves
A-Ci curves are the function of net carbon assimilatory rate [A] versus calculated sub stomatal CO 2 concentration [Ci]. As per the Farquhar model, the action of RuBisCO was calculated as depicted in Fig. 5E. The plants can be grouped into three clusters: one was exhibiting high RuBisCO activity, which includes Oryza sativa, Saccharum officinarum, and Pholidota pallida; the second bunch with a moderate RuBisCO activity displayed by Spathoglottis plicata, Sarcanthus pauciflorus and Aloe vera and the third cluster including the remaining species presenting low RuBisCO activity.

Tropical Orchids and the Factors That Contribute to Their Effective Decarboxylation
Malate decarboxylation efficiency in tropical orchids indicated that PEPCK is the dominant decarboxylation enzyme whose efficiency is reinforced by the activity of NADP-ME or NAD-ME. The enzyme analysis proved that Pholidota leaves displayed high decarboxylation efficiency and shared similar traits with Saccharum. Studies suggested that high NADP-ME activity indicates high RuBisCO activity (Ghannoum et al. 2005). We expected that Pholidota leaf, Acampe aerial root, and Vanda leaf would be the most tolerant species to display high photosynthetic efficiency based on the enzyme activity. The quantitative parameters, such as vein density, intercellular airspaces, and epidermal tissue proportions, can discriminate between C3 and C4 species but not within C4 subtypes (Muhaidat et al. 2007). CAM plants always displayed an enigmatic light-responsive curve, occasionally behaving very close to C4 and sporadically falling below C3. The CO 2 exchange in CAM species is based on at least four distinct phases of autotrophic and heterotrophic CO 2 fixation biochemistry (Osmond 1978). CO 2 fixation processes are expected to quicken at the low light intensity and infrequently pose difficulty in computing the light-limited rate of photosynthesis using net CO 2 fixation (Osmond et al. 1979).

Indications from Stomatal Behaviour and Quantum Yield
Stomata also tend to be closed during the de-acidification process, resulting in negligible CO 2 fluxes. Stomatal closure can lead to negative photosynthetic rates. The diversity in the photosynthetic light response curve in orchids chosen for the study is attributed to various factors. The factors that affect the light-responsive curves include-(a) location of the leaf (horizontal or vertical) and its phyllotaxy, (b) shape of the leaf, (c) area of the leaf (broad or narrow), (d) the number of stomata in the upper (adaxial) and the lower (abaxial) surfaces and (e) photosynthetic enzymes and their behaviour (Evans 1993). Photosynthesis in the intermediate light range (600-700 μmol m −2 s −1 ) is the most efficient region where the convexity of the photosynthetic light response curve is maximum.   In our study, the quantum yield of photosynthesis is chosen as a measure of photosynthetic efficiency expressed in moles of PAR absorbed per mole of CO 2 fixed. Depending on the measurement conditions and techniques adopted, Φ measurements ideally reflect different aspects of the photosynthetic apparatus. The maximum quantum yield (Φ max) was obtained when photosynthesis is lightlimited, a state inferred by a linear relationship between photosynthesis and PAR. Theoretically, when Φ max = x, x moles of photons are required to reduce one mole of CO 2 without photorespiration. Because there is some cyclic photophosphorylation operational, Φ maxima are inferred to be closer to 0.112 in most plants (Long et al. 1993).

Evidence Obtained from Light Response Curves on CAM Photosynthetic Efficiency
Some plants, predominantly wild genera growing in a compromised milieu, may intrinsically display low photosynthetic efficiency (Singsaas et al. 2001). It is evidenced that the higher the quantum yield, the higher will be the photosynthetic efficiency. Quantum yield was calculated from the photosynthetic light response data using linear regression across three or more points, where net CO 2 assimilation was linearly related to PAR (von Caemmerer and Farquhar 1981). Photoinhibition is a light-induced reduction in photosynthesis, which may either result from photodamage or photoprotective chloroplast processes. These could probably be induced by the growth locale of the plant or by the experimental protocol (Bongi and Long 1987;Groom and Baker 1992), often caused by shallow temperatures or excess PAR (Sharkey 1988).
The largest of these sinks is photorespiration, which usually occurs at 10-30% of the photosynthesis rate at 25 °C with 350 μmol mol −1 CO 2 at 21% oxygen (Sharkey 1988;Peterson 1990;Yeo et al. 1997). This sink can be eliminated by measuring ΦCO 2 under a reduced 1-2% O 2 atmosphere. Many plant physiologists suggest that photorespiration cannot adequately define variance in quantum yield. Since photoinhibition and photorespiration cannot clarify the variation in quantum yield, we based our analysis of the photosynthetic light response curve on the calculation of Φ max (Singsaas et al. 2001).

Photorespiration Influences Photosynthetic Energy Transduction
The primary difference between C3 and C4 plants is that photorespiration occurs in C3 plants, even at high levels of CO 2 . Leverenz (1987) suggested that the light-responsive curve can be subdivided into four regions-the first is the Kok Effect region, where the mitochondrial respiration increases at minimal light. We observed a similar effect in the present study, where we found that < 10 μmol m −2 s −1 PAR affected net photosynthesis and thereby increased the slope of the light-responsive curve. The second is the region, where net photosynthetic rate data points are aligned in linear mode with increasing PAR. The third is the transition region between the linear and light-saturated region, and the fourth and the last are the light saturating region.
When the photosynthetic rate is just about to take a linear phase, it implies that light is restrictive and respiration is constant for calculating Φ max. It is often seen that the inclusion of data points from the Kok Effect region would cause an overestimation of ΦCO2. In contrast, the inclusion of data points from the transition region would cause an underestimation of Φ CO 2 . It is suggested that improvement in PE played only a minor role in increasing productivity achieved in the last half-century (Zhu et al. 2010). However, assessment of PE remains a crucial factor because it portrays inadequacies in photosynthetic energy transduction. The immediate goal includes improving the demonstration of leaves in flora (notably in crops and threatened species) to evade light saturation and additional investigations on the successful photorespiratory bypass, which demonstrated enhanced productivity in model species.

Variables that Affect CAM Photosynthetic Efficiency
The analysis and interpretation of the A-Ci curve depend on some underlying assumptions (Farquhar et al. 1980). 'A-Ci' curve (assimilation rate plotted against intercellular CO 2 concentration) can provide some insights into the biochemistry of a leaf or plant. For example, the value of Ci where photosynthesis and respiration are equipoised is designated as the CO 2 compensation point. The initial data points of slope deliver an in vivo measure of the activity of RuBisCO in the leaf suggests the plants' carboxylation efficiency, termed 'mesophyll conductance.' The reaction function of 'A-Ci' curve represents the mechanical basis behind many models reported to date in plant physiology (Manter et al. 2003;Harley et al. 1992). According to Farquhar et al. (1980), carboxylation rates are regulated by three processes. These are (a) amount, activity and kinetics of RuBisCO, (b) rate of RuBP restoration sustained by the electron transport system, and (c) infrequent availability of Triose Phosphate. The A-Ci curve gives values on estimates of the maximum rate of RuBisCO-mediated carboxylation is defined merely as RuBisCO activity.
In the present study, nonlinear regression techniques, based on the equations of Farquhar et al. (1980), which were later modified in some studies (Sharkey 1985;Harley and Sharkey 1991), were used to estimate the maximum rate of RuBisCO activity for each A-Ci curve. Our results indicated that although 'A' is a primary confounding factor for RuBisCO activity, it is not the sole variable that controls it. Besides 'A', mesophyll conductance (g m ) also plays a critical role in determining the RuBisCO activity. Our study indicated low ΦCO 2 for the CAM cluster compared to C3 and C4, which could be attributed to photorespiration. We adopted the C3 Farquhar model for computing RuBisCO activity for all plants except Saccharum officinarum-the C4 control. RuBisCO exists both in storage and functional form in plant systems. Since RuBisCO might exist as a storage protein and in inactivated form (Cheng and Fuchigami 2000;Warren et al. 2000;Warren et al. 2003), its activation is probably restricted in CAM plants due to the time the readings were taken. RuBisCO activity is highest during the third phase in CAM plants, whilst the readings were taken in the second phase.
Tropical Orchids' Ability to Mitigate CO 2 and the Associated Causes Fischer and Turner (1978) hypothesised that in dry habitats, natural selection would favour plants with high water use efficiency. The Y/PCR ratio (factor 'X') as per the histomorphometric analysis agrees with the photosynthetic measurements and enzyme studies. The thriving ability of CAM orchids, notably in Acampe praemorsa, Bulbophyllum neilgherrense and Vanilla planifolia, is due to its high WUE. These orchids cover the canopies of the forest land and play a crucial role in retaining the fixed CO 2 much longer than the C4 groups. The data derived from tissues, such as the aerial root, the pseudo bulb, and the leaf lamina, prove efficient carbon retention by orchids. This feature tags them as right candidates for relocating in areas where mitigation of greenhouse gases is of paramount importance. High adaptability of CAM plants to CO 2 enriched areas is termed as 'Downregulation' as it negates photosynthetic apparatus, which is visualised as low RuBisCO activity. However, they possess a functional PEPC (Phosphoenolpyruvate carboxylase) which displays a higher affinity for CO 2 than RuBisCO. The PEPC-driven CAM mechanism provides them with an intrinsic ability to mitigate CO 2 superfluously. CO 2 influx results in nocturnal acidification of mesophyll cells, the transpiration ratio is 3-10-fold with an innate ability to evade ROS-mediated damage.

Conclusion
The present study emphasises the significance of the widespread prevalence of tropical orchids in the Western Ghats and also Orchidaceae being the second largest plant family in the globe. There is a demand for increased crop production brought about by the rise in population. Emissions from agronomy necessitate novel strategies to incentivize sustainable agronomic practices (Godfray and Robinson 2015;Carlson et al. 2016). Total CO 2 influx through Saccharum crops in an area of 68,541 ha, monitored at 2006/2007 crop season with 5,133,212 tonne of CO 2 , came to approximately 74.9 tonne of CO 2 ha −1 (de Figueiredo et al. 2010). However, emissions from post-harvest burning resulted in CO 2 efflux that often exceeds the carbon influx. The present study indicated that propagation of CAM topical orchids, such as Vanilla planifolia, Dendrobium Sonia (white variety), Bulbophyllum neilgherrense, Acampe praemorsa, Pholidota pallida on trees and hedges, would alleviate GHG emissions, notably CO 2 , in a micro-niche equivalent to Saccharum. Orchids being an ornamental group, there is no apprehension of postharvest carbon efflux as well. Increasing the area apportioned for essential indigenous plant species to prevent competition from non-native plants could help local carbon assimilation. Because of high water use efficiency, propagation of CAM orchids is one of the winning landscaping strategies that ecologists could adopt to mitigate CO 2 in a micro-ecological niche showing steady temperature rise.