Introduction

Capturing atmospheric carbon dioxide (CO₂) using photosynthesis is crucial for the production of plants (Friend et al. 2009). Future changes in the global climate should affect the key processes involved in photosynthetic productivity. CO₂ enrichment promotes growth in cultivated plants. Increased CO₂ uptake hastens growth and facilitates the production of some combination of more and larger plant leaves, branches, flowers, and fruits. This greater yield occurs because increased CO₂ concentrations promote photosynthesis (Dong et al. 2016;Nederhoff 2004). CO₂ concentrations are higher during cold temperatures when the greenhouse is not vented during the nighttime and early morning hours (Reddy et al. 2010). Crassulacean acid metabolism (CAM) is a process of fixation of atmospheric CO₂ during nighttime and early morning hours in some plants. CO₂ has positive and negative effects on CAM plants. Biomass increases by 35% in CAM plants exposed to enriched CO₂ treatment (Drennan and Nobel 2000;Xu et al. 2014). Increasing CO₂ concentrations from 350 to 650 μmol·mol^-1 in CAM plants increases the dry weight production of Agave deserti, Ferocactus acanthodes, and Kalanchoe hybrids (Endo and Ikusima 1997). Compared with plant responses at 380 μmol·mol^-1 CO₂, Selenicereus megalanthus (CAM plant) had 129%, 73%, and 68% increases in total daily net CO₂ uptake, shoot elongation, and shoot dry mass, respectively, at 1,000 μmol·mol^-1 CO₂ (Weiss et al. 2010). However, growing Doritaenopsis at 700 – 1,000 μmol·mol^-1 CO₂ was not more effective than growing the plants at atmospheric CO₂ conditions during the vegetative growth stages (Endo and Ikusima 1997). The excessive carbohydrates produced by elevated CO₂ leads to photosynthetic acclimation, which limits production. The biomass of the CAM plant Ananas comosus grown at 712 μ mol·mol^-1 CO₂ was decreased by 10% compared with that of plants grown at atmospheric CO₂ (Drennan and Nobel 2000).

The CAM plant Phalaenopsis is a popular and valuable potted ornamental orchid because the grower can easily schedule growth to meet specific market dates (Lu et al. 2016). It has attractive and long lasting flowers in a variety of colors, sizes, shapes and a high wholesale value. During vegetative growth, cultivation of Phalaenopsis requires temperatures above 28°C for 18 – 20 months for leaf development (Cho et al. 2019;Wang 2007); the high temperature (> 28°C) also inhibits flowering (Blanchard and Runkle 2006). This long production time is expensive for commercial producers because every stage requires use of a controlled environment (Holley 2016). During vegetative growth, rapid production of new shoot and leaf production is essential (Tremblay and Gosselin 1998). CO₂ enrichment in the greenhouse nursery might be used as a method to shorten the plant growth period.

Plant ages can be divided into juvenile, young, and mature stages of vegetative growth in Phalaenopsis (Guo and Lee 2006). Different cultivation conditions are required at different growth stages. Commercial growers usually purchase immature plants contained in flasks. At the juvenile stage, 1- to 20-week-old Phalaenopsis are not competent to flower, even at flower induction conditions of temperatures < 26°C (Hong 2017;Lee et al. 2015). At the young stage, small bare-root plants of 24-week-old Phalaenopsis can produce flowers at relatively low temperatures (i.e., < 26°C). At the mature stage, 36-week-old Phalaenopsis (20 cm leaf span) are transplanted into the final 12 cm pots for continued vegetative growth (Blanchard et al. 2007). Growth responses are different depending on the species and plant growth stages. In Cymbidium, rates of photosynthesis in 1-year-old plants are greater compared with rates in 2-year-old plants grown at the same CO₂ concentrations (Kim et al. 2013).

In previous study, 36-week-old Doritaenopsis (Phalaenopsis × Doritis) grown under 800 and 1,600 μmol·mol^-1 CO₂ had increased net CO₂ uptake and leaf initiation compared to the plants grown under 450 μmol·mol^-1 CO₂ concentration (Yun et al. 2018). Leaf span was lower in the 36-week-old Doritaenopsis grown under the 800 and 1,600 μmol·mol^-1 CO₂ concentration compared to the plants grown under 450 μmol·mol^-1 CO₂. However, no reports have been published on the effects of growth and photosynthetic response on the different growth stages in vegetative period of Phalaenopsis. In present study, we determined the photosynthetic characteristics and growth responses of 2-, 24-, and 36-week-old (i.e., juvenile, young, and mature vegetative stages, respectively), Phalaenopsis Queen Beer ‘Mantefon’ at difference CO₂ concentrations.

Materials and Methods

Plant materials and growth conditions

Phalaenopsis Queen Beer ‘Mantefon’ was propagated by tissue culture, 2-, 24-, and 36-week-old plants were purchased from Orchid Nursery (Goyang, Korea; 38°N latitude, 127°E longitude). The 2-week-old Phalaenopsis was cultivated after deflasking. Plants had one leaf (mean, 3.0 cm width, 6.8 cm length, and 6.8 cm leaf span) at 2 weeks, two leaves (mean, 3.3 cm width, 9.0 cm length, and 15.2 cm leaf span) at 24 weeks, and three leaves (mean, 5.0 cm width, 9.6 cm length, and 20.1 cm leaf span) at 36 weeks. The 2-, 24-, and 36-week-old plants were transplanted into their respective 6 cm, 6 cm, or 12 cm diameter pots filled with sphagnum moss (Lonquen Ltda., Puerto Montt, Chile). The air temperature was maintained at 29°C with 70% relative humidity. Three wave cool white fluorescent lamps (Phosphor fluorescent lamp, Dulux L 36W, Osram Co., Seoul, Korea) were used to maintain the photosynthetic photon flux at 160 μ mol·m^-2·s^-1 during 06:00 - 18:00 h at the plant canopy. The plants were fertigated by hand once every 10 days with solution of water-soluble fertilizer (electrical conductivity (EC) 1.0 ± 0.1 dS·m-1, pH 6.0 – 6.5; Newgold 20-8.7-16.7 (N-P-K), Neufarm Co., Ltd., Munster, Germany). All plants were grown in growth chamber (HB-303DHC, Hanbaek Scientific Co., Suwon, Korea).

CO₂ treatments

The CO₂ concentration was maintained using automated continuous injection of pure CO₂ at 400 (control, atmospheric CO₂), 800, or 1,600 μmol·mol^-1 CO₂. Purified CO₂ (CO₂ cylinders, Seoul Gas Co., Ltd, Seoul, Korea) was supplied through flowmeters at a rate of approximately 50 mL/min. The CO₂ treatments were applied for 6 hour (00:00 – 06:00 h) dark periods for 32 weeks. The CO₂ concentration in the chamber was monitored at 1 min intervals using automatic sensor (Air Quality Monitor Am^-21, Wise Sensing Inc., ‘Younin’, Korea).

Growth measurements

Time to leaf initiation from the start of the CO₂ treatment was measured when the first visible leaf was 0.5 cm long. Leaf number longer than 0.5 cm were counted for each plant every 4 weeks. Leaf length was measured for the first leaf initiated after CO₂ treatment began. Leaf span was measured as the longest length from one leaf tip to the opposing leaf tip. Number of leaves, leaf length, and leaf span were measured for 32 weeks after the CO₂ treatment began. A completely randomized design with 8 plants per treatment was used for the growth measurements.

Photosynthetic characteristics

Net CO₂ uptake, stomatal conductance (g_s), and transpiration rate (t_r) were measured at 01:00 h (nighttime) and 11:00 h (daytime), r espectively. M easurements were t ak en w ith a portable photosynthesis system (Li-6400XT, Soldan Co., Seoul, Korea) using the instrument’s built-in ‘auto-program’ function. The atmospheric CO₂ concentrations in the plant growth chambers were approximately 400, 800, or 1,600 μ mol·mol^-1, and the same amounts of CO₂ were supplied into the leaf chambers. Photosynthetic characteristics were measured at uppermost mature leaf onto 6 cm² leaf chamber for each plant, with a light condition of 160 μ mol·m^-2·s^-1. Measurements were obtained at a 29°C leaf temperature and at 300 μmol·s^-1 flow rate. Net CO₂ uptake, gs, and tr of the 24- and 36-weeks old plants were measured at 12 weeks after the CO₂ treatments began. Due to small leaf s ize of 2 -week -old p lants, n et CO₂ u ptak e, g_s, and t_r were recorded at 32 weeks. Each measurement followed a completely randomized design with three replications per treatment.

Statistical analysis

Statistical analyses were performed using SAS system for Windows version 9.3 (SAS Inst. Inc., Cary, NC, USA). Differences among treatment groups were assessed using Tukey’s honestly significant difference (HSD) tests. p-values < 0.05 were considered to indicate statistically significant results. Graph module analyses were performed using Sigma Plot version 10.0 (Systat Software Inc., San Jose, CA, USA).

Growth parameters of leaves

The time to leaf initiation of 2- and 36-week-old plants was reduced to 35.0 and 55.0 days in plants grown at 1,600 μmol･mol^-1 CO₂, compared with the control plants, 58.8 and 67.7 days, respectively. (Figs. 1A and C). In 24-week-old plants, time to leaf initiation was reduced to 39.6 and 38.0 days in plants grown at 800 and 1,600 μmol･ mol^-1 CO₂, compared with the controls (58.4 days) (Fig. 1B). After 28 weeks, leaf numbers in 2- and 24-week-old plants grown at 1,600 μmol·mol^-1 CO₂ were significantly greater compared with that of plants grown at 400 μ mol·mol^-1 CO₂ (Figs. 2A and B). In 36-week-old plants, the numbers of leaves in plants grown at 800 and 1,600 μ mol·mol^-1 CO₂ were significantly greater compared with plants grown at 400 μmol·mol^-1 CO₂ at 20, 28, and 32 weeks (Fig. 2C). Leaf initiation and leaf number were promoted when plants were grown at the 800 - 1,600 μ mol·mol^-1 CO₂ range, regardless of differences in vegetative growth stage. Our previous study revealed that 60-week-old Phalaenopsis grown at 1,600 and 2,400 μ mol·mol^-1 CO₂ have significantly enhanced leaf numbers, leaf lengths, and leaf widths compared with plants grown at 450 and 800 μmol·mol^-1 CO₂ (Kim et al. 2017). More leaves are produced in plants grown at 800 and 1,600 μ mol·mol^-1 CO₂ compared with plants grown at 450 μmol･ mol^-1 CO₂, and leaves are initiated at a faster rate at 1,600 μmol·mol^-1 CO₂ in 36-week-old Doritaenopsis (Yun et al. 2018). Elevated CO₂ concentrations promote growth in cultivated plants (Friend et al. 2009). CO₂ stimulates cell division and cell expansion, which has been associated with increased plant growth rates (Huang and Xu 2015;Pritchard et al. 1999). Exposure to elevated CO₂ concentrations results in shorter cell cycling in plants in both the root and shoot meristems (Kinsman et al. 1997). Shortened cell cycling appears to be supported by increased transport of assimilates and carbohydrate availability in shoot apical meristems. Compared with plants in the control group, plants grown at 1,600 μmol·mol^-1 CO₂ conditions had greater leaf numbers; exposure to the elevated CO₂ concentration likely resulted in shortened cell cycling. New leaves in plants grown at 1,600 μmol･mol^-1 CO₂ also had faster initiation; the time to leaf initiation of plants grown at 1,600 μmol･mol^-1 CO₂ conditions was shorter than in plants grown at 400 μmol･mol^-1 CO₂ conditions (Figs. 1 and 2A - C). Leaf length was longer in plants grown at the 1,600 μ mol·mol^-1 CO₂ conditions compared with plants grown at 400 μmol･mol^-1 CO₂ conditions until 16 weeks of CO₂ treatments in the 2-, 24-, and 36-week-old plants (Figs. 2D - F). After 16 weeks, leaf length was longer in the 2-, 24-, and 36-week-old plants grown at 400 μmol･mol^-1 CO₂ compared with plants grown at 1,600 μmol･mol^-1 CO₂. The growth of leaf length that was exposed to high CO₂ concentration stopped after 16 weeks. The reason of the low growth rate of leaf length after 16 weeks is that higher CO₂ concentration conditions promote new leaf development rather than the growth of leaf length. After 28, 20, and 16 weeks, leaf span in 2-, 24-, and 36-month-old plants grown at 400 μmol·mol^-1 CO₂ was greater than in plants grown at 800 or 1,600 μmol·mol^-1 CO₂ (Figs. 2G - I). Leaf span of 2-week-old plants were not statistically significant differences among different CO₂ conditions until 24 weeks (Fig. 2G), because leaf length of plants in flask was longer than 2-week-old plant grown under 800 or 1,600 μ mol·mol^-1 CO₂ treatment (data not shown).

Plants can be divided into source and sink components (Hofius and Börnke 2007). The source components are the locations where net fixation of CO₂ occurs (e.g., older leaves). The sink components are the sites where assimilates are stored or used (e.g., younger leaves, roots, and fruits). Increased carbohydrate production associated with elevated CO₂ exceeds the plant’s capacity to produce new sinks as it matures, and net photosynthetic rates decline to balance source activity and sink capacity (Thomas and Strain 1991). Acclimatization to elevated atmospheric CO₂ concentrations, as is observed for many species, is usually attributed to altered source-sink relationships. Accumulation of excess soluble sugars and starch results in feedback inhibition of photosynthesis. Feedback inhibition may result in sugar and phosphate build-up, and reduced rates of starch and sucrose synthesis. A build-up of sugars may also suppress ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and result in a lowered photosynthetic rate. An imbalanced source-sink might indicate that the amounts of nutrients were inadequate to support photosynthesis and growth. Nutrient limitation is more likely to disrupt the balance between source and sink in plants growing under conditions of elevated CO₂ concentrations (Baker and Thomas 1992). To alleviate nutrient deficiencies, plants should be supplied with different nutrients as CO₂ concentrations vary. The small leaf span in Phalaenopsis grown at greater CO₂ concentrations will be affected by nutrient deficiencies. The Phalaenopsis were fertigated using water-soluble fertilizer at EC 1.0 d·s^-1, regardless of the different CO₂ concentrations. In this study, the Phalaenopsis ‘Mantefon’ grown at a range of 800 - 1,600 μmol·mol^-1 CO₂ conditions had stimulation of leaf initiation, but the effect diminished with time and smaller-sized leaves resulted.

Photosynthetic characteristics

In 2-, 24-, and 36-week-old plants, during the nighttime hours net CO₂ uptake was 1.95 to 5.88 μmol·m^-2·s^-1 compared with -0.54 to 0.61 μmol·m^-2·s^-1 during the daytime (Figs. 3A - C). All vegetative growth stages of Phalaenopsis ‘Mantefon’ performed CAM (i.e., CO₂ u ptak e during the nighttime). Net CO₂ uptake in 2-week-old plants grown at 1,600 μmol·mol^-1 CO₂ conditions was significantly greater than in plants grown at 400 μmol·mol^-1 CO₂ conditions at nighttime (Fig. 3A). In 24-week-old plants, net CO₂ uptake was significantly greater under 800 - 1,600 μ mol·mol^-1 CO₂ conditions compared with plants grown at 400 μmol･mol^-1 CO₂ conditions at nighttime (Fig. 3B). Net CO₂ uptake in 36-week-old plants grown at 800 μ mol·mol^-1 CO₂ was significantly greater than in plants grown at 400 μmol·mol^-1 CO₂ (Fig. 3C). During daytime, net CO₂ uptake in 2-, 24-, and 36-week-old plants was not significantly different between different CO₂ concentrations (Figs. 3A - C). During photosynthesis, CO₂ is fixed and reduced to carbohydrate for plant growth (Huang and Xu 2015).

Increasing photosynthesis can be due to the greater carboxylation capacity in elevated CO₂ conditions. The CAM plant Selenicereus megalanthus grown at 1,000 μmol·mol^-1 CO₂ has a 129% increase in total daily net CO₂ u ptak e compared with plants grown at atmospheric CO₂ concentrations (Weiss et al. 2010). The net CO₂ u ptak e increased with older plant growth stages; 2-, 24-, and 36-week-old plants grown at 400 μmol･mol^-1 CO₂ had 1.95, 2.30, and 3.50 μmol·m^-2·s^-1 uptake, respectively, at nighttime (Figs. 3A - C). Because of the increased net CO₂ uptake with older plant growth stages, in 2-, 24-, and 36-week-old plants, 3.3, 5.6, and 6.8 leaves, respectively, were produced at 400 μmol･mol^-1 CO₂ by 32 weeks (Figs. 2A - C). During nighttime, net CO₂ uptake in Phalaenopsis TS97 is lower in juvenile plants compared with young and mature plants (Guo and Lee 2006). In CAM plants, increases in net daily CO₂ uptake under elevated atmospheric CO₂ conditions reflect increases in both Rubisco-mediated daytime CO₂ uptake and phosphoenolpyruvate carboxylase-mediated nighttime CO₂ u ptak e; t he l atter results in increased nocturnal malate accumulation (Drennan and Nobel 2000). In 2-week-old plants, gs and tr were higher in the plants grown at 800 μmol·mol^-1 CO₂ than in plants grown at the other CO₂ concentrations during nighttime (Figs. 3D and G). The gs and tr values in 24-week-old plants were higher in plants grown at 800 and 1,600 μmol·mol^-1 CO₂ compared with the plants grown at 400 μmol･mol^-1 CO₂ during nighttime (Figs. 3E and H). In 36-week-old plants, gs and tr values were not significantly different between different CO₂ concentrations (Figs. 3F and I). At the cellular level, elevated CO₂ concentrations induce stomata closure for increased water use efficiency (Wang et al. 2015). In 2-week-old Phalaenopsis ‘Mantefon’, gs and tr were decreased under 1,600 μmol·mol^-1 CO₂ conditions compared with plants grown at 800 μmol·mol^-1 CO₂. Decreased gs is an interactive factor and low water availability might benefit plant productivity under increased atmospheric CO₂ concentrations. t_r was reduced due to a lower g_s (Wang et al. 2015). Maximum t_r and maximum stomatal opening occur in younger leaves of tobacco plants (Václavýk 1973). gs decreases with increasing leaf age in the chaparral shrub, Lepechinia calycina, growing in its natural habitat (Field and Mooney 1983). In Phalaenopsis ‘Mantefon’, t_r decreased with increasing plant growth stage, independent of CO₂ concentration.

In our present study, in 36-week-old Phalaenopsis Queen Beer ‘Mantefon’ (i.e., mature stage), leaf number were significantly greater in plant grown at 800 and 1,600 μ mol·mol^-1 CO₂ conditions compared with plants grown at 400 μmol·mol^-1 CO₂. We found that Phalaenopsis Queen Beer ‘Mantefon’ in 2- and 24-week-old (i.e., juvenile and young stages) plants grown at 1,600 μmol·mol^-1 CO₂ had increased net CO₂ uptake, leaf number, and shorten the time to leaf initiation compared with the plants grown at 400 μmol·mol^-1 CO₂. With these results, using 800 - 1600 μmol·mol^-1 CO₂ range for 36-week-old (i.e., mature stage) and 1,600 μmol·mol^-1 CO₂ condition for 2- and 24-week-old (i.e., juvenile and young stages) of Phalaenopsis Queen Beer ‘Mantefon’, growers may be able to promote leaf growth faster with increasing leaf number and reducing the time to leaf initiation.