Introduction

Potassium (K) is one of the major mineral nutrients impacting orchids’ growth, flower initiation, and flower development of Phalaenopsis (Wang 2007), Cymbidium (An et al. 2012), and Dendrobium (Bichsel et al. 2008). Phalaenopsis ‘Taisuco Kochdian’ fertigated with 300 mg·L^-1 K had longer leaf length and spike and more flowers than those with 50 or 100 mg·L^-1 K (Wang 2007). Translocation from source to sink in Cymbidium was promoted more in the plants with 100 and 200 mg·L^-1 K than in the K-deficient plants at the flower initiation stage (An et al. 2012). Symptoms of the K deficiency in Phalaenopsis when fertigating 50 or 100 mg·L^-1 K included rusty or bronze patches, leaf yellowing, and necrosis on leaf tips (Wang 2007). K-deficient leaves of cotton plants decreased photosynthesis to partitioning assimilates in plant tissue, resulting from low chlorophyll content and poor chloroplast ultrastructure (Zhao et al. 2001). Plants show K deficiency symptoms much earlier under limited nitrogen (N) levels (Gartner 1969). Salt stress causes nutrient deficiencies due to the competition of Na⁺ and Cl^- (Abdullakasim et al. 2018). Still, the application of potassium sulfate (SOP) counteracted salt-induced reduction in the growth and yield of rice plants (Din et al. 2001). Foliar application of SOP significantly improved the growth, achene yield, photosynthetic and transpiration rates, stomatal conductance, and water-use efficiency of the salt-stressed sunflower plants (Akram et al. 2009).

Elevated atmospheric CO₂ enhances leaf and canopy photosynthesis and productivity in C₃ crops because the present atmospheric CO₂ concentration is insufficient to saturate Rubisco and elevated CO₂ inhibits the competing process of photorespiration (Bowes 1991). CAM (crassulacean acid metabolism) induced Rubisco can be activated more in CAM Mesembryanthemum crystallinum than in C₃M. crystallinum (Davies and Griffiths 2012). Numerous studies have demonstrated that CAM plants’ growth and yield are favored by elevated CO₂ (Croonenborghs et al. 2009;Nobel and Israel 1994;Weiss et al. 2010). The cladodes of Opuntia ficus-indica were developed more when the CO₂ concentration was doubled (Nobel and Israel 1994). Aechmea ‘Maya’, ornamental bromeliads, showed at both CO₂ concentrations an equal biomass enhancement throughout the experimental period. Still, the total leaf area and thickness of Guzmania ‘Hilda’ increased in the plants grown under elevated CO₂ than those under ambient CO₂ (Croonenborghs et al. 2009). Fruit fresh mass of Selenicereus megalanthus increased by 63% in response to elevated CO₂ than those exposed to ambient CO₂ (Weiss et al. 2010).

Like other CAM ornamental plants, Phalaenopsis growth, flowering, and photosynthesis respond well to elevated atmospheric CO₂ concentration (Cho et al. 2019;Song et al. 2019;Yun et al. 2018). Yun et al. (2018) found that Phalaenopsis grown under 1600 μmol·mol^-1 CO₂ had a higher number of leaves, net CO₂ uptake, transpiration rate, and stomatal conductance than plants grown under 450 μmol·mol^-1 CO₂ conditions. However, rapid growth and increased productivity of plants grown under elevated CO₂ require more K supply. The leaf critical K level of cotton was 12 g·kg^-1 under ambient CO₂ for photosynthesis, main-stem elongation, and biomass accumulation, whereas 18-19 g·kg^-1 under elevated CO₂ of 720 μL·L^-1 (Reddy and Zhao 2005). The K uptake was significantly reduced in potato tubers under elevated CO₂ of 680 μL·L^-1 (-6.7%) compared with ambient CO₂ (3.2%) (Fangmeier et al. 2002). The photosynthesis rate under elevated CO₂ of 600 ± 10 μmol·mol^-1 increased by 15.6% in the plants under 200 mg·L^-1 K fertilization than those under K deficiency in wheat (Asif et al. 2017). The potential yield, the sum of the number of visible floral buds and flowers, number of lateral branches, and water-use efficiency of Phalaenopsis Queen Beer ‘Mantefon’ were higher in the plants exposed to nutrient electrical conductivity (EC) of 2.0 dS·m^-1 (N 98 mg·L^-1, P 66 mg·L^-1, K 202 mg·L^-1) than those to EC 1.0 dS·m^-1 (N 220 mg·L^-1, P 149 mg·L^-1, K 454 mg·L^-1) under elevated CO₂ (Cho et al. 2020).

We hypothesize that elevated atmospheric CO₂ concentration may increase the growth and development of Phalaenopsis plants to the CO₂. A sufficient K supply using SOP fertilizer is needed for the synergistic effect of elevated atmospheric CO₂ concentration. The objectives were to determine the interactive effects of elevated CO₂ concentration and SOP supply on Phalaenopsis Queen Beer ‘Mantefon’ flowering, biomass, and photosynthesis characteristics under controlled environmental conditions.

Materials and methods

Plant materials and growth conditions

Clones of 18 months after deflasking, Phalaenopsis ‘Queen Beer ‘Mantefon’ plants were purchased in June 2018 and transplanted into 120 mm pots (0.5L volume) with a medium containing a 100% dried Chilean sphagnum moss (Lonquen Ltd., Puerto Montt, Chile). The plants were grown in a Phalaenopsis nursery in Guangzhou, China, during the vegetative stage (from January 2017 to June 2018). During this period, average temperatures, relative humidity, and photosynthetic photon flux in the greenhouse were 30 ± 2°C, 70 ± 10%, and 250 ± 50 μmol·m^-2·s^-1, respectively.

The experiment was conducted at the plastic greenhouse of Bulmuri Orchid Nursery in Paju, Korea (37°N, 126°E) from 8 Jun to 7 Dec 2018. Plants were cultivated at an average temperature of 23 ± 2/18 ± 1°C (day/night) and the relative humidity of 50/80%, monitored by a data logger (AM-21A, WISE Sensing Inc., Yongin, Korea). A 50% shading screen surrounded the plants in the greenhouse with an eave height of 2.5 m and a ridge height of 3.0 m. PPF of full sunlight at Bulmuri Orchid Nursery was 1,400 μmol·m^-2·s^-1 in June and 1,000 μmol·m^-2·s^-1 in December 2018.

CO₂ and potassium sulfate treatments

Eight treatments included two levels of CO₂: ≈ 400 μmol·mol^-1 (Ambient CO₂) and ≈ 720 μmol·mol^-1 (Elevated CO₂) during 22:00 - 04:00 HR (Fig. 1), which were controlled using a CO₂ analyzer (SH-VT250, Soha-Tech Co. Ltd., Seoul, Republic of Korea), and four levels of potassium sulfate (SOP) supply. The CO₂ treatments were imposed from spike emergence through final harvest 20 weeks after treatments (WAT). The stock solutions used for all four different SOP treatments were prepared with commercial agricultural fertilizers. The four SOP treatments were used by mixing 13.7N-0P-46K (Krista K, Yara, Oslo, Norway) 267 mg·L^-1, 12N-61P-0K (Krista MAP, Yara) 733 mg·L^-1, 15.5N-0P-0K (Calcinit, Yara) 466 mg·L^-1, and four different concentrations of 0N-52P-43K (SOP, Yara) at four additional (SOP) concentrations: (1) SOP1 level (Control, 100 mg·L^-1 K) throughout the experiment; (2) SOP2 level (300 mg·L^-1 K); (3) SOP3 level (500 mg·L^-1 K); and (4) SOP4 level (700 mg·L^-1 K), until final harvest (20 WAT). The EC, pH, and molecular components of each SOP level are indicated in Table 1. The magnesium and phosphorus concentrations were controlled at 0.00 and 2.74 mmol·L^-1. Fertigation was applied 200 mL per pot by hand-drip irrigation once a week.

Measurements

The first and second flowers spike length, the number of lateral branches, and floral buds were recorded every two weeks for 20 WAT on 11 plants in each treatment. Flower spikes, lateral branches, and floral buds with lengths of ≥ 5 mm were counted for each plant. Spike length was measured from the node at the base to the topmost. The number of flower spikes and flowers was recorded only at 20 WAT. Then, plants were divided into three parts leaves, spikes, and roots, which were dried at 80°C for seven days and weighed for biomass.

Gas exchange was measured from 22:00 to 01:00 HR in the uppermost mature leaves at 20 WAT on three plants in each treatment. Net CO₂ uptake (P_N), stomatal conductance (g _s), transpiration rate (t _r), and water-use efficiency (WUE) were determined using a portable infrared gas analyzer (LI-6400, Li-Cor, Lincoln, NE, USA) equipped with a LED lighting system (6400-02B, Li-Cor). The gas exchange parameters were set up as 0.6 ± 0.4 kPa vapor pressure deficit and 500 mL·min^-1 air flow rate. In the leaf chamber, the relative humidity ranged from 55 to 70%, and the temperature was kept at 18°C. The CO₂ concentration inside the leaf chamber was maintained at approximately 400 and 720 μmol·mol^-1, which equals the level in the greenhouse for ambient and elevated CO₂ treatments, respectively. The value of WUE was obtained by dividing the value of P_N by E (Xu et al. 2014).

The petal color of the surface of three spots of two different petals from each plant was measured using a color meter (CR-10 Plus, Minolta Co., Osaka, Japan) and recorded using the CIE (L*, a*, and b*) from uniform color space at 20 WAT. The L* scale ranged from no reflection (L* = 0; black) to perfect diffuse reflection (L* = 100; white), the a* scale ranged from negative values for green to positive values for red, and the b* scale ranged from negative values for blue to positive values for yellow.

Experimental design and data analysis

The experiment was a completely randomized block design with 11 plants. Eight treatments were randomly arranged in the greenhouse during the experiment. All flowering and physiological measurements were made on 3 or 11 plants for each treatment.

Data were statistically analyzed by the ANOVA procedures in the SAS system for Windows version 9.4 (SAS Inst. Inc., Cary, NC, USA) to determine the interactive effects of the two factors of CO₂ and SOP nutrition on plant flowering, dry matter accumulation, gas exchange, and petal color. The means were compared by Duncan’s multiple range test at p < 0.05 and showed using Sigma Plot version 10.0 (Systat, Software, Inc., San Jose, CA, USA).

Results

Flower spike length and number of branches, floral buds, flower spikes, and flowers

The first flower spike length was 1.08 – 1.34 and 1.08 – 1.19 times increased more in the plants grown under ambient and elevated CO₂ with SOP2 than those with SOP1, respectively, from 8 to 20 WAT (Fig. 2A). The first and second flowers spike length did not differ among CO₂ treatments from 8 to 20 WAT (Figs. 2A and B). The first and second flowers spike length were increased by 8.3% and 13.9% in the plants grown under elevated CO₂ with SOP3 than those with SOP1 at 20 WAT, respectively. The number of lateral branches and floral buds had significant (p < 0.05) CO₂ × SOP interactive effects (Figs. 2C and D). The number of lateral branches increased more in the plants grown under elevated CO₂ with SOP1-3, ranging from 1.0 to 1.9, than those grown under ambient CO₂, ranging from 0.5 to 0.8 at 12 WAT (Fig. 2C). The number of lateral branches was not significantly different between CO₂ treatments from 16 to 20 WAT, regardless of SOP levels. The plants grown under ambient CO₂ with SOP3 had more floral buds than those under ambient CO₂ with SOP1 at 12 WAT. The number of floral buds increased more in the plants grown with SOP3 than those grown with SOP1 at ambient CO₂ conditions, but it decreased when plants were grown under elevated CO₂ conditions with SOP3 at 20 WAT (Fig. 2D).

There were no significant differences between the number of flower spikes according to CO₂ and SOP treatments (Table 2). However, the number of flowers decreased in the plants grown under elevated CO₂ with SOP2-4 than those grown under ambient CO₂.

Biomass partitioning

Elevated CO₂ and SOP treatments not affected not only shoot biomass but also spike biomass, but the interactive effects of CO₂ and SOP treatments on root biomass were significant (p < 0.01) (Fig. 3). Plants receiving SOP1 under ambient CO₂ had a higher root and spike biomass than those grown with SOP2-4 under ambient CO₂ (Figs. 3B and 3C). Elevated CO₂ significantly increased biomass accumulation in roots, and the root biomass had the greatest increase in the plants grown with SOP2, while shoot and spike biomass altered least by SOP level (Fig. 3).

Photosynthesis

Both CO₂ and SOP treatments significantly impacted P_N of Phalaenopsis plants (p < 0.001 and p < 0.05, respectively), and their interactive effects on P_N were also detected (p < 0.05) (Fig. 4A). The stimulation of P_N due to elevated CO₂ increased as SOP supplied more from SOP1 to SOP3. Plants grown under elevated CO₂ with SOP1-4 had a 33, 174, 82, and 36% higher P_N, respectively. g _s and t _r did not differ between plants grown under ambient and elevated CO₂ (Figs. 4B and 4C). A significant difference (p < 0.05 and p < 0.001) was detected in WUE among CO₂ and SOP treatments, respectively (Fig. 4D). The plants receiving SOP3 under elevated CO₂ had a higher WUE than those under ambient CO₂.

Petal color

No significant differences existed between L* according to CO₂ treatments (Table 2). However, a* decreased in the plants grown under elevated CO₂ with SOP1, SOP2, and SOP4 than those under ambient CO₂, indicating that the petal color was less red. High values for a* were observed in plants grown under elevated CO₂ with SOP4 treatments.

Discussion

CO₂ is the main component of biomass in plants, and an elevated CO₂ level increases the total biomass in plants (Walia et al. 2022). The root biomass was 2 - 16% increased more in Phalaenopsis ‘Fuller’s Pink Swallow’ under elevated CO₂ of 800, 1600, and 2400 μmol·mol^-1 than those grown under ambient CO₂ of 450 μmol·mol^-1 (Kim et al. 2017). Using elevated CO₂ and K conditions in cotton, biomass accumulation and portioning are promoted by higher K levels in leaves, which act synergistically with elevated CO₂ (Reddy and Zhao 2005). The WUE improved at a higher K supply of 10 mM K than those at 0 mM K in Hibiscus (Egilla et al. 2005). The P_N and WUE were improved more in Phalaenopsis Queen Beer ‘Mantefon’ grown under elevated CO₂ of 800 μmol·mol^-1 than those under ambient CO₂ of 450 μmol·mol^-1 at the vegetative growth stage (Yun et al. 2018). As WUE of plants affected by CO₂ and SOP levels at p < 0.05 and < 0.001, respectively (Fig. 4), the elevated CO₂ with SOP2 and SOP3 used here increased root biomass in Phalaenopsis Queen Beer ‘Mantefon’ at the reproductive growth stage.

Elevated CO₂ increases P_N in CAM plants because higher CO₂ increases both Rubisco-mediated daytime CO₂ uptake and phosphoenolpyruvate carboxylase (PEPCase)-mediated nighttime CO₂ uptake, nocturnal malate accumulation, and carbon assimilated into carbohydrates for plant growth and development (Drennan and Nobel 2000). Elevated CO₂ of 700 – 800 μmol·mol^-1 induces floral transition with enhanced sugar through increased photosynthesis in ornamental flowering plants: 6 days in Gailardia pulchella, 7 days in Lotus corniculate and Phytolacca americana, and 20 days in Phalaenopsis at a light level of 260 μmol·m^-2·s^-1 (Carter et al. 1997;Cho et al. 2019;He and Bazzaz 2003;Reekie and Bazzaz 1991). Excessive sugars delay the onset of flowering and control the flowering mechanism (Springer and Ward 2007). The flowering was delayed by 8 to 10 days in the plants on media containing 5% sucrose compared with those on media containing 1% sucrose in Arabidopsis (Ohto et al. 2001). In this study, Phalaenopsis had a higher P_N under elevated CO₂, which might have influenced the flowering signals of plants and delayed flowering time. Excessive sugars may delay flowering time through 20 weeks of CO₂ treatments (Cho et al. 2019), but the flowering time results through 16 weeks of CO₂ treatments differed from previously reported. The photosynthetic rate, total carbohydrate, and foliar starch content of pigeon pea increased under elevated CO₂ of 550 μmol·mol^-1 and delayed flowering by 8 to 9 days but had no adverse effect on final yield (Sreeharsha et al. 2015). The number of flowers was 8.7, 7.3, 2.2, and 3.3 more under elevated CO₂ than those under ambient CO₂ with SOP1 to SOP4, respectively (data not shown), which indicated that Phalaenopsis Queen Beer ‘Mantefon’ also had no adverse effects on final production.

Plants mobilized the excessive soluble sugars to potential sinks of roots at reproductive stages. The enhancement of plant root biomass by elevated CO₂ was related to higher leaf photosynthetic capacity (Figs. 3 and 4). Besides representing a greater sink strength, the higher root biomass under elevated CO₂ in Phalaenopsis could lead to greater nutrient uptake, enhancing P_N. Elevated CO₂ increases the number of fine and lateral roots and subapical root hair development (Li et al. 2012). The changes in root morphology may enlarge the root biomass and thus aid the nutrient and water uptake. K uptake from the soil under elevated CO₂ may be reduced due to decreased t _r or ameliorate the effects of drought stress by reducing the stomatal resistance (Asif et al. 2018). S is mainly bounded to carbon to form organic molecules along with N and P in plants (Li et al. 2020). While K decreased with S in shoots, the changes in K and S contents in Chinese cabbage were correlated with linear regression (Reich et al. 2016). This can explain why elevated CO₂ with SOP2 and SOP3 triggered a significant increase in plant root biomass and P_N. The increased WUE in elevated CO₂ with SOP3 is attributed to the higher net photosynthesis compared with ambient CO₂, underscoring its potential for higher productivity of Phalaenopsis.

We detected a lower a* value in elevated CO₂ than in ambient CO₂. The a* of strawberry under the elevated CO₂ treatment decreased by 20% compared to those under the ambient air during the storage due to lower anthocyanin concentration and down-regulated phenylpropanoid and flavonoid biosynthesis under CO₂ stress (Li et al. 2019). Also, elevated CO₂ reduced a* of petal color in the leaves of pigmented lettuce ‘Oat leaf’ to a greater extent in saline conditions than in non-salinity conditions (Pérez-López et al. 2013); this scenario would explain the lowest a* of Phalaenopsis Queen Beer ‘Mantefon’ under elevated CO₂ with SOP4. However, the anthocyanins in strawberry fruit accumulated dramatically after removing CO₂ stress, suggesting that elevated CO₂ didn’t have a residual effect on anthocyanins synthesis if CO₂ elevation was removed (Li et al. 2019). Since long-term exposure to elevated CO₂ concentrations results in reduced flower numbers and increased floral bud abortion, Phalaenopsis subjected to elevated CO₂ should display before flower spike induction (Kim et al. 2017). Therefore, Phalaenopsis Queen Beer ‘Mantefon’ flowers are sensitive to elevated CO₂, but the redness of the petal color itself could be restored after appropriate control of CO₂ supply term.

In conclusion, elevated CO₂ levels stimulated Phalaenopsis root biomass production with a nutrient range from SOP2 to SOP3 but did not affect flower spike development in this study. As a consequence of higher P_N and WUE, there were increases in root biomass production which acted as a sink organ. Plants grown under elevated CO₂ conditions required greater amounts of SOP2 to SOP3, consisting of 300 to 500 mg·L^-1 K, than those under ambient CO₂. Consequently, nutrients ranging from SOP2 to SOP3 should be supplied for Phalaenopsis Queen Beer ‘Mantefon’ cultivation to improve the photosynthesis during the reproductive growth stage under elevated CO₂ condition of 720 μmol·mol^-1.