Introduction

Greenhouse systems manipulate the growth environment to enhance plant production and flowering quality (Proietti et al. 2022). However, heating and cooling in greenhouses often generate a large amount of surplus CO₂ (Cho et al. 2022). Developed in Korea, rooftop greenhouses are installed on empty spaces on building rooftops to reduce the building’s energy load and decrease the energy consumption (cooling/heating/CO₂) required for crop production (KIMM 2019). Moreover, rooftop greenhouses utilize building energy for stable cultivation regardless of climate change and take advantage of discarded energy in the city, thus reducing cultivation energy (Kim et al. 2019). The surplus CO₂ can be used in the production of ornamental plants in greenhouses by a high-efficiency distributed power generation system (Lee et al. 2015). Plants are divided into C₃, C₄, and Crassulacean acid metabolism (CAM) types based on their photosynthetic processes (Drennan and Nobel 2000). CAM plants use surplus CO₂ generated by cooling and heating at night when ventilation is not needed in a greenhouse.

Schlumbergera truncata is a multi-flowering cactus that exhibits a variety of flower colors and splendid flowers, and it is a popular ornamental plant in Europe, America, and Korea (Lee et al. 2020). S. truncata grows in phylloclade units and it takes 30 to 60 days for a phylloclade to develop (GARES 2020). S. truncata is a short-day plant with flowers blooming at the apical end of 3 to 5 phylloclades (Lee et al. 2020). Cactus and succulents are known to be representative CAM plants (Cushman and Bohnert 1999). However, research on cactus photosynthetic characteristics is insufficient due to their morphological characteristics (Lee et al. 2003), and studies are mainly conducted on edible cactus such as Opuntia ficus-indica and Hylocereus undatus. Therefore, we conducted a study on the CO₂ uptake pattern of S. truncata, in a growth chamber and a greenhouse according to phylloclade maturity. The both the growth chamber and a greenhouse, mature phylloclades exhibited night CO₂ uptake (Jung et al. 2023). S. truncata absorbs CO₂ at night when ventilation is unnecessary.

In the previous studies, Phalaenopsis grown under elevated CO₂ level of 800 μmol·mol^-1 exhibited increased biomass production (Kim et al. 2017). Elevated CO₂ levels of either 800 or 1200 μmol·mol^-1 enhanced flower production and shortened the flowering time in Phalaenopsis (Cho et al. 2019). However, the response of ornamental CAM plants to elevated CO₂ was inconsistent and did not always yield positive outcomes (Croonenborghs et al. 2009). Phalaenopsis grown under high CO₂ (1600 μmol·mol^-1) levels resulted in flower bud abortion, which negatively impacted flower production (Kim et al. 2017). This suggests that there exists an optimal elevated CO₂ level and duration for growth and flowering. Elevated CO₂ can alter plant nutrient conditions and limited nutrients can reduce plant leaf area and photosynthesis (Mu et al. 2016). The decrease in leaf size under elevated CO₂ and the non-significant changes of the net CO₂ uptake under elevated CO₂ with light control (Cho et al. 2019;Song et al. 2019;Yun et al. 2018) may be interpreted as sink limitation of long-term elevated CO₂ supply. To alleviate the sink limitation of plants, manipulation of light level or fertility needs to be re-adjusted under elevated CO₂ (Frantz and Ling 2011).

The elevated CO₂ has been widely used as a gas fertilizer in greenhouse cultivation for vegetable cultivation, particularly recently, as greenhouse technologies have improved (Bisbis et al. 2018). However, research on ornamental CAM plants is scarce. This study was conducted on the growth and photosynthetic response of Schlumbergera truncata under nighttime elevated CO₂.

Materials and Methods

Plant materials and growth conditions

On December 2022, the cuttings were planted, 12 cuttings per 10 cm pot, at a commercial greenhouse (‘Sang Ah Farm’, Goyang, Korea, latitude 37°N, longitude 126°E). 8-months after cutting, Schlumbergera truncata ‘Red Candle’ was transported to Seoul Women’s University in Seoul, Korea (latitude 37°N, longitude 127°E). The experiment was conducted at the information communication technologies (ICT) smart greenhouse at Seoul Women’s University from 14 August 2023 to 25 December 2023. Plants were transplanted into a 15 cm plastic pot filled with a soil mixture (perlite:peatmoss = 7:3, v/v). The air temperature of the cultivation periods was controlled at 23/20°C (day/night) and the relative humidity was maintained at 71/82% (day/night). Temperatures and relative humidity were automatically recorded at 1-minute intervals using a data logger (SH-VT250, Soha Tech, Korea), and the data are shown in Figs. 1A and 1B. Black-out curtains were used to shade the plants from 17:00 to 08:00 hours between 9 October to 8 November 2023, to induce flower bud formation. After 10 days under a short-day photoperiod, benzylamino purine 100 mg·L^-1 was sprayed once at a rate of 10 mL per pot.

CO₂ supply

Two levels of ambient (≈ 400 μmol·mol^-1) and elevated CO₂ (≈ 1000 μmol･mol^-1) were applied to the plants for 10 h from 20:00 to 06:00 hours for 8 weeks after treatment (WAT). Elevated CO₂ was supplied with pure CO₂ from compressed gas cylinders (Bottle CO₂ ≥ 99%) (Seoul Specialty Gases Co., Ltd., Seoul, Korea). CO₂ was emitted upward from the ground through the polyvinyl chloride (PVC) pipe with holes drilled. The greenhouse was divided into an ambient CO₂ section and an elevated CO₂ section for CO₂ treatment. The volumes of the ambient and elevated CO₂ treatment spaces were 104.85 m³ and 69.9 m³, respectively. The two treatment areas were not completely sealed, so the ambient CO₂ concentration was affected by nighttime CO₂ enrichment. The average nighttime CO₂ concentration in the ambient CO₂ treatment section was 406 μmol·mol^-1, which does not differ significantly from the average atmospheric CO₂ concentration of 419 ppm in 2023 (NOAA 2024). The CO₂ concentrations were monitored using a CO₂ analyzer (SH-VT250AS, Aion Inc, Daejeon, Korea). The data are shown in Fig. 1C.

Data collection

The number of branches and phylloclades, mature phylloclade length and width, and immature phylloclade length and width were measured from 9 plants every 2 weeks during 8 WAT. The length and width of mature and immature phylloclades were measured continuously on the same phylloclade from 0 WAT. The number of phylloclades was measured with the phylloclade having lengths of ≥ 5 mm. For the dry mass measurements, the plants were cut into shoots, and roots at 8 WAT. The dry mass of shoots and roots was measured after drying the fresh plants in an oven at 70°C for 3 days.

Photosynthetic characteristics were measured on the mature and immature phylloclade using a portable photosynthesis system (Li-6400XT, Li-Cor Inc., Lincoln, NE, USA) equipped with a clear chamber (6400-08, Li-Cor., NE, USA). The measurements were taken at 10:00 to 14:00 hours (mid-day) and 22:00 to 02:00 hours (mid-night) at 6 and 8 WAT. The photosynthetic characteristics were measured by selecting the most immature phylloclade at the time of measurement and the mature phylloclade connected to it. The length and width of mature and immature phylloclade using photosynthesis measurements were at 6 and 8 WAT are shown in Table 1. The air temperature in the chamber was set to match ambient and relative humidity, ranging from 55 to 70%. The CO₂ concentration inside the chamber was maintained at 400 μmol·mol^-1 CO₂ (ambient CO₂) and 1000 μmol·mol^-1 CO₂ (elevated CO₂), which equals the level in the greenhouse. The net CO₂ uptake, water-use efficiency (WUE), stomatal conductance (g_s), and transpiration rate were measured. Gas exchanges were measured with 3 replicated plants per treatment. The value of WUE is calculated using the equation.

WUE (water-use efficiency) = net CO₂ uptake ÷ transpiration rate

The chlorophyll fluorescence parameter the maximum photochemical efficiency of PSII (F_v/F_m) was measured by using a portable pulse amplitude modulation fluorometer (JUNIOR PAM, Walz, Effeltrich, Germany). Before the measurements, the phylloclades were adapted to darkness for 15 min. The matured phylloclade was selected for measurement. F_v/F_m values were measured from 10:00 to 12:00 hours on 5 plants per treatment after 12 WAT.

The number of flower buds of ≥ 5 mm was counted for each plant every 2 days. Days to flowering were measured from the start of CO₂ enrichment to flowering. Flowering was defined as the full expansion of the petals of the first flower per pot.

Statistical analysis

Statistical analyses were performed using the R program for version 4.2.3 (Development Core Team, Vienna, Austria). Statistical significance was determined using the Student’s t-test with p-values < 0.05 and one-way analysis of variance (ANOVA) with a post hoc Tukey test (p < 0.05). Graphical analysis was performed using SigmaPlot program version 10.0 (Systat Software Inc., CA, USA).

Results

Phylloclade growth and plant biomass

The immature phylloclade width was significantly increased in plants grown under ambient CO₂ than those under elevated CO₂ (Table 2). At 0 to 8 WAT, the mature phylloclade width and length, and the immature phylloclade length, did not show significant differences among the CO₂ treatments. The number of branches and phylloclade were not significantly different at 0 and 2 WAT than those of CO₂ treatments. At 4 to 8 WAT, the number of branches and phylloclades were significantly greater in plants grown under ambient CO₂ than those under elevated CO₂ (Figs. 2A and 2B). The shoot and root dry mass was not significantly different among the CO₂ treatments (Table 3). The immature phylloclade width, number of branches, and number of phylloclades were negatively correlated with increasing CO₂ levels (r = -0.6810, -0.6020, and -0.6766) (Table 5).

Photosynthetic characteristics and maximum quantum yield of PSII (F_v/F_m)

The net CO₂ uptake rate was significantly affected by phylloclade age and the interaction between phylloclade age and CO₂ uptake time at 6 and 8 WAT (Table 4). At 6 WAT, net CO₂ uptake was the highest in mature phylloclades of plants grown under both ambient and elevated CO₂ at night, at 2.51 and 1.30 μmol·CO₂·m^-2·s^-1, respectively, but there was no statistical significance (Fig. 3A). Immature phylloclades exhibited CO₂ uptake only during the day at 6 WAT but showed uptake during both day and night at 8 WAT (Fig. 3B). The gs did not show a significant difference among the CO₂ treatments at 6 and 8 WAT (Figs. 3C and 3D). The gs was significantly affected only by CO₂ uptake time at 6 and 8 WAT (Table 4). The WUE did not significantly differ among the CO₂ treatments at 6 and 8 WAT during the day (Figs. 3E and 3F). The WUE was significantly affected by phylloclade age, net CO₂ uptake time, and the interaction between phylloclade age and CO₂ uptake time at 6 and 8 WAT (Table 4). The WUE of mature and immature phylloclades at night increased as CO₂ levels increased (r = 0.7462 and 0.9312, respectively) (Table 5). The F_v/F_m, an indicator of PSII’s maximum quantum yield, was 0.760 in plants grown under elevated CO₂, which was not significantly different from the ambient CO₂ of 0.735 (Table 6).

Flowering

The floral buds first appeared at 77 days after treatment (DAT) in plants grown under elevated CO₂ and at 81 DAT in those under ambient CO₂ (Fig. 4). At 123 DAT, plants grown under elevated CO₂ had 82.7 floral buds, compared to 72.1 buds for those under ambient CO₂. However, this difference was not statistically significant. The flower length and width did not show significance among the treatments (Table 7). The days to flowering were 121.7 days for plants grown under ambient CO₂ and 123.7 days for those grown under elevated CO₂, but the difference was not significant.

Discussion

Elevated CO₂ benefits ornamental plant production through plant growth to improve the yields and qualities of flowers, but some plant species may exhibit unresponsive or opposite effects in response to elevated CO₂ (Hussain et al. 2021;Xu et al. 2014). Leaf senescence or visible injuries, such as chlorosis, necrosis, and leaf curling, are observed in some plants at higher CO₂ levels (Mortensen 1987). In this study, no visible phylloclade injuries were observed in plants grown under elevated CO₂. However, there was a decrease in phylloclade growth in plants grown under elevated CO₂ compared to those grown under ambient CO₂ (Fig. 2 and Table 2).

During 4 years of CO₂ enrichment, the growth of beech and the amount of its nutrients decreased (Hagedorn et al. 2002). Limited nutrients can reduce plant leaf area and photosynthesis (Mu et al. 2016). The number of leaves increased in the plants grown under 800 and 1600 μmol·mol^-1 during 32 or 36 weeks of treatments with increased net CO₂ uptake, and Phalaenopsis was fertigated with water-soluble fertilizer of 20N-8.7P-16.7K at electrical conductivity ranged from 0.8 to 1.0 dS･m^-1 (Song et al. 2019;Yun et al. 2018). Naing et al. (2016) suggested that the leaf growth rate increased in the plants grown under CO₂ enrichment (1000 μmol·mol^-1) and with a light level of 250 μmol·m^-2·s^-1. Cho et al. (2019) observed an increase in floral buds and flowers in Phalaenopsis Queen Beer ‘Mantefon’ when plants were grown under a light level of 260 ± 40 μmol·m^-2·s^-1 with 800 μmol·mol^-1 CO₂ compared to those grown under 400 μmol·mol^-1 CO₂. The enhanced photosynthetic rates due to elevated CO₂ more than doubled under high-light conditions compared to low-light conditions in sweetgum trees (Herrick and Thomas 1999). However, this study did not provide additional fertilizer or supplemental light. These results suggest that environmental conditions (e.g., light and nutrients) should be considered when cultivating plants under elevated CO₂ to maintain high plant quality.

Elevated CO₂ increases carbohydrate production in plants, but as they mature, the limited ability to create new storage leads to a decline in net photosynthesis (Thomas and Strain 1991). Similarly, the mature phylloclade of S. truncata showed higher net CO₂ uptake at night in plants grown under ambient CO₂ compared to those grown under elevated CO₂ at 6 WAT (Fig. 3A). Long-term elevated CO₂ could lead to photosynthetic acclimation, potentially reducing the photosynthetic machinery and leaf photosynthetic potential (Griffin and Seemann 1996). Studies support these responses: in Opuntia ficus-indica, the stomatal frequency decreased by 20% under elevated CO₂ compared to ambient CO₂. Consequently, CO₂ uptake and water vapor conductance were affected (Nobel 1999). At 8 WAT, immature phylloclades grown under ambient CO₂ did not uptake CO₂ at night, while immature phylloclades grown under elevated CO₂ did uptake CO₂ at night (Fig. 3B). Once the facultative CAM plants, photosynthesis can switch from C₃ to CAM under abiotic stress (such as atmospheric CO₂ concentration, drought, salinity, photoperiod, light, etc.) (Qiu et al. 2023). During the daytime in Clusia pratensis at its C₃ state, the 800 ppm CO₂ treatment showed no effect on nocturnal CO₂ exchange, indicating the absence of CAM induction (Winter and Holtum 2014). These data suggest that CO₂ concentration may not be the CAM-inducing factor. However, previous studies treated CO₂ during the daytime, so there is a difference between this study, which treated CO₂ at night. The gs of mature phylloclades exhibiting CAM characteristics were lower in plants grown at elevated CO₂ compared to those grown at ambient CO₂. Stomatal behavior is another specific feature of CAM plants. Stomatal movement is controlled by many different factors, including light and CO₂ concentration (Guan et al. 2020). Guard cells form epidermal stomatal gas-exchange valves in plants and regulate the aperture of stomatal pores in response to changes in the CO₂ concentration in leaves (Engineer et al. 2016). CAM plants exhibit 2 to 5-fold higher WUE compared to C₃ and C₄ plants (Nobel and Barrera, 2004). In this study, at 8 WAT, nighttime CO₂ uptake was observed exclusively in the immature phylloclades of the elevated CO₂ treatment. The WUE was significantly higher elevated CO₂ than those grown under ambient CO₂ at 8 WAT (Fig. 3F).

Measured chlorophyll fluorescence can be used to predict, monitor, and identify stress in plants (Kalaji et al. 2016). The presence of low F_v/F_m will indicate substantial photoinhibition or down-regulation of PSII (Murchie and Lawson 2013). Pulse-amplitude modulated fluorescence measurements have been utilized to detect alterations in plant physiological status in reaction to shifting environments before observable morphological changes occur (Zha et al. 2017). F_v/F_m is not significantly different among the CO₂ treatments; however, there were differences in plant growth among the CO₂ treatments observed during the vegetative growth stage (Table 5). Reduced values of F_v/F_m indicate photoinhibition, decreased photosynthetic capacity, and a reduced number of triplet states in functional reaction centers when plants are exposed to stress (Na et al. 2014). F_v/F_m measurements were conducted after discontinuing CO₂ treatment, suggesting that stress was induced during the vegetative growth stage in plants treated with 1000 μmol·mol^-1 of CO₂ treatment.

The effects of elevated CO₂ on flowering time are not as well understood and vary widely among species (Springer and Ward 2007). flowering characteristics were not significant among the CO₂ treatments. Despite the photosynthetic acclimation of wheat plants to high levels of CO₂, total soluble sugars in grains increased (Sinha et al. 2011). Although photosynthetic rates decreased, it is possible that the increase in sugars led to an increase in the number of floral buds.

In conclusion, this study showed that elevated CO₂ significantly decreased the growth of S. truncata. The immature phylloclade width, number of phylloclades, and branches were significantly higher plant grown under ambient CO₂ than those of the elevated CO₂. The flowering characteristics did not significantly differ among the CO₂ treatments.