Introduction

Light/dark cycle generated by the Earth’s rotation is a significant factor for the growth of plant. It influences the biomass accumulation, photosynthesis, seed number, and seed viability of plants closely related to plant physiology and agronomic characters. Thus offering a proper daylight condition is critical to the efficient plant vegetation growth. However, adaptation to light in plant is not a simple process but accompanies more sophisticated endogenous biological cycle adjustments known as ‘circadian rhythms’. The main functionality of circadian rhythms is that plants can persist under steady environmental conditions so they can anticipate their external alterations (Dodd et al. 2015). Hereby, circadian rhythms do a momentous role to enhance smooth growth of plant (Dodd et al. 2005).

The first scientific research on plant circadian rhythm was reported by de Marian who observed persistent daily leaf movements of heliotrope plant. After a century, studies revealed that plants can have their own running rhythms which are shorter than 24 h (De Candolle 1832; Pfeffer 1915) and these endogenous rhythms were not operated by other exogenous factors. Then, the idea of heritable circadian rhythms by gene was suggested (Darwin and Darwin 1880). The heritable circadian rhythms concept opened the era of circadian rhythms investigation by genetic analysis in 1970s (McClung 2006). Until now, the main research stream of plant circadian rhythms focuses on genetic analysis and gene modification. On the other hand, only a few manuscripts reported physiological aspects of circadian rhythms. Withrow and Withrow (1949) reported that normal cycle (12/12 h) had advantages over short (6/6 h) and long (24/24 h) cycles for the growth of tomato. An Arabidopsis mutant with elongated circadian rhythms accumulated more biomass at a longer cycle (Green et al. 2002; Dodd et al. 2005). However, because solid mechanism of circadian rhythms feedback loop has not been well understood, it is necessary to approach from fundamental aspects of plant physiology.

Most of plants take CO₂ for their photosynthesis during the light period, however, some plants take CO₂ during the dark period while they do their photosynthesis during the light period with captured CO₂. This type of photosynthesis is called CAM (Crassulacean Acid Metabolism) which has a distinguishable CO₂ uptake pattern that affects exogenous daylight cycle. One of the plants processing CAM is orchids. Orchidaceae has more CAM species than other plants. Because they usually are known to take CO₂ during the dark period, which is used for their photosynthesis, they have their own CO₂ uptake patterns. Therefore, external light has an effect to regulate CAM photosynthesis (Kluge and Ting 1978). From 12/12 h light/dark cycle, relatively short light period makes nocturnal stomata open and increases total CO₂ fixation during the dark period in Kalanchoe blossfeldiana (Queiroz 1974). By contrast, a longer light period forwards daytime CO₂ uptake and inhibits fixation during the dark period, and finally results in limited growth (Nose et al. 1986; Sekizuka et al. 1995).

Orchids are distributed all regions of the world except Antarctica and are found growing in many different habitats and elevation gradients (Morrison and Pridgeon 2000). Despite the diversity of orchids in nature, only a small number of genera including Phalaenopsis, Cymbidium, Dendrobium, and Oncidium are cultivated in large quantities as commercial ornamental crops. In particular, Phalaenopsis hybrids that include Doritaenopsis Queen Beer ‘Mantefon’ have become popular potted orchids because of their ease of scheduling to meet specific market dates, high wholesale value, and long post-harvest life (Wang and Lee 1994).

As many Orchidaceae plants do, Dtps. Queen Beer ‘Mantefon’ also has a typical CAM process for its photosynthesis. Length of light period has been known to regulate CAM photosynthesis (Kluge and Ting 1978). However, there has been little studies to investigate the effect of shortened light/dark cycle instead of 24 h cycle on the vegetative growth and CO₂ uptake of orchid plants

Therefore, the objective of this study was to determine the effect of shortened light/dark cycle on the CO₂ uptake and vegetative growth of immature Dtps. Queen Beer ‘Mantefon’ in order to reduce the production period.

Materials and Methods

Plant and growth conditions

Clones of tissue cultured 4-month-old Dtps. Queen Beer ‘Mantefon’ were purchased from Sang Mi Orchids (Taean, Korea) and transported to Seoul National University Farm (Suwon, Korea). The plants were transplanted into 10 cm pots filled with 100% sphagnum moss. The plants were irrigated every week with a water-soluble fertilizer (EC 0.8 mS·cm-1; Hyponex professional 20N-20P-20K, Hyponex Japan Corp., LTD., Osaka, Japan). Twelve uniform plants were chosen per each treatment for the experiment. The average leaf span was 4.3 cm measured from stretched leaves and the number of total leaves was 11 at the beginning of the experiment. Leaf span was measured by extending the longest leaves to a horizontal position, measuring the length from tip point of one leaf to another tip point of opposite leaf (Blanchard and Runkle 2006).

Photoperiod and temperature treatments

Dtps. Queen Beer ‘Mantefon’ clones of uniform size were grown in a closed plant factory maintained at 28°C. Each compartment provided four different light/dark cycles: 06/06 h, 08/08 h, 10/10 h, and 12/12 h. A photosynthetic photon flux density (PPFD) of 160 ± 10 μmol·m^-2·s^-1 was provided during the light period by warm-white LED lamps (GMG Korea Co., Ltd., Ansan, Korea). The PPFD was measured by using a spectrum solar electric quantum meter (Spectrum Technologies, Inc., Aurora, IL, USA) at the plant canopy. Twelve plants with 3 - 4 fully expanded leaves were placed in each compartment and grown for 17 weeks. Locations of plants were randomly rotated every week to maintain uniform light conditions.

Data collection and statistics

The number of new leaves (longer than 1.0 cm), the number of total leaves, leaf span, and leaf chlorophyll content were measured every 4 weeks. Length, width, and thickness of the uppermost matured leaf were also measured. The leaf chlorophyll content was measured with the uppermost matured leaf by using a chlorophyll meter (SPAD 502, Konica Minolta Sensing, Inc., Sakai, Osaka, Japan). Three plants were randomly collected from each light/dark cycle, the youngest mature leaves were used for photosynthetic gas exchange measurement for 24 h, and leaf conductance was measured at 5 weeks and 9 weeks after treatment with an infrared gas analyzer system (LI-6400, Li-Cor, Inc., Lincoln, NE, USA). External air was scrubbed of CO₂ and mixed with a supply of pure CO₂ to create a standard concentration of 400 μmol･mol^-1 and flow rate was 500 μmol･m^-2･s^-1. Each plant had been measured for 25 h and data was logged every 10 min for detecting unique photosynthetic patterns of CAM. Statistical analysis for vegetative growth was performed by using SAS system for Windows version 9.3 (SAS Institute, Inc., Cary, NC, USA). Differences among the treatment means were assessed by Tukey’s honestly significant difference test at p < 0.05. Regression and graph module analyses were performed by using Sigma Plot version 10.0 (Systat Software Inc., Chicago, IL, USA).

Results and Discussion

Effects of light/dark cycle manipulation on CO₂ uptake patterns

Under various light/dark cycle manipulations, ‘Mantefon’ exhibited typical CAM photosynthesis patterns (Fig. 2) and stomatal conductance patterns (Fig. 3). The patterns of CO₂ uptake in each light/dark cycle were similar to those in 2016 and 2017, where both experiments were processed with the same schematic time table (Fig. 1). The CO₂ uptake rate of 12/12 h cycle showed typical patterns of CAM plants ( Fig. 2 A). T here w as a gradual increase with no abrupt increase in the stomatal conductance after the dark period started. After 5 and 10 weeks of treatment, the net CO₂ uptake rate under 06/06 h treatment showed sinusoidal pattern in which it started to increase from the midpoint of dark period and peaked at dark/light transition point (Fig. 2D). Then, it started to decrease and flattened at midpoint of light period.Fig. 4Fig. 5

From the first and second cycle, there was a fluctuation in the maximum CO₂ uptake rate value after 10 weeks of treatment. A similar pattern was observed from the stomatal conductance rate in 06/06 h treatment (Fig. 3D). The net CO₂ uptake rate in 08/08 h treatment started to increase linearly at light/dark transition point. Then, it dropped abruptly and flattened during the light period (Fig. 2C). The stomatal conductance in 08/08 h treatment also showed the similar trend, but started to increase from the mid dark period instead of light/dark transition point (Fig. 3C). The net CO₂ uptake in 10/10 h treatment started at light/dark transition points and peaked at the mid dark period. Then, it started to decrease and flattened at early light period (Fig. 2B).

In many cases, CAM is known as an adjustable metabolic system. Therefore, CAM plants quickly respond to the changes in environmental conditions (Franco et al. 1990; Haagkerwer et al. 1992; De Mattos and LÜttge 2001; Dodd et al. 2002; Lüttge 2004; Chen et al. 2008). In 10/10 h treatment, CO₂ uptake patterns were similar to those in 12 /12 h treatment. In 10/10 h treatment CO₂ uptake began soon after dark period began (Fig. 2). CO₂ uptake continued even after the light period began. However, when the light/dark cycle were shortened to 06/06 h (Fig. 2B) and 08/08 h (Fig. 2C), the start point of CO₂ uptake moved forward by 3 and 4 h, respectively, to the midpoint of dark period. In 06/06 h treatment, CO₂ uptake pattern showed different patterns compared with that in 12/12 h treatment (Fig. 2A and D). CAM plants usually close their stomata during the light period to minimize the loss of water and CO₂ in plants (Black 2003).

CO₂ and water must be taken up by the open stomata during the dark period. Furthermore, the onset of darkness not only stops CO₂ uptake, but also starts the degradation of chloroplast starch (Dodd et al. 2002). The amount of starch in the chloroplasts falls through the dark period because breakdown products flow to the cytosol to sustain the export of sucrose to other organs (Taiz and Zeiger 2006).

Dtps. Queen Beer ‘Mantefon’ is a CAM plant (Cui et al. 2004) which fixes atmospheric CO₂ via open stomata during the dark period and does photosynthetic assimilation via the Calvin cycle during the light period (Chen et al. 2008). Because CAM plants possess a cyclical and reciprocal fluctuation of CO₂ uptake (Osmond 1978), those patterns of CO₂ uptake affected by in vitro light/dark cycle affects photosynthetic characteristics in CAM. Altered light/dark cycle also affected the plant’s own circadian rhythms because circadian rhythms are adjusted by external environmental stimuli throughout the light period (Greenham and McClung 2015). Therefore, if light/dark cycles are shortened, circadian rhythms in plants also change many physiological responses.

This kind of disparity in CO₂ uptake could appear obviously in the total amount of CO₂ uptake. Based on these patterns (Fig. 2), total amount of CO₂ uptake was calculated in their own light/dark cycle manipulation (Fig. 4). Except 06/06 h treatment, total CO₂ uptake during the dark period occupied the most of the diel amount. Both total amount and uptake amount during the dark period decreased as light/dark cycle length was shortened. Especially in 06/06 h treatment, CO₂ uptake amount during light period was larger than that of dark period. This aspect of total CO₂ amount seemed to stem from stomata opening during light period. The maximum CO₂ uptake amount during the dark period was found from 12/12 h treatment followed by 10/10 h, 08/08 h, and 06/06 h treatment (Fig. 4). Conversely, the maximum CO₂ uptake amount during light period was found from 06/06 h treatment followed by 08/08 h, 10/10 h, and 12/12 h treatment. Sekizuka et al. (1995) reported that relationship between photoperiod and CAM does have relationship with energy and another control factor of CAM. In CAM plants, a short period of light would result in incomplete decarboxylation of malate which can inhibits stomata opening during the dark period, and therefore, CO₂ uptake lowers. (Lüttge 2007). The similar results were observed in ‘Mantefon’ leaves in which 06/06 h treatment resulted in low amount of CO₂ uptake during the dark period. Also 08/08 h treatment showed low amount of CO₂ uptake during the dark period compared with those of 10/10 h and 12/12 h treatment. These results indicated that CO₂ uptake of plants could be completely influenced by external stimuli.

Effects of light/dark cycle manipulation on vegetative growth

Overall growth showed differences to each other (Table 1). From the youngest mature leaf, no significant difference was found in leaf length. However, in leaf width and thickness, there were significant differences among treatments. 12/12 h and 10/10 h treatments showed wide and thick leaves than 06/06 h treatments. Especially in leaf thickness, 06/06 h treatments showed thinner leaf than other treatments, with high leaf length/width ratio with no significance. There was no significant difference in the number of new leaves. However, as light/dark cycle treatment duration increased, the total number of leaves significantly increased. In fresh weight and dry weight, 06/06 h treatment showed the lowest values in both leaves and roots (Table 2). Contrary to the amount of CO₂ uptake, 10/10 h treatment resulted in the highest values in both leaf and root in dry weight.

In an early study of the effects of photoperiod on plants vegetative growth, many researchers found that long light period promoted dry weight in many species of temperate grass (Heide et al. 1985, Hay 1990, Solhaug 1991). This study also showed that 12/12 h and 10/10 h treatments resulted in heavier dry weight than other two treatments. However, dry weight of 10/10 h treatment was higher than that of 12/12 h treatment. In some CAM plants, it was reported that short photoperiod could make the cell of plants get bigger so that the plants could absorb more CO₂ (Sipes 1985). Sekizuka et al. (1995) reported that hybrid of Dendrobium and Phalaenopsis had changes in its capacity for CO₂ uptake by photoperiod. Since we applied only four different light/dark cycles, more detailed research is needed to determine more appropriate light and dark period to promote vegetative growth more effectively.

In conclusion, after manipulating light/dark cycle of Dtps. Queen Beer ‘Mantefon’, total CO₂ uptake amounts were the highest in 12/12 h treatment (control group), followed by 10/10 h, 06/06 h, and 08/08 h treatments. Vegetative growth of 10/10 h treatment closely followed that of 12/12 h treatment, while fresh weight and dry weight of leaves and roots were the heaviest at 10/10 h treatment.

Because the deviation originated from the inherent characteristics of CAM photosynthesis pathway, detailed research is required to determine more effective ways to find appropriate light/dark cycle condition to enhance the vegetative growth of Dtps. Queen Beer ‘Mantefon’. As 10/10 h treatment resulted in the similar vegetative growth to 12/12 h treatment, more detailed studies are needed to determine the optimum length of light and dark cycle in order to reduce overall vegetative growth period.