Introduction

The additional photosynthetic daily-light integral associated with low-intensity, day-extension, or night-interruption (NI) lighting is often assumed to have a negligible impact on net photosynthesis (Hong et al. 2014). The absorption of excess light can, however, lead to the increased production of highly reactive intermediates and byproducts that can cause photo-oxidative damage and inhibit photosynthesis (Li et al. 2009). Plants grown under low light intensity or different light quality can develop physiological and anatomical abnormalities such as low photosynthesis rates or irregular functioning of the stomata (Lawlor 2001).

The photosynthetic capacity of leaves depends on a number of factors including the characteristics of the photosynthetic machinery and the availability of nutrients. Nitrogen is particularly important for the synthesis of cellular components including chlorophyll and ribulose-1,5- bisphosphate carboxylase/oxygenase (RuBisCO) (Lawlor 2001). Nitrogen deficiency reduces both source and sink capacities of leaves by reducing the formation of photosynthetic components and thus the photosynthetic rate decreased, and it can consequently shorten the productive lifespan of leaves.

Commercially produced Cymbidium is globally popular because of its large and showy flowers and its long postharvest life. Its growth is rather slow, however, and it takes several years to commercially produce flowering plants. There is, therefore, demand for commercial cultivars with a shorter production period that can be economically produced within 2 years, compared with the 3 – 4 year production period of conventional Cymbidium. Kim et al. (2011, 2015) reported such rapid production in two cultivars of ‘Red Fire’ and ‘Yokihi’ using low-light intensity or high-light intensity night-interruption (NI) lighting. In those studies, without additional nitrogen fertilization, the leaves of Cymbidium plants grown with NI lighting turned yellow. In a previous report, nitrogen fertilization resulted in depression of the midday photosynthetic rate and decreased chlorophyll contents in plants grown under high-intensity NI lighting (Kim et al. 2015). The effects of the photoperiod, including those of NI lighting on flowering, have been studied extensively (Adams et al. 2008; Oh et al. 2009), but few studies have attempted to exploit the effects of the photoperiod on vegetative growth. No study has yet examined the photosynthetic characteristics of Cymbidium plants grown under NI lighting with regard to nitrogen nutrient conditions.

We postulated that the physiological performance and photosynthetic characteristics of Cymbidium depend not only on the photosynthetic photon flux (PPF) but also on the nutrient availability. We hypothesized that plants receiving both a higher PPF during NI lighting and a greater nitrogen supply would accumulate more biomass than plants receiving lower levels of either or both factors. To test that hypothesis, we examined the key photosynthetic traits of Cymbidium plants grown under NI lighting conditions with nitrogen fertilization.

Materials and Methods

Plant materials and growth conditions

Cymbidium ‘Red Fire’ (Mukoyama Orchids Co., Ltd., Japan) plants were transplanted at the 4 weeks of mericlonal stage into 10 cm pots, cultured for 4 months, and then transplanted again into 16 cm pots. The pots contained 100% chopped coconut husk. The plants were grown in a greenhouse at the experimental farm of the College of Agriculture and Life Sciences, Seoul National University in Suwon, Republic of Korea. The average day/night temperatures in the greenhouse were measured by a thermocouple (0.127 mm type E) in an aspirated chamber every 30 s. Hourly averages were recorded by a CR-10 datalogger (Campbell Scientific, Logan, Utah, USA). The hourly average PPF inside the greenhouse was 561 μmol·m^-2·s^-1 (Li 190; Li-Cor Co., Inc., Lincoln, NE, USA) during the day (10:00 – 14:00 HR) throughout the experimental period. The plants were irrigated daily with tap water. Five grams of water-soluble, controlled-release 13N-5.7P-10.8K fertilizer (Mukoyama Orchids Co., Ltd.) was placed on the top of each pot at the time of transplantation and again when the first and second pseudobulbs emerged. Water soluble micronutrient fertilizers were applied bimonthly using a sprinkler. The micronutrient fertilizer provided 472, 3.4, 316, 1.6, 1.2, 1.2, 0.1, and 0.1 g·m^-3 (EC 1.0 dS·m-1) Ca(NO₃)₂·4H₂O, Fe-EDTA, MgSO₄·7H₂O, MnSO₄, ZnSO₄·7H₂O, H₃BO₃, CuSO₄·5H₂O, and Na₂MoO₄·2H₂O, respectively.

Light treatments

Twenty eight plants were divided into three groups for experiments. All plants received a natural daylight that was truncated to 9 h by retractable, opaque, black cloth that covered the greenhouse from 17:00 to 08:00 HR. The three treatment groups were grown with photoperiods consisting of a 1) 9 h short-day (SD), 2) an SD with additional low-intensity (3 - 7 μmol·m^-2·s^-1) NI (LNI) lighting, and 3) an SD with additional high-intensity (120 μmol·m^-2·s^-1) NI (HNI) lighting, r espectively. T he N I lighting w as provided from 22:00 to 02:00 HR by high-pressure sodium lamps (SKL-01; GEO, Hwasung, Republic of Korea). The plants were exposed to the respective experimental conditions for 16 weeks starting 1 year after transplanting (November 2011 to February 2012). The environmental conditions such as air temperature, relative humidity, and CO₂ concentration were kept consistent among the sections of the greenhouse. The CO₂ concentration in the greenhouse was ambient during the day, approximately 400 μmol·mol^-1, and elevated by a CO₂ supply system (SH-MVG250, Soha-tech, Seoul, Republic of Korea) to 800 μmol·mol^-1 during the night to maximize the effects of the NI lighting.

Nitrogen treatments

During the light treatments, the plants were supplied with one of four different concentrations of supplemental nitrogen (0, 100, 200, or 400 mg·L^-1). Thus, there were 12 different experimental treatments: SD+0N, SD+100N, SD+200N, SD+400N, LNI+0N, LNI+100N, LNI+200N, LNI+400N, HNI+0N, HNI+100N, HNI+200N, and HNI+400N. The supplemental nitrogen was supplied by the addition of 0, 0.28, 0.56, or 1.12 g·L^-1 NH₄NO₃ at a 2:8 ratio to the micronutrient fertilizer solutions. The K, P, Ca, and Mg levels were held constant at 100, 100, 100, and 50 mg·L^-1, respectively. The plants were given 300 – 350 mL nutrient solution per day by a drip irrigation system. The average electrical conductivity values for the solutions were 0.7, 0.9, 1.3, and 1.7 dS·m^-1, depending on the N level, and the pH of all the solutions was adjusted with 1N KOH and 2N H₂SO₄ to 6.1 ± 0.1.

Measurements and data collection

Gas exchange was measured in three replicate plants per treatment in January 2012 with 1 h interval daily fluctuation using a portable photosynthesis system (Li 6400; Li-Cor Co., Inc., Lincoln, NE, USA) equipped with an infrared gas analyser. The fourth mature leaf from the base of the flowering pseudobulb was placed inside a 6 cm² top-clear chamber. The light intensity during the day ranged between 400 and 600 μmol·m^–2·s^–1 PPF with natural light intensity through the open chamber, and the block temperature was kept at 20°C and 15°C during the day and night, respectively. The relative humidity in the leaf chamber ranged between 55 and 75%. The same amount of CO₂ that was in the greenhouse was supplied to the leaf chamber during the measurement. Diurnal changes in the net CO₂ assimilation rate (A_n), stomatal conductance (g_s), and transpiration rate (E) were recorded for 24 h using a built-in autoprogram with 6 equipments for 6 days.

The number of leaves, leaf length, leaf width, pseudobulb diameter, and the SPAD value were measured monthly. Leaf length was measured from the longest leaf from the base of the pseudobulb. The pseudobulb diameter was measured at the widest point of the flowering pseudobulb using a digital caliper (ABS Digimatic Caliper; Mitutoyo Co., Ltd., Tsukuba, Japan). The dry weight (DW) of the shoots (leaves and pseudobulbs) and that of the roots were determined after drying the shoots and roots in an oven at 80°C for 3 days after the end of the experiment.

Statistical analysis

The experiment was arranged in a factorial design with seven replicates plants per treatment. Statistical analyses with two-way ANOVA were performed using the SAS system for Windows version 9.3 (SAS Institute Inc., Cary, NC, USA). Differences among the treatment means were assessed by Tukey’s significant difference test at P < 0.05. Regression analyses were performed using Sigma Plot (SYSTAT Inc., Chicago, IL, USA).

Results

The A_n of the leaves in the different treatment groups increased from nearly zero at dawn to a maximum between 11:00 and 13:00 HR (Fig. 1). Under the SD conditions, the A_n was higher during the day (from approximately 11:00 to 17:00 HR) in the SD+100N, and especially the SD+400N, plants compared with the SD+0N and SD+200N plants (Fig. 1A). Under the LNI conditions, the A_n was positive, but the nitrogen treatments had no effect on the A_n during the day or night (Fig. 1B). Under the HNI conditions, the A_n in the HNI+0N plants was significantly lower during the day than that in the HNI+100N, HNI+200N, and HNI+400N plants (Fig. 1C). The HNI+0N plants exhibited a lower rate of A_n throughout the day compared with the plants grown under the LNI or SD conditions. The peak A_n during the HNI illumination was no significantly different that compared with A_n of the day among the plants receiving the additional nitrogen.

The trend in g_s during the day was similar to that in A_n among the plants grown under the SD and LNI conditions (Fig. 2). There was, however, no midday depression of g_s in the HNI+0N plants (Fig. 2C). During the NI treatments, the g_s increased more in the plants grown under the LNI and HNI conditions. Similar to the A_n and g_s, the E declined in the SD+200N plants (Fig. 3A). The night-time E increased during both the LNI and the HNI illuminations. In the plants grown under the HNI conditions, a midday depression of E was observed in the HNI+0N and HNI+100N plants (Fig. 3C).

The leaf number had significance in light and N concentration. HNI with 100N and 200 N treatments had more leaves than other treatments, but SD and LNI had the most leaves with 400 N. The leaf width, pseudobulb diameter, and shoot and root DW were similar among the different photoperiods and nitrogen treatments (Table 1). The chlorophyll concentration was significantly different among the photoperiods and nitrogen treatments. N effect was different depending on NI treatment.

Discussion

One potential way to enhance plant growth is to increase the photosynthetic rates of source leaves by providing higher growth irradiances when other factors (e.g. water and nutrient status) are not limiting (He et al. 1998). Such an approach is sometimes not effective, however, especially in shade plants (Hew and Yong 2004). For instance, light saturation of Oncidium ‘Goldiana’ leaves at all the different stages of development occurred at a PPF between 80 and 100 μmol·m^–2·s^–1. Cymbidium is also classified as a partial shade plant, which limits the ability to stimulate its growth and flowering by increasing the light intensity (Menard and Dansereau 1995).

A small increase in photosynthesis has a greater impact when the ambient light levels are low rather than high (Kim et al. 2015). We observed positive net photosynthesis in Cymbidium plants during LNI illumination (Fig. 1B), suggesting that the increased carbohydrate contents (Kim et al. 2013) and promotion of growth and flowering (Kim et al. 2011) observed during NI illumination in previous studies, might have been caused by increased photosynthesis, which contradicts the paradigm that light of approximately 3 - 4 μmol·m^–2·s^–1 is unlikely to have any impact on net photosynthesis (Lichtenthaler et al. 1981). Adams et al. (2008) reported that the relationship between PPF and net photosynthesis is nonlinear; low-intensity, long-day lighting can offset respiration very efficiently, even in the shade-avoiding petunia (Petunia × hybrida). Furthermore, Hofstra et al. (1969) reported that low-intensity light can be used to offset respiration, demonstrating that a PPF of 13 μmol·m^–2·s^–1 during the night was five times more efficient than that during the day in promoting photosynthesis in orchardgrass (Dactylis glomerata).

The decline in photosynthetic gas exchange during the day in the plants that received the HNI treatment could be linked to decreased chlorophyll content in the leaves (Table 1), possibly resulting from nitrogen deficiency associated with increased leaf-area production and plant energy conversions in photosynthesis during the HNI illumination (Table 1). Increased plant growth requires greater nutrition, and during nutrient deficiency, the photosynthetic apparatus is protected from ‘over-energization’ (Dewir et al. 2005). Lower rates of photosynthesis resulting from nitrogen limitation are often attributed to reductions in leaf chlorophyll content and RuBisCo activity. Nitrogen deficiency has been shown to reduce biomass production by reducing leaf chlorophyll content and A_n in crops such as spinach (Bottrill et al. 1970), sunflower (Cechin and Fumis 2004), and maize (Khamis et al. 1990).

The high-irradiance NI lighting likely exceeded the plants photo-protective capacity, and the plants suffered high irradiance-induced damage to the photosynthetic apparatus. Such damage typically manifests as decreases in the quantum yield of CO₂ assimilation and in the rate of light-saturated photosynthesis, and is often linearly related with an inhibition of PSII photochemistry (Long et al. 1994). For example, flower production declined in Oncidium when the annual total hours of sunshine were high (Ding et al. 1980). We did not expose the plants in our study to high-intensity light during the day or night; however, the plants g rown u nder t he H NI c onditions r educed t heir photosynthetic capacity.

We did not observe greater biomass accumulation as a result of higher net photosynthesis and additional nitrogen supply. We assumed that the duration of the treatments was not enough to affect biomass characteristics in Cymbidium, which takes longer for morphological changes to occur compared with herbaceous plants. Although supplemental lighting can increase growth in orchids, fertilization must also be optimized based, in part, on the light intensities that the plants receive. The plants grown under the HNI conditions required more than 100 mg·L^-1 nitrogen to protect them against depression of photosynthesis (Fig. 1). Previous studies (Evans 1993; Hikosaka et al. 1998) have examined the consequences to photosynthesis of the re-allocation of nitrogen within the leaf or of changes in the leaf nitrogen content under different growth irradiances.

In conclusion, Cymbidium photosynthesized even at a low PPF of 3 - 4 μmol·m^–2·s^–1 during NI illumination. Prolonging the photoperiod with high-irradiance NI illumination can result in a reduction of photosynthesis, depending on the plant species and other abiotic factors, such as nutrition. Plants grown under HNI conditions required more nitrogen than plants grown under SD and LNI conditions to support their increased rates of photosynthesis. We therefore suggest using an additional nitrogen supply greater than 100 mg·L^-1 when HNI l ighting is used to cultivate Cymbidium.