Effect of CO₂ Enrichment on the Photosynthesis and Dry Matter Accumulation in the Oriental Hybrid Lily ‘Siberia’

Abstract

We investigated the effects of CO₂ enrichment on photosynthesis, growth, and dry matter accumulation in the Oriental hybrid lily ‘Siberia’. The photosynthetic rate increased as the CO₂ concentration was elevated compared to the ambient level. The increase in the photosynthetic rate was greater in the low concentration range and lower in the high concentration range. The relationship between the light intensity-photosynthetic rate and temperature-photosynthetic rate was investigated under different CO₂ concentrations. The entire light-photosynthesis and temperature-photosynthesis curves moved toward a considerably higher photosynthetic rate when the CO₂ concentration was increased from 380 ppm to 1000 ppm. In contrast, when the CO₂ concentration was increased from 1000 ppm to 2000 ppm, the increase in the entire light-photosynthesis curve was small. The relationship between the CO₂ concentration and the maximum temperature point of the photosynthetic rate was unclear. We also cultured ‘Siberia’ lily plants with and/or without CO₂ enrichment (1500 ppm) altered before (the early stage) and after (the late stage) the visible flower bud stage. The CO₂ enrichment increased dry weights dry weight/fresh weight ratios of whole plants, and individual parts at flowering, resulting in improved cut flower quality and enlargement of the mother bulb and daughter bulblets. The effective period of CO₂ enrichment was after the visible flower bud stage. In cases where CO₂ enrichment was effective for dry matter accumulation, the dry matter distribution ratios of the mother bulb and daughter bulblets to the whole plant were high, and those of the leaves, stem, and flower buds were low. The relative growth rate and net assimilation rate from planting to the flowering stage were increased with CO₂ enrichment applied after the visible flower bud stage, indicating that the dry matter accumulation and photosynthesis were enhanced. Finally, the issues that need to be addressed for applying practical CO₂ enrichment technology to various lilies are discussed.

Introduction

Lilies (Lilium spp.) are popular in Japanese markets and the production volume ranks second only to the chrysanthemum. Lilies have gorgeous and colorful flowers, and are traded at high unit prices, so high-quality production is required. The quality of horticultural crops is based on their dry matter accumulation. We have reported the effects of cultivation temperature (Inamoto et al., 2013, 2016) and light intensity at different growth stages (Inamoto et al., 2015) on dry matter accumulation in whole plants and its distribution to different plant parts using the Oriental hybrid lily ‘Siberia’ as a material. We also evaluated the relationship between temperature (Inamoto et al., 2016) and light intensity (Inamoto et al., 2015), and the photosynthetic rate at different developmental stages of ‘Siberia’ plants. In addition to temperature and light intensity, CO₂ concentration is another factor that strongly affects dry matter accumulation in plants. CO₂ enrichment technology for ornamental plants, including lilies, has been examined to improve productivity and quality (Mortensen, 1987). However, there has been little research on the basic aspects of photosynthesis, as well as dry matter production and distribution, in lilies.

The present study first investigated the effects of CO₂ on the characteristics of photosynthesis in the Oriental hybrid lily ‘Siberia’. Righetti et al. (2007) indicated that the expression of CO₂ assimilation data on a per leaf area basis made it difficult to determine how the photosynthesis efficiency changed as plants matured. Therefore, we measured the photosynthetic rate of intact potted plants on a whole-plant basis using an open gas-exchange system.

In addition to investigating photosynthesis, we also cultured ‘Siberia’ lily plants under two levels of CO₂ concentrations to evaluate the effects of photosynthetic CO₂ enrichment in the early and late developmental stages. Because the quality of horticultural products is determined largely by the dry matter accumulation, expressed as dry weight, we focused on the dry matter weights of individual plant parts. Moreover, we were interested in the effect of CO₂ enrichment on dry matter partitioning in lilies. Most cut flower production of lilies, which is classified as a bulbous plant, starts from bulbs that are rich in nutrients. In tulip, a typical bulbous plant, most of the dry matter is derived from the stored nutrients in the mother bulb (Ho and Rees, 1975; Inamoto et al., 2000a, b; Rees, 1969). However, bulbous plants need to form new bulbs for the next growth cycle. In tulips, we found that the duration of cold treatment of mother bulbs affected the competitive partitioning of dry matter between shoots (equivalent to cut flowers) and daughter bulblets at flowering, and significantly affected the quality of cut flowers. Next, we applied growth analysis as previously applied to other ornamental plants such as rose (Ushio et al., 2008) and tulip (Inamoto et al., 2000a), and as we applied to lily (Inamoto et al., 2013, 2015, 2016), to clarify the relationship between photosynthetic efficiency and CO₂ enrichment.

Finally, the effects of CO₂ enrichment on the growth and flowering of lilies obtained from the experimental results are summarized, and the issues that need to be addressed for the application of practical CO₂ enrichment technology to various lilies are discussed.

Materials and Methods

All experiments were conducted at the Tohoku Agricultural Research Center, NARO, Morioka, Japan. Bulbs of the Oriental hybrid lily ‘Siberia’, (circumference 18–20 cm) produced in the Netherlands and stored at subzero temperature, were obtained from an importer.

Experiment 1: Relationship between CO₂ concentration and photosynthetic rate

The bulbs were thawed at 2–5°C for 1–2 weeks before pre-rooting treatment. On May 28, 2008, the pre-rooting treatment of bulbs at 10°C with sphagnum peat moss to encourage root development (Abe et al., 1998) started. The bulbs were planted in pumice in pots (25 cm diameter, 30 cm deep) on July 9, 2008. The planting depth was approximately 10 cm, to ensure adequate growth of stem roots (Beattie and White, 1993). A slow-release fertilizer (35 g per pot; Micro-Long Total 201-100; JCAM AGRI. Co., Ltd., Tokyo, Japan) containing 12:10:11:2:0.1:0.06 (w/w) N:P₂O₅:K:Mg:Mn:B was applied to the surface of the pumice in each pot. The plants were cultured in a plastic house, which was covered with a sheet of black cheesecloth providing 40% shading to prevent temperature rise, ventilated at 25°C.

Equipment, measurement procedures, and calculations for estimating photosynthetic rate were almost same as those described in our previous report (Inamoto et al., 2015). Photosynthesis was estimated between August 30 (52 days after planting) and September 10, 2008 (63 days after planting). Stem lengths, numbers of leaves, leaf areas, and fresh weights at this time are shown in Table 1 and the length of flower buds was approximately 5 cm. An open polycarbonate chamber system (120 cm long × 50 cm wide × 90 cm high) for the photosynthesis measurements was placed in a programmable temperature room (LP-1PP; Nippon Medical & Chemical Instruments Co., Ltd., Osaka, Japan). The light was supplied by fluorescent tubes (FPR96EX-D/A; Panasonic Electric Works Co., Ltd., Osaka, Japan) set above (six tubes) and on both sides (four tubes on each side) of the chamber. For photosynthetic rate measurements, photosynthetic photon flux density (PPFD) of 700 to 850 μmol photons·m⁻²·s⁻¹ was supplied at the plant canopy. Air in the chamber was humidified to > 70% relative humidity with a supersonic humidifier (HM-303N; TGK Co., Ltd., Tokyo, Japan).

Table 1

Stem lengths, numbers of leaves, leaf areas and fresh weights of Oriental hybrid lily ‘Siberia’ plants used to measure the net photosynthetic rate in Experiment 1.

Four plants were arranged in the chamber at a distance of 25 cm between the plant center. At the time of measuring the photosynthetic rate, flower buds were removed to avoid any overestimation of the respiratory rate. This action was performed because we previously observed that the respiratory rate was extremely high in flower buds compared to all other plant parts at the flowering stage (data not shown). To correct the effect of the delay in temperature change of the underground parts to air temperature in the chamber, the instantaneous respiratory rate of underground parts was calculated from the medium temperature and the temperature-respiration curve using previous data from Inamoto et al. (2016). The instantaneous photosynthetic rate of the aerial part was calculated by subtracting the respiratory rate of underground parts from the photosynthetic rate of the whole plant.   

$${\mathrm{P}}_{\mathrm{a}}={\mathrm{P}}_{\mathrm{w}}-{\mathrm{R}}_{\mathrm{u}}$$[1]

where, P_a is the net photosynthetic rate of the aerial parts, P_w is the photosynthetic rate of the whole plant, and R_u is the respiratory rate of the underground parts. The following description describes the net photosynthetic rate of this aerial part.

The curve of CO₂ concentration-photosynthetic rate was fitted with a hyperbola curve.   

$${\mathrm{P}}_{\mathrm{n}}=\frac{\mathrm{a}·\mathrm{x}+\mathrm{b}}{\mathrm{c}+\mathrm{x}}$$[2]

where P_n is the net photosynthetic rate (μmol CO₂/plant/s), x is the CO₂ concentration (ppm), and a, b, and c are constants.

The light intensity-photosynthetic rate relationships were fitted with non-rectangular hyperbola curves following Marshall and Biscoe (1980), Hirose and Werger (1987), and Lieth and Pasian (1990).   

$$\begin{array}{l}{\mathrm{P}}_{\mathrm{n}}={\mathrm{P}}_{\mathrm{g}}-\mathrm{R}\\ =\frac{\mathrm{f}·\mathrm{x}+{\mathrm{P}}_{\max }-\sqrt{{\left(\mathrm{f}·\mathrm{x}+{\mathrm{P}}_{\max }\right)}^2-4\mathrm{ }\mathrm{f}·\mathrm{x}·{\mathrm{P}}_{\max }·\mathrm{q}}}{2\mathrm{ }\mathrm{q}}-\mathrm{R}\end{array}$$[3]

where P_n (μmol CO₂/plant/s) is the net photosynthetic rate, P_g (μmol CO₂/plant/s) is the gross photosynthetic rate, f is the initial slope of the curve (μmol CO₂/plant·μmol photon⁻¹·m²), x is the PPFD (μmol photon·m⁻²·s⁻¹), P_max is an asymptotic maximum value of P_g for large values of I (μmol CO₂/plant/s), q is a convex degree (a dimensionless parameter indicating the degree of curvature; 0 ≤ q ≤ 1), and R is the respiratory rate (μmol CO₂/plant/s).

The curve of temperature-photosynthetic rate relationships on a whole-plant basis was fitted with cubic curves.   

$${\mathrm{P}}_{\mathrm{n}}=\mathrm{a}·{\mathrm{x}}^3+\mathrm{b}·{\mathrm{x}}^2+\mathrm{c}·\mathrm{x}\mathrm{ }+\mathrm{d}$$[4]

where P_n is the net photosynthetic rate (μmol CO₂/plant/s), x is the leaf temperature (°C), and a, b, c, and d are constants.

The constants, solutions, and extrema in these equations were calculated using DeltaGtaph ver. 7 (Red Rock Software Inc., Salt Lake City, UT, USA) and Grapher Ver 2.7 (Apple Inc., Cupertino, CA, USA).

Experiment 2: Influence of CO₂ enrichment during different plant developmental stages on dry matter accumulation

Bulbs stored at subzero temperature were used. On August 19, 2009, the pre-rooting treatment of bulbs at 10°C was started. The bulbs were planted in pots on September 2, 2009. One bulb was planted in one 21 cm-diameter plastic pot filled with a growth medium (Premier PRO-MIX BX Mycorise; Premier Tech Ltd., Rivière-du-Loup, QC, Canada) containing 10 g of the same slow-release fertilizer as in Experiment 1. The planting depth was approximately 10 cm. At the planting time, plants were sampled, and their fresh weight (FW) and dry weight (DW) were measured.

We prepared two natural light constant temperature growth chambers (GC; Koitotron S153A; Koito Electric Industries, Ltd., Shizuoka, Japan). One GC was used for CO₂ enrichment treatment; CO₂ gas was provided to the GC daily from 4:00 to 16:00 every day (CO₂+GC) and the CO₂ condition was ambient in the other GC (CO₂−GC). For the feeding of CO₂ to CO₂+GC, pure CO₂ gas was provided from gas cylinders via a solenoid valve. The concentration of CO₂ in the GCs was measured and controlled by a CO₂ transmitter (eSence Disp; Senceair AB., Delsbo, Sweden), and a data logger system (XL100; Yokogawa Electric Corporation., Tokyo, Japan). In preliminary operations of the CO₂ supply device, a decrease in the concentration of CO₂ by about 500 ppm from the set value was observed due to the influence of ventilation and a delay in CO₂ supply. Therefore, considering the results of Experiment 1, the set value of the CO₂ concentration of the CO₂+GC was at 1500 ppm.

On the day of bulb planting the plants were settled in the GCs. Thirty plants were arranged in each GC. The temperature in all GCs was set to 25°C during the day (06:00–18:00) and to 20°C during the night (18:00–06:00). The relative humidity (RH) in the GCs was maintained at > 70%. The plants were grown under natural day length.

We have previously shown that the visible flower bud stage switched to self-sustaining growth via photosynthesis in ‘Siberia’ lily in an experiment with light shading (Inamoto et al., 2015). Similarly, in this experiment, we defined the alternation in the presence or absence of CO₂ enrichment as the visible flower bud stage. Therefore, on October 2, when the flower buds became visible at the end of the stem (30 days after planting: day 30), 15 plants grown in CO₂+GC were transferred to CO₂−GC, and 15 plants grown under CO₂−GC were transferred to CO₂+GC. Therefore, the four experimental plots were set based on CO₂ enrichment before or after the visible flower bud stage as follows: CO₂−/CO₂−, CO₂−/CO₂+, CO₂+/CO₂−, and CO₂+/CO₂+.

On November 16, just before the top of the flower buds opened (75 days after planting: day 75), all plants from those plots were simultaneously harvested for measurement at the flowering stage. This was done because once the flower buds open, the FW of the flower organs changes very rapidly, and this can affect the values related to FW. In another experiment with CO₂ enrichment of lily ‘Siberia’, in which the flowering date was defined as just before the opening of the top of the flower buds, there was no difference in days from planting to flowering (Inamoto et al., 2012).

The sampled plants were then separated into their various parts, and their size, FW, DW, leaf area (LA), and leaf color were recorded. Next, the dry matter (DM) distribution ratios, defined as the DW of individual part/DW of the whole plant, were calculated. The DW/FW ratios of the whole plant and individual parts were also calculated.

From the DW and LA data, we calculated the leaf area ratio (LAR, LA per unit of total plant DW, cm²·g⁻¹), leaf mass per area (LMA, leaf DW per LA), relative growth rate (RGR, rate of increase in total plant DW per unit of plant DW, mg·g⁻¹·d⁻¹), and net assimilation rate (NAR, rate of increase in total plant DW per unit of LA, g·m⁻²·d⁻¹), as described in our previous reports (Inamoto et al., 2013, 2015, 2016).

Results

Experiment 1: Relationship between CO₂ concentration and photosynthetic rate

Figure 1 shows the relationship between CO₂ concentration and photosynthetic rate when the temperature in the chamber was set at 25°C and the light intensity at the plant canopy was approximately 850 μmol·m⁻²·s⁻¹ PPFD. As the CO₂ concentration was elevated, the photosynthetic rate increased. When the CO₂ concentration was raised to 2000 ppm, the photosynthetic rate was two times higher than the ambient level. However, the increase in photosynthetic rate was greater in the lower concentration range and lower in the higher concentration range, i.e., the photosynthetic rate at a temperature of 25°C and a light intensity of 850 μmol·m⁻²·s⁻¹ PPFD was calculated from the regression equation to be 1.86 μmolCO₂/plant/s at 380 ppm, 3.62 μmolCO₂/plant/s at 1000 ppm, and 4.03 μmolCO₂/plant/s at 2000 ppm.

Fig. 1

Relationship between the CO₂ concentration and the net photosynthetic rate of the Oriental hybrid lily ‘Siberia’ in Experiment 1. The temperature in the chamber was set at 25°C and the light intensity at the plant canopy was approximately 850 μmol·m⁻²·s⁻¹ PPFD. The curve was fitted with a hyperbola curve. See equation [2] in the Materials and Methods section of the text. The coefficient of determination was 9.994.

Figure 2 shows the relationships between light intensity and photosynthetic rate under different CO₂ concentrations when the temperature in the chamber was set at 25°C. Figure 3 shows the relationships between leaf temperature and photosynthesis under different CO₂ concentrations with a light intensity at the plant canopy of approximately 850 μmol·m⁻²·s⁻¹ PPFD. The entire light-photosynthesis and temperature-photosynthesis curves rose toward considerably higher photosynthetic rates when the CO₂ concentration was increased from 380 ppm to 1000 ppm (Figs. 2 and 3). In contrast, when the CO₂ concentration was increased from 1000 ppm to 2000 ppm, the rise in these curves decreased. Under the three CO₂ concentrations, the light-photosynthesis curves showed that the photosynthetic rate increased with increasing light intensity (Fig. 2). The calculated constants of the light-photosynthesis non-rectangular hyperbola curves for each CO₂ concentration are shown in Table 2. The P_max-value increased significantly when the CO₂ concentration was increased from 380 ppm to 1000 ppm, but it was almost the same for 1000 ppm and 2000 ppm (Table 2). The q-value at 2000 ppm was considerably higher than those at 380 and 1000 ppm CO₂ (Table 2). The temperature-photosynthesis curve showed that the photosynthetic rate increased with increasing temperature and decreased after reaching its peak (Fig. 3). The maximum photosynthetic rate at 850 μmol·m⁻²·s⁻¹ PPFD was calculated to be 28.7°C at 380 ppm CO₂, 26.7°C at 1000 ppm CO₂, and 30.0°C at 2000 ppm CO₂ (Fig. 3).

Fig. 2

Relationships between the light intensity and the net photosynthetic rate of the Oriental hybrid lily ‘Siberia’ under different CO₂ concentrations in Experiment 1. The temperature in the chamber was set at 25°C. The curves were fitted with non-rectangular hyperbola curves. See equation [3] in the Materials and Methods section of the text. Constants in the equation are shown in Table 2. Coefficients of determination were 0.990 for 380 ppm, 0.988 for 1000 ppm, and 0.978 for 2000 ppm.

Fig. 3

Relationships between the leaf temperature and the net photosynthetic rate of the Oriental hybrid lily ‘Siberia’ under different CO₂ concentrations in Experiment 1. The light intensity at the plant canopy was approximately 850 μmol·m⁻²·s⁻¹ PPFD. The plots are weighted average every 1°C. The curves were fitted with cubic curves. See equation [4] in the Materials and Methods section of the text. Coefficients of determination were 0.997 for 380 ppm, 0.988 for 1000 ppm, and 0.980 for 2000 ppm. Arrows indicate the maximum value points of each curve.

Table 2

Constants in the light-photosynthetic non-rectangular hyperbola curves under three CO₂ concentration conditions in Experiment 1.

Experiment 2: Influence of CO₂ enrichment during different plant developmental stages on dry matter accumulation

The light intensity (PPFD and DLI: daily light integral) during the experiment at the experimental field is shown in Figure 4. The daily average CO₂ concentration from 4:00 to 16:00 in the CO₂+GC was in the range of 1150–1650 ppm (average over the experimental period: 1470 ppm), while that in the CO₂−GC was in the range of 400–580 ppm (average over the experimental period: 480 ppm).

Fig. 4

The light intensity (PPFD and DLI) during Experiment 2 (September 1–November 16, 2009). The solid lines and open circles indicate the PPFD and DLI, respectively. On October 2 (the dotted line; visible flower bud stage) the plants of CO₂+/CO₂− and CO₂−/CO₂+ plots were transferred from chambers with and without CO₂ to another chamber.

Table 3 shows the growth status of the plants grown under different CO₂ enrichment treatments at the flowering stage. There was no significant effect of CO₂ enrichment on the number of leaves, number of flower buds, or stem length. The stem diameter just below the inflorescence was larger in the CO₂−/CO₂+ and CO₂+/CO₂+ treatments, and smaller in the CO₂−/CO₂− and CO₂+/CO₂− treatments. Leaf color (SPAD) was higher in the CO₂−/CO₂− plots than in the other plots.

Table 3

Numbers of leaves, flower buds, and daugher bulblets, leaf color, and stem lengths and diamter of Oriental hybrid lily ‘Siberia’ plants at the flowering stage grown under different CO₂ enrichment treatments in Experiment 2.

Table 4 shows the FWs and DWs of whole plant and aerial parts (leaves, stem, and flower buds), the DM distribution ratios of individual aerial parts, the DW/FW ratios of the whole plant and individual aerial parts, and increase in DW of the whole plant from planting at the flowering stage. In the following descriptions, large and small relationships with a significant difference are indicated by >, those without significant difference are indicated by ≥, and nearly identical values (without significant difference) are indicated by =. The FWs of whole plants were greater in the order of CO₂+/CO₂+ ≥ CO₂−/CO₂+ ≥ CO₂+/CO₂− ≥ CO₂−/CO₂− and the DWs of whole plants were CO₂+/CO₂+ = CO₂−/CO₂+ > CO₂+/CO₂− = CO₂−/CO₂−. The increase in DW of the whole plants from planting to flowering was 44.5 g over the entire-period CO₂ enrichment (CO₂+/CO2+), which was more than twice as high as the 21.8 g in the no CO₂−enrichment plants (CO₂−/CO₂−) (Table 4). Furthermore, there was no significant difference in DWs of whole plants between the CO₂+/CO₂+ and CO₂−/CO₂+ plots, or between the CO₂+/CO₂− and CO₂−/CO₂− plots respectively. The FWs and DWs of leaves, and the FWs of stems were not significantly different among the experimental plots, but the DWs of stems were greater in the order of CO₂+/CO₂+ ≥ CO₂−/CO₂+ ≥ CO₂+/CO₂− ≥ CO₂−/CO₂−. The order of the FWs and DWs of flower buds were as follows: CO₂+/CO₂+ = CO₂−/CO₂+ > CO₂+/CO₂− = CO₂−/CO₂−. The DM distribution ratios of leaves and stems were lower in the CO₂−/CO₂+ and CO₂+/CO₂+ plots compared with those in CO₂+/CO₂− and CO₂−/CO₂−. The order of the DM distribution ratio of flower buds was CO₂−/CO₂− ≥ CO₂+/CO₂− = CO₂−/CO₂+ = CO₂+/CO₂+. The DW/FW ratios of the whole plant, leaves, and stems were significantly higher in the CO₂−/CO₂+ and CO₂+/CO₂+ compared with those in CO₂−/CO₂− and CO₂+/CO₂−. The DW/FW ratios of flower buds was not significantly different among the four experimental plots.

Table 4

Fresh and dry weights, dry weight distribution ratios to whole plant, and dry weight/fresh weight ratios of whole plant and aerial parts of Oriental hybrid lily ‘Siberia’ plants at the flowering stage grown under different CO₂ enrichment treatments in Experiment 2.

Table 5 shows the FWs, DWs, the DM distribution ratios, and the DW/FW ratios of the underground parts (bulb roots, stem roots, mother bulb, and daughter bulblets). No consistent trend was observed for the bulb roots. The DWs of stem roots were greater in the order of CO₂−/CO₂+ = CO₂+/CO₂+ ≥ CO₂+/CO₂− ≥ CO₂−/CO₂−. The FWs and DWs of mother bulbs were as follows: CO₂+/CO₂+ = CO₂−/CO₂+ > CO₂+/CO₂− = CO₂−/CO₂−. The DM distribution ratios and DW/FW ratios of mother bulbs were also in the order of CO₂+/CO₂+ > CO₂−/CO₂+ = CO₂+/CO₂− > CO₂−/CO₂−. The values of FWs and DWs of the daughter bulblets were markedly smaller than those of other parts of the plant, and the order was CO₂+/CO₂+ ≥ CO₂−/CO₂+ = CO₂−/CO₂− = CO₂+/CO₂−.

Table 5

Fresh and dry weights, dry weight distribution ratios to whole plant, and dry weight/fresh weight ratios of underground parts of Oriental hybrid lily ‘Siberia’ plants at the flowering stage grown under different CO₂ enrichment treatments in Experiment 2.

The growth analysis results are shown in Table 6. The LAs of the whole plant and individual leaves were not significantly different among the four experimental plots. The order of LARs was CO₂−/CO₂− > CO₂+/CO₂− > CO₂−/CO₂+ = CO₂+/CO₂+, and the LMAs were significantly greater in the CO₂+/CO₂+ than in the others. The RGRs and NARs from planting to flowering were as follows: CO₂+/CO₂+ = CO₂−/CO₂+ > CO₂+/CO₂− = CO₂−/CO₂−.

Table 6

Leaf areas, leaf area ratios, leaf mass per areas, relative growth rates, and net assimilation rates of Oriental hybrid lily ‘Siberia’ plants at the flowering stage grown under different CO₂ enrichment treatments in Experiment 2.

Discussion

In Experiment1, as in many plant species, the photosynthetic rate of ‘Siberia’ lily was enhanced by increasing the CO₂ concentration (Figs. 1, 2, and 3). The photosynthetic characteristics of Oriental hybrid lilies, including ‘Siberia’, have already been reported by Chang et al. (2008). Their results were generally consistent with our evaluation of photosynthetic characteristics, although there were some differences. When our whole plant photosynthetic rate values (Figs. 2 and 3) were converted to leaf area-based values (data not shown), they were lower than those of Chang et al. (2008), especially in the low light intensity range. This difference may be because they measured the photosynthesis of individual leaves, while we measured the individual plant. Individual plants contain non-photosynthetic parts other than leaves, reducing the apparent photosynthetic rate due to respiration. In addition, there may be an effect of mutual shading of the leaves. Sorrentino et al. (1997) mentioned that the contribution to total photosynthesis in ‘Casa Blanca’ lily plants depends on the leaf position and apical leaves made a large contribution to total photosynthesis. Standard photosynthesis measurements in lily have been made on a per-unit leaf-area basis (Sorrentino et al., 1997; Wang, 1990; Zhang et al., 2011). As mentioned in the introduction, Righetti (2007) insisted that the photosynthetic rate on a whole plant basis is more indicative of the actual aspects of photosynthetic assimilation in plants than the photosynthetic rate of an individual leaf.

The relationship between CO₂ concentration and photosynthetic rate can be adapted to a hyperbola, where the increase rate in the photosynthetic rate was greater in the low concentration range and lower in the high concentration range when the CO₂ concentration was elevated from the ambient level (about 400 ppm) (Fig. 1). The relationships between light intensity- and temperature-photosynthetic rate were examined under different CO₂ concentrations (Figs. 2 and 3). The entire light-photosynthesis and temperature-photosynthesis curves moved toward a considerably higher photosynthetic rate when the CO₂ concentration was increased from 380 ppm to 1000 ppm. In contrast, when the CO₂ concentration was increased from 1000 ppm to 2000 ppm, the increase in the entire light-photosynthesis and temperature-photosynthesis curves was small. These results indicate that the effect of CO₂ enrichment is more effective under growing conditions with a low CO₂ concentration range.

Table 2 shows the values of the constant terms in the non-rectangular hyperbolas used to approximate the light-photosynthesis curves shown in Figure 2. The P_max-value increased significantly when the CO₂ concentration was increased from 380 ppm to 1000 ppm, and was almost the same for 1000 ppm and 2000 ppm (Table 2). However, the measured photosynthetic rate was only the range up to 1000 μmol·m⁻²·s⁻¹ PPFD in this experiment (Fig. 2). Since extrapolation of function curves to higher light levels should be avoided, the P_max-value will not be discussed here. On the other hand, the q-value at 2000 ppm was considerably higher than those at 380 and 1000 ppm CO₂ (Table 2). It is noteworthy that the q-values indicate the convexity of the curves; the larger the q-value, the steeper the curvature becomes (Fig. 2). This indicates that the photosynthetic rate at 2000 ppm increased sharply with an increase in light intensity, then suddenly peaked, i.e., the photosynthesis is saturated (Fig. 2). Moreover, this may indicate some morphological or biochemical responses and limitations to elevated CO₂. The causes of limited photosynthesis under high CO₂ concentration include mechanical factors such as stomatal closure (Haworth et al., 2016; Morison and Gifford, 1983; Swalls and O’Leary, 1976; Tanigawa and Kobayashi, 1991) and biochemical factors such as limitation by Rubisco status, which is involved in the uptake of CO₂ into the Calvin circuit (Besford, 1993; Besford et al., 1990; Wong, 1979), limitation of electron transport on photon flux reception (Farquhar et al., 1980), and triosephosphate inhibition, which is involved in the transfer of assimilates from chloroplasts to the cytoplasm (Sage et al., 1989; Sharkey, 1985). Further research is needed to determine which factors limit CO₂ uptake by lilies.

The temperature-photosynthesis curves indicate that there is a temperature range that is suitable for photosynthesis in lilies (Fig. 3). In tomato (Nilsen et al., 1983) and carnation (Enoch and Hurd, 1977), the temperature at which maximum photosynthesis shifted was higher at high CO₂ concentrations. Conversely, the photosynthetic rate of chrysanthemum was decreased at high temperatures under high CO₂ concentration (Tanigawa and Kobayashi, 1991). In contrast to the results in these horticultural plants, in the range of 20–35°C, the degree of change in the photosynthetic rate was small in ‘Siberia’ lily (Fig. 3). In addition, the relationship between CO₂ concentration and the maximum temperature point of the photosynthetic rate was unclear (Fig. 3). Sorrentino et al. (1997) reported that the Oriental hybrid lily ‘Casablanca’ can be maintained high under high temperatures of 31–32°C, probably in experiments under ambient CO₂ concentration.

In Experiment 2, when CO₂ was applied for the entire period from planting to flowering (CO₂+/CO₂+), the FW of flower buds was 83.9 g (Table 4) and the diameter of the stem just below the inflorescence was 8.64 mm (Table 3), which was significantly greater than the FW of flower buds of 69.4 g (Table 4) and stem diameter of 7.58 mm (Table 3) in the no CO₂ enrichment plants (CO₂−/CO₂−), indicating the desirable traits of cut flowers in terms of larger flowers and thicker stems. In addition, the FW of the mother bulb was doubled from 25.2 g in the no CO₂ enrichment plot to 50.5 g in the entire period applied plots (Table 5), which is desirable for bulb reuse for the next cultivation season. Furthermore, the increase in the DW/FW ratios of the leaves, stem (Table 4), and mother bulb (Table 5) due to CO₂ enrichment are also desirable because it results in firm and good quality cut flowers and bulbs. Moreover, the well-developed stem roots (Table 5) may improve the quality of cut flowers and bulbs through nutrient absorption.

The CO₂ enrichment for the entire period (CO₂+/CO₂+) resulted in greater DWs and DW/FW ratios of the whole plant and most individual plant parts compared to no CO₂ enrichment (CO₂−/CO₂−) (Tables 4 and 5). We also applied CO₂ enrichment at two different growth stages, that is, from planting to the visible flower bud stage and from the visible flower bud stage to the flowering stage, and found that CO₂ enrichment only after the visible flower bud stage (CO₂−/CO₂+) showed results similar to those of entire period enrichment (CO₂+/CO₂+) (Tables 4 and 5). In our previous report (Inamoto et al., 2015), when the shading rate was varied in these two stages, the light intensity from planting to the visible flower bud stage did not affect the final DWs of whole plants or individual plant parts at flowering, and high light intensity only after the visible flower bud stage increased the final DWs. Therefore, the effect of supply at each growth stage is the same for both light and CO₂, and it was reconfirmed that the visible flower bud stage is the time of switching to autonomous photosynthetic growth. The reason for these results is that in ‘Siberia’ lily, the LA is sufficiently enlarged after the visible flower bud stage for a well-established photosynthetic system (Inamoto et al., 2015).

The CO₂ enrichment not only increased the dry matter accumulation in whole plants, but also affected the distribution of dry matter among the plant parts (Tables 4 and 5). As observed in tulips (Inamoto et al., 2000a, b), a competitive relationship was found between aerial and underground parts in ‘Siberia’ lily. The DM distribution ratios of leaves, stems, and flower buds were lower in CO₂+/CO₂+ and CO₂−/CO₂+ plots in which CO₂ enrichment was effective for total dry matter accumulation (Table 4). Corresponding to this, in these experimental plots, the DM distribution ratios were considerably higher in the mother bulb and to some extent in the daughter bulblets (Table 5). The distribution of assimilated products of photosynthesis was higher in the plant parts that were actively growing at that time. For example, it has been shown that the translocation rate of assimilates from bent shoots (source) to upright flowering shoots (sink) in “arching” rose cultivation depends on the sink strength associated with the size and autotrophic growth of upright shoots (Isobe et al., 2020). In Lilium speciosum, which is one of the breeding materials for Oriental hybrid lilies, enlargement of the mother bulb and daughter bulblets begins just after the visible flower bud stage (Ohkawa, 1977), and the photosynthetic assimilates of the leaves may be strongly drawn to the bulb and bulblets. In addition, a reduction in the overall sink intensity of plants may even result in a decrease in photosynthesis itself (Arp, 1991).

The absolute and relative reduction in leaf size with CO₂ enrichment, as measured by LA and LAR, have been reported for several plant species (Kelly et al., 1991; Wong, 1979). The abundant supply of CO₂ may have caused a kind of adaptation to the smaller size of leaves for CO₂ absorption, which in turn affected the distribution of dry matter. In Experiment 2, the CO₂ enrichment did not affect the LA, but reduced the LAR (Table 6) of the lily ‘Siberia’. Moreover, the increased LMA, especially in the CO₂+/CO₂+ treatment (Table 6), indicates an increase in leaf thickness. The phenomenon of increased leaf thickness due to CO₂ enrichment has been reported in other C₃ plant species such as rose (Hayashi et al., 2021), poplar (Radoglou and Jarvis, 1990), sweet gum (Thomas and Harvey, 1983), and soybean (Leadley et al., 1987; Thomas and Harvey, 1983). The RGRs and NARs from planting to flowering of CO₂+/CO₂+ and CO₂−/CO₂+ plots were considerably higher than those of CO₂+/CO₂− and CO₂−/CO₂− plots (Table 6). Since NAR is an index of the increase in DW per LA, i.e., the photosynthetic efficiency, this result is consistent with the results of Experiment 1 in which CO₂ concentration and photosynthetic rate were evaluated.

This study clarified the response of ‘Siberia’ lily, which is a typical Oriental hybrid variety, to CO₂. CO₂ enrichment increased photosynthetic rate and dry matter accumulation in lilies, resulting in improved cut flower quality and enlargement of the mother bulb and daughter bulblets. Furthermore, CO₂ enrichment applied after the visible flower bud stage was significantly more effective. CO₂ enrichment up to 1000 ppm is assumed to be sufficiently effective. However, the variety of lilies, and their growth and physiological characteristics are very diverse. For example, Chang et al. (2008) reported wide diversity of photosynthetic characteristics among Oriental hybrid lily cultivars. In this study, we found that the average time from planting to flowering of Oriental hybrid lilies was about 1.5 times longer than that of Asiatic hybrids (Inamoto, unpublished data). Therefore, for the practical enrichment of CO₂, it is necessary to study varieties or at least groups of varieties with similar characteristics. Considering the results of this report and previous reports (Inamoto et al., 2015), we consider that the key points are the period up to flowering (period for assimilation by photosynthesis) and leaf development (leaf-area expansion).

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