CAM-physiology and carbon gain of the orchid Phalaenopsis in response to light intensity, light integral and CO₂

2.1 Plant material and general growth conditions

Two experiments were conducted: a lighting experiment and an additional CO₂-experiment. Both experiments were performed using Phalaenopsis “Sacramento” (further referred to as Phalaenopsis).

For the lighting experiment, young plants were potted in a bark medium, grown in a commercial greenhouse and transferred to the climate chambers of Plant Lighting (Bunnik, the Netherlands) 17 weeks after potting. The plants were divided into six climate-units of 2 m² each, all situated within one climate chamber and 130 plants per unit (112 plants per unit after destructive measurements). The plants were grown at 29°C average diel temperature, relative humidity (RH) was ±70% and the CO₂ concentration was 800 ppm. This relatively high CO₂ concentration was to maximize malate storage, which increases distinctiveness between effects of lighting-treatments on malate (de)carboxylation. Every 4 to 5 days plants were watered with a nutrient solution (EC ≈ 1.2, pH ≈ 5.8). To ensure that all measurements (photosynthesis, malate and citrate samples) were under conditions without any drought stress, individual plants were sometimes watered an additional time before measuring. In the first 3 weeks, the conditions were equal in all six climate units, allowing the plants to acclimate to the new conditions.

Light was provided by solar simulators emitting a spectrum nearly identical to that of natural sunlight within the range 360–780 nm (Artificial Sunlight Research Module (ASRM), SLHolland, Bunnik, the Netherlands). The spectrum is shown in Figure 1. The ASRM is dimmable with retention of the spectrum, and the UV (360–400 nm), PAR (400–700 nm) and far-red (700–780 nm) spectral ranges are also dimmable separately via three 0–10 V signals. Different lighting schedules could be programmed using a programmable DMX-interface (SLESA UE7, Nicolaudie, France) connected to a multiple-channel DMX to 0–10 V signal converter. Throughout the lighting experiment, the spectrum as shown in Figure 1 was maintained, whereas light intensity patterns were different for the different treatments (details below).

2.2 Experimental conditions used for the lighting experiments

Throughout the experiment, all plants received a 16 h photoperiod in which at certain times light intensities changed (Table 1). These settings were tuned to the expected CAM-phases based on explorative photosynthesis measurements: 6:00–7:30 (expected CAM-phase II), 7:30–17:30 (expected CAM-phase III) and 17:30–22:00 (expected CAM-phase IV). After the initial 3 weeks acclimation (exposed to MML, Table 1), the light settings were varied between all six climate-units for 1 day, to measure the impact of different light intensities on the CAM pattern (see gas-exchange and biochemical analysis methods below). The different lighting treatments are in Table 1. Expected CAM-phase III is roughly split into PAR setting I and II for, respectively, the first and the second half of CAM-phase III. Only for the expected CAM-phase III, which was determined at the end of the acclimation period, the lighting differed between treatments. Note that the lighting treatments may change the duration of phase III.

After determining the short-term (1 day) effect of the changes in light regime, the plants were grown for another 10 weeks under six different lighting regimes (Table 2). The long-term effect of these treatments on the CAM-photosynthesis pattern was measured on newly developed, fully expanded leaves: photosynthesis was measured 6 to 10 weeks after starting the treatments and leaf samples for biochemical analysis were taken 9 to 10 weeks after starting the treatments. The photoperiod (16 h) and average daily light integral were equal for all six treatments (5.6 mol m⁻²). In treatment MML, MML/T, HLL and LHL the light integral within CAM-phase III was equal, however, according to a different pattern. This was to resolve the question of whether light intensity regulates the rate of malate decarboxylation, and if there are any consequences of different lighting patterns during CAM-phase III over the long term. LLH received only half the light intensity of the control over the time-course for CAM-phase III in the MML treatment, after which the light intensity was increased. This was to resolve the question if malate storage overnight is decreased, or if CO₂ is released from the leaf during phase III and/or if CAM-phase III is extended over a longer period. The alternating light treatment received an illumination pattern of 4 days low light (LLL), and 4 days high light (HHL). Over 8 days this results in an equal average daily light integral as the MML treatment. This was to resolve the question of how photosynthesis patterns respond to fluctuating light conditions, and if such fluctuations have result in a penalty regarding the total carbon gain.

2.3 Photosynthesis measurements

All photosynthesis measurements were performed on fully grown, unshaded leaves of well-watered plants. Measurements were performed with a Li-6400 infra-red gas-analyser with a head Fluorometer and a 2 cm² net area leaf chamber (LI-COR, Nebraska, USA). In all measurements the light spectrum in the leaf chamber was set on 10% blue and 90% red, air flow was 200 μmol s⁻¹, block temperature was 28°C, matching sample and reference gas was done every 20 min and the RH was similar to that in the plant growth chamber. Data were logged every 2 min for gas exchange measurements and chlorophyll fluorescence was measured every 20 minutes during daytime and not during night time. Photosystem II operating efficiency (Fq′/Fm′; where Fq′ = Fm′ − F′) (Baker, 2008)) was determined by recording F′ at steady-state and Fm′ after applying a saturating light pulse (>7,000 μmol m⁻² s⁻¹ for 0.8 s).

Initially, diel patterns of gas exchange and fluorescence were measured to determine the time periods of the four CAM-phases (n = 8). CO₂ was set at 800 ppm and the leaf chamber lighting was programmed such that the light intensity pattern matched with that in the growth chamber. For all diel measurements in all experiments, each leaf was clamped in the leaf chamber for approximately 20 h. Measurements always started and ended during CAM-phase III, allowing quantitative analysis of leaf CO₂ uptake during phases I, II and IV. Although leaf transpiration is measured and stomatal conductance (g_s) is calculated by the Li-6400, these measurements are often below the detection limit (g_s < 0.01 mol m⁻² s⁻¹) at the start of phase IV (stomata open) and end of phase II (stomata close). Therefore, we present atmospheric CO₂-uptake > 0 as the moment that the stomata open.

After changing the light settings in the growth chamber (Table 1), gas-exchange was measured for ±5 min per leaf at a light intensity of 120 μmol m⁻² s⁻¹ PAR measuring light. These screening measurements were done on 29–39 leaves per treatment for the LLL, MML, HHL and VVL treatments (the treatments with a constant light intensity during PAR setting I and II) and served to determine how the growth-light intensity during CAM-phase III affects the time of malate depletion, and if that results in a lower electron transport rate (ETR) and an earlier start of CAM-phase IV (start of CO₂-uptake). In addition, these measurements show if a lower light intensity (LLL) results in an extended CAM-phase III or possibly CO₂ not assimilated being released from the leaf. By turning off the measuring light for 2 min per leaf in CAM-phase IV following the ±5 min light intensity of 120 μmol m⁻² s⁻¹ PAR, we could determine whether CO₂ taken up by the leaf resulted in carboxylation via Rubisco (CO₂-uptake drops at light off) or via PEP-C (light-independent CO₂-uptake).

After 6 weeks acclimation to the long-term light treatments (Table 2), diel patterns of gas exchange and fluorescence were measured on six leaves per treatment for treatment MML, MML/T, HLL, LHL, LLH, and for the alternating light treatment six leaves on the first and on the fourth day of the alternating low and high light periods (LLL D1, LLL D4, HHL D1, HHL D4; N = 6 per measuring period). The leaf chamber lighting was programmed such that the light intensity pattern matched with that of the growth light treatment.

2.4 Malate and citrate measurements

Changes in malate and citrate concentration in the leaf during the light period were measured in the lighting experiments. On the day that the light settings in the growth chamber were changed (Table 1) and after long-term adaptation (Table 2), leaf samples were cut at approximately 2-h intervals between 06:00 and 22:00 (nine times per day). Four leaf discs (1 cm diameter) were cut from pairs of fully grown unshaded leaves from two plants (two discs per leaf) each time, and the complete time-series was made from the same pair of leaves, starting from the leaf tip towards halfway the leaf length in the direction of the leaf base (three pairs of leaves per treatment, n = 3). To exclude if the sampling position on the leaf affects the malate concentration, samples were also taken at the different positions on the leaf at the same time. No difference between the leaf tip and the middle leaf position was measured. To quantify the effect of taking samples from the same leaf, samples were taken for 4 days in a row at 08:30 from the same leaves (N = 3). The malate concentration decreased from 125 to 83 mmol/m² (p < .05) on the second day and remained stable on the third and fourth day. This implies that the sampling method may have quantitatively affected the malate response measured in the time-series over the day. Nonetheless, it is highly unlikely that this has interacted with the clear differences in malate decarboxylation in response to light shown in the results section. The method does not affect the malate concentration measured at the start of the day as those leaves were always fully intact.

The samples were placed in Eppendorf tubes, frozen in liquid nitrogen and stored at −80°C. Subsequently, the samples were freeze-dried, weighed, powdered with a ball-mill (1 minute with two metal balls of 3 mm), after which 15 mg was weighed. The 15 mg was subsequently dissolved in 5 mL 75% ethanol, heated in a shaking water bath (80°C, 20 min) and centrifuged for 5 min (8,500 rpm, 4°C). Next, 1 mL of supernatant was dried in the Speed Vac at 55°C, evaporated ethanol was replaced by 1 mL MiliQ water and mixed for 10 min in an ultrasonic bath (50 Hz) followed by vortex and centrifuge for 10 min. Samples were diluted fivefold with MiliQ water before the malate and citrate concentrations were finally measured with a Dionex HPAEC system (Dionex Corporation, Sunnyvale, CA, USA) equipped with a GS50 pump, an ED50 detector and with an IonPac AS11-HC (250x2mm) column, and eluted with a gradient starting at 16 mM NaOH. Total malate and citrate were calculated in mmol m⁻² using leaf mass area of dried leaf discs.

2.5 Experimental conditions used for the CO₂ experiment

To assess the effect of elevated CO₂ on carbon gain, a separate experiment was performed. Phalaenopsis “Sacramento” plants were grown for 29 weeks in four climate-controlled glass cabins situated in a greenhouse in the Netherlands. The cabins allowed accurate control of temperature, humidity and CO₂. Daylight was supplemented with high-pressure sodium (HPS) lamps if necessary, to reach the required daily light integral (DLI) and a 15-h photoperiod. In the vegetative stage, the temperature was 28.5°C, relative humidity was 70% and the average DLI was 6.4 mol PAR m⁻². In the flowering stage, the temperature was lowered to 19.5°C and the average DLI was 8.3 mol PAR m⁻². Throughout the experiment, in two cabins the CO₂ concentration was 400 ppm, and in two cabins it was 800 ppm.

After 12 to 16 weeks growth in the cabins (end vegetative stage), and again after 25 to 29 weeks (flowering stage), a selection of plants were transferred to a climate chamber with a similar climate and DLI as in the cabins, however with stable lighting conditions provided by a combination of ASRM (Figure 1) and HPS lamps. The 400 and 800 ppm grown plants were further grown in the climate chamber at 400 ppm and the 800 ppm, respectively. The plants were allowed to acclimate for 4 days and over the following days, the diel patterns of gas exchange and fluorescence were measured on fully expanded leaves of six different plants per cabin (12 leaves per CO₂ treatment for both the vegetative and flowering stage; n = 12). The leaf chamber lighting was programmed according to the light intensity pattern in the growth chamber. Both in the climate chamber and in the leaf chamber the same CO₂ concentration was set.

To assess if long-term growth at elevated CO₂ results in acclimation of diel CO₂-uptake capacity and CO₂-uptake in phase I (as an indicator of the malate-pool), 400 ppm grown plants were also placed in a climate chamber with 800 ppm CO₂ and photosynthesis was measured at 800 ppm CO₂ (leaves of six plants per cabin, so n = 12 leaves for both the vegetative and flowering stage).

2.6 Statistics

Statistical analyses were carried out on the malate and citrate measurements via a paired t-test when comparing two timesteps within a treatment and a normal t-test when comparing for differences in-between the treatments at a certain timestep. The malate decarboxylation rate was analysed by comparing the slope of the curve within a certain time interval via a normal t-test. The data from all diel photosynthesis measurements were analysed through one-way ANOVA analyses.

3.1 CAM pattern before changing light intensity

During the last 4 days of the initial 3-week acclimation period in the climate chamber, the diel gas-exchange and ETR were measured (Figure 2). During CAM-phase II (06:00–07:12) ETR was rising and during CAM-phase IV (17:00–22:00) ETR decreased gradually. A considerable part of the diel CO₂-uptake was during CAM-phase IV, which lasted 5 h (17:00–22:00).

3.2 Effect short-term (1 day) change in light intensity on CAM pattern

After changing the light intensity settings during 07:30–17:30 (Table 1), leaf samples were collected for malate and citrate analysis, and photosynthesis measurements were carried out on as many leaves as possible (±5 min at 120 and 2 min 0 μmol m⁻² s⁻¹ PAR measuring light) in treatments LLL, MML, HHL and VVL (i.e., the treatments with a constant light intensity of 60, 120, 180 and 300 μmol m⁻² s⁻¹ PAR during PAR setting I and II). The higher the light intensity, the earlier the opening of the stomata (CO₂-uptake > 0, Figure 3a) and the earlier ETR started to decrease (Figure 3c), both indicating that the higher the light intensity, the earlier malate is depleted. There is some variation in the exact moment that net atmospheric CO₂-uptake started (indication stomata started opening) between leaves, which is most visible for the MML treatment (Figure 3a). Notably, when the measuring light was switched off, CO₂-uptake dropped to 0 or lower until 18:00, which points to CO₂ being fixed via Rubisco until 18:00 (VVL a bit earlier). However, after 18:00 CO₂-uptake became independent of light, which points at atmospheric CO₂ being fixed via PEP-C later in CAM-phase IV (compare Figure 3a,b). Only the LLL treatment does not start taking up CO₂ until after ±19:30. This points at a prolonged CAM-phase III and therefore delayed and shortened CAM-phase IV.

The biochemical measurements of the malate concentration are in line with the patterns measured with gas-exchange and chlorophyll fluorescence, and give more insight into the underlying processes. Figure 4a,b shows the effect of the different light intensities during CAM-phase III (Table 1) on the malate concentration in the leaves. Over the first 1.5-h interval after the start of the day (i.e., CAM-phase II, 06:00–07:30) the malate concentration still increases slightly but significantly (p < .01; analysed for all treatments together as the treatments did not differ until 7:30). This is in line with the CO₂-uptake measured during that time interval (Figure 2). Apparently, at least part of the CO₂ taken up during CAM-phase II is fixed via PEP-C and stored as malate. After 07:30, CAM-phase III had started and the malate concentration decreased. Clearly, the higher the light intensity, the earlier malate is depleted (Figure 4a). In addition, the rate of malate depletion changed significantly in response to a change in light intensity halfway through CAM-phase III (switch of PAR setting I and II; p < .01; Figure 4b). The time at which malate was depleted was largely in line with the time that net CO₂-uptake started (stomata opened) for the MML, HHL and VVL treatments (compare Figures 3a and 4a). Hours after that moment the malate concentration started increasing again (Figure 4a; p < .01 except for the LLL treatment), which is in line with the start of the light-independent CO₂-uptake (Figure 3b). In the LLL treatment, not all malate was decarboxylated during daytime: as suggested with the gas-exchange measurements, indeed CAM-phase III was prolonged and CAM-phase IV was shortened in this treatment with half the light intensity during CAM-phase III compared with the previous growth weeks. Nevertheless, despite not all malate being depleted by the end of the day, the leaves still have a CAM-phase IV with some small amount of CO₂ uptake (Figure 3a,c). These results reveal that the rate of malate decarboxylation is light-regulated in Phalaenopsis. For the Δ_citrate measurements, only a minor response was measured compared with Δ_malate (Figure 4c). However, citrate still did decrease significantly (p < .01). There was no effect of light intensity on the decarboxylation rate of citrate (p > .25). The minor response of citrate is in line with Pollet et al. (2010) and Pollet, Vanhaecke, Dambre, Lootens, and Steppe (2011).

3.3 Effect of a long-term (>6 weeks) change in light intensity on the daily cycle of CAM

After at least 6 weeks of growth under the different lighting schedules (Table 2), leaf samples were collected for malate and citrate analysis, and CO₂-uptake was measured over 24 h, starting before CAM-phase IV. In four treatments (MML, MML/T, HLL and LHL), the light integral during CAM-phase III was equal, however, according to different patterns (respectively medium = control, trapezoid, high/low, low/high). Between these treatments, no significant differences in diel CO₂-uptake were measured (Table 3). As found for the short-term treatments (Figure 4b), the higher the light intensity, the faster malate was decarboxylated (p < .05, Figure 5a) and malate was depleted at approximately the same time in all four treatments. Only the difference in malate depletion rate between HLL and MML was not significant. During CAM-phase IV, the malate concentration increased again (p < .01), except for LLH, which did not deplete its malate pool. However, it may be that the moment that LLH reached the lowest malate concentration was between two sampling time points (at 18:09 phase IV started, see Table 3), and therefore, in reality, malate may have increased from this moment onwards.

In treatment LLH, the DLI was equal to treatments MML, MML/T, HLL and LHL, however, during the first 11 h after the start of the day (i.e., approximately the natural hours for CAM-phase III in tropical Asia), the light integral was lower, and the next 5 h the light intensity was much higher. In this treatment, the diel CO₂-uptake was significantly lower (−25%) compared with the other four treatments (Table 3), and the malate concentration depleted at a slower rate (p < .01; Figure 5a). The difference between the minimum and maximum daily malate concentration represents the daily amount of malate supplying the leaf with CO₂ available for growth. This difference is 20% lower for treatment LLH, compared with the average difference between the minimum and maximum daily malate concentration of the other four treatments. This is approximately in line with the difference in CO₂-uptake measured between treatment LLH and the other four treatments (Table 3). These results show that there are limitations in the plasticity of the time CAM-phase III can take, even for the newly developed leaves after a > 6 weeks adaptation period.

The change in citrate concentration over the day was minor with a Δ_citrate in the order of 10 mmol m⁻² day⁻¹, whereas Δ_malate was 10-fold (Figure 5c). As measured for malate, the citrate concentration decreased during the day (p < .01) and increased again in CAM-phase IV (p < .01).

The alternating light treatment did have a DLI that was equal to the other treatments when averaged over 8 days. However, there were periods with higher and lower light during PAR setting I and II (Table 2). The first day with low light during PAR setting I and II, following 4 days with high light, malate was not depleted by the end of the day, with approximately 30% of the concentration at the start of CAM-phase III remaining (Figure 6). The CO₂-uptake during the following phases IV, I and II was low (0.15 mol m⁻² day⁻¹) compared with the control (MML) treatment (0.27 mol m⁻² day⁻¹; Table 3). This lower CO₂-uptake does not necessarily imply a lower malate availability on the following day due to the malate remaining from the day before. However, after 4 days low light, the malate concentration at the start of CAM-phase III was lower (p < .01) and depleted (or nearly so) by the end of the day (Figure 6), and the CO₂-uptake was 0.21 mol m⁻² day⁻¹, which was similar to the LLH treatment and significantly lower than the control (MML) treatment (Table 3). During the first day of high light during CAM-phase III, following 4 days of low light, malate was depleted rapidly (Figure 6) and CO₂-uptake over the following phases IV, I and II were immediately as high as after the fourth day of high light (Table 3). Alternating 4 days low and 4 days high light resulted in an average CO₂-uptake of 0.24 mol m⁻² day⁻¹, which is slightly lower than the MML treatment (0.27 mol m⁻² day⁻¹), suggesting a somewhat lower growth potential under alternating light conditions compared to constant conditions at an equal DLI.

3.4 Effect of CO₂ on diel carbon gain

The CO₂ concentration had a significant effect on diel net CO₂-uptake of Phalaenopsis (Table 4). At 800 ppm, both the diel CO₂ uptake and the CO₂-fixation during phase I increased 25–31% compared with 400 ppm, for both the vegetative and flowering stages. CO₂-uptake also increased significantly during phase IV. During phase II, CO₂-uptake was small, however, significantly higher for the 400 ppm treatment. The diel CO₂-uptake was overall higher in the flowering stage than in the vegetative stage. The leaves of plants grown at 400 ppm CO₂ which were subsequently exposed to and measured at 800 ppm CO₂, responded with a similar increase in CO₂-uptake as the 800 ppm grown leaves measured at 800 ppm. Notably, the maximum g_s decreased with an increase in CO₂ concentration. The effect of CO₂ concentration on the start of CAM-phase III (end of net CO₂-uptake, indicating stomatal closure) and CAM-phase IV (opening of stomata) was not significant or only small, resulting in a similar duration of the combined phases IV, I and II.

4.1 Regulation of malate decarboxylation in CAM-phase III

Clearly, it is light intensity regulating malate decarboxylation in CAM-phase III, either directly or, more likely, indirectly via the CO₂ assimilation rate. At a light intensity in CAM-phase III of only half of that during growth in the weeks before, no excessive CO₂ leaking out of the leaf was measured, as for instance found by Chen and Lin (2012), and malate was not depleted by the end of the day (LLL in Figure 4a). In addition, the malate decarboxylation rate responded proportionately to changes in light intensity (Figures 4a,b and 5). The length of CAM-phase III and start of CAM-phase IV were adjusted to the moment malate was depleted and were therefore also influenced by the light intensity during phase III. In this respect, malate decarboxylation of Phalaenopsis responds in a very similar way to the response to light intensity reported for several Kalanchoe species (Barrow & Cockburn, 1982; Kluge, 1968; Lüttge, 2004).

4.2 Carboxylation pathway in CAM-phase IV

At higher light intensities CAM-phase III was shorter, resulting in an earlier start of CAM-phase IV (i.e., start of direct atmospheric CO₂-uptake in the light period; Figure 3), which is in line with previous studies on Kalanchoe daigremontiana and Kalanchoe blossfeldiana (Barrow & Cockburn, 1982; Thomas et al., 1987). CO₂ fixation was via Rubisco until about 18:00 (VVL a bit earlier), which was 12 h after lights on. This was shown by turning the measuring light off, in response to which CO₂-uptake dropped to zero or below (Figure 3b). However, beyond the natural daylength for Phalaenopsis of about 12 h, CO₂ was fixed mainly via PEPC: CO₂-uptake did not decrease anymore when turning the measuring light off (compare Figure 3a,b), ETR dropped to lower values (Figure 3c), and the leaf malate concentration started rising again (p < .01; Figure 4a). Even in the 1-day treatment with LLL, where malate was not depleted by the end of the day (Figure 4a), direct atmospheric CO₂-uptake started around 20:00 and the CO₂ was taken up independent of light (Figure 3a,b). This indicates that the start of phase IV (opening stomata) is not necessarily associated with malate depletion and the subsequent decrease in internal CO₂ concentration alone, which is considered an important factor in stomatal rhythms of CAM plants (Males & Griffiths, 2017). This normally occurs under stable lighting conditions (compare the control MML with LLL in Figure 4a). This is consistent with previous statements that the length of CAM-phase III, and therefore the start of CAM-phase IV, are not only regulated (indirectly) by light intensity and the drawdown of internal CO₂ but also by the circadian clock imposing limitations on the plasticity of the length of CAM-phase III (Dodd et al., 2002; Hartwell, 2005; Von Caemmerer & Griffiths, 2009).

4.3 Regulation of diel carbon gain by light integral

The response of CAM-metabolism to the DLI has not been studied in much detail yet. Lüttge and co-workers have shown that Clusia responded to a higher DLI by increasing Δ_malate (Franco, Ball, & Lüttge, 1991; Haag-Kerwer, Franco, & Lüttge, 1992). Nocturnal acidification was increased in several cactus species in response to an increase in DLI up to 30 mol m⁻² (Nobel, 1988). In our study on Phalaenopsis, the average light integral was equal for all six long-term (>6 weeks) treatments (Table 2). The four treatments (MML, MML/T, HLL and LHL) with an equal light-sum according to different patterns during CAM-phase III, all showed a similar diel CO₂-uptake (Table 3) and Δ_malate during daytime (Figure 5a). The treatment with alternating periods of 4 days low and 4 days high light, elegantly shows how the Δ_malate responds to the light integral (Figure 6 and Table 3): 1 day lower light following a high light period resulted in malate not being depleted by the end of the day, and a very low CO₂-uptake the following night. However, after 4 days low light, malate was depleted by the end of the day, and the concentration per area of leaf at the start of the day was markedly lower (LLL D4) than after a high light period (HHL D4 and LLL D1). Only 1 day high light following 4 days low light immediately resulted in a much higher diel CO₂-uptake. After the fourth day of high light, the subsequent diel CO₂-uptake was higher than the control (MML; Table 3). Previous studies by Guo et al. (2012) showed that exposing a commercial Phalaenopsis cultivar to a full day of very low light conditions (25 μmol m⁻² s⁻¹) resulted in CO₂ release during the day and higher pre-dusk malate levels compared to plants grown at higher light intensities. In the following night, CO₂ uptake as well as pre-dawn malate levels in these plants were significantly lower, underpinning our conclusion that plasticity of these plants allows for short-term adaption to changes in light intensity.

Notably, LLH with only lower light during CAM-phase III (60 μmol m⁻² s⁻¹ PAR) followed by 180 μmol m⁻² s⁻¹ PAR, resulted in a significantly lower CO₂-uptake over 24 h and a similarly smaller Δ_malate, compared to the results for the MML control treatment. Apparently, it is the light integral during CAM-phase III or during the “natural photoperiod” of the first ±12 h of the day, and not the total DLI that regulates the daily carbon gain (i.e., the diel net CO₂-uptake). This proposition is underpinned by comparing LLH and the alternating light treatment. After 4 days LLL the diel CO₂-uptake was similar to that of LLH (Table 3), and also Δ_malate over the day was similar (compare Figures 5a and 6). The light integral during CAM-phase III was also the same in LLH and the alternating light treatment (during 4 days LLL), but the light integral over the whole day was markedly higher in LLH (5.6 vs. 3.5 mol m⁻² day⁻¹ PAR for LLH and the alternating light treatment LLL respectively). From this, it can be concluded that light after the first ±12 h of the day is not used as effectively for carbon gain relative to the light in the first ±12 h.

4.4 Regulation of diel carbon gain by CO₂

Despite PEP-C having a high affinity for CO₂ (Lüttge, 2001), our results clearly show that a CO₂ concentration well-above atmospheric levels strongly increased the diel net CO₂-uptake (25–31%; Table 4). As the CO₂-uptake also increased proportionally during phase I, our results support the conclusion that neither the storage capacity for malate in the vacuole nor the supply of PEP as substrate for CO₂-fixation into malate via PEPC, were limiting factors for an increased carbon gain at elevated CO₂ concentration under the conditions used in this study. Notably, the duration of CAM-phase III was not significantly, or only slightly longer for the plants grown at 800 ppm CO₂ compared with the 400 ppm plants. This demonstrates that the rate of malate decarboxylation in the leaves grown at 800 ppm was increased. Long-term acclimation to elevated CO₂ (up to 25 weeks) did not result in acclimation of CO₂ storage capacity, as the leaves grown at 400 ppm CO₂ exposed to 800 ppm had a similar increase in CO₂-uptake as the leaves grown and measured at 800 ppm CO₂, compared with the leaves grown and measured at 400 ppm CO₂.

The decrease in g_s in response to a higher CO₂ concentration (Table 4) is seen more often in CAM plants, for example, for bromeliad Aechmea (Ceusters et al., 2008). In the same study elevated CO₂ from 380 to 700 ppm did not increase CO₂-uptake during phase I, possibly due to the decreased g_s. It is known that stomatal behaviour of CAM plants can be sensitive to changes in internal CO₂ concentrations (Wyka, Duarte, & Lüttge, 2005). When the internal CO₂ concentration in the leaf rises at the start of CAM-phase III, stomatal closure serves to prevent CO₂ leakage out of the leaf (Males & Griffiths, 2017). Possibly the stomatal closure response at the start of CAM-phase III is regulated by both the internal CO₂ concentration and the circadian clock (Males & Griffiths, 2017). Notably, the diel CO₂-uptake was higher for plants in the flowering stage than for plants in the vegetative stage (Table 4), which may be due to the higher DLI for the flowering stage plants.

4.5 Practical implications for Phalaenopsis growers

Besides the new insights into the physiological plasticity of CAM in Phalaenopsis, this study provides useful practical insights for growers, especially for those growing Phalaenopsis in high-tech production systems with control over lighting and CO₂. In CAM-phase II the malate concentration continued to rise (Figure 4), and light use efficiency (ETR) gradually increased (Figure 2). This is likely to be because PEPC remained active in phase II until Rubisco was sufficiently activated (Griffiths et al., 2002; Roberts, Borland, & Griffiths, 1997). In practical terms, this implies that starting the day at full light intensity is wasting energy. Note that under natural conditions, light intensity also rises gradually after dawn. For CAM-phase III we found that malate decarboxylation responded strongly to light intensity. In addition, carbon gain was regulated by the light integral during CAM-phase III rather than the light integral over the whole day. This implies that growers need to assure that enough light is available within CAM-phase III, and have flexibility in their choice of how to reach a high enough light integral in CAM-phase III. In addition, growers should be aware of light-stress induced injury in CAM-phase IV, as the ETR decreased after malate was depleted (Figure 3). During CAM-phase IV, high light intensity was not utilized fully, so lights can be dimmed in a commercial greenhouse. To what extent phase IV then contributes to additional growth and productivity remains to be determined. CO₂ fertilization is useful to increase carbon gain, but only during CAM-phase IV and I (Table 4).

In conclusion, our results show how Phalaenopsis “Sacramento” responds to light (conclusions 1 and 2) and provide novel insights into the regulation of CAM (conclusions 3 and 4):