Introduction

People spend the majority of their time (80–90%) indoors, where indoor environmental quality (IEQ) significantly impacts occupants’ health1, cognitive functions2, and productivity3. Biophilic design4, an architectural method that integrates natural elements into the built environment, has emerged as a significant factor influencing IEQ. This approach, increasingly adopted by architects, often involves incorporating plants into indoor settings. Numerous studies have shown that indoor plants improve occupants' well-being and health5,6, workplace satisfaction7, perceived productivity8,9,10,11, and cognitive functions, including creativity12,13,14 and working memory12. However, despite the increasing incorporation of indoor greenery, there remains a scarcity of information on the ideal lighting conditions that balance energy efficiency with the sustainable growth of plants in such environments. For instance, a field study conducted by Tan et al.15 in an office building in Singapore revealed that excessive lighting for indoor plants can lead to significant energy losses. Maintaining healthy growth of indoor plants is essential for minimizing maintenance and replacement costs associated with poor growth. On the contrary, excessive growth of plants, requiring substantial pruning efforts, can also be undesirable. Additionally, energy loss resulting from excessive lighting plans for plants is not favorable for building owners.

Although literature has long existed specifying recommended light intensities for various species of crops and ornamental plants16,17, the recommended ranges are broad and often assume daily exposures of 12–16 h, leaving the relationship between viable light intensity and exposure time unclear. The minimum light intensity necessary for sustaining photosynthesis can be referenced through the light compensation point (LCP), defined by photosynthetic photon flux density (PPFD). Past literature has identified the LCPs for several types of ornamental plants18; however, lighting design for indoor plants must also consider the required day length. Daily light integral (DLI), an index representing the cumulative PPFD over a day, is utilized in agriculture as a metric for assessing crop yield and quality19,20. However, examples of using DLI for the growth assessment of ornamental plants are rare, except for experiments conducted by Tan et al.15, which demonstrated that the light compensation points for Philodendron erubescens and Dracaena surculosa range from 0.50 to 1.00 mol m−2 day−1. Research on how tropical foliage plants adapt to the low light levels typical of architectural lighting remains scarce, with Chen et al.21 being a notable exception. Their findings revealed that Ficus benjamina adapts to indoor conditions (16 μmol m−2 s−1) through increases in specific leaf area (SLA), internode length, and chlorophyll (Chl) content, compared to growth in a shade house with a light level of 300 μmol m−2 s−1. However, the light intensity used in experiments designed to simulate indoor conditions (approximately 1200 lx from white fluorescent lights) is more than twice the standard illuminance of modern offices (300–500 lx)22,23, and the impact of day length was not incorporated into the research scheme.

As the use of plants in indoor environments increases, understanding whether ornamental plants can maintain growth in low-light and short-daylength conditions, such as offices without daylight, becomes a crucial first step in considering architectural lighting design for plants. Additionally, indoor greening in offices may use spotlights to increase day length and light intensity for plants. Clarifying the effects of these lighting strategies on the physiological responses of ornamental plants is beneficial from a practical standpoint.

In the present study, a test chamber experiment was conducted to explore the lighting requirements of three common indoor plant species in Japan (Epipremnum aureum, Pachira aquatica, and Rhaphidophora tetrasperma), given the increased promotion of plant use in workplaces. This research established three lighting conditions simulating indoor workplaces, with the following objectives: (1) To analyze the possibility of survival of ornamental plants under low DLI conditions, which simulate an indoor workplace environment lacking access to daylight; (2) To analyze the impact of extending the day length or increasing the intensity of supplementary lighting on the growth of the studied plants. Plant materials were grown under three different lighting conditions for 191 days. Additionally, growth rate, morphology, and photosynthetic performance were evaluated to explore the intrinsic reactions to different lighting conditions. This approach seeks to contribute to optimal architectural lighting design for indoor greening and enhance understanding of plant physiology in indoor built environments.

Results

Test chamber conditions

Table 1 presents a summary of the environmental conditions measured under three experimental setups. Case 1 represents the lowest DLI condition, simulating the minimal light level in an office space without access to daylight. Here, the PPFD at canopy height was set to 6.8 µmol m−2 s−1 (approximately 500 lx with white LED), with a day length of 9 h per day (from 9:00 am to 6:00 pm). Case 2 represents a middle DLI condition, assuming extended lighting hours for indoor plants. The PPFD at canopy height was maintained at 6.7 µmol m−2 s−1 (approximately 500 lx with white LED), with a day length of 18 h per day (from 6:00 am to 12:00 am). Case 3 features the highest DLI, with the greatest light intensity and the longest duration of lighting. The PPFD at canopy height was about 20.1 µmol m−2 s−1 (approximately 1500 lx with white LED), with a day length of 18 h per day (from 6:00 am to 12:00 am). White LED lights were used as the light source to simulate office lighting. For all the cases, the temperature was set at 22 °C, relative humidity at 50%, and CO2 was supplied from 8:00 am to 8:00 pm across all conditions, targeting a simulated office CO2 concentration of 800 ppm. The calculated DLIs were 0.22 mol m−2 day−1 for Case 1, 0.43 mol m−2 day−1 for Case 2, and 1.30 mol m−2 day−1 for Case 3.

Table 1 Measured environmental conditions for each experimental condition (mean ± SD).
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Growth analysis

Figure 1 shows images of the plant materials before and after the chamber treatment under each experimental condition. Figure 2 illustrates the comparisons of the total dry weights and calculated relative growth rate (RGR) for the plant materials under each condition. The initial physiological parameters of the plants are illustrated in Table S1. The outcomes of the statistical analyses are detailed in Table S2. One P. aquatica in Case 1 succumbed to root rot during the experiment and was subsequently excluded from the analysis that followed. All other plant materials sustained growth. For E. aureum and R. tetrasperma, statistically significant differences in total dry weight and RGR were observed between Cases 1 and 3 and Cases 2 and 3. The total dry weight and RGR of P. aquatica did not show significant differences across the experimental conditions. This outcome could be attributed to the substantial mass of its stem, which included dead tissue, potentially masking variations in growth performance attributable to the different light conditions.

Figure 1
figure 1

Plant materials after 190 days of cultivation under different experimental conditions. For each species, three samples were randomly selected from each condition. The scale bars represent 10 cm, adjusted for the plants in the foreground.

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Figure 2
figure 2

Comparisons of total dry weight (A) and relative growth rate (B) for the three species of plant materials after 190 days of cultivation under the three different experimental conditions. The daily light integral for Cases 1, 2, and 3 are 0.22, 0.43, and 1.30 mol m−2 day−1, respectively. Box plots illustrate the median (horizontal line), mean (triangles), 25th and 75th percentile (box), 10th and 90th percentile (whiskers), and each dot represent an individual value. n.s., not significant.

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Plants’ morphology

Figure 3 presents the comparisons of the number of leaves, leaf area, SLA, and leaf area ratio (LAR) of the plant materials across each experimental condition. The outcomes of the statistical analyses are detailed in Table S3. For E. aureum, a significant difference in the number of leaves was observed among all experimental condition combinations, with an increase in DLI correlating to a higher leaf count. Furthermore, the leaf area for E. aureum in Case 3 was significantly larger compared to Cases 1 and 2. P. aquatica and R. tetrasperma showed no significant differences in the number of leaves and leaf area under the three different experimental conditions.

Figure 3
figure 3

Comparisons of number of leaves (A), leaf area (B), specific leaf area (C), and leaf area ratio (D) for the three species of plant materials after 190 days of cultivation under the three different experimental conditions. The daily light integral for Cases 1, 2, and 3 are 0.22, 0.43, and 1.30 mol m−2 day−1, respectively. Box plots illustrate the median (horizontal line), mean (triangles), 25th and 75th percentile (box), 10th and 90th percentile (whiskers), and each dot represent an individual value. n.s. not significant.

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For all three plant species studied, the SLA in Case 3 was significantly lower than in Cases 1 and 2, indicating that plants subjected to Case 3 developed thicker leaves. This observation confirms that the difference in light intensity between 6.8 and 20.1 µmol m−2 s−1 directly affects leaf morphology, with leaves developing denser structures under higher light conditions.

Figure S1 shows the proportion of leaves, stems, and roots in the total dry weight of plant materials. The LWR of E. aureum was higher in Case 2 compared to Cases 1 and 3. The extended daily light exposure in Case 2 may have contributed to greater assimilation in the leaves compared to Case 1. Additionally, the proportion of assimilates allocated to the stem in Case 3 was higher than in Case 2, resulting in a lower LWR in Case 2. The LWR of P. aquatica was higher in Case 3 compared to Cases 1 and 2. This suggests that exposure to higher light intensity contributed to an increased assimilation in the leaves. The LWR of R. tetrasperma was higher in Cases 1 and 2 compared to Case 3. Similar to E. aureum, the reason for this in Case 3 is due to a greater allocation of assimilates to the stem, which can be observed in the differences in plant form among the cases, as shown in Fig. 1. These results suggest that the distribution of assimilates within the plant is influenced by the duration of light exposure and the intensity of light, affecting the growth dynamics between the stem and leaves.

SPAD value

Figure 4 illustrates the comparisons of Soil Plant Analysis Development (SPAD) values after 182 days of cultivation under the three different experimental conditions. The outcomes of the statistical analyses are detailed in Table S4. Across all examined plant species, SPAD values in Case 3 were significantly higher compared to Cases 1 and 2, indicating an increase in Chl content under higher light conditions. Specifically, for P. aquatica, the SPAD values were higher in Case 2 than in Case 1, suggesting that extending light exposure from 9 to 18 h at 6.8 µmol m−2 s−1 can lead to an increase in the Chl content of the leaves.

Figure 4
figure 4

Comparisons of SPAD values for the three species of plant materials after 182 days of cultivation under the three different experimental conditions. The daily light integral for Cases 1, 2, and 3 are 0.22, 0.43, and 1.30 mol m−2 day−1, respectively. Box plots illustrate the median (horizontal line), mean (triangles), 25th and 75th percentile (box), 10th and 90th percentile (whiskers), and each dot represent an individual value. n.s. not significant.

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Photosynthetic performance

Figure 5 shows the light response curves of P. aquatica for each condition studied. When the PPFD was set at 500, 1000, and 2000 µmol m−2 s−1, Case 3 exhibited higher photosynthetic rates compared to Case 1. However, at light intensities of 250 µmol m−2 s−1 or lower, there was no significant difference among the conditions. The outcomes of the statistical analyses are detailed in Table S5. Table S6 lists the estimated maximum net photosynthetic rate, dark respiration rate, and leaf LCP. The mean estimated maximum net photosynthetic rate was highest in Case 3, which was significantly higher than those in Case 1. No significant differences were observed in the estimated dark respiration rate and estimated LCP among the experimental conditions. Table S7, S8, and S9 show the stomatal conductance, intercellular carbon dioxide concentration, and transpiration rate of P. aquatica for each PPFD level under the three experimental conditions. Stomatal conductance and transpiration rate tend to be lowest in Case 1 under all PPFD conditions, indicating that photosynthesis is more suppressed compared to the other cases. On the other hand, although there is no significant difference in stomatal conductance between Case 3 and Case 2, the photosynthetic rate is higher in Case 3 at high PPFD. Case 3 shows a lower intercellular carbon dioxide concentration than Case 2, suggesting that the efficiency of CO2 utilization within the leaves might be higher, likely due to the higher chlorophyll content.

Figure 5
figure 5

Comparison of the light response curves (photosynthesis per leaf area) of P. aquatica among the three experimental conditions. Mean ± SE. Asterisks indicated the significant differences (*p < 0.05, **p < 0.01).

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Discussion

This study revealed that all three plant species sustained growth throughout a 191-day experiment under a condition of 6.8 µmol m−2 s−1 for 9 h per day (Case1, DLI = 0.22 mol m−2 day−1). The European Committee for Standardization defines illuminance requirements in offices for writing, typing, reading, and data processing at 500 lx (approximately 6.8 µmol m−2 s−1 with white LEDs)22. Additionally, the lighting duration of 9 h per day (9:00–18:00) used in this study is shorter than the typical lighting hours found in standard offices. Therefore, the results of this experiment indicate a low likelihood of light deficiency in the three plant species in weekday office spaces where lighting for occupants is adequately planned. A previous growth chamber study by Tan et al.15 showed that the required DLI for P. erubescens and D. surculosa was between 0.50 and 1.00 mol m−2 day−1. As the three plant species used in this study have survived in a DLI of 0.22 mol m−2 day−1, they might have a higher shade tolerance than P. erubescens and D. surculosa. Additionally, Tan et al.15 reported a high correlation between the DLI provided using fluorescent lights and electricity consumption (R2 = 0.9989). This study showed that extra energy consumption due to supplementary lighting might be unnecessary when selecting plant species that do not need high DLI and choosing an appropriate placement position in workplaces. Meanwhile, it's worth considering that, unlike this experiment, real office settings may not maintain lighting or air conditioning during off days. Additionally, in actual workplace environments, daily fluctuations in temperature and humidity occur, and the air velocity conditions may differ from those in the chamber, where air is circulated 24 h a day. In this experiment, chambers were used to analyze the response of plants to different lighting conditions within a building. However, future research should involve placing plants in actual office environments for long-term observations to comprehensively analyze the combined effects of indoor light, heat, and airflow on plants.

This study demonstrated that the use of supplementary white LED lighting, particularly when increasing light intensity from 6.8 to 20.1 µmol m−2 s−1, impacts the growth rate and morphology of ornamental plants (Figs. 2, 3). E. aureum and R. tetrasperma showed significantly higher RGR in Case 3 than in Cases 1 and 2. The elevated DLI affected the amount of carbon assimilation and resulted in a higher increase in dry weight in Case 3. In particular, E. aureum had more leaves under higher DLI conditions at the final measurement, and their appearance was visibly different among the conditions (Fig. 1). In addition, the LWR of E. aureum and R. tetrasperma were the lowest in Case 3 compared to other conditions. This result is related to the higher ratio of stem weight to whole weight in Case 3 (Fig. S1). The climbing plants such as E. aureum and R. tetrasperma showed significant stem elongation and increased spatial volume under 20.1 µmol m−2 s−1 conditions compared to 6.8 µmol m−2 s−1, indicating the practical effectiveness of fine-tuning light intensity for those wishing to reduce pruning efforts. In contrast, P. aquatica might assimilate carbon mainly in leaves, and LWR was higher in Case 3 than in Cases 1 and 2 (Fig. 3). These findings suggest that the appearance of ornamental plants can vary significantly when the DLI is adjusted with indoor supplementary lighting, especially through the augmentation of light intensity. Architects and designers can select lighting plans tailored to their specific greening objectives, whether to save energy by limiting plant growth or encourage more vigorous growth through intense lighting. In managing climbing plants, for instance, keeping light levels at around 6.8 µmol m−2 s−1 can moderate their growth and minimize the need for frequent pruning. Conversely, the use of supplementary lighting could promote stem elongation, potentially allowing for the faster and more effective greening of large spaces. A recent study introduced an accurate simulation approach for calculating indoor PPFD and DLI employing three-dimensional architectural models and spectral irradiance simulations24. Consequently, architects can now craft lighting environments for indoor plants, informed by an understanding of the plants' physiological responses to various light intensities.

Evaluation of leaf physiology and photosynthetic performance was conducted to investigate the intrinsic reactions to different lighting conditions. The SLA was significantly smaller in Case 3 than in Cases 1 and 2 for all three plant species. This trend matches the response observed by Chen et al.21 in F. benjamina adapting from greenhouse light intensity (300 µmol m−2 s−1) to architectural illuminance of 1000 lx (16 µmol m−2 s−1). The experiment further demonstrates that clear differences in SLA can also be observed across a narrower range of PPFD (6.7 µmol m−2 s−1 to 20.1 µmol m−2 s−1) adjusted by architectural lighting. Under a lighting environment of 6.8 µmol m−2 s−1, plants exhibited morphological adaptation strategies for more efficient light capture than under conditions of 20.1 µmol m−2 s−1. SLA is generally known to correlate positively with mass-based nitrogen content25,26. However, in this experiment, all plants exhibited their highest SPAD values in Case 3, where SLA was lowest. This suggests that the increased light exposure from 6.8 µmol m−2 s−1 to 20.1 µmol m−2 s−1 led to an increase in leaf mass and, consequently, an increase in Chl content per unit leaf area. The photosynthetic rate per leaf area of P. aquatica was significantly higher in Case 3 than in Case 1 only at high light intensities above 500 µmol m−2 s−1, but no significant differences were observed between the Cases at low light levels below 250 µmol m−2 s−1. These findings indicate that plants acclimated to the low light intensity of 6.8 µmol m−2 s−1 can achieve area-based photosynthetic rates in low-light conditions comparable to those of plants grown under higher light intensity (20.1 µmol m−2 s−1), demonstrating the effectiveness of physiological adaptation strategies to dimly lit indoor environments. Chen et al.21 reported that F. benjamina acclimated to light levels of indoor environments (16 µmol m−2 s−1) exhibited a decrease in the Chl a/b ratio and an increase in total Chl content per leaf area. Similarly, in our experiment, P. aquatica under low PPFD conditions (Case 1 and 2) might have increased the proportion of Chl b to enhance light absorption efficiency in low light conditions. Additionally, in Case 3, the SPAD values were significantly higher than in Case 1 and 2, and the light saturation point was also higher, suggesting an increase in the synthesis of Chl a. While our study compared SPAD values, which serve as a relative indicator of total Chl a and b, future studies should analyze the specific contents of Chl a and b using methods such as the spectrophotometric method27 to better interpret plant acclimation to different lighting conditions.

In conclusion, this study has yielded insights applicable to the practical design of indoor greening and identified strategies for the adaptation of ornamental plants to low-light environments within buildings. The present study showed that E. aureum, P. aquatica, and R. tetrasperma sustained growth for 191 days under lighting conditions simulating a workplace without access to daylight (500 lx ≈ 6.8 µmol m−2 s−1 of white LED lighting for 9 h day−1). This result indicates that extra energy consumption due to supplementary lighting is unnecessary when selecting plant species with sufficient shade tolerance and choosing an appropriate placement position in workplaces. Additionally, increasing the lighting intensity in buildings from 6.8 to 20.1 µmol m−2 s−1 significantly affects the appearance of ornamental plants, as confirmed by the substantial increase in RGR and the decrease in LWR due to an increase in biomass allocation to the stems of climbing plants. Therefore, lighting plans that utilize supplementary lighting to promote healthy and rapid growth of plants can be effective, especially when greening large-volume spaces. Lastly, it was observed that ornamental plants adapt to low-light conditions of 6.8 µmol m−2 s−1 by increasing their SLA, with photosynthetic measurements supporting the increased SLA as a means to efficiently utilize light in low-light environments. Insights into the adaptation of ornamental plants to low-light conditions, such as in buildings without direct daylight, can be effectively applied in the design of architectural spaces where humans and plants coexist within the context of biophilic design.

Methods

Plant materials

All plant materials were sourced from plant shops in Japan. The plants were cultivated in potting compost comprised of peat moss, red ball earth, cocopeat, Kanuma soil, and slow-release fertilizer, contained within plastic pots measuring 120 mm in diameter. Throughout the duration of the experiment, no extra fertilizer was administered.

Experimental procedure

The experiment was carried out at the Institute for Rural Engineering, National Agriculture and Food Research Organization in Tsukuba, Japan, spanning from October 2020 to June 2021. Prior to the treatments in the growth chambers, plants underwent a 47-day acclimatization period in an air-conditioned glass greenhouse under a shade cloth. The greenhouse maintained an air temperature of 20 °C, with the maximum indoor solar radiation reaching approximately 120 W m−2 on clear days. Following this acclimatization period, six plants of each species were harvested to measure their fresh weight, dry weight, and leaf area, establishing a baseline for subsequent growth analysis. The remaining plants were then relocated to three distinct growth chambers designed with varied lighting conditions. In each experimental setup, six plants per species were maintained and watered on a weekly basis. After a six-month period, the SPAD values were recorded to assess Chl content without destruction. Additionally, measurements evaluating photosynthetic performance were conducted between April 2021 and May 2021. At the conclusion of the 191-day treatment period within the chambers, all plants were harvested for a final assessment of fresh weight, dry weight, and leaf area to complete the growth analysis.

Test chamber conditions

Plant growth chambers S10H (CONTROLLED ENVIRONMENTS Inc., Pembina, ND, U.S.A.) were used to control environmental conditions with different DLI levels. The monitoring of air temperature, relative humidity, and illuminance was facilitated by a wireless data logging system RTR-574 (T&D Corporation, Nagano, Japan), while CO2 concentrations were measured with a CO2 meter RTR-576 (T&D Corporation, Nagano, Japan). The data were collected at 20-min intervals throughout the experimental period. To convert the measured illuminance values into PPFD, the spectral distribution was assessed using a portable spectroradiometer MS-720 (EKO Instruments Co., Ltd., Tokyo, Japan). Figure S2 illustrates the spectral irradiance measured at canopy height in each chamber. Based on the obtained spectral distribution, the conversion factor from illuminance to PPFD was established as 0.01365 (µmol m−2 s−1)/lx. This conversion factor was used to derive the PPFD shown in Table 1 from the recorded illuminance values. The calculation of DLI employed the Eq. (1):

$$DLI=\frac{PPFD\times 3600\times \tau }{{10}^{6}}$$
(1)

where DLI is the daily light integral (mol m−2 day−1), PPFD is the photosynthetic photon flux density (µmol m−2 s−1), and τ is the duration of light exposure per day (h).

Growth analysis and plants’ morphology

The substrate surrounding the roots was gently washed, and the plants were divided into shoots (stems and leaves) and roots to calculate the weight change before and after treatment in the chambers. Fresh weights were measured using an electronic balance, and leaf areas were measured using a leaf area meter Li-3100 (LI-COR Inc., Lincoln, NE, U.S.A.). The tissues were then oven-dried at 80 °C until they reached a constant mass to estimate their dry weights. Relative growth rate (RGR) was calculated as {[ln (final plant dry mass) – ln (initial plant dry mass)] ÷ 190 d}. SLA was calculated by dividing the total leaf area of a plant by the total leaf dry weight, and LWR was calculated by dividing the dry weight of leaves by the total dry weight of the plants.

SPAD value

The Chl meter SPAD-502 (KONICA MINOLTA Inc., Tokyo, Japan) was used to evaluate the differences in the nitrogen status of plants among the three experimental conditions. The SPAD meter quantifies the absorbance discrepancy between red light (λ = 650 nm) and near-infrared light (λ = 940 nm) and provides a proxy for Chl28,29. For each plant species, fully opened leaves from 5 to 6 plants subjected to each experimental condition were selected for analysis, resulting in a total of 60 measurements. Specifically for the leaves of E. aureum, which feature a white speckled pattern, there was a potential variation in chloroplast content based on their position on the leaves. To address this, areas of uniform green color were chosen for the measurement of SPAD values.

Photosynthesis measurement and leaf-based light response curves

To evaluate the differences in photosynthetic performance among plants under the three experimental conditions, photosynthetic rates were tested using a leaf-chamber infra-red gas analyzer LI-COR 6400 (LI-COR Inc., Lincoln, NE, U.S.A.) with an enclosed leaf area of 6.0 cm2. The photosynthetic rates of E. aureum and R. tetrasperma were found to be too low for accurate measurement, limiting the ability to obtain light response curves for P. aquatica only. The relative humidity and leaf temperature in the chamber were continuously monitored, and they ranged from 45 to 51% and from 22 to 27 °C. The initial CO2 concentration in the chamber was set to 500 ppm. Measurements were performed between 10:00 am and 6:00 pm. Seven young, fully opened mature leaves were tested under each experimental condition. The light intensity provided to the leaves was gradually increased steadily at intervals of 0, 2, 5, 10, 20, 50, 100, 250, 500, 1000, and 2000 µmol m−2 s−1. Light of variable intensity was provided by built-in red/blue LEDs. Each intensity level was maintained for 180–300 s to allow the photosynthetic response to stabilize before increasing to the next intensity. Before starting the measurement with PPFD = 0, a dark acclimation time of ten minutes was set by placing a leaf in the leaf chamber. The IRGA was calibrated prior to each set of measurements.

The following hyperbola Eq. (2) was used to fit the measured photosynthetic rate to the light response curves29.

$${P}_{n}(I)=\frac{\alpha I+{P}_{max}-\sqrt{{\left(\alpha I+{P}_{max}\right)}^{2}-4\theta \alpha I{P}_{max}}}{2\theta }-{R}_{d}$$
(2)

where Pn (I) is the net photosynthetic rate, I is the PPFD, θ is the curve curvature, and α is the gradient of the plant photosynthesis to the light response curve in the case of I = 0, that is the initial gradient of the light response curve. Pmax is the maximum net photosynthetic rate, and Rd is the dark respiration rate.

Statistical analysis

A one-way analysis of variance (ANOVA) was performed to compare plant growth parameters and leaf morphology across three experimental conditions. Given the small sample size within each group, population normality and homogeneity of variances at each level were assumed. Tukey’s honestly significant difference (HSD) test was utilized for post hoc multiple comparisons. For the comparison of SPAD values among the experimental conditions, the Kruskal–Wallis H test, followed by the Dunn-Bonferroni post hoc test, was employed due to the lack of normality and homogeneity of variances in some groups. All statistical analyses were facilitated using the statistical software SPSS (version 28).