Synergistic Effects of Elevated CO₂ and Enhanced Light Intensity on Growth Dynamics, Stomatal Phenomics, Leaf Anatomy, and Photosynthetic Performance in Tomato Seedlings

Abstract

Elevated [CO₂] enhances light interception and carboxylation efficiency in plants. The combined effects of [CO₂] and photosynthetic photon flux density (PPFD) on stomatal morphology, leaf anatomy, and photosynthetic capacity in tomato seedlings remain unclear. This study subjected tomato seedlings (Solanum lycopersicum Mill. cv. Jingpeng No.1) to two [CO₂] (ambient [a[CO₂], 400 µmol·mol⁻¹] and enriched [e[CO₂], 800 µmol·mol⁻¹]) and three PPFD levels (L; low[Ll: 200 µmol·m⁻²·s⁻¹], moderate[Lm: 300 µmol·m⁻²·s⁻¹], and high[Lh: 400 µmol·m⁻²·s⁻¹]) to assess their interactive impacts. Results showed that e[CO₂] and increased PPFD synergistically improved relative growth rate and net assimilation rate while reducing specific leaf area and leaf area ratio. Notably, e[CO₂] decreased stomatal aperture (−13.81%) and density (−27.76%), whereas elevated PPFD promoted stomatal morphological adjustments. Additionally, Leaf thickness increased by 72.98% under e[CO₂], with Lm and Lh enhancing this by 10.79% and 41.50% compared to Ll. Furthermore, photosynthetic performance under e[CO₂] was further evidenced by improved chlorophyll fluorescence parameters (excluding non-photochemical quenching). While both e[CO₂] and increased PPFD Photosynthetic performance under e[CO₂] was further evidenced by improved chlorophyll fluorescence parameters (excluding non-photochemical quenching). Moreover, e[CO₂]-Lh treatment maximized total dry mass and seedling health index. Correlation analysis indicated that synergistic optimization of stomatal traits and leaf structure under a combination of e[CO₂] and increased PPFD enhanced light harvesting and CO₂ diffusion, thereby promoting carbon assimilation. These findings highlight e[CO₂]-Lh as an optimal strategy for tomato seedling growth, providing empirical guidance for precision CO₂ fertilization and light management in controlled cultivation.

1. Introduction

Tomato is a globally preeminent horticultural crop [1], driven by its dual nutritional and economic value [2]. Notably, China cultivates 1.16 million hectares of tomato, yielding 70.21 million tons annually (Food and Agriculture Organization of the United Nations [FAO], 2023) [3], highlighting its critical role in global agriculture [4]. Vigorous seedling establishment is essential for optimal vegetative growth, fruit yield, and quality [5]. However, seedling development is highly sensitive to environmental cues [6,7,8,9,10], with photosynthetic photon flux density (PPFD) and CO₂ concentration ([CO₂]) being key regulators of energy capture and carbon assimilation, respectively [11]. Although numerous studies have examined the individual and combined effects of [CO₂] and PPFD on greenhouse vegetables [12,13,14,15,16], their synergistic impacts on leaf anatomical plasticity, stomatal responses, and photochemical efficiency are not well understood.

Atmospheric [CO₂] has reached unprecedented modern levels (≈425 ppm in 2024), though geological records show higher concentrations in ancient eras [17]. Projections indicate a potential doubling by 2100, exceptional given its long-term atmospheric stability (comprising ~0.04% of the atmosphere) [18]. Elevated [CO₂] (e[CO₂]) consistently promotes seedling development, critical for optimizing crop productivity and fruit quality [13,16]. Mechanistically, in C3 plants, this results from e[CO₂]-enhanced carboxylation via ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), coupled with reduced photorespiratory losses through ribulose 1,5-bisphosphate (RuBP) regulation, boosting net photosynthesis (Pn) and biomass accumulation [19]. By contrast, C4 and crassulacean acid metabolism (CAM) plants show muted responses due to anatomical/biochemical adaptations that minimize photorespiration regardless of [CO₂] [20]. Concomitantly, these physiological improvements in C3 plants drive higher relative growth rate (RGR), linked to sink-source dynamics and root-zone conditions [21]. Additionally, e[CO₂] reduces stomatal conductance (gs) and transpiration (Tr), improving water use efficiency [16], while enhancing chlorophyll fluorescence parameters (e.g., maximum PSII photochemical efficiency (F_v/F_m), PSII quantum yield (ΦPSII)) to boost light-use efficiency [22]. Structural modifications, including increased mesophyll thickness, altered stomatal density (SD), and reduced stomatal aperture (SA), often accompany these functional changes [23]. Notably, responses vary by species and context, influenced by developmental stage, root microenvironment, and experimental duration [24]. Given tomato seedlings’ sensitivity to environmental cues, characterizing e[CO₂]-driven morphophysiological adaptations is vital for refining precision management in controlled cultivation.

Light—encompassing intensity, spectral quality, and photoperiod—is a fundamental regulator of plant ontogenesis [25,26]. Among these, light intensity not only serves as the energy substrate for carbon fixation but also initiates photoreceptor-mediated signaling pathways governing morphogenesis and stress acclimation [27]. Specifically, plants harness light energy via photosynthesis, generating adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH) during light-dependent reactions [26]. However, this process inherently involves solar radiation-induced reactive oxygen species (ROS) generation, particularly under high light, where excessive excitation energy can overwhelm the photosynthetic apparatus and induce oxidative stress [28]. Light influences seedling photosynthetic performance by modulating chloroplast development, anatomical traits, and key Calvin cycle enzymes [16,29]. Optimal light intensity fosters robust growth, enhances light energy conversion, and improves seedling health indices [6]. This threshold varies across C3, C4, and CAM plants due to divergent growth habits [30]. Notably, it promotes leaf structural integrity and hydraulic efficiency, while supraoptimal intensities induce photoinhibition via PSII destabilization [31]. Conversely, light intensity fluctuations drive dynamic stomatal adjustments: high light elevates stomatal index and conductance for maximal CO₂ uptake, whereas low light promotes stomatal expansion to enhance light interception [32]. This dual role is evident in gas exchange: increasing light intensity boosts Pn while often reducing gs [12]. Within physiological thresholds (below 450 µmol·m⁻²·s⁻¹), elevated light intensity improves ΦPSII and F_v/F_m in tomato seedlings, optimizing light energy absorption and carbon assimilation [33]. Additionally, plants also employ photoreceptor-mediated photoprotection mechanisms under excessive light, including signaling-driven heat dissipation pathways [34]. Collectively, this phenotypic plasticity underscores the need for precision light management in greenhouses to balance growth promotion and stress mitigation.

Stomatal dynamics, regulated by interactive [CO₂] and light intensity, mediate the balance between carbon assimilation and transpirational water loss in plants [35,36]. Stomata exhibit acute behavioral adjustments and chronic morphological adaptations to environmental fluctuations, including CO₂ variations [23,37]. These responses are often coordinated, though species- and genotype-specific differences exist [37]. In C3 plants like Arabidopsis, high light (175 µmol·m⁻²·s⁻¹) triggers phytochrome-mediated increases in stomatal density [38], while elevated [CO₂] suppresses stomatal aperture via anion channel modulation and abscisic acid signaling crosstalk [39]. These opposing effects suggest a complex regulatory network, yet mechanistic insights into their combined impacts on stomatal morphology and photosynthetic performance remain limited. Concurrently, leaf anatomical traits are key phenotypic adaptations to environmental change [29]. Although tomato seedling leaf anatomy is sensitive to environmental cues, comprehensive data on [CO₂]-light intensity interactions are scarce. Chlorophyll fluorescence analysis provides a powerful tool for dissecting photosynthetic stress responses, underpinning agricultural management strategies in changing climates [22,40]. Previous studies show that elevated [CO₂] and light intensity individually enhance F_v/F_m, indicating improved light capture [32]. However, their interactive effects on photochemical efficiency and stomatal-anatomical correlations require systematic investigation.

The objective of this research is to investigate the synergistic effects of elevated e[CO₂] and enhanced light intensity on leaf anatomical plasticity, stomatal morphology, and photosynthetic physiology in tomato seedlings. Specifically, we hypothesize that combined optimization of [CO₂] and light environments modulates mesophyll tissue architecture and stomatal patterning, thereby improving carbon assimilation efficiency through enhanced light interception and CO₂ diffusion. Integrating morphological, anatomical, and physiological measurements, this study aims to elucidate how multi-factor environmental optimization balances growth promotion and resource use efficiency, providing guidelines for sustainable tomato seedling production in horticulture.

2. Materials and Methods

2.1. Plant Material Preparation

Tomato (Solanum lycopersicum Mill. cv. ‘Jingpeng No. 1’) seeds were surface-disinfected by soaking in 55 °C water for 15 min, followed by a 6–8 h soak in room-temperature water. Seeds were then incubated in a dark incubator (28 °C, 95% RH) for 72 h to promote germination, after which they were sown in 72-cell trays filled with a peat:vermiculite:perlite (3:1:1, v/v/v) seedling substrate. Seedlings were cultivated for 35 days in an illumination incubator (PRX-450C-30, Ningbo, China) under controlled conditions: 25 °C/18 °C day/night temperature, 250 µmol·m⁻²·s⁻¹ PPFD [41], 70% RH, and a 14-h photoperiod. At the 4-leaf stage, they were transplanted into 20 cm (diameter) × 16 cm (height) plastic pots with commercial compost (Jiahui Co., Yangling, China). Artificial illumination was provided by metal halide lamps (HQI, 400 W; Osram Co., Munich, Germany), with spectral outputs validated using a calibrated spectroradiometer (Figure 1). Pure CO₂ gas (99.9% purity; Shaanxi Meiqi Trading Co., Ltd., Yangling, China) was used for enrichment treatments.

2.2. Experimental Design

The experiment employed a full factorial design with two factors: [CO₂] (ambient [a[CO₂]: 400 µmol·mol⁻¹] and enriched [e[CO₂]: 800 µmol·mol⁻¹]) and light intensity (L; low[Ll: 200 µmol·m⁻²·s⁻¹], moderate[Lm: 300 µmol·m⁻²·s⁻¹], and high[Lh: 400 µmol·m⁻²·s⁻¹]). Notably, prior research [32] showed 300 µmol·m⁻²·s⁻¹ with red-blue light supplementation optimizes tomato seedling growth, thus defining our moderate light level, with 200 and 400 µmol·m⁻²·s⁻¹ as the low and high values. Six treatments (a[CO₂]-Ll, a[CO₂]-Lm, a[CO₂]-Lh, e[CO₂]-Ll, e[CO₂]-Lm, e[CO₂]-Lh) were replicated 20 times across two climate chambers (QIUSHI Environment, Zhejiang, China). Environmental parameters were rigorously controlled as follows: (1) Infrared CO₂ detectors [16] maintained target concentrations during the 12-h photoperiod, with a[CO₂] restored at night. (2) Light levels were calibrated using a quantum sensor (MQ-100, Apogee Instruments, Logan, UT, USA) by adjusting lamp-to-canopy distances. Electric fans ensured uniform [CO₂] distribution and temperature stability. (3) Day/night temperatures of 23 °C/18 °C and 65% humidity. Plants were randomized every 48 h to minimize positional bias and irrigated biweekly with half-strength Hoagland solution (pH 6.5, EC 1.3 mS·cm⁻¹), with irrigation volume adjusted according to growth status to ensure adequate nutrient supply. Substrates were covered with aluminum foil to reduce evaporation. The experiment lasted 15 days post-transplant.

2.3. Growth Parameter Quantification

At 0 and 15 days after treatment (DAT), six plants per treatment were destructively sampled. Total leaf area was measured using a Li-3000 leaf area meter (LI-COR, Lincoln, NE, USA), and dry mass was determined via oven-drying (80 °C, 72 h) followed by gravimetric analysis (ME204E balance, Mettler-Toledo, ±0.0001 g, Greifensee, Switzerland). The formulas for calculating RGR, net assimilation rate (NAR), leaf area ratio (LAR), and specific leaf area (SLA) were as follows [42]: RGR = [ln(W₂) − ln(W₁)]/(t₂ − t₁); NAR = (W₂ − W₁)/[(t₂ − t₁) × (L₁ + L₂)/2]; LAR = L₂/W₂; SLA = L₂/WL₂. Note: W_1,2 and L_1,2 denote total dry mass and leaf area of the plant at 0 (t₁) and 15 DAT (t₂), respectively, while WL₂ denotes the leaf dry mass at 15 DAT (t₂).

2.4. Determination of Stomatal Traits

At 10 DAT, leaf abaxial surfaces were gently cleared of debris using a rubber bulb. From the third fully expanded tomato leaf, 4–6 leaflets were excised and immersed in 4% glutaraldehyde. Primary fixation proceeded in fresh glutaraldehyde: first at 25 °C (≥2 h), then at 4 °C (≥6 h). Samples underwent four 10-min rinses in 0.1 M phosphate buffer (pH 6.8). Ethanol dehydration followed graded concentrations (30% to 90%, 15 min/step) with three final 100% ethanol washes (30 min each). Critical point drying employed isoamyl acetate substitution (1 mL, 10–15 min). Dried specimens were mounted on stubs, gold-sputter coated, and imaged using a JSM-6360LV tungsten SEM (JEOL, Tokyo, Japan). Stomatal density and morphology (length/width/aperture) were determined by analyzing 15 fields per treatment in ImageJ (v1.53, NIH, Baltimore, MD, USA).

2.5. Determination of Leaf Anatomical Structure

At 10 DAT, the third fully expanded apical leaf underwent paraffin sectioning. Six leaf segments were fixed in FAA (50% ethanol, 10% formaldehyde, 5% glacial acetic acid, 35% distilled water) for 24 h. Samples were dehydrated through an ethanol series (70%, 85%, 90%, 95%; 3 h per step), then absolute ethanol (≥16 h). Clearing proceeded in xylene/absolute ethanol (1:1, v/v), followed by pure xylene (4 h total). Tissues were embedded in paraffin (55–60 °C, 24 h) and solidified on a cooling stage. Sections (8–10 µm) cut with a rotary microtome were stained with safranin-fast green and imaged using an Olympus BX51 microscope (OlympusOptical Co., Tokyo, Japan). For each treatment, 4–6 leaves were sectioned, with 15 fields analyzed per section to quantify leaf thickness, palisade mesophyll tissue (PM), spongy mesophyll tissue (SM), and upper/lower epidermis thickness.

2.6. Determination of Gas Exchange and Chlorophyll Fluorescence

From 11 to 12 DAT, diurnal photosynthesis was profiled using two synchronized LI-6400XT systems (Li-COR Inc., Lincoln, NE, USA) measuring gas exchange in third fully-expanded leaves within controlled-environment chambers. LED-illuminated leaf chambers maintained treatment-specific [CO₂] and PPFD levels (e.g., 400 µmol·mol⁻¹ CO₂ + 200 µmol·m⁻²·s⁻¹ PPFD for a[CO₂]-Ll conditions). Following 60–120 s stabilization to achieve steady-state, photosynthetic parameters (Pn, gs, transpiration rate (Tr)) were recorded at 2-h intervals (09:00–19:00 h) with five biological replicates. This parallel measurement design minimized environmental variability while capturing diurnal physiological responses.

Dark-adapted yields (initial fluorescence, F₀; maximum fluorescence, F_m) and light-adapted yields (minimum fluorescence under actinic light, F₀′; maximum fluorescence under actinic light, F_m′; steady-state fluorescence, F_s) were recorded. Key photochemical parameters (F_v/F_m, ΦPSII, electron transport rate (ETR), photochemical quenching coefficient (qP), non-photochemical quenching (NPQ), and effective photochemical quantum efficiency (F_v′/F_m′)) were calculated using standard equations: F_v/F_m = (F_m − F₀)/F_m; ΦPSII = (F_m′ − F_s)/F_m′; ETR = ΦPSII × PAR × 0.85 × 0.5; qP = (F_m′ − F_s)/(F_m′ − F₀′); NPQ = (F_m − F_m′)/F_m′; F_v′/F_m′ = (F_m′ − F₀′)/F_m′.

2.7. Determination of Dry Mass Accumulation and Seedling Health Index

After 15 DAT, growth parameters were quantified in four randomly selected plants per treatment. Plant height and stem diameter were measured using a metric ruler and digital caliper (0.01 mm resolution), respectively. Whole plants were rinsed with deionized water, surface-dried, and oven-dried at 65 °C (48 h) to constant mass. Total dry mass (TDM) was determined using an analytical balance (ME203, Mettler-Toledo; ±0.001 g, Greifensee, Switzerland). Thereafter, seedling health index (SHI) was calculated as: SHI = (stem diameter × total dry mass)/plant height, providing an integrated assessment of seedling vigor.

2.8. Statistical Analysis

Statistical analyses employed SAS 9.4 (SAS Institute, Cary, NC, USA) for two-way ANOVA (excluding diurnal gas exchange data), evaluating [CO₂] and light intensity main effects and interactions. Data met ANOVA assumptions (normality, homoscedasticity) verified through SAS procedures. Tukey’s HSD tests (p < 0.05) determined treatment differences. Pearson correlations among morphological, anatomical, stomatal, photosynthetic, and SHI parameters were computed in Origin 2024 (OriginLab Corp., Northampton, MA, USA). Results expressed as mean ± SE. Figures generated with Origin 2024 and GraphPad Prism 6.01 (GraphPad Software, Inc., La Jolla, CA, USA).

3. Results

3.1. Growth Traits

RGR, NAR, SLA, and LAR of tomato seedlings exhibited strong correlations (p < 0.001) with [CO₂] and light intensity (Figure 2), with significant CO₂ × light interactions for RGR and NAR (p < 0.05). Elevated [CO₂] increased RGR by 7.49–23.10% and NAR by 14.06–28.44% across light gradients (low to high), while reducing SLA (10.97–16.74%) and LAR (11.75–18.42%) (Figure 2A–D). Independently, higher light intensities enhanced RGR (19.59–40.11%) and NAR (24.46–61.74%) but decreased SLA and LAR versus low light. The e[CO₂]-Lh treatment produced maximal growth metrics, demonstrating distinct synergistic interaction.

3.2. Stomatal Traits

All stomatal traits responded significantly to light and [CO₂] (p < 0.05;

Table 1. Stomatal morphological responses of tomato seedlings under a factorial design of two CO₂ levels and three PPFD treatments in growth chambers. Data: mean ± SE (n = 15). Lowercase letters indicate significant differences among treatments (Tukey’s HSD, p < 0.05). NS-not significant, * p < 0.05, ** p < 0.01, *** p < 0.001 (two-way ANOVA).

Treatments	SL (µm)	SW (µm)	SA (µm)	SD (No.mm−2)
a[CO2]-Ll	22.52 ± 0.56 c	10.50 ± 0.67 cd	2.05 ± 0.11 bc	302.61 ± 18.59 d
e[CO2]-Ll	18.87 ± 0.62 d	10.50 ± 0.36 cd	1.96 ± 0.12 c	229.30 ± 15.51 e
a[CO2]-Lm	23.15 ± 0.33 c	11.50 ± 0.30 c	2.29 ± 0.10 b	440.11 ± 19.73 b
e[CO2]-Lm	26.30 ± 0.48 b	14.64 ± 0.28 b	2.11 ± 0.10 bc	335.96 ± 10.99 c
a[CO2]-Lh	19.10 ± 0.26 d	10.03 ± 0.20 d	2.41 ± 0.08 a	543.30 ± 15.44 a
e[CO2]-Lh	27.53 ± 0.51 a	16.64 ± 0.67 a	2.15 ± 0.17 bc	351.01 ± 11.62 c
Analysis of variance
[CO2]	**	**	**	**
Light	*	**	*	***
[CO2]:light	NS	NS	*	**

/Figure 3). Significant CO₂ × light interactions occurred for SA (p < 0.05) and SD (p < 0.01). Elevated [CO₂] increased stomatal length (SL) by 13.61–44.14% and width (SW) by 27.30–65.90% versus ambient counterparts under moderate-high light, while reducing SA (4.39–10.78%) and SD (23.66–35.39%) across all light levels. Within e[CO₂] treatments, SL and SW showed light-responsive increases. Under ambient [CO₂], moderate-high light boosted SA (6.63–9.68%) and SD (45.98–66.31%), peaking at a[CO₂]-Lh conditions.

3.3. Leaf Anatomical Structure

Leaf anatomical parameters responded significantly to [CO₂], light intensity, and their interaction (p < 0.05;

Table 2. Leaf anatomical characteristics of tomato seedlings under a factorial design of two CO₂ levels and three PPFD treatments in growth chambers. Data are presented as mean ± SE (n = 15). Lowercase letters indicate significant differences among treatments (Tukey’s HSD, p < 0.05). * p < 0.05, ** p < 0.01 (two-way ANOVA).

Treatments	LT (μm)	PM (μm)	SM (μm)	UE (μm)	LE (μm)	PM/SM
a[CO2]-Ll	133.39 ± 3.19 d	39.83 ± 0.83 e	76.89 ± 0.85 f	12.50 ± 0.21 e	4.17 ± 0.07 e	0.52 ± 0.01 a
e[CO2]-Ll	295.46 ± 2.55 a	102.08 ± 1.95 a	166.30± 1.81 b	20.83 ± 0.43 b	6.25 ± 0.12 a	0.53 ± 0.02 c
a[CO2]-Lm	158.82 ± 2.39 c	49.17 ± 2.00 d	88.93 ± 1.65 e	16.29 ± 0.42 d	4.43 ± 0.10 d	0.55 ± 0.02 a
e[CO2]-Lm	299.71 ± 8.98 a	66.67 ± 1.25 c	208.33 ± 1.97 a	19.48 ± 0.43 bc	5.23 ± 0.14 c	0.32± 0.02 b
a[CO2]-Lh	243.17 ± 5.04 b	79.33 ± 0.75 b	141.58 ± 3.46 d	18.75 ± 0.30 c	3.59 ± 0.23 f	0.56 ± 0.01 b
e[CO2]-Lh	283.84 ± 9.30 a	96.26 ± 4.08 a	156.11 ± 2.77 c	25.71 ± 0.36 a	5.76 ± 0.21 b	0.62 ± 0.02 a
Analysis of variance
[CO2]	**	**	**	*	*	**
Light	*	*	**	*	**	**
[CO2]:light	**	*	*	**	**	*

/Figure 4). Elevated [CO₂] augmented palisade mesophyll (PM, +71. 07%), spongy mesophyll (SM, +86.94%), and epidermal thickness (upper, UE + 41.11%; lower, LE + 42.79%), collectively increasing total leaf thickness (LT) by 75.65% versus ambient [CO₂]. The ratio of PM to SM (PM/SM) responded non-uniformly to e[CO₂], increasing at low/high light (+1.92–10.71%) but decreasing at moderate light (−41.82%). Across both CO₂ regimes, LT, SM, UE, and LE thicknesses increased with light intensity. Under ambient [CO₂], PM/SM ratio increased linearly with light intensity, whereas e[CO₂] induced nonlinear responses peaking at high light. The e[CO₂]-Lh treatment yielded maximal LT, PM, UE, and PM/SM values.

3.4. Diurnal Variation in Photosynthesis, Transpiration Rate, and Stomatal Conductance

To characterize the interactive effects of elevated [CO₂] and light intensity on plant physiology, diurnal dynamics of Pn, Tr, and gs were systematically analyzed. All treatments exhibited a bimodal diurnal pattern with distinct peaks and troughs (Figure 5). Elevated [CO₂] significantly enhanced daytime Pn by 10.06–18.22% (Figure 5A) but reduced Tr and gs by 20.32–25.88% (Figure 5B) and 3.78–18.43% (Figure 5C), respectively. Moderate and high light intensities increased Pn by 20.39–29.37% and 50.95–58.95% compared to low light (Figure 5A), with concurrent Tr enhancements of 15.66–39.97% (moderate) and 30.93–62.38% (high) (Figure 5B). Stomatal conductance followed a similar trend, increasing by 8.55–28.88% and 42.43–81.87% under moderate and high light, respectively (Figure 5C). The highest Pn was observed under e[CO₂]-high light (Lh), while a[CO₂]-Lh elicited maximal Tr and gs across all conditions.

3.5. Chlorophyll Fluorescence Parameters

Chlorophyll fluorescence parameters showed significant associations with [CO₂], light intensity, and their interactions (p < 0.05, 0.01, or 0.001;

Table 3. Chlorophyll fluorescence parameters were measured in tomato leaves under a factorial design of two CO₂ levels and three PPFD treatments in growth chambers. Values are means ± SE (n = 5). Lowercase letters indicate significant differences among treatments (Tukey’s HSD, p < 0.05). * p < 0.05, ** p < 0.01, *** p < 0.001 (two-way ANOVA).

Treatments	Fv/Fm	ΦPSII	ETR	qP	NPQ	Fv′/Fm′
a[CO2]-Ll	0.54 ± 0.01 d	0.51 ± 0.00 d	125.85 ± 1.15 c	0.50 ± 0.01 c	0.68 ± 0.01 b	0.52 ± 0.02 c
e[CO2]-Ll	0.63 ± 0.01 c	0.58 ± 0.01 c	158.54 ± 0.94 b	0.61 ± 0.01 b	0.59 ± 0.00 c	0.66 ± 0.00 b
a[CO2]-Lm	0.70 ± 0.02 b	0.63 ± 0.01 b	144.56 ± 2.54 ab	0.56 ± 0.02 b	0.65 ± 0.01 c	0.61 ± 0.01 ab
e[CO2]-Lm	0.78 ± 0.04 a	0.71 ± 0.00 a	167.63 ± 3.98 a	0.66 ± 0.01 a	0.53 ± 0.01 d	0.70 ± 0.00 a
a[CO2]-Lh	0.69 ± 0.02 b	0.64 ± 0.01 b	153.54 ± 6.37 b	0.58 ± 0.02 ab	0.71 ± 0.01 a	0.63 ± 0.01 b
e[CO2]-Lh	0.74 ± 0.01 a	0.69 ± 0.01 a	169.93 ± 5.23 a	0.65 ± 0.03 a	0.64 ± 0.02 c	0.69 ± 0.02 a
Analysis of variance
[CO2]	*	**	*	*	**	**
Light	**	***	*	*	**	*
[CO2]:light	*	**	*	*	**	*

). Across [CO₂] levels, F_v/F_m, ΦPSII, ETR, qP, and F_v′/F_m′ increased with rising light intensity. Moderate and high light intensities enhanced F_v/F_m by 26.72% and 22.62%, respectively, and ΦPSII by 22.97% and 22.23%, relative to low light. NPQ exhibited a U-shaped pattern, decreasing at moderate light before increasing under high light. Elevated [CO₂] further improved photochemical efficiency, boosting F_v/F_m (+11.78%), ΦPSII (+11.41%), ETR (+17.54%), qP (+16.64%), and F_v′/F_m′ (+17.07%), while reducing NPQ by 13.85%. The e[CO₂]-Lh treatment maximized all fluorescence parameters except NPQ, highlighting synergistic enhancements in light harvesting and energy conversion under combined high CO₂ and light intensity.

3.6. Total Dry Mass and Seedling Health Index

To evaluate the combined effects of e[CO₂] and enhanced light intensity on seedling quality, TDM and SHI were measured (Figure 6). Both parameters were significantly influenced by [CO₂], light intensity, and their interactions (p < 0.05–0.001), following parallel response trends. Specifically, e[CO₂] increased TDM and SHI by 40.36% (Figure 6A) and 5.95% (Figure 6B) versus ambient [CO₂]. Moderate and high light intensities further boosted TDM by 32.20% and 73.29%, and SHI by 31.66% and 64.96%, respectively, relative to low light. The e[CO₂]-Lh treatment achieved the highest TDM (6.28 g·plant⁻¹) and SHI (0.79), surpassing all other combinations. These results highlight synergistic benefits of e[CO₂] and high light intensity in promoting biomass accumulation and seedling vigor, with e[CO₂]-Lh representing an optimal strategy for controlled-environment tomato seedling production.

3.7. Correlations Among Plant Physiological and Morphological Traits

Pearson correlation analysis (Figure 7) revealed a near-perfect positive correlation between RGR and NAR (r = 0.99). Notably, both RGR and NAR were strongly negatively correlated with SLA (r = −0.95 to −0.97) and LAR (r = −0.97), indicating a trade-off between leaf expansion and carbon allocation efficiency. Moreover, PM and SM thicknesses showed strong positive correlations with total leaf thickness (LT; r = 0.85–0.95), supporting their combined role in optimizing light interception. Furthermore, UE thickness positively correlated with RGR (r = 0.82) and Pn (r = 0.98), suggesting that epidermal reinforcement enhances photosynthetic capacity through structural support. Pn positively correlated with stomatal dimensions (length [SL], r = 0.64; width [SW], r = 0.83) and growth metrics (RGR, r = 0.98; NAR, r = 0.98; TDM, r = 0.96; SHI, r = 0.97), thereby indicating that enlarged stomatal apertures facilitate CO₂ diffusion and carbon assimilation. Conversely, SD weakly negatively correlated with Pn (r = −0.22), potentially reflecting a trade-off between gas exchange and water conservation under high vapor pressure deficit. In addition, chlorophyll fluorescence parameters showed F_v′/F_m′ strongly correlated with ETR (r = 0.94), while NPQ negatively correlated with SLA and LAR, suggesting enhanced photoprotection in low-SLA leaves. SHI robustly correlated with TDM (r = 0.87), Pn (r = 0.97), RGR (r = 0.91), and NAR (r = 0.92), thus indicating that e[CO₂] and increased light intensity promote seedling vigor via enhanced carbon assimilation and biomass accumulation.

Collectively, these results show that elevated [CO₂] and PPFD synergistically enhance growth traits (RGR and NAR) while reducing leaf area parameters (SLA and LAR; Figure 2), thereby facilitating light absorption and organic matter accumulation (Figure 6). Notably, these treatments optimize stomatal morphology (Table 1/Figure 3) and leaf anatomy (Table 2/Figure 4), enhancing light harvesting, CO₂ diffusion, and energy utilization. At the physiological level, e[CO₂] improves net photosynthesis (Pn; Figure 5) and photochemical parameters (F_v/F_m, ΦPSII, ETR), while decreasing NPQ (Table 3)—findings indicative of enhanced light energy use and photoprotective acclimation under high light. Mechanistically, Pn correlates positively with stomatal dimensions (e.g., SL, SW), leaf anatomical traits (e.g., UE), and growth metrics (RGR, NAR, TDM, SHI; Figure 7). Moreover, SHI shows a robust association with TDM, RGR, and NAR, reinforcing its role as a key vigor indicator. By integrating morphological, anatomical, and photosynthetic data—particularly TDM and SHI—the e[CO₂]-Lh treatment emerges as optimal for promoting seedling growth and vigor in this study. These findings validate the study’s hypotheses, providing empirical support for enhanced plant growth under combined CO₂ levels and PPFD treatments.

4. Discussion

Recent advances in understanding the interactions between [CO₂] and PPFD have substantially influenced plant physiological ecology, attracting considerable research attention [11,12,13,14,15,16]. Leaf phenotypic traits, including anatomical architecture, stomatal density, and leaf area, exhibit high sensitivity to environmental variables such as [CO₂] [43] and light intensity [44], thereby affecting carbon assimilation and plant growth [45]. This study explores the short-term effects of e[CO₂] and increased light intensity on these traits, employing correlation heatmaps to decipher trait interrelationships. Our results shed light on how tomato seedlings adjust their growth, leaf anatomy, stomatal characteristics, and photosynthetic performance under ambient and elevated [CO₂] conditions across a light intensity gradient in controlled environments.

Growth parameters, pivotal for vegetative and reproductive development, are deeply influenced by environmental factors such as light availability [46] and [CO₂] [47]. These parameters—including SLA, NAR, and LAR—integrate eco-physiological signals, reflecting carbon allocation and resource use efficiency. RGR, a key vigor indicator, arises from the synergistic interaction between NAR and LAR. Elucidating these relationships enhances our understanding of plant-environment adaptation, providing theoretical grounds for agricultural and ecological research [48]. Notably, elevated [CO₂] substantially increased RGR and NAR while reducing SLA and LAR (p < 0.001, Figure 2), consistent with prior studies [47,49]. This suggests e[CO₂] enhances carbon assimilation via optimized light interception efficiency rather than increased leaf mass per unit area. Conversely, reduced light intensity decreased NAR but upregulated SLA and LAR (p < 0.001, Figure 2), a phenotypic plasticity strategy to maximize light capture, in line with earlier findings [13,49]. These results highlight plant adaptive strategies through trait modulation and photosynthetic enhancement [50]. RGR and NAR exhibited significant [CO₂]-light intensity interactions, driving enhanced dry mass accumulation and seedling robustness (p < 0.05, Figure 7), which correlated with increased net photosynthesis (Figure 5). The greater RGR and NAR increments under high light (Figure 2A,B) likely stem from coupled light/dark reaction enhancements, adaptive leaf structural modifications (Table 1 and Table 2, Figure 3 and Figure 4), alleviated RuBisCO activity limitations (Table 3) [16], and optimized carbon allocation (Figure 2). Collectively, these findings underscore the potential of CO₂ fertilization to enhance C3 plant (e.g., tomato) growth via light environment synergies, offering insights for precision agriculture.

Stomata serve as primary conduits for water and gas exchange with the external environment, their behavior and morphology regulated by [CO₂] and light intensity, thereby influencing Pn and gs [23,51]. Short-term exposure to e[CO₂] and increased light intensity elicits opposing effects on stomatal morphology: e[CO₂] induces stomatal closure to conserve water, whereas enhanced light intensity promotes stomatal opening to facilitate CO₂ uptake for photosynthesis [52]. Stomatal aperture and density are critical determinants of photosynthetic performance, balancing gas exchange efficiency with water conservation [53]. In this study, e[CO₂] significantly reduced stomatal aperture and density (Table 1, Figure 3), minimizing water loss and enhancing water use efficiency, consistent with prior research [16]. Conversely, increased light intensity tended to elevate these parameters, aligning with studies showing moderate light improves Pn, Tr, and gs [32,54]. Notably, e[CO₂]-Lh induced a marked reduction in stomatal aperture relative to low/moderate light conditions. This may reflect a potential protective mechanism under combined high CO₂ and light stress, involving increased LT and carbon allocation to structural tissues, as evidenced by elevated leaf and plant dry masses in tomato seedlings. A potential confounding factor is heat emission from metal halide lamps, which may have indirectly influenced stomatal dynamics [23,32]. Future studies using LED lighting systems could dissociate light quality/intensity effects from thermal influences, enabling precise characterization of stomatal responses to photic stimuli.

Leaf anatomical structure is dynamically modulated by environmental factors, including light intensity and [CO₂] [23,43,44,55], with profound effects on photosynthetic efficiency and drought tolerance via carbon partitioning and water retention mechanisms. Consistent with prior studies [56], e[CO₂] substantially enhanced LT, PM, and SM (Table 2, Figure 4), indicating that elevated CO₂ optimizes leaf architecture to facilitate CO₂ diffusion and dissolution, thereby boosting photosynthetic capacity and biomass accumulation. Concurrently, this structural optimization likely synergized with nutrient sufficiency to enhance carbon assimilation, as elevated CO₂ has been shown to upregulate plant nutrient demand [57]. Furthermore, increased light intensity enhanced leaf mass and PM across [CO₂] levels, with significant interactions promoting LT. This suggests higher light intensities improve light interception efficiency [58], acting synergistically with e[CO₂] to enhance light energy absorption and CO₂ fixation [16]. Notably, e[CO₂] reduced the PM/SM ratio under moderate light intensity, contrasting with previous reports [57]. This discrepancy likely arises from divergent environmental contexts and the optimal nutrient management in our study, where elevated CO₂-treated plants prioritized balancing photosynthetic efficiency with resource utilization [24], reflecting phenotypic resilience and plasticity in response to climate variables—a phenomenon warranting further mechanistic investigation [58].

Gas exchange dynamics (Figure 5) corroborate leaf structural and stomatal observations, indicating that e[CO₂] significantly augmented Pn while reducing gs and Tr. These findings validate the structure-function paradigm, whereby e[CO₂] enhances photosynthesis through leaf architectural adjustments, including increased thickness and stomatal trait modifications, in line with prior studies [23,45]. Enhanced Pn, gs, and Tr under elevated light intensity align with established mechanisms of light-driven stomatal activation and carbon fixation [31,51,59]. Notably, diurnal fluctuations in photosynthetic parameters reflect dynamic environmental cues and plant water status, modulating the balance between carbon fixation and transpirational water loss [60]. In this study, e[CO₂] significantly increased F_v/F_m, ΦPSII, and ETR (Table 3), consistent with earlier reports [40,61]. This suggests elevated CO₂ enhances photosynthetic performance by optimizing light energy partitioning—augmenting photochemical efficiency while reducing NPQ—and improving photosynthetic machinery functionality [62].

Plant phenotypic traits—including leaf area, anatomical structure, TDM, and SHI—serve as direct indicators of adaptability to environmental variables [23,32]. In this study, e[CO₂], increased light intensity, and their interactions substantially influenced leaf area-related traits (SLA, LAR; Figure 2) and leaf structural parameters (stomatal, mesophyll, and epidermal characteristics; Table 1 and Table 2/Figure 3 and Figure 4). Notably, TDM and SHI showed significant positive correlations with [CO₂], light intensity, and their interaction (Figure 6), supporting our prior hypothesis: combined optimization of CO₂ and light intensity enhances mesophyll architecture and stomatal patterning, thereby boosting carbon assimilation capacity. Given the rising demands of the vegetable industry for high-quality seedlings [5], scientifically optimized environmental control strategies—such as CO₂ fertilization and light regulation—will become pivotal for intensive seedling production systems [16]. While short-term e[CO₂] is widely acknowledged to enhance photosynthetic performance and growth, its efficacy may decline across developmental stages due to metabolic reprogramming [16,23]. Thus, future research should explore the dynamic adaptive mechanisms of tomato plants to concurrent [CO₂] and light intensity increases, enabling sustainable optimization of greenhouse cultivation protocols.

The correlation matrix (Figure 7) reveals key associations among growth, anatomical, and photosynthetic traits in tomato plants, highlighting adaptive strategies under environmental gradients. In this study, RGR and NAR showed strong negative correlations with SLA and LAR, indicating resource allocation trade-offs in light-limited environments where reduced leaf expansion prioritizes carbon assimilation efficiency [52,63]. Notably, RGR and NAR were positively associated with Pn, TDM, and SHI, consistent with prior findings [64], suggesting that e[CO₂] and increased PPFD enhance biomass accumulation by boosting carbon assimilation. Leaf anatomical traits (e.g., UE, LT, SM) correlated positively with Pn, aligning with studies demonstrating structural optimization for carbon assimilation [65]. Pn exhibited a positive correlation with SW but a negative correlation with SA [66], reflecting the interplay between CO₂-driven carboxylation efficiency and light-induced photoprotection mechanisms. NPQ negatively correlated with growth traits, likely balancing photoprotection and resource allocation: e[CO₂] promoted low-SLA leaf structures, while high light induced NPQ upregulation, collectively limiting leaf expansion (low LAR) [24]. These dynamics align with research on CO₂ fertilization and photoprotective acclimation [65], emphasizing the trade-off between light harvesting and carbon gain as a critical acclimation strategy [13].

This study comprehensively analyzed morphological, physiological, anatomical, and photosynthetic responses to e[CO₂] and increased PPFD. Importantly, transferring tomato seedlings to differing light intensities triggered stepwise activation of photoprotective pathways and morphological adjustments [41,67,68]. Prior research shows 240 µmol·m⁻²·s⁻¹ optimizes seedling growth, photosynthetic efficiency, and quality traits, while suboptimal or excessive light reduces chlorophyll content and stomatal conductance [67]. O’Carrigan et al. [68] further documented that prolonged high-light exposure (≥6 weeks) alters guard cell/stomatal morphology, induces stomatal closure, and suppresses photosynthesis. Whether pre-culture enhances seedling resilience to fluctuating light environments remains unaddressed. Moreover, its short-term design limits insights into long-term dynamics (e.g., full growth cycles). Long-term acclimation of stomatal behavior, photosynthetic apparatus, and leaf anatomy—along with yield and quality outcomes—warrants further investigation [24,49]. Future studies should characterize these parameters over extended periods to develop robust tomato production strategies under climate-smart greenhouse management.

5. Conclusions

Elevated [CO₂] and increased light intensity significantly enhanced RGR and NAR while reducing SLA and LAR. Concurrently, e[CO₂] decreased stomatal aperture and density, whereas increased light intensity modulated stomatal morphology to enhance gas exchange. These treatments collectively optimized leaf anatomical traits, thereby enhancing light interception and CO₂ diffusion efficiency. Elevated [CO₂] improved photochemical parameters (F_v/F_m, ΦPSII, ETR) while reducing NPQ, indicative of enhanced light energy utilization and potential photoprotective acclimation under high light. Individually and synergistically, e[CO₂] and increased PPFD promoted TDM and SHI, with the highest values observed under e[CO₂]-Lh. Collectively, these findings, coupled with correlation analysis, demonstrate that e[CO₂] and increased light intensity optimize leaf structural and stomatal traits, thereby enhancing light capture, CO₂ assimilation, and energy conversion efficiency. Based on these results, the e[CO₂]-Lh treatment (800 µmol·mol⁻¹ [CO₂] and 400 µmol·m⁻²·s⁻¹ PPFD) is recommended for tomato seedling production in controlled growth environments to maximize seedling vigor and resource use efficiency.