Effects of light intensity on the growth and photosynthetic physiology of Zelkova schneideriana seedlings

Abstract

Zelkova schneideriana is an important timber species used in the transformation of monoculture coniferous forests in southern China. However, the optimal light conditions for cultivating Z. schneideriana under forest canopies remain to be determined. In this study, one-year-old container seedlings of Z. schneideriana were used in a field experiment under four artificially simulated light intensities: full sunlight (CK, 100% PAR), low shade (L, 70% PAR), medium shade (M, 51% PAR), and high shade (H, 22% PAR). The results showed significant differences in growth, morphology, and photosynthetic physiology of seedlings under different light intensities. Z. schneideriana seedlings exhibited a certain degree of shade tolerance during early establishment, achieving optimal growth performance under medium shade conditions. Compared to CK, the net increments in height and diameter at root collar of seedlings under M increased by 197.69% and 145.27%, respectively. Specific leaf area was greatest under high shade, indicating that leaves tend to utilize a larger leaf area to acquire carbon resources. Seedlings under M exhibited the highest values for photosynthetic pigment content, key photosynthetic enzyme activities, and photosynthetic nitrogen- and phosphorus-use efficiencies, which were consistent with their response to light-saturated photosynthetic rate. The phenotypic plasticity indices of the seedlings ranked in the following order: photosynthetic physiological traits > light response traits > morphological traits. Photosynthetic pigment content was identified as the physiological parameter most sensitive to changes in light intensity. Therefore, for the understory introduction of Z. schneideriana seedlings, it is recommended that canopy thinning be implemented to maintain understory light intensity at approximately 50% of full sunlight, thereby facilitating rapid seedling establishment and growth.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08937-5.

Introduction

Light availability is a critical environmental factor controlling the survival and early growth of understory seedlings [31]. Excessively high light intensity can damage the photosynthetic apparatus of tree seedlings, leading to photoinhibition. Conversely, insufficient light reduces photosynthetic capacity, severely limiting seedling growth and potentially causing mortality [5, 36]. In response to heterogeneous light conditions, tree seedlings exhibit significant phenotypic plasticity by adjusting their morphological and physiological traits [50]. Previous studies have demonstrated that under low-light stress, trees enhance light capture and utilization through morphological adaptations, such as increased leaf area and reduced leaf thickness [23, 27]. They also implement physiological adjustments, including lowering the light saturation point (LSP), light compensation point (LCP), and dark respiration rate (R_d). These adjustments promote net organic matter accumulation by increasing carbon assimilation and reducing respiratory consumption, thereby sustaining seedling growth [44, 48]. However, a contrasting pattern has been observed in Quercus virginiana seedlings, which under heavy shading showed higher LSP, LCP, and R_d compared to those under full sunlight conditions [38]. This indicates that the response strategies of seedlings vary significantly among tree species under heterogeneous light environments. Therefore, clarifying the light requirements at the seedling stage, as well as species-specific response and adaptation mechanisms to varying light conditions, is essential for guiding light environment selection and management during early-stage afforestation or forest regeneration projects.

In recent years, China has implemented the National Reserve Forest Construction Program to improve forest quality and enhance ecological functions. The program focuses on thinning existing coniferous forests and introducing native valuable tree species through understory planting, thereby establishing uneven-aged, multi-layered coniferous and broad-leaved mixed forests [19, 42]. The suitability of ecological niches for understory planting is largely determined by the light requirements and adaptive capacity of the tree species [33, 53]. Therefore, investigating the responses and adaptations of native valuable tree species seedlings to varying light environments, and clarifying their light adaptation thresholds, are essential for scientifically matching tree species to site conditions and ensuring the success of understory planting.

Zelkova schneideriana is a deciduous broad-leaved tree belonging to the genus Zelkova in the family Ulmaceae. Valued for its superior wood properties, this species is widely used in high-end furniture and panel production, which gives it considerable economic importance. Therefore, Z. schneideriana has been recognized as a premium native broad-leaved species selected for promotion in plantation programs in Guizhou Province. To date, research on Z. schneideriana has mainly focused on optimizing container seedling systems and exploring basic cultivation techniques. For example, Luo et al. [21] determined the optimal growing substrate for its container seedlings, and Lu et al. [20] investigated fertilization aimed at improving seedling growth after afforestation. These studies, however, were conducted exclusively in pure stands. There has been no scientific research on cultivation techniques for Z. schneideriana within the context of existing stand transformation. The success of such transformations depends largely on the performance of underplanted seedlings—specifically their survival, growth, and development—which are influenced by a complex interaction of intrinsic and extrinsic factors [31]. Currently, little is known about how underplanted Z. schneideriana seedlings respond and adapt to understory light environments. Furthermore, practical guidelines for matching seedling light requirements to suitable forest site conditions are currently lacking. To address this, one-year-old Z. schneideriana container seedlings were planted in the field following standard afforestation protocols. Four light levels were established using shade nets to simulate a range of understory light environments, allowing for the examination of associated changes in seedling growth, morphology, and photosynthetic physiology. This study aimed to (1) characterize the physiological responses and adaptations of Z. schneideriana seedlings to different light intensities, and (2) identify the optimal light conditions for their growth. The results are expected to offer both a theoretical basis and practical guidance for matching Z. schneideriana seedlings with appropriate forest site conditions during stand transformation.

Materials and methods

Study site

This study was conducted at the Yunguanshan State-owned Forest Farm (106° 44′ 29″ E, 26° 32′ 17″ N) in Guizhou Province, China. The experimental site is located at an elevation of 1163.7 m above sea level. The climate is classified as a humid subtropical monsoon climate, with a mean annual precipitation of 1174.7 mm. The mean temperature and relative humidity during the experimental period were 19.9 °C and 79%, respectively. The topography of the study site is gentle. The soil is classified as yellow earth, and its pH was 5.2. Key soil chemical properties were determined as follows: organic matter, 28.48 g kg⁻¹; total nitrogen, 1.49 g kg⁻¹; total phosphorus, 0.34 g kg⁻¹; total potassium, 6.6 g kg⁻¹; alkali-hydrolyzable nitrogen, 55.57 mg kg⁻¹; available phosphorus, 2.97 mg kg⁻¹; and available potassium, 56.81 mg kg⁻¹.

Experimental design

One-year-old Zelkova schneideriana container seedlings were sourced from a nursery in Rongjiang County, Guizhou Province, China. Seedlings of uniform size and free from visible damage or disease were selected for the experiment. These seedlings were transplanted into the experimental plots on 10 April 2024 and allowed to acclimate for one month before light treatments began. The average seedling height and diameter at root collar were 86.58 ± 1.85 cm and 6.13 ± 0.13 mm, respectively.

The experimental site was located on a single flat area with relatively consistent environmental conditions. Four light level treatments were established by suspending black shade nets of different mesh densities 2.5 m above the ground. Each treatment had three replicates, with 20 seedlings planted per replicate at a spacing of 0.5 m × 0.5 m to minimize mutual shading. The light treatments were applied from May 10 to December 10, 2024. To accurately assess differences among treatments, photosynthetically active radiation (PAR) was measured simultaneously on both clear and cloudy days at a height of 5 cm above the seedling apex within each treatment and in adjacent open areas under full sunlight, using two high-resolution spectroradiometers (LI-180, Li-Cor Inc., USA). The proportion of PAR (PAR%) was then calculated. Three sets of measurements were taken per replicate, and the PAR value for each treatment was calculated as the average across the three replicates. These results are presented in Table 1. The formula used to calculate PAR (%) was as follows:

Table 1.

PAR and relative PAR percentage at the apex of Z. schneideriana seedlings under different light intensity

Treatments	Clear day		Cloudy day		Mean PAR (%)
	PAR (μmol m−2 s−1)	PAR (%)	PAR (μmol m−2 s−1)	PAR (%)
Full sunlight (CK)	1683.6 ± 36.7	100	645.2 ± 5.0	100	100
Low shade (L)	1206.8 ± 12.2	72	443.9 ± 7.9	69	70
Medium shade (M)	850.6 ± 10.6	51	325.8 ± 3.5	51	51
High shade (H)	389.1 ± 8.1	23	132.1 ± 3.0	20	22

Data are presented as mean ± standard error (n = 3)

PAR Photosynthetically active radiation

Measurement of growth and morphological traits

Seedling height and diameter at root collar

Seedling height and diameter at root collar were measured for all seedlings across treatments and replicates at both the beginning and end of the experiment. Height was measured using a steel tape (precision: 0.1 cm), and diameter at root collar was measured using a digital vernier caliper (precision: 0.01 mm). The net increments in height and diameter at root collar were subsequently calculated.

Leaf morphological traits

In September 2024, three representative healthy seedlings were selected from each replicate, and three mature leaves were collected from the middle to upper part of the canopy of each seedling. Leaf fresh mass (LFM, g) was measured using an electronic balance with a precision of 0.001 g. Leaf length (LL, cm), leaf width (LW, cm), and leaf area (LA, cm²) were measured by scanning the leaves with a flatbed scanner (Epson Perfection V700 Photo, Japan) and analyzing the resulting images with ImageJ (version 1.51i NIH, USA). The leaf samples were then oven-dried at 105 °C for 30 min to deactivate enzymes, followed by drying at 80 °C until a constant weight was achieved. Leaf dry mass (LDM, g) was measured using an electronic balance with a precision of 0.001 g. Specific leaf area (SLA, m² kg⁻¹) and leaf dry matter content (LDMC, g g⁻¹) were calculated as follows:

Leaf anatomical structure

Leaf samples were collected for both morphological and anatomical analyses, with three mature leaves per treatment allocated specifically for the latter. From each leaf, a segment (0.5 cm × 0.5 cm) was excised from the basal one-third region along the midrib using fine scissors. The segments were immediately fixed in FAA fixative in the field and subsequently transported to the laboratory for further processing. Transverse Sects. (10 μm thick) were prepared using a standard paraffin-embedding method and stained following established protocols [30]. The sections were examined under a light microscope (Nikon Eclipse E100, Japan), and digital images were acquired with an attached camera system (Nikon DS-U3, Japan). The following anatomical traits were measured using image analysis software (Case Viewer, v. 2.3, 3DHISTECH Ltd., Hungary): leaf thickness (LT), upper epidermal thickness (UET), lower epidermal thickness (LET), palisade tissue thickness (PT), and spongy tissue thickness (ST). Total epidermal thickness (ET) was calculated as UET + LET.

Measurement of light-response curves

In early September 2024, one representative healthy seedling was selected from each replicate for photosynthetic measurements. Light-response curves were measured on fully expanded, sun-exposed leaves between 09:00 and 11:00 on consecutive clear days using a portable photosynthesis system (LI-6800, LI-COR Inc., USA). The measurements were conducted under the following constant conditions: CO₂ concentration was maintained at 400 µmol mol⁻¹ and photosynthetic photon flux density (PPFD) stepwise decreased from 1800, 1600, 1400, 1200, 1000, 800, 500, 200, 150, 100, 70, 50, 20 to 0 μmol m⁻² s⁻¹. At each light level, the minimum and maximum wait times were set to 120 s and 200 s, respectively. When the coefficient of variation of net photosynthetic rate (P_n) and stomatal conductance remained below 1% over a 60-s interval, leaf photosynthesis was considered to have reached a steady state and the instrument automatically recorded the data. The light-response curves were fitted using the mechanistic model proposed by [49]. The fitting was performed using an online photosynthetic calculation tool (http://zipiao.tech/g/12ye). A coefficient of determination (R²) greater than 0.98 was considered acceptable, and parameters including light-saturated net photosynthetic rate (A_max), light saturation point (LSP), light compensation point (LCP), dark respiration rate (R_d), and apparent quantum yield (AQY) were derived. The model expression is as follows:

where P_n is the net photosynthetic rate, I is the photosynthetic photon flux density (PPFD), α is the initial slope of the light-response curve, and β and γ are coefficients.

Measurement of photosynthesis-related physiological parameters

Photosynthetic pigment content

Three representative healthy seedlings were selected from each replicate. For photosynthetic pigment determination, one mature leaf was collected from the middle to upper part of the canopy of each seedling. From each leaf, leaf discs (approximately 1 cm² in area) were punched from interveinal regions and then finely cut into strips. The strips were placed into sealed test tubes containing 5 mL of 80% (v/v) acetone and extracted in darkness at 25 ℃ for 24 h, until the leaf segments became fully discolored [46]. The absorbance of the supernatant was measured at 663, 646, and 470 nm using a UV–Vis spectrophotometer (UNICO UV-2100, USA) to obtain A₆₆₃, A₆₄₆, and A₄₇₀, respectively. The concentrations of chlorophyll a (Chl_a), chlorophyll b (Chl_b), total chlorophyll (Chl_T), and carotenoids (Car) were calculated using the following equations [41]:

where V is the volume of the extraction solution (mL) and S is the leaf sample area (cm²).

Activities of key photosynthetic enzymes

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the primary carbon-fixing enzyme in C₃ plants and is recognized as one of the rate-limiting enzymes in photosynthesis. Its activity directly determines CO₂ fixation capacity [52]. Rubisco activase (RCA) regulates the transition between the activated and deactivated states of Rubisco, thereby modulating its catalytic activity under varying environmental conditions [1]. Therefore, in this study, the activities of Rubisco and RCA were measured to more accurately assess the effects of light intensity variation on the carbon assimilation process of seedlings. The activities of Rubisco and RCA were measured in leaf tissue extracts using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) according to the method of Li et al. [12]. The ELISA kits were obtained from Shanghai Keshun Biotechnology Co., Ltd.

Photosynthetic nitrogen and phosphorus use efficiencies

Sampling was performed following the same method as used for photosynthetic pigment determination, with nine leaves selected from each replicate. Samples from the same treatment were pooled and oven-dried to constant weight at 80 °C. The dried materials were ground and passed through a 0.15-mm sieve to obtain a fine, homogeneous powder. Three analytical replicates were prepared per treatment. Total nitrogen (N) and total phosphorus (P) contents were determined using an automatic discontinuous chemical analyzer (DeChem-Tech. GmbH, Hamburg, Germany) after H₂SO₄-H₂O₂ digestion. Photosynthetic nitrogen-use efficiency (PNUE) and photosynthetic phosphorus-use efficiency (PPUE) were calculated according to Song et al. [29] as follows:

where A_max is the light-saturated net photosynthetic rate (μmol m⁻² s⁻¹) and SLA is the specific leaf area (m² kg⁻¹).

Statistical analysis and other

Microsoft Excel 2019 was used for preliminary data processing. The mean values of each indicator for each replicate (n = 3) were calculated and served as the basis for analysis. Two-way analysis of variance (ANOVA) was performed using SPSS 26.0 (IBM Corp., USA). The results showed no significant differences among replicates for the measured indicators (Table S1), indicating high consistency of conditions across replicates. Therefore, subsequent analyses considered only the effect of light intensity. When ANOVA indicated significant effects (p < 0.05), post hoc mean comparisons were conducted using Duncan’s multiple range test. Relationships among leaf traits under different light treatments were examined using principal component analysis (PCA) and Pearson correlation analysis. All figures were prepared using Origin 2024 (OriginLab Corporation, USA). The phenotypic plasticity index (PPI) for each trait was calculated following Valladares et al. [35] as PPI = (V_max – V_min)/V_max, where V_max and V_min represent the maximum and minimum mean values of a trait across all treatments, respectively. This index allows for the comparison of changes in traits expressed in different units.

Results

Effects of light intensity on the growth of Z. schneideriana seedlings

The net increments in seedling height and diameter at root collar (Fig. 1) were lowest under the full sunlight (CK) and highest under the medium shade (M), with a significant difference between these two treatments (p < 0.05). In contrast, no significant difference was observed between the low shade (L) and high shade (H) (p > 0.05). Compared with CK, the height increment under L, M, and H treatments increased by 73.29%, 197.69%, and 69.69%, respectively. Similarly, the diameter at root collar increment under these treatments increased by 75.75%, 145.27%, and 86.86%, respectively. These results suggest that moderate shading during the first year after afforestation promotes both height and diameter growth of Z. schneideriana seedlings, with the M treatment exhibiting the most pronounced beneficial effect.

Fig. 1.

Net increments in height (a) and diameter at root collar (b) of Z. schneideriana seedlings under different light intensity. Data are presented as mean ± standard error (n = 3). Different lowercase letters indicate significant differences among treatments (p < 0.05). CK, full sunlight; L, low shade; M, medium shade; H, high shade

Effects of light intensity on the leaf morphology of Z. schneideriana seedlings

Leaf characteristics

Leaf area (LA) was greatest under the M treatment and lowest under CK (Fig. 2). Compared with CK, LA under the M treatment increased significantly by 101.05% (p < 0.05). Specific leaf area (SLA) showed a gradual increase with decreasing light intensity. Significant differences were observed among most treatment pairs (p < 0.05), except between the CK and L treatments, where the difference was not significant (p > 0.05). SLA under the H treatment was 27.41%, 26.82%, and 14.07% higher than that under the CK, L, and M treatments, respectively. Leaf dry matter content (LDMC) showed a trend opposite to that of SLA in response to light intensity, with significant differences among all treatments (p < 0.05). Compared with CK, LDMC under the H treatment decreased by 20.50%. These findings indicate that Z. schneideriana seedlings are capable of adjusting their leaf morphological traits in response to varying light environments.

Fig. 2.

Leaf characteristics of Z. schneideriana seedlings under different light intensity. a Leaf area (LA); b Specific leaf area (SLA); c Leaf dry matter content (LDMC). Data are presented as mean ± standard error (n = 3). Different lowercase letters indicate significant differences among treatments (p < 0.05). CK, full sunlight; L, low shade; M, medium shade; H, high shade

Leaf anatomical structure

Analysis of leaf anatomical structure (Table 2) showed that leaf thickness (LT) and palisade tissue thickness (PT) varied significantly among light intensity (p < 0.05), with both decreasing significantly as light intensity declined. Compared with CK, LT under the L, M, and H treatments decreased by 5.99%, 12.62%, and 21.37%, respectively. Similarly, PT decreased by 12.63%, 19.66%, and 39.55% under the corresponding treatments. Epidermal thickness (ET) was highest under CK and was significantly higher than that under all shading treatments, although no significant differences were observed among the L, M, and H treatments. Spongy tissue thickness (ST) was significantly greatest under L, with no significant differences occurred among the CK, M, and H treatments. The ratio of palisade to spongy tissue (P/S) declined with decreasing light intensity. Significant differences in the P/S ratio were observed across most treatment pairs, except for the comparison between L and M treatments. Anatomical sections (Fig. 3) further revealed that leaves under the H treatment were notably thinner and exhibited a looser arrangement of palisade tissue, enlarged intercellular spaces, a higher relative proportion of spongy tissue, and a markedly reduced P/S ratio. These structural adjustments suggest that reductions in LT and changes in the P/S ratio represent key adaptive responses of Z. schneideriana seedlings to low-light environments.

Table 2.

Leaf anatomical structure indices of Z. schneideriana seedlings under different light intensity

Treatments	LT (μm)	ET (μm)	PT (μm)	ST (μm)	P/S
CK	170.22 ± 0.53a	32.85 ± 0.94a	74.85 ± 0.68a	62.42 ± 1.15b	1.20 ± 0.03a
L	160.02 ± 0.75b	25.68 ± 0.67b	65.40 ± 0.65b	68.70 ± 0.88a	0.95 ± 0.02b
M	148.73 ± 1.21c	23.93 ± 0.59b	60.13 ± 0.63c	64.48 ± 0.91b	0.93 ± 0.01b
H	133.83 ± 1.39d	25.53 ± 0.39b	45.25 ± 0.94d	63.23 ± 0.95b	0.72 ± 0.02c

Data are presented as mean ± standard error (n = 3). Different lowercase letters indicate significant differences among treatments (p < 0.05)

CK Full sunlight, L Low shade, M Medium shade, H High shade, LT Leaf thickness, ET Epidermal thickness, PT Palisade tissue thickness, ST Spongy tissue thickness, P/S Palisade to spongy tissue ratio

Fig. 3.

Leaf anatomical structures of Z. schneideriana seedlings under different light intensity. CK, full sunlight; L, low shade; M, medium shade; H, high shade; UET, upper epidermal thickness; LET, lower epidermal thickness; PT, palisade tissue thickness; ST, spongy tissue thickness

Light-response curves of Z. schneideriana seedlings under different light intensities

The net photosynthetic rate (P_n) of Z. schneideriana seedlings rose with increasing photosynthetically active radiation (PAR) under all light intensity (Fig. 4). Below 500 μmol m⁻² s⁻¹ PAR, P_n increased sharply across all treatments. The PAR level at which P_n peaked varied among treatments, reaching 895.53, 1075.92, 1113.72, and 1216.10 μmol m⁻² s⁻¹ under CK, L, M, and H, respectively. Overall, P_n was significantly higher under the M treatment than under the others. Photosynthesis of Z. schneideriana seedlings was inhibited under both high-light (CK) and extremely low-light (H) conditions.

Fig. 4.

Light-response curves of Z. schneideriana seedlings under different light intensity. Data are presented as mean ± standard error (n = 3). CK, full sunlight; L, low shade; M, medium shade; H, high shade; P_n, net photosynthetic rate; PAR, photosynthetically active radiation

The light-saturated net photosynthetic rate (Aₘₐₓ) and apparent quantum yield (AQY) were highest under the M treatment and lowest under CK (Table 3), with a significant difference between these two treatments (p < 0.05). No significant differences in these parameters were observed between the L and H treatments. Compared with CK, A_max and AQY under the M treatment increased by 68.30% and 56.67%, respectively. The light saturation point (LSP), light compensation point (LCP), and dark respiration rate (R_d) all declined significantly with decreasing light intensity (p < 0.05). However, LSP did not differ significantly between the L and M treatments, nor did LCP between the CK and L treatments.

Table 3.

Light-response parameters of Z. schneideriana seedlings under different light intensity

Treatments	Amax (μmol m−2 s−1)	LSP(μmol m−2 s−1)	LCP (μmol m−2 s−1)	Rd (μmol m−2 s−1)	AQY (mol mol−1)
CK	6.36 ± 0.30c	1216.10 ± 14.55a	42.05 ± 1.02a	2.07 ± 0.02a	0.028 ± 0.001c
L	9.86 ± 0.39b	1113.72 ± 16.85b	39.68 ± 0.89a	1.87 ± 0.01b	0.035 ± 0.001b
M	17.07 ± 0.54a	1075.92 ± 14.12b	32.73 ± 0.56b	1.81 ± 0.01c	0.044 ± 0.001a
H	8.96 ± 0.30b	895.53 ± 15.15c	30.19 ± 0.43c	1.70 ± 0.01d	0.036 ± 0.001b

Data are presented as mean ± standard error (n = 3). Different lowercase letters indicate significant differences among treatments (p < 0.05)

CK Full sunlight, L Low shade, M Medium shade, H High shade, A_max Light-saturated net photosynthetic rate, LSP Light saturation point, LCP Light compensation point, R_d Dark respiration rate, AQY Apparent quantum yield

Effects of light intensity on photosynthetic pigment content in Z. schneideriana seedlings

The contents of chlorophyll a (Chl_a), chlorophyll b (Chl_b), and total chlorophyll (Chl_T) differed significantly among light intensity (p < 0.05; Fig. 5a, b, d), following the order M > H > L > CK. Under the M treatment, the contents of Chl_a, Chl_b, and Chl_T were 2.93, 4.12, and 3.20 times higher, respectively, than under CK. Carotenoid (Car) content was highest under M and lowest under CK (Fig. 5c). Significant differences in Car content were observed among all treatment pairs except between L and H. Both the chlorophyll a/b ratio (Chl_a/b) and the carotenoid-to-total chlorophyll ratio (Car/Chl_T) declined with decreasing light intensity. The Chl_a/b ratio differed significantly across all light treatments (p < 0.05); compared with CK, L, and M, it was reduced by 29.71%, 22.40%, and 8.33%, respectively, under the H treatment. The Car/Chl_T ratio was significantly higher under CK and L than under M and H (p < 0.05). Specifically, relative to the M treatment, the Car/Chl_T ratio was 50.57% higher under CK and 55.52% higher under L. Similarly, compared with the H treatment, it was 19.60% higher under CK and 23.54% higher under L.

Fig. 5.

Photosynthetic pigment content in leaves of Z. schneideriana seedlings under different light intensity. a Chlorophyll a (Chl_a); b chlorophyll b (Chl_b); c carotenoids (Car); d total chlorophyll (Chl_T); e chlorophyll a/b ratio (Chl_a/b); f carotenoid-to-total chlorophyll ratio (Car/Chl_T). Data are presented as mean ± standard error (n = 3). Different lowercase letters indicate significant differences among treatments (p < 0.05). CK, full sunlight; L, low shade; M, medium shade; H, high shade

Effects of light intensity on key photosynthetic enzyme activities and photosynthetic nitrogen- and phosphorus-use efficiencies

The activities of Rubisco and Rubisco activase (RCA), as well as photosynthetic nitrogen-use efficiency (PNUE) and photosynthetic phosphorus-use efficiency (PPUE), were highest under the M treatment and lowest under CK (Fig. 6). Rubisco activity differed significantly among all treatments (p < 0.05). Under the M treatment, it was 21.60%, 12.88%, and 5.27% higher than under CK, L, and H, respectively. Compared with CK and L, RCA activity in M increased significantly by 55.38% and 30.37%, respectively, but did not differ significantly between the M and H treatments. Both PNUE and PPUE were significantly higher under the M treatment than under the other three treatments (p < 0.05). Compared with CK, PNUE and PPUE under the M treatment increased by 88.73% and 166.07%, respectively. Relative to L, the increases were 56.51% and 73.87%; and relative to H, the increases were 35.08% and 55.86%. These results show that medium light conditions effectively enhance the activity of key photosynthetic enzymes and improve photosynthetic nitrogen- and phosphorus-use efficiencies in Z. schneideriana seedlings, thereby promoting rapid growth.

Fig. 6.

Activities of key photosynthetic enzymes and photosynthetic nitrogen- and phosphorus-use efficiencies in leaves of Z. schneideriana seedlings under different light intensity. a Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco); b Rubisco activase (RCA); c Photosynthetic nitrogen-use efficiency (PNUE); d Photosynthetic phosphorus-use efficiency (PPUE). Data are presented as mean ± standard error (n = 3). Different lowercase letters indicate significant differences among treatments (p < 0.05). CK, full sunlight; L, low shade; M, medium shade; H, high shade

Principal component analysis of leaf traits in Z. schneideriana seedlings

Principal component analysis (PCA) performed on 21 leaf traits across the light intensity (Fig. 7) showed that the first two principal components (PC1 and PC2) explained 75.23% and 15.49% of the total variance, respectively, cumulatively accounting for 90.72%. Along PC1 axis, CK and L exhibited positive distribution, whereas M and H exhibited negative distribution. On PC2 axis, M exhibited positive distribution, whereas H exhibited negative distribution. Among the determinants of PC1, leaf dry matter content (LDMC), leaf thickness (LT), epidermal thickness (ET), palisade to spongy tissue (P/S), light compensation point (LCP), dark respiration rate (R_d), chlorophyll a/b ratio (Chl_a/b), and carotenoid-to-total chlorophyll ratio (Car/Chl_T) exhibited positive distribution. In contrast, apparent quantum yield (AQY), chlorophyll a (Chl_a), chlorophyll b (Chl_b), Rubisco and RCA activities, photosynthetic nitrogen-use efficiency (PNUE), and photosynthetic phosphorus-use efficiency (PPUE) exhibited negative distribution. Light saturation point (LSP), light-saturated net photosynthetic rate (A_max), leaf area (LA), specific leaf area (SLA), and palisade tissue thickness (PT) were the determinants of PC2. Based on the vector angles, A_max was positively correlated with LA, PPUE, PNUE, AQY, Chl_a, Chl_b, Rubisco activity, and RCA activity, but negatively correlated with ET and Car/Chl_T. These results indicate that variation in carbon assimilation capacity is not driven by a single leaf trait but is co-regulated by multiple traits related to leaf morphology, anatomy, and photosynthetic physiology.

Fig. 7.

Principal component analysis of leaf traits in Z. schneideriana seedlings under different light intensity. Abbreviations: A_max, light-saturated net photosynthetic rate; LSP, light saturation point; LCP, light compensation point; R_d, dark respiration rate; AQY, apparent quantum yield; LA, leaf area; SLA, specific leaf area; LDMC, Leaf dry matter content; LT, leaf thickness; ET, epidermal thickness; PT, palisade tissue thickness; ST, spongy tissue thickness; P/S, palisade to spongy tissue ratio; Chl_a, chlorophyll a; Chl_b, chlorophyll b; Chl_a/b, chlorophyll a/b ratio; Car/Chl_T, carotenoids-to-total chlorophyll ratio; Rubisco, Ribulose-1,5-bisphosphate carboxylase/oxygenase; RCA, Rubisco activase; PNUE, photosynthetic nitrogen-use efficiency; PPUE, photosynthetic phosphorus-use efficiency

Phenotypic plasticity indices of traits in Z. schneideriana seedlings

Under varying light intensities, the phenotypic plasticity indices (PPI) of the examined traits ranged from 0.091 to 0.805 (Fig. 8). Among photosynthetic physiological traits, PPUE, Car, Chl_a, Chl_T, and Chl_b all showed high phenotypic plasticity, with PPI values exceeding 0.5. In contrast, Rubisco activity exhibited relatively low plasticity. Regarding light-response traits, Aₘₐₓ displayed the highest PPI, whereas R_d exhibited the lowest. Among leaf morphological traits, LA had the highest PPI, followed by PT and the P/S ratio, while ST demonstrated the lowest PPI and minimal variation. Overall, phenotypic plasticity across light intensity ranked in the following order: photosynthetic physiological traits > light response traits > morphological traits. Photosynthetic pigment content, photosynthetic nitrogen-use efficiency (PNUE), and photosynthetic phosphorus-use efficiency (PPUE) were the most responsive to light variation and therefore can serve as key physiological indicators for monitoring light adaptation in Z. schneideriana seedlings.

Fig. 8.

Phenotypic plasticity indices of traits in Z. schneideriana seedlings under different light intensities. Abbreviations: Chl_b, chlorophyll b; Chl_T, total chlorophyll; Chl_a, chlorophyll a; Car, carotenoid; PPUE, photosynthetic phosphorus-use efficiency; PNUE, photosynthetic nitrogen-use efficiency; Car/Chl_T, carotenoids to total chlorophyll ratio; RCA, Rubisco activase; Chl_a/b, chlorophyll a/b ratio; Rubisco, Ribulose-1,5-bisphosphate carboxylase/oxygenase; A_max, light-saturated net photosynthetic rate; AQY, apparent quantum yield; LCP, light compensation point; LSP, light saturation point; R_d, dark respiration rate; LA, leaf area; P/S, palisade to spongy tissue ratio; PT, palisade tissue thickness; ET, epidermal thickness; SLA, specific leaf area; LT, leaf thickness; LDMC, Leaf dry matter content; ST, spongy tissue thickness

Discussion

Growth and leaf morphological responses of Z. schneideriana seedlings to light intensity

Morphological traits are the most intuitive external expression of tree adaptation to environmental changes. Among these, adjustments in seedling height and diameter at root collar are particularly pronounced [50] and serve as key indicators of tree adaptability to varying light conditions. Under low-light stress, most tree species tend to increase height growth at the expense of diameter growth [27, 37]. In contrast, this study shows that both height and diameter at root collar increments in Z. schneideriana seedlings peaked under medium shade. This response may be attributed to the enhanced carbon assimilation capacity observed under medium shade, a pattern also reported for Pinus yunnanensis [18], Acer mono, and Acer pseudosieboldianum [53].

Leaves are highly sensitive and plastic organs that readily adjust to environmental changes during tree growth and development. In heterogeneous light environments, they can adjust their external and internal traits through a range of adaptive pathways [26]. Studies have demonstrated that under low-light conditions, specific leaf area (SLA) tends to increase, leading to thinner leaves with a larger leaf area [14, 16]. This morphological adjustment improves light capture and utilization under resource-limited conditions, thereby facilitating the accumulation of photosynthetic products. In the present study, leaf length, width, and area of Z. schneideriana seedlings were significantly greater under the medium shade treatment than under the other treatments (Fig. 2, S1), indicating that medium shade effectively increases leaf area to enhanced light capture. Under the high shade treatment, SLA increased significantly, while LDMC decreased significantly, reflecting a trade-off between resource acquisition and structural investment in leaves. Under high shade treatment, Z. schneideriana seedlings prioritize rapid light acquisition by reducing investment in leaf structure and constructing greater leaf area with lower dry matter input, thereby alleviating the adverse effects of low-light stress. This strategy is consistent with the resource-acquisitive end of the leaf economics spectrum (LES) [43]. Anatomical changes also reflect adaptive responses to varying light intensities. With decreasing light intensity, leaves of Z. schneideriana seedlings became significantly thinner, showing a pronounced reduction in palisade tissue thickness (PT), looser cell arrangement, enlarged intercellular spaces, a relative increase in spongy tissue thickness (ST), and a marked decline in the palisade to spongy tissue ratio (P/S). By modifying their internal leaf structure, Z. schneideriana seedlings may reduce light quantum loss, thereby improving light capture and utilization efficiency to support growth under low-light conditions. A similar phenomenon was also observed in shaded Emmenopterys henryi [11]. In general, thicker leaf epidermis is associated with greater drought resistance [3]. In this study, epidermal thickness (ET) was significantly greater under full sunlight than under shaded treatments, an adaptation that likely helps alleviate water stress caused by stomatal closure under high-light intensity. Conversely, the thinner epidermis under low-light conditions may reduce the barrier to light penetration, thereby enhancing light capture within the leaf.

Responses of leaf photosynthetic traits in Z. schneideriana seedlings to light intensity

Light-response curves describe the relationship between net photosynthetic rate and light intensity, providing a useful tool for assessing plant photosynthetic capacity [8]. Among the derived parameters, the light-saturated net photosynthetic rate (A_max) serves as a direct indicator of photosynthetic capacity. In this study, A_max of Z. schneideriana seedlings showed a unimodal response to decreasing light intensity, increasing initially before decreasing, a pattern consistent with reports for other tree species, such as Pinus koraiensis [55] and Euterpe edulis [34]. The light saturation point (LSP) and light compensation point (LCP) reflect a plant's ability to utilize light; lower values allow more efficient photosynthesis under low light, thereby promoting organic matter accumulation [44, 53]. Adjustments in LCP are often accompanied by changes in dark respiration rate (R_d), with a lower R_d indicating reduced metabolic maintenance costs [6, 25]. In the present study, LSP, LCP, and R_d all decreased with declining light intensity. These responses suggest that Z. schneideriana seedlings can initiate carbon assimilation at lower light levels while limiting respiratory consumption of photosynthetic products, thereby helping to maintain carbon metabolic balance and support growth under shaded conditions. The apparent quantum yield (AQY) reflects the efficiency of light energy conversion in photosynthesis, with a higher AQY value indicating a greater capacity to utilize low-intensity light [17]. Typically, AQY in plants ranges from 0.03 to 0.05 mol CO₂ mol⁻¹ photons [48]. In this study, the AQY of Z. schneideriana seedlings was relatively low under full sunlight, suggesting photoinhibition under high-light intensity. Conversely, AQY under medium shade was significantly higher than under the other treatments, indicating an enhanced ability to use low light. These results suggest that one- to two-year-old Z. schneideriana seedlings possess a certain degree of shade tolerance. However, studies have shown that the light requirements of trees increase with age [2]. For example, moderate shading benefits the early development of Phoebe chekiangensis seedlings, but as the seedlings grow, the light requirement of saplings older than three years increases, and shading instead inhibits their growth [54]. As trees grow larger, they tend to adopt more conservative resource allocation strategies, investing more resources into non-photosynthetic tissues to enhance competitiveness [29].

Our experiment simulated afforestation under varying light environments while holding other conditions constant, capturing only the growth and physiological responses of Z. schneideriana seedlings during the initial year of planting. Based on the findings, it can be preliminarily recommended that when establishing Z. schneideriana seedlings under a forest canopy, uniform thinning be applied according to the stand’s canopy structure and closure to maintain light intensity at approximately 50% of full sunlight, thereby supporting seedling survival and rapid early growth. However, in actual forest stands, the light adaptability of Z. schneideriana is not only influenced by the interaction between site conditions and light environment but also shifts as the trees age. Therefore, understory afforestation practices using this species require further experimental trials across a range of canopy closure levels to assess how the interaction between site conditions and light environment affects its growth and physiology. In addition, long-term monitoring is essential to track age-related changes in light demand and adaptability, enabling dynamic adjustments of the light environment in stand management to support the robust growth and development of Z. schneideriana at all stages.

Photosynthetic pigments mainly consist of chlorophylls and carotenoids. Chlorophylls are responsible for light absorption, transfer, and conversion, whereas carotenoids perform dual functions of light harvesting and photoprotection, playing crucial roles in photosynthesis [32]. In this study, Aₘₐₓ was significantly correlated with chlorophyll content in Z. schneideriana seedling leaves (Fig. S2), indicating that chlorophyll content can determine the photosynthetic rate in seedlings [4]. As light decreased, Chlorophyll a (Chl_a), chlorophyll b (Chl_b), and total chlorophyll (Chl_T) initially increased and then declined, suggesting that Z. schneideriana seedlings enhance chlorophyll synthesis to capture more light under low-light conditions. However, under high shade, chlorophyll content decreased significantly, an effect that may be attributed to damage to chloroplast structure caused by insufficient light, resulting in a reduction in the number of chloroplasts and grana [37]. This decrease may also be associated with the inhibition of light-dependent protochlorophyllide oxidoreductase (LPOR) activity. LPOR catalyzes the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), a key step in chlorophyll biosynthesis, and reduced LPOR activity under low-light conditions limits chlorophyll synthesis [9]. Additionally, shading generally reduces the proportion of red light absorbed by Chl_a and increases the proportion of blue light absorbed by Chl_b [39]. The present results support this pattern. As light intensity decreased, the increase in Chl_b in Z. schneideriana leaves was proportionally greater than that of Chl_a, leading to a decline in the Chl_a/b ratio. This shift can be regarded as an adaptive response of Z. schneideriana seedlings to low-light conditions. An increase in the carotenoid-to-total chlorophyll ratio (Car/Chl_T) contributes to enhanced photoprotective capacity [7]. In this study, the Car/Chl_T ratio of Z. schneideriana seedlings was significantly higher under full sunlight and low shade than under the other treatments, indicating that under high-light conditions the seedlings increase the relative content of carotenoids to dissipate excess light energy and reduce potential damage to the photosynthetic apparatus, a response consistent with that reported for Camptotheca acuminata [22].

In addition to photosynthetic pigments, the activities of key photosynthetic enzymes—such as Rubisco and RCA—as well as photosynthetic nitrogen-use efficiency (PNUE) and photosynthetic phosphorus-use efficiency (PPUE) are critical determinants of plant photosynthetic efficiency [15, 47]. This view is further supported by the highly significant positive correlations observed between A_max in Z. schneideriana seedlings and Rubisco activity, RCA activity, PNUE, and PPUE (Fig. S2). Rubisco, one of the most abundant enzymes in plant, is a central target for improving plant photosynthetic efficiency and yield potential [15], while RCA regulates the transition between Rubisco's activated and inactive states [1]. In this study, the activities of Rubisco and RCA in Z. schneideriana seedlings showed a unimodal response to decreasing light intensity, peaking under medium light intensity. This pattern suggests that Z. schneideriana seedlings downregulate the activities of both enzymes under either high-light or extremely low-light conditions as an acclimation to light stress, consistent with previous reports [13]. Furthermore, the partitioning ratio of nitrogen between the carboxylation system (Rubisco) and the thylakoid light-harvesting system (chlorophyll) is a key regulatory factor affecting plant photosynthetic efficiency [51]. Previous studies have shown that plants tend to allocate more nitrogen to Rubisco under high light to increase its carboxylation rate, whereas under low light, they prioritize allocating nitrogen to chlorophyll to enhance light capture capacity [45]. However, in the present study, Rubisco activity in Z. schneideriana seedlings under full light treatment was reduced, which may be attributed to decreased nitrogen investment in Rubisco as a protective mechanism for leaves [10, 28]. The trends in PNUE and PPUE were generally consistent with the activities of key photosynthetic enzymes, indicating that under medium light conditions, Z. schneideriana seedlings allocated a higher proportion of nitrogen to Rubisco to enhance photosynthetic efficiency without limiting nitrogen investment in light capture. This resulted in a balanced distribution of nitrogen between carboxylation and light capture, thereby maximizing light use efficiency and carbon assimilation capacity in the seedlings. Sustaining higher photosynthetic efficiency also requires more energy, which may further increase leaf PPUE.

Phenotypic plasticity of Z. schneideriana seedlings under different light intensities

During their lifecycle, plants experience fluctuations in abiotic and biotic factors, such as light, water, nutrients, and natural enemies [40]. In response to these variations, plants exhibit plasticity in morphology, structure, and photosynthetic traits. The phenotypic plasticity index (PPI) serves as a key measure for quantifying the degree of such adaptive responses. The magnitude of PPI reflects the strength of a plant's ability to adjust to environmental change [24]. Under different light intensity treatments, the PPI values for Z. schneideriana seedling leaves followed this order: photosynthetic physiological traits > light response traits > morphological traits. Among photosynthetic physiological traits, the PPI for chlorophyll a and b contents, as well as photosynthetic phosphorus-use efficiency (PPUE), all exceeded 0.59, indicating a high degree of plasticity. Principal component analysis (PCA) further revealed that chlorophyll content and PPUE are the most responsive physiological parameters to changes in the light environment. Under low-light conditions, Z. schneideriana seedlings can rapidly increase chlorophyll content and enhance phosphorus-use efficiency, thereby improving their light capture and absorption. Furthermore, Rubisco activity was a determinant of PC1 in the PCA and exhibited a highly significant positive correlation with A_max (Fig. 7 and Fig. S2). Despite this strong association, Rubisco activity itself demonstrated low plasticity. This pattern may be explained by the high plasticity observed in RCA activity. Z. schneideriana seedlings likely adjust RCA activity to promote the activation of Rubisco, thereby efficiently enhancing carbon assimilation capacity. Among the leaf morphological traits, leaf area, palisade tissue thickness, and the ratio of palisade to spongy tissue displayed high plasticity. In contrast, epidermal thickness and leaf thickness showed relatively low plasticity. These findings are consistent with the adaptive strategies of leaf traits in Z. schneideriana seedlings under varying light intensities, as revealed by PCA. Specifically, under high-light intensity, the seedlings adopt a conservative resource allocation strategy by investing in leaf structural reinforcement to enhance stress resistance. In contrast, under low-light conditions, they produce larger leaf areas and thinner leaves to rapidly capture light resources. Thus, the coordinated rapid adjustment of leaf morphology and photosynthetic traits represents a key strategy for Z. schneideriana seedlings to improve light capture and utilization efficiency, enabling adaptation to variable light environments.

Conclusion

Light intensity significantly affected the growth, leaf morphology, and photosynthetic physiology of Z. schneideriana seedlings during their first year following afforestation. Growth was optimal under medium light conditions (51% PAR), while both high light and high shade inhibited seedling development. Under medium light conditions, seedlings displayed maximal leaf area, elevated chlorophyll content, higher activities of key photosynthetic enzymes, and improved photosynthetic nitrogen- and phosphorus-use efficiencies. These traits collectively supported higher light-use efficiency and carbon assimilation capacity. Therefore, for the understory establishment of one-year-old Z. schneideriana seedlings in forest structure adjustment and national reserve forest projects, it is recommended that understory light intensity be maintained at approximately 50% of full sunlight through selective thinning to promote rapid early growth. Nevertheless, the long-term optimal light environment for Z. schneideriana afforestation requires further study.

Abbreviations

Z. schneideriana

Zelkova schneideriana

PAR

Photosynthetically active radiation

CK

Full sunlight

L

Low shade

M

Medium shade

H

High shade

LL

Leaf length

LW

Leaf width

LA

Leaf area

LDM

Leaf dry mass

SLA

Specific leaf area

LDMC

Leaf dry matter content

LT

Leaf thickness

UET

Upper epidermal thickness

LET

Lower epidermal thickness

ET

Epidermal thickness

PT

Palisade tissue thickness

ST

Spongy tissue thickness

P/S

Palisade to spongy tissue ratio

P_n

Net photosynthetic rate

A_max

Light-saturated net photosynthetic rate

LSP

Light saturation point

LCP

Light compensation point

R_d

Dark respiration rate

AQY

Apparent quantum yield

Chl_a

Chlorophyll a

Chl_b

Chlorophyll b

Chl_T

Total chlorophyll

Car

Carotenoids

Chl_a/b

Chlorophyll a/b ratio

Car/Chl_T

Carotenoids-to-total chlorophyll ratio

Rubisco

Ribulose-1,5-bisphosphate carboxylase/oxygenase

RCA

Rubisco activase

PNUE

Photosynthetic nitrogen-use efficiency

PPUE

Photosynthetic phosphorus-use efficiency

PPI

Phenotypic plasticity indices

Authors’ contributions

Conceptualization, FY.J., SC.S., XL.W.; Methodology, FY.J., SC.S., XL.W.; Formal analysis and investigation, FY.J., XL.W.; Writing-original draft preparation, FY.J., XL.W.; Writing—review and editing, FY.J., SC.S., XL.W.; Funding acquisition, XL.W.; Resources, XL.W. All authors reviewed the manuscript.

Funding

This research was supported by Science and Technology Innovation Talent Team Building Project for Seedling Breeding and Plantation Cultivation of Precious Tree Species in Guizhou Province (CXTD [2023] 006); National Natural Science Foundation of China (grant No. 31870613), and Guizhou Province High-level Innovative Talents Training Plan Project [2016] 5661.

Data availability

Data will be made available on request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.