Light is the material basis with which all plants in nature carry out various life-sustaining activities (Kami et al., 2010). Light is a primary determinant of the geographical distribution and growth of plants through light intensity, light quality, photoperiod and other factors (Jason et al., 2018). As one of these important factors, light intensity has an important impact on plant growth, morphology, physiological metabolism, signal transduction and photosynthetic physiology (Fan et al., 2013; Lu et al., 2018). The relationship between plants and the light environment has always been a hot topic in ecology.

When the light intensity in the environment changes, the growth state of plants will inevitably change to a certain extent, and this change is mainly caused by the influence of the active light capture ability of the plant and the ability to synthesize photosynthates (Chen et al., 2014). Studies have found that in a weak light environment, the leaf area, total biomass, specific leaf area, and leaf area ratio of plants will increase, thus making leaves larger and thinner, increasing the area of light interception, improving the utilization efficiency of limited light resources, increasing stomatal conductance, and maintaining a high photosynthetic rate to ensure normal plant functions in low-light environments (Guo et al., 2019; Pan et al., 2020). Moreover, the absorption and utilization of light energy can be enhanced in plants growing in low-light conditions through increases in chlorophyll content, apparent quantum efficiency, actual photochemical quantum yield of PSII (Φ_PSII) and electron transfer rate (ETR) (Guenni et al., 2018; Shi et al., 2018) and reductions in energy waste by reducing the light compensation point, dark respiration rate, photorespiration rate and heat dissipation ratio (Cai et al., 2017; Ghorbanzadeh et al., 2020). However, in a strong light environment, plants can reduce the capture of excess light and the damage caused to plants by increasing leaf thickness, reducing leaf area, reducing chlorophyll content, and reducing the transpiration of leaf water by closing stomata (Mokany et al., 2006).

Through the long evolutionary process, plants have formed a series of protective mechanisms that enable them to adapt to the complex and dynamic light environment. Studies have found that when plants grow in an unsuitable light environment, osmotic regulatory substances such as soluble sugar (SS), soluble protein (SP) and proline (Pro) can maintain the balance of cell osmotic potential and reduce the damage to the cell membrane caused by osmotic stress through changes in content (Yin and Shen, 2016). Antioxidant systems also play an important role in plants coping with unfavorable light conditions, resulting in the accumulation of intracellular reactive oxygen species (ROS) when plants are exposed to environmental stress; thus, the activities of protective enzymes such as superoxide dismutase (SOD), catalase (POD) and peroxygenase (CAT) and the content of nonenzymatic antioxidants increase (Liu Y. et al., 2018; Zushi et al., 2020), and the contents of active oxygen species such as O^·-2, H₂O₂, OH^- and malondialdehyde (MDA) decrease. These changes can reduce peroxidation damage to the cell membrane and improve the adaptability of plants to unfavorable light environments (Ellouzi et al., 2011). Plants tolerate shade stress in a variety of ways (Israeli et al., 1995; Koike, 2010; Modrzyński et al., 2015). Some studies have found that moderate shade can benefit plants not only by preventing them from being burned by light but also by allowing them to meet their light requirements for growth (Petritan et al., 2007). Therefore, it is important to study how plants tolerate shade stress to more effectively establish stable, diverse and stratified planting structures when constructing landscape gardens.

Carpinus betulus L. is a deciduous tree belonging to the Betulaceae family with beautiful foliage and golden leaves in autumn. It plays an important role in forest communities in temperate regions of Europe and Asia Minor (Sikkema et al., 2016) and has been introduced and cultivated in many places in China with good ecological adaptability (Shi et al., 2017). In landscapes, C. betulus is often planted in the form of street trees or hedgerows and is well-known in private and public green areas for its autumn leaves. It is a suitable species for both ornamental and urban habitats and has been used for biological monitoring of particulate matter (Prinz and Finkeldey, 2015). Additional studies have shown that the antioxidant- and anticancer-related properties of C. betulus leaf extracts are exceptional (Cieckiewicz et al., 2012; Hofmann et al., 2016) and that compared to other common coniferous and deciduous trees, these extracts contain very high amounts of total phenols and phenolic acids (Kuiters and Sarink, 1986). All these results indicate that C. betulus leaves are a promising renewable resource for the biorefinery and utilization of its antioxidant extractives in the future for healthcare and medical products. The high ornamental and economic value of C. betulus makes it extremely popular globally. In recent years, C. betulus has been widely used in China, and its adaptability is good. Recent studies on C. betulus have mostly concentrated on breeding (Chalupa, 1990), seed biology (Zhu et al., 2013), heat resistance (Shi and Zhu, 2015), drought and salt tolerance (Stojnić et al., 2016; Zhou et al., 2018). At present, with the rapid development of urban construction in China, the urban shade space is becoming larger, air pollution and smog get more severe in big cities (Xue et al., 2022). It is of great significance to select the plant resources with high ornamental value and shade tolerance to carry out scientific and effective greening of the shade space and give play to the best ecological environmental benefits of green space. Light has a great influence on the growth of C. betulus. However, information on the response of C. betulus to different light intensities is scarce.

Therefore, the main objective of this study is to comprehensively assess the influence of shade stress on C. betulus and to determine the mechanisms of adaptation employed by this plant to tolerate low-light stress. We hypothesized that the growth, biomass accumulation and leaf gas exchange rate of C. betulus seedlings would decrease under the effects of light stress. We examined the growth and physicochemical changes in seedlings grown under different degrees of shading created through artificial shading treatment, explored the adaptability of C. betulus seedlings to different light conditions, and sought to determine the most suitable light conditions to promote the growth and development of C. betulus seedlings. The aim of this study was to provide scientific data and findings related to the light management of C. betulus and to make better use of Carpinus species in landscaping and industrial production.

Light intensity is an important environmental factor that is closely related to plant growth and development. When the external light environment changes, the growth, biomass and material distribution of plants will be affected to a certain extent. Under normal circumstances, the biomass and relative growth rate of plants will become lower in low-light environments (Pik et al., 2020). To adapt to shaded environments, plants usually undergo growth and morphological adjustments, a process called morphological plasticity, to adapt to the complex light environment. Some studies have shown that when plants are exposed to shade stress, they tend to have a decreasing base diameter and increasing plant height; that is, they show the characteristics of being ‘slender’. This may be because to obtain the maximum amount of light, the plant reduces the carbon used for basal diameter growth and allocates more assimilated carbon to vertical plant growth to capture more light energy (Liu C. et al., 2018). It was found in this study that with the decrease in light intensity, the plant height growth increased, but the ground diameter increment, taproot length, root, stem, leaf and total biomass decreased continuously. These results indicated that shading had a great effect on the growth of C. betulus, and the lower the light intensity was, the more restricted the growth. Biomass allocation patterns and their changes in aboveground and belowground parts are important representations of tree adaptation strategies to the environment (Guenni et al., 2018). Usually, when restricted by light conditions, trees allocate more resources to aboveground plant parts (Xue et al., 2022). By reducing the allocation of resources to root biomass and increasing the allocation of resources to leaf and stem biomass, trees will obtain more light resources, which is conducive to improving their ability to compete for light and therefore survive (Ma et al., 2014). The study of Toledo-Aceves and Swaine (2007) showed that with the weakening of light intensity, the allocation ratio of the root biomass of pioneer tree species in tropical rainforests decreased, while the allocation ratio of aboveground biomass increased. In this study, we found that the root:shoot ratio of C. betulus decreased with decreasing light intensity, which supported the above view. This finding indicated that the biomass allocation of the underground plant parts gradually became lower than that of the aboveground parts with the weakening of light to improve the ability of the plant to capture light and maintain plant growth.

Since leaves are the largest organ by which plants interact with the external environment, their morphological structure is very sensitive to changes in the ecological environment; therefore, changes in morphological and physiological characteristics can best reflect the influence of the light environment on plants and the ability of plants to adapt to the light environment (Fan et al., 2013; Yao et al., 2017). Studies have shown that most plants in a weak light environment can change blade shape, such as by increasing leaf length, leaf width, leaf area, leaf number and plant height to adapt to an environment with insufficient light intensity; in bright light, plants will adapt by increasing blade thickness, secreting a waxy layer and increasing leaf area to cope with strong light inhibition (Mokany et al., 2006; Klavins and Klavins, 2020). This study found that the leaf area and SLA of C. betulus increased with decreasing light intensity. This result showed that when the outside light intensity weakened, in order to capture more limited energy, plant height increased and the leaf area enlarged, thus reducing the consumption of plant nutrient substances by increasing the SLA, reducing the leaf thickness within a limited scope, and expanding the leaf area. These changes allowed the plant to more efficiently capture light energy and lower the compensation to the decline in the photosynthetic rate of the photon flux density phenomenon, thus enabling survival in the weak light environment, which is consistent with the view of Moreira et al. (2009). Leaves are the main site of photosynthesis, and changes in the anatomical characteristics of leaves will inevitably affect the photosynthetic efficiency of plants (Jumrani and Bhatia, 2020; Knauer et al., 2020). It was found that the thinness of the upper and lower epidermis and palisade tissues, the presence of palisade tissue cells with a low aspect:axis ratio, and the loose arrangement of the spongy tissue cells could improve light transmission and chloroplast light capture ability (Barbosa et al., 2021). This conclusion was further confirmed by the anatomical observation of leaves of C. betulus under different light intensities; that is, with the decrease in light intensity, the leaf thickness and presence of palisade tissue and spongy tissue of C. betulus decreased significantly, and the arrangement of cells in the spongy tissue gradually loosened, gradually increasing the gap between cells. This finding suggested that plants can adapt to different light environments by adjusting their leaf structures. The plasticity of plant leaf anatomical characteristics in response to changes in the light environment has been widely studied and has been confirmed in Corylus avellana (Catoni et al., 2015), Sesleria nitida (Puglielli et al., 2015) and other plants, and the patterns are basically consistent across species.

Photosynthesis by plants is a physiological process that is sensitive to environmental factors, and light intensity is one of the main factors affecting photosynthesis. Chlorophyll content, chlorophyll fluorescence parameters and photosynthetic parameters are important indexes for measuring the strength of plant photosynthesis (Dai et al., 2009; Zhang et al., 2017). Although different plants have different characteristics in response to changes in the light environment, under adversity and stress, plants will develop in the direction that is conducive to photosynthesis (Jutamanee and Rungwattana, 2017). In general, the decrease in the photosynthetic rate among plants under stress is mainly caused by stomatal limitation and nonstomatal limitation (Rezai et al., 2018). Stomatal limitation is due to the decrease in stomatal conductance, and CO₂ from the external environment is restricted from entering the plant through the leaf stomata, which leads to a decrease in the photosynthetic rate. However, the reduction in the photosynthetic rate caused by nonstomatal limitation is mainly caused by the reduction in photosynthesis-related enzyme activities and the damage to photosynthetic organs caused by stress, and intercellular carbon dioxide may still be maintained at a high level at this time. Ci is an important basis for judging whether stomatal factors cause changes in the photosynthetic rate (Takeda and Maruta, 2008). Ci is positively correlated with Pn, Tr and Gs, indicating that the decline in photosynthesis is caused by stomatal factors hindering CO₂ utilization; otherwise, it is caused by nonstomatal factors. The results of this study showed that with the decrease in shading, the trend of Pn in C. betulus was basically consistent with that of Gs and Tr, which showed overall decreasing trends, and the lowest value was reached at 25% light intensity. However, the content of Ci was negatively correlated with the Pn, Tr and Gs, which indicated that photosynthesis in C. betulus was affected by nonstomatal factors, and the decrease in the leaf photosynthetic rate was caused by the restriction of Rubp carboxylation and photochemistry. A similar phenomenon has been observed in Asarum forbesii (Lu et al., 2022) and Emmenopterys henryi (Li et al., 2020). This further indicated that when the light intensity was too low, the plants had a light deficit and could not obtain the necessary light energy for life-sustaining activities, resulting in the lowest photosynthetic rate.

Chlorophyll fluorescence, part of the photosynthetic process, mainly reflects the efficiency of light absorption, transmission and utilization by plants and is commonly used to characterize the level of photosynthetic efficiency of plants (Genty et al., 1989; Pashkovskiy et al., 2018). Fv/Fm is an indicator of the photoconversion efficiency of PSII, while Fv/Fo indicates the potential activity of the PSII photoreaction center. The higher the Fv/Fm and Fv/Fo are, the higher the photoconversion efficiency of PSII (Zhang, 1999). Baker (2013) found that the Fv/Fm of plants is generally 0.75~0.85 under normal conditions. In this experiment, the Fv/Fm and Fv/Fo in the treatment groups were lower than those in the control group with the increase of shading degree (P < 0.05), indicating that the open proportion of the PSII reaction center in C. betulus under the shading treatment decreased and the light energy utilization efficiency was reduced. NPQ reflects nonphotochemically dissipated energy, which is one of the photoprotective mechanisms of plants (Heraud and Beardall, 2000). In this experiment, Φ_PSII, qP, ETR and NPQ of C. betulus leaves increased first and then decreased with increasing shading degree, and under the severe shading treatment (T3), Φ_PSII, qP, ETR and NPQ were significantly lower than those under the other shading treatment groups, indicating that the photosynthetic apparatus of C. betulus leaves may be partially inactivated or damaged under severe shading conditions. The primary photochemical activity of PSII decreased, the photosynthetic electron transport rate was inhibited, the proportion of heat energy dissipation in the absorbed light energy increased, and the utilization efficiency of absorbed light energy of PSII decreased, resulting in a decrease in the photosynthetic rate. These results indicated that C. betulus had a certain adaptability to low light, but this ability was limited. Light intensity that is too low reduces the photosynthetic efficiency of plants and affects the normal growth of plants.

Chlorophyll has the function of absorbing and transferring light photons and is an important component in the maintenance of normal photosynthesis of plants. Its content is closely related to the site conditions of plant growth and the characteristics of plants (Agathokleous et al., 2020). Chl a and Chl b are the main photosynthetic pigments, and carotenoids can absorb light energy and transfer it to chlorophyll to assist plants in photosynthesis (Liu et al., 2014). The content and proportion of these pigments are important indicators for plants to cope with environmental stress (Chai et al., 2018). In this study, the contents of Chl a, Chl b, Chl (a+b) and carotenoids in C. betulus increased gradually compared with the CK with decreasing light intensity, which indicated that low light intensity may beneficial to the synthesis of chlorophyll and carotenoid, and made the treatment groups show greener leaf color than the control ( Figure 1 ). C. betulus seedlings could improve the efficiency with which it captured light radiation by increasing the chlorophyll content in low-light environments, thus adapting to different low-light environments. The chlorophyll a/b value is one of the important indexes to measure the shade tolerance of plants, and plants can adapt to shading by reducing chlorophyll a/b (Dai et al., 2009). Some studies have suggested that the chlorophyll a/b value of shade plants is below 3.0 (Lichtenthaler et al., 1981; Hoflacher and Bauer, 1982). The Chl a/b of C. betulus was positively correlated with light intensity, decreasing with the decrease in light intensity, and the Chl a/b values were all higher than 3.0, indicating that C. betulus is a typical plant with high requirements for light intensity in the growing environment.

Osmotic regulatory substances play an important role in plant resistance to abiotic stress. When reactive oxygen species are produced rather than antioxidants, the cytoplasmic membrane can perform substantial membrane lipid peroxidation; at this time, the permeability of the plant cell membrane will increase, leading to cytoplasmic extravasation and physiological and metabolic plant disorders (Ramanjulu and Bartels, 2002). Then, the plants will reduce the cell water loss caused by stress by adjusting the contents of osmotic regulatory substances such as SS, SP and Pro to maintain the balance of cell osmotic pressure and thus reduce the degree of damage caused by stress (Hamouda et al., 2015). SS and SP are not only important nutrients but also participate in the osmoregulation process of plant cells, and SS can also remove ROS in vacuoles and chloroplasts and indirectly activate the antioxidant enzyme system (Van and Valluru, 2009). Pro has been shown to be an effective free radical scavenger that plays an important role in stabilizing protoplasmic colloids and cell metabolic processes and maintaining osmoregulatory balance (Mutava et al., 2015). This study found that the contents of SS and SP in C. betulus leaves first increased and then decreased with decreasing light intensity, suggesting that under mild shade stress, C. betulus can remove ROS from vacuoles and chloroplasts by increasing SS and SP contents and maintain turgor pressure by osmotic regulation through the synergistic effect of carbon and nitrogen metabolism pathways. However, under moderate and severe shading stress, the SS and SP contents gradually decreased to their lowest levels in the T3 treatment, which may be due to the lack of light. The rate of photosynthesis in the leaves decreased sharply, and the synthesis of SS and SP was blocked; these effects will lead to a shortage of nutrients in plants, causing adverse effects on growth. The Pro content in C. betulus increased under mild and moderate levels of shade (i.e., the T1 and T2 treatments) but decreased significantly under heavy shade conditions (the T3 treatment), suggesting that the Pro content of C. betulus can still be maintained at a relatively high content under appropriate shading conditions, scavenging free radicals in plant cells and counteracting the effect of light intensity factors on cell osmoregulation, while its synthesis is blocked in extremely low light, thus limiting the effect of osmoregulation.

ROS are one-electron reduction products of a class of oxygen, which are important signal transduction substances in plants (Kaushik and Aryadeep, 2014). A study found that when plants suffer adversity stress, on the one hand, ROS material will stimulate plants to produce a series of physiological and biochemical reactions and resistance to environmental stresses of the outside world; on the other hand, when the generation and removal of ROS in plant cells are in an unbalanced state, membrane lipid peroxidation will occur in the cell membrane, and protein oxidation will cause damage to the cell membrane, resulting in the imbalance of normal physiological metabolism of plants and even leading to the death of plants (Pareek et al., 2017). MDA is the final product of membrane lipid peroxidation and one of the important signs of membrane system damage (Sofo et al., 2004). This study found that the MDA content and relative conductivity of C. betulus leaves increased with decreasing light intensity, which was the lowest under natural light intensity and the highest under the T3 condition. These results indicated that shading caused low-light stress in C. betulus, which led to damage to membrane function or membrane structure, an increase in membrane permeability, and serious membrane lipid peroxidation, and the same phenomenon was also found in Quercus wutaishanica (Zhang et al., 2021). SOD, POD and CAT are important antioxidant enzymes (Zhao et al., 2019). When plants are under environmental stress, ROS can induce their activity to increase and remove the accumulated O^·-2 and H₂O₂ in the cells, thus playing a protective role for plant cells (Koca et al., 2006). SOD first converts O^·-2 into H₂O₂ and O₂ and then dissociates the generated H₂O₂ and endogenous H₂O₂ to stimulate POD and CAT activities, and the three synergistic processes eliminate excessive reactive oxygen species in plants (Neto et al., 2006). Studies have found that shading, drought, salinity and other stresses can induce the activities of SOD, POD, CAT and other antioxidant enzymes and increase the ability of plants to remove reactive oxygen species, but this process is species-specific and varies with different stress types (Favaretto et al., 2011; Bharuth et al., 2020; Liu et al., 2022). This study found that the activities of SOD, POD, CAT and APX in leaves of C. betulus first increased and then decreased with increasing shading intensity, but the activities of protective enzymes were higher than those of the control under different shading conditions. This indicated that under a low-light environment, C. betulus can regulate the activity of protective enzymes in the body to remove the O^·-2 and H₂O₂ produced by shading stress. However, under severe shading conditions, the metabolic balance in plants was disrupted, and the enzyme activity and free radical scavenging function were irreversibly decreased, resulting in severe membrane lipid peroxidation in C. betulus. With the decrease in light intensity, the protective enzyme activities of C. betulus showed the same trend, indicating that there was an obvious synergistic effect of antioxidant enzymes in vivo.

Auxin (IAA), gibberellin (GA₃) and abscisic acid (ABA) are important endogenous hormones in plants. Studies have shown that light is not only an important environmental factor for plant photosynthesis but can also regulate plant growth and development through endogenous hormones by using genes related to photoreceptor signaling (Jiang et al., 2021). Cui et al. (2014) found that shading decreased the contents of IAA and GA₃ and increased the contents of ABA in the leaves of summer maize. In this study, the content of IAA decreased with decreasing light intensity, but the contents of GA₃ and ABA showed the opposite trend, which differed from the above research results. ABA mainly inhibits cell division and elongation and inhibits the growth of roots, stems, leaves and other organs (Khorshidi and Nojavan, 2006). The ABA content in leaves of C. betulus increased under shaded conditions, which strengthened the inhibitory effect of shading on leaf growth. GA₃ can promote leaf growth and maintain leaf function, and an increase in GA₃ content can promote the growth of leaves (Ulger et al., 2004). GA₃ would also promote the seedling height, the increase of plant height increment in low light conditions may be due to the increase of GA₃ content. What’s more, studies have also shown that GA₃ in leaves can inhibit chlorophyll decomposition, and the two have a synergistic effect (Li et al., 2010), that may be the reason of why chlorophyll content in C. betulus increased as the degree of shading increased. Therefore, on the one hand, low light intensity increased GA₃ and maintained leaf development. On the other hand, reducing the IAA content and increasing the ABA content resulted in leaf thinning and reduced dry matter accumulation. Therefore, the synergistic effect of different endogenous hormones promoted the elongation and broadening of leaves and increased the leaf area of C. betulus under low light conditions.

Shade tolerance is determined by genetic characteristics of plants and many biological and abiotic factors. In this study, the morphological, physiological and ecological response of C. betulus seedlings under different light intensities were studied ( Figure 8 ). In the future, further studies can be conducted from the perspective of proteins and molecules levels to comprehensively analyze the mechanism of shade tolerance in plants.

This study presents new information on the growth and ecophysiological responses of C. betulus seedlings to different light intensities. C. betulus was affected by shading stress through the inhibition of plant biomass accumulation, photosynthetic capacity and stomatal behavior, and increas ing MDA content and relative electrolytic conductivity, suggesting a limited degree of shade tolerance. Our results found that C. betulus seedlings can activate relevant mechanisms to adapt to low light stress, such as adjusting growth and leaf morphology, accumulating relatively high concentrations of organic solutes to maintain cellular osmotic balance and improving the activity of antioxidant enzymes as a defense against shading stress. A reduction in the growth of C. betulus seedlings was observed under severe shading conditions (T3). C. betulus seedlings can adapt to sunlight, grow best in natural light, and can tolerate certain degrees of shading, which is capable of growing under shading with 75% or 50% light transmittance. However, under heavy shading (25% light transmittance), C. betulus develops insufficient resistance to low-light environments and has a difficult time growing. Our study provides a better understanding of the responses of C. betulus to low-light stress at the physiological and biochemical levels. This ornamental tree species has the potential to be cultivated in the middle and upper layers of landscape gardens in China.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

QZ and ZZ conceived and designed the experiments; QZ, FZ, and HZ performed the experiments and analyzed the data; and QZ wrote the paper. All authors read and approved the final paper.

This research was funded by the National Natural Science Foundation of China (31770752), the Jiangsu Science and Technology Support Program (BY2015006–01), the 333 Projects of Jiangsu Province (BRA2018065), and the Natural Science Foundation for Universities of Jiangsu Province (20KJB220006) and is a project of the 13th Five-Year Plan of Jiangsu Open University (2020-D-01).

We would like to thank Jing Yang, a laboratory specialist at the Advanced Analysis Testing Center (AATC), Nanjing Forestry University, China, for assistance with the observations of the microstructures of the plants. We express our gratitude to Feibing Wang, a research assistant at the College of Landscape Architecture, Nanjing Forestry University, China, for her help cultivating the seedlings.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer YX declared a shared affiliation with the author HZ, ZZ to the handling editor at the time of review.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.