Cyclocarya paliurus (Batal) Iljinskaja is a multiple function tree species that belongs to the Juglandaceae family and is mainly distributed in the sub-tropical highlands of China (Fang et al., 2006). Leaves of this species have been widely used as tea production and ingredient of food industry for a long time in China (Fang et al., 2011; Xie et al., 2015). Diverse biological activities, including antioxidant, antidiabetic and antimicrobial activities, have been found in the extracts of the leaves, which are attributed to the abundant phytochemicals such as flavonoids, triterpenoids, polyphenolics, and polysaccharides (Kurihara et al., 2003; Xie et al., 2015; Wu et al., 2017; Liu et al., 2018a,b). Because of these beneficial effects on the human health, an increased demand for the production of C. paliurus leaves is now required for medical and commercial uses. Thus, large-scale leaf-harvesting plantations of this species have been established in recent years, and cultivation technologies including optimizing soil and light environment have been carried out (Deng B. et al., 2012; Liu et al., 2016).

Light is considered the most direct environmental factor affecting plant growth and development (Ma et al., 2015; Yamori, 2016). Specifically, changes in light wavelengths due to the different properties of light-harvesting pigments can directly affect plant morphological, anatomical, biochemical, and physiological parameters (Haliapas et al., 2008; Fan et al., 2013). Red light has been shown to control the development of photosynthetic apparatus and up-regulate the integrity of starch in some species (Sæbø et al., 1995; Shimizu et al., 2011). Blue light is reported to control the integrity of chloroplast proteins, which are important for chloroplast development and plant photosynthesis (Hogewoning et al., 2010). Meanwhile, distinct effects of green light on leaf hyponasty, stem elongation, apical dominance, leaf expansion, and photosynthesis have been reported (Schmitt and Wulff, 1993; Wang and Folta, 2013). Previous studies also reported that responses of plants to differential light quality are species specific (Hogewoning et al., 2010; Nanya et al., 2012; Yu et al., 2017). For example, Ouyang et al. (2003) reported that blue light resulted in significantly higher biomass accumulation of Cistanche deserticola than red light. However, Chen et al. (2014) showed that fresh weight and dry weight of lettuce were lowest under blue light when compared with other light treatments. Yu et al. (2017) also reported that red light improved Camptotheca acuminata growth by reducing the accumulation of reactive oxygen species (ROS) and activating photosynthetic processes. Additionally, Johkan et al. (2012) reported that short-wavelength green LED light with high intensity was effective to active leaf photosynthetic rate and plant growth of Lactuca sativa.

Plants have developed several useful photoprotective mechanisms to cope with the diverse light stresses such as excess UV light and other non-beneficial light wavelengths (Yamori, 2016). The first photoprotective mechanism is known as avoiding exposure to light through some simple methods like leaf movement, changing of leaf area (LA), and altering chloroplast positions (Corlett et al., 1994; Suetsugu and Wada, 2007). Photoprotective mechanisms involved in the photosynthetic electron transport have also been proposed, including (1) thermal energy dissipation to protect PSII [also called non-photochemical quenching (NPQ)], (2) plastoquinol terminal oxidase (PTOX) as an alternative electron transport around PSI, and (3) ROS scavenging mechanisms through non-enzymatic antioxidants (ascorbate, carotenoids, and tocopherol) and enzymatic antioxidants [including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)] (Baena-Gonzalez and Aro, 2002; Holt et al., 2004; Takahashi and Badger, 2011). Among these protective systems, NPQ mechanism and ROS scavenging abilities are often measured to identify the adaptation level of plants under light stress, as they can effectively reduce the damage caused by excessive light energy (Yamori, 2016; Yu et al., 2017). It should be noted that some plants also produce other macromolecular compounds like phenolics or flavonoids to scavenge ROS caused by light stress, especially when UV or short-wavelength green and blue lights increased (Cruces et al., 2017; Liu et al., 2018c).

Nowadays, some spectrum LEDs or films have been used in indoor plant pre-cultivation (or in greenhouse) at seedling developmental stage to promote the growth traits and the accumulation of bioactive compounds in economic or medicinal trees, such as Quercus ithaburensis and Anoectochilus roxburghii (Smirnakou et al., 2017; Ye et al., 2017). Previous studies have reported the effects of differential light quality and light intensity on the development of C. paliurus, such as biomass and polysaccharide accumulation (Yang et al., 2017). Moreover, it was demonstrated that blue light promoted the production of functional flavonoids (kaempferol, isoquercitrin, and quercetin) and related gene expression in C. paliurus seedlings (Liu et al., 2018c). However, little information is known about how varying light quality affects the photosynthetic process and the photoprotective mechanisms in C. paliurus seedlings. We hypothesized that the difference in photosynthetic efficiency of plants under differential light quality was related to the degree of photoinhibition and the activation of photoprotection, which occur at both morphology and photochemical levels including anatomical changes, the regulation of photochemistry activity and photosynthetic pigments, as well as antioxidant balance including enzymes systems and non-enzymes systems. Thus, the objective of this study was to investigate the response mechanisms of growth and photosynthesis of C. paliurus to varying light qualities (white, blue, green and red LED lights) through measuring biomass accumulation, morphology changes, photosynthetic capacity, stomata ultrastructure, pigments content, chlorophyll fluorescence parameters, ROS production, antioxidant enzymes, and phenolic content. Findings from this study will give insights into the response mechanisms of plant photosynthesis to differential light quality and provide scientific basis for cultivation of this plant species with supplementary artificial light, especially at seedling developmental stage.

Light quality is a critical factor regulating photosynthetic capacity which directly affects the final yield and quality of plants. However, the responses to differential light quality are often species specific (Haliapas et al., 2008; Fan et al., 2013; Yu et al., 2017). Plants have evolved a system of sensory photoreceptors to accept changes of light signals in the environment (Gyula et al., 2003). The perception and regulation of these signals are controlled by a network of photoreceptors, such as cryptochromes (blue/UV-A light receptors, 340–520 nm), phytochromes (red/far-red receptors, 680–735 nm), and phototropins (phot1 and phot2) (Lin, 2000). Thus, when plants are grown under light with single wavelength band, some of the photoreceptors responsible for plant growth and morphogenesis will not receive signal, and this could lead to violations in plant development. In the present study, GL and RL significantly inhibited plant growth of C. paliurus, whereas positive effects were observed from WL and BL. Results of this study support our hypotheses that the difference in photosynthetic efficiency of C. paliurus plants under differential light quality is related to the degree of photoinhibition and the activation of photoprotection, which occur at both morphology and photochemical levels. And these photoprotective mechanisms are mainly related to better morphogenetic occurrences like leaf growth and stomata development, effective regulation of photosynthetic pigments and photochemistry activity, as well as reduced ROS accumulation.

Light quality affects a range of plant characteristics, such as seedling height, biomass accumulation, leaf size and anatomical structure (Yu et al., 2017). Leaf movement, such as leaf rolling, is an efficient strategy to protect leaves from photodamage (Corlett et al., 1994). However, leaf movement wasn’t observed in C. paliurus plants under differential light quality in this study. Compared with GL and RL, C. paliurus plants under WL and BL performed much better with higher values of total biomass, SLM, seeding height increment (SHI), leaf thickness and palisade length (Tables 1, 2 and Figure 2). Considering the white LED used containing blue light (445nm), we can conclude that blue light is more important for C. paliurus growth at seedling developmental stage than other lights. This result was inconsistent with those reported in other plants like strawberry, Cistanche deserticola and Anoectochilus roxburghii (Ouyang et al., 2003; Miranda and Williams, 2007; Ye et al., 2017). Some previous studies have revealed that a higher biomass allocation to the root system and a higher value of root/shoot ratio can increase the ability of plants to survive under stress conditions (Aranda et al., 2001; Tsakaldimi et al., 2005; Luis et al., 2010). Thus, the significantly higher values of root/shoot ratio under BL, GL, and RL than that under WL indicated that these lights caused varying degrees of stress on C. paliurus seedlings, among which the highest was observed under GL (Table 1). Compared with WL and BL, RL used in this study had a significantly inhibitory effect on plant height, LA, and biomass of C. paliurus seedlings, which was inconsistent with that of cucumber (Su et al., 2014). However, RL treatment caused a significant increase in GDI of C. paliurus than other lights used (Table 2). This result was different from what is reported on Sinapis alba, Pelargonium and Camptotheca acuminata, of which RL resulted in a higher allocation to plant shoot growth (Cosgrove, 1982; Appelgren, 1991; Yu et al., 2017). These contrast findings confirm that the responses of plant morphological characteristics to changes of light quality are species specific.

Stomata regulate water vapor diffusion and carbon dioxide uptake in plants. Stomatal density, distribution and opening thus affect plant photosynthesis, which are often influenced by many factors including light, temperature, and CO₂ (Ye et al., 2017). Blue light has been reported to cause the voltage-dependent plasma membrane K⁺ channels to open, which then enhances water and K⁺ flow into the guard cells, and finally forces the stomata to open (Schroeder et al., 2001; Shimazaki et al., 2007). In the current study, values of leaf stomatal length, width and opening were observed largest under BL (Table 3 and Figure 3), which were similar to those reported in grape, Tagetes erecta, and Anoectochilus roxburghii (Heo et al., 2002; Poudel et al., 2008; Ye et al., 2017). The decreases in Pn associated with the reduced Tr, Gs, and Ci under RL and GL indicated that stomatal limitation occurred (Figure 4), which was inconsistent with the lower values observed in stomatal length, width, single stomatal pore area, and number of stoma per unit LA (Table 3 and Figure 3). The lower stomatal opening and number per unit LA under RL and GL might help plants to reduce the burden of the PSII reaction center and protect tissues from light stress (Lawson et al., 2011). The percentage absorption of GL (70–80%) by plant leaves has been reported not much smaller than that of BL and RL (90%), GL is absorbed very weakly by pigments and chlorophylls but it can reach the deeper layers of the leaf (Terashima et al., 2009). Thus, it is possible that the excess energy (when PPFD kept the same at 850 μmol m^-2 s^-1) that can’t be utilized by photochemistry then causes photoinhibition. A decrease in growth with an increase in the proportion of GL has also been demonstrated by Kim et al. (2004a,b). The highest ratio of palisade to spongy in GL treatment (Table 2 and Figure 2) also suggested that plants under green light changed their leaf anatomy to adapt to the light stress especially when stomatal closure happened and Ci became limited (Figures 3, 4). However, we also noticed that although plants under RL showed much higher Gs than under GL, the net rate of CO₂ assimilation did not differ between them (Figure 4). As a result, the differences in Pn among all types of plants cannot be explained by the change in Gs, and non-stomatal limitations might exist. Thus, the quantitative analysis of photosynthetic limitations under different light qualities should be carried out in the future.

Chlorophyll content is another important factor for photosynthesis and plant growth. In this study, differential light quality significantly affected chlorophyll pigments and their ratios (Table 4). The decreases in total chlorophyll content were observed under BL, GL and RL, suggesting that these lights caused damages to photosynthetic pigments. Yu et al. (2017) showed that RL strongly promoted Chlb synthesis in Camptotheca acuminata. Similar results were also reported in Cattleya loddigesii and Triticum aestivum (Galdiano et al., 2012; Manivannan et al., 2015). However, compared with RL, BL treatment in this study resulted in a significantly higher leaf chlorophyll content, which was inconsistent with that reported in lettuce, Toona sinensis and Anoectochilus roxburghii (Zhang et al., 2010; Kobayashi et al., 2013; Ye et al., 2017). Chla is mainly concentrated around PSI and PSII, whereas Chlb is most abundant in light-harvesting complexes (LHC) (Venema et al., 2000). Thus, a change in the Chla/b ratio often indicates a change in the ratio between the light-harvesting complexes (LHC) and the reaction centre complexes of the photosystem. The significantly increased values of Chla/b ratio under BL, GL and RL showed that plants under these lights changed the ratio of photosynthetic pigments to maintain a good photoelectron transfer system.

Carotenoids play an essential photoprotective role in chloroplasts by scavenging ROSs (Schmitt et al., 2014). Furthermore, the photoprotective role of xanthophylls for dissipation of excess energy has been demonstrated (Tefler, 2002). Thus, the significantly higher carotenoids contents of C. paliurus plants under WL and BL can lead to the lower levels of ROSs compared to other light quality, which might help plants to maintain a better growth rate. Car/Chl is another important indicator for the adaptation of plants to light conditions, as it is known that increases in this parameter are often observed when plants are grown under stress (Penuelas et al., 1995). Thus, compared with WL and BL, GL and RL have caused the unhealthy development of C. paliurus plants. Since Car values between WL and BL did not differ significantly and were significantly lower under RL and GL, we could conclude that the decreases in total chlorophyll contents contributed the most to the significantly higher Car/Chl ratios under BL, GL and RL.

Chlorophyll fluorescence serves as a subtle reflection of the photochemical activities of photosynthetic systems in plant physiological experiments (Pashkovskiy et al., 2018). Generally, plants grown under light stress typically have lower Fv/Fm values than non-stressed ones (Baker, 2008). In the present study, Fv/Fm, PI_ABS and Pn were significantly reduced under RL and GL compared with WL and BL (Figures 4, 6), indicating the occurrence of significant photoinhibition under these light wavelengths. The light energy absorbed by one active reaction center (ABS/RC) usually reflects the ratio between chlorophyll pigments in the fluorescence-emitting complexes and in the active reaction centers (Strasser et al., 2010). The increase in ABS/RC in plants under GL and RL can be explained by a decrease in the number of active reactive centers of PSII, which might serve as a defense mechanism to reduce the burden of its systems when light stress occurred. Another effective protective mechanism is the dissipation of the absorbed light energy into heat (NPQ mechanism) (Baena-Gonzalez and Aro, 2002), which is confirmed by the much higher values of non-photochemical quenching per one reaction center of PSII (DIo/RC) or per cross section (DIo/CSm) in C. paliurus plants under GL and RL (Figure 6). Furthermore, the energy used to reduce Q_A by the unit reaction center of PSII (TRo/RC) was observed significantly higher under GL and RL, which indicated that C. paliurus plants under these lights improved their efficiency of the remaining active reaction centers to dissipate the energy in the electricity chain. It seems that plants grown under GL in this study have developed most of the characteristics of so called “shade plants” – they have thinner leaves, larger LA, thin palisade tissue, lower number of stomata, and higher chlorophyll a/b ratio. These changes in morphological, chlorophyll content and proportion provide an effective photoprotective strategy for plant growth, as mentioned above. However, the question remains if the observed decrease of PSII capability is reversible, i.e., it is photoinactivation, or irreversible - photoinhibition, but this needs to be further studied.

ROS, such as O₂•^-, H₂O₂ and •OH, can be generated by the direct transfer of the excitation energy from chlorophyll, electron transport chains and the chloroplasts (Sriram et al., 2005), which can cause photodamage to PSII directly (Pashkovskiy et al., 2018). Higher values of MDA (a factor which reflects the level of lipid peroxidation) and significantly higher values of H₂O₂ were observed from plants under GL and RL (Figure 7), which indicated that a high level of oxidative damage had been caused by excessive ROS. It is interesting to find that C. paliurus plants under BL also accumulated more phenolics to help maintain the ROS balance (Figure 7F). The neighboring hydroxyl groups of phenolics allow them to act as an effective role to reduce ROS (Cruces et al., 2017; Liu et al., 2018c). ROS scavenging abilities in plants under GL and RL were confirmed mainly through other methods such as increasing enzymatic antioxidants [peroxidase (POD), and catalase (CAT)] (Figures 7D,E), and also phenolics (Figure 7F). These results indicated that increasing enzymatic antioxidants and phenolics was an effective measure to scavenge ROS especially when light stress occurred.

Growth and photosynthesis of C. paliurus plants showed highest performances under WL and BL, which were attributable to the better morphogenetic occurrences like leaf growth and stomata development, regulations of photosynthetic pigments and photochemistry activity, as well as reduced ROS accumulation. Plants subjected to GL and RL suffered significant photoinhibition but also effectively developed photoprotective mechanisms mainly through changes of leaf anatomy and pigment ratios, NPQ mechanism and increasing enzymatic antioxidants and phenolics contents. This study will help uncover the response mechanisms of plant photosynthesis to light quality, and provide a theoretical basis for improving the cultivation of C. paliurus seedlings. The underlying molecular mechanism remains unknown and more tests in both greenhouse and field conditions, such as adding suitable plastic films or LEDs with spectral characteristics at seedling developmental stage, are needed for applications to improve the yield and quality of this species.

YL is responsible for the whole process of experimenting and writing the paper. TW and SF provide experimental guidance and amend the manuscript. MZ and JQ help to do the experiment.

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.