Effects of shading on lignin biosynthesis in the leaf of tea plant (Camellia sinensis (L.) O. Kuntze)

Shading can effectively reduce photoinhibition and improve the quality of tea. Lignin is one of the most important secondary metabolites that play vital functions in plant growth and development. However, little is known about the relationship between shading and xylogenesis in tea plant. To investigate the effects of shading on lignin accumulation in tea plants, ‘Longjing 43’ was treated with no shading (S0), 40% (S1) and 80% (S2) shading treatments, respectively. The leaf area and lignin content of tea plant leaves decreased under shading treatments (especially S2). The anatomical characteristics showed that lignin is mainly distributed in the xylem of tea leaves. Promoter analysis indicated that the genes involved in lignin pathway contain several light recognition elements. The transcript abundances of 12 lignin-associated genes were altered under shading treatments. Correlation analysis indicated that most genes showed strong positive correlation with lignin content, and CsPAL, Cs4CL, CsF5H, and CsLAC exhibited significant positively correlation under 40% and 80% shading treatments. The results showed that shading may have an important effect on lignin accumulation in tea leaves. This work will potentially helpful to understand the regulation mechanism of lignin pathway under shading treatment, and provide reference for reducing lignin content and improving tea quality through shading treatment in field operation.


Introduction
Light is an important factor that regulates plant growth and development. The light radiation could affect plant morphogenesis, photosynthesis, and phototropism (Monteith 1965;Chory 1997), which established itself as the driving force of the photosynthetic processes that convert carbon dioxide into organic products (Monteiro et al. 2014). The physiological and molecular basis of light-induced process has been well elaborated in higher plant (Castillon et al. 2007). The mechanism of light radiation regulates phenylpropanoid biosynthesis has been reported in some model plants (Li et al. 1993;Albert et al. 2009;Matus et al. 2009;Robin et al. 2009). Many metabolites are derived from downstream branches of the phenylpropanoid pathway, including lignin.
Lignin is a widely exist phenolic biopolymer in plants, which constitute the second most abundant organic compound after cellulose (Rogers and Campbell 2004;Liu et al. 2018b). Lignin play critical roles in the growth and development of plants, promoting water transport, enhancing plant cell wall rigidity, and plant pathogen defense and various environmental stresses (Ithal et al. 2007;Shadle et al. 2007;Jullyana et al. 2010;Zheng et al. 2017). Lignin monomers are synthesized through a series of aromatic hydroxylation and O-methylation reactions. Generally, the lignin polymers are primarily derived from mainly three monomers: coniferyl alcohol (G unit), p-coumaryl alcohol (H unit), and sinapyl alcohol (S unit) (Ruben et al. 2012). Lignin biosynthetic pathway has been elucidated in previous studies, and the corresponding enzymes and genes participating in the metabolic pathway of lignin biosynthesis have also been elucidated (Boerjan et al. 2003;Qiao and Dixon 2011). Phenylalanine ammonia lyase (PAL), 4-coumarate-CoA ligase (4CL), and cinnamate Communicated by Stefan Hohmann.

Electronic supplementary material
The online version of this article (https ://doi.org/10.1007/s0043 8-020-01737 -y) contains supplementary material, which is available to authorized users.
* Jing Zhuang zhuangjing@njau.edu.cn 1 Tea Science Research Institute, College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China 4-hydroxylase (C4H) participated in general phenylpropanoid pathway (Goujon et al. 2003;Riboulet et al. 2009). Hydroxycinnamoyl-CoA transferase (HCT), 4-coumarate 3-hydroxylase (C3'H), caffeoyl-CoA O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H), caffeic acid O-methyltransferase (COMT), cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD), peroxidase (PER) and laccase (LAC) participated in specific lignin pathways (Ruben et al. 2010;Cheng et al. 2017). Tea plant (Camellia sinensis (L.) O. Ktze), an important economic crop species of theaceae, is widely grown in the world, which provide raw materials to produce nonalcoholic beverage "tea". Tea leaf is rich in flavonoid, theanine, vitamins, polysaccharides, and minerals (Liang et al. 2010;Li et al. 2018). A large number of studies have proved that tea flavonoids (especially catechins) are beneficial to human health. Studies have shown that shading treatment can effectively reduce photoinhibition and improve the quality of tea (Qin et al. 2011;Tian et al. 2017). Tea plants grown in the shading conditions shown higher amino acid contents but lower catechins (Mo et al. 2010). Theanine is a main and special kind of amino acid components in tea, which mainly contributes to sweetness and umami, whereas catechins and caffeine contribute to astringency. There was a negative correlation between lignin accumulation and shoot tenderness. The degree of lignification reflects the tender condition of tea plant leaves and determines the quality of tea plant leaves. In addition, lignin metabolism and flavonoid metabolism share phenylpropane metabolic pathways. The synthesis of lignin and tea polyphenols (flavonoids) and other main functional components of tea have a competitive substrate, which will also affect the accumulation of functional components of tea. Shading in tea garden is an effective measure to improve the tenderness of tea buds and leaves (Wang et al. 2012a;. In the past years, studies on the effects of light conditions on the synthesis of tea plant secondary metabolites mainly focus on the biosynthesis of flavonoids (Koretskaya and Zaprometov, 1975;Saijo 1980;Zagoskina et al. 2003;Wang et al. 2012a). However, few researches have been reported on the effect of shading treatment on the molecular mechanisms of lignin synthesis in tea plant.
'Longjing 43', originating in Hangzhou city, Zhejiang Province, China, is a well-known diploid tea plant cultivar due to its high yield as well as for its high quality. This study aims to investigate the effects of shading treatments on morphological, lignin accumulation, anatomical characteristics, and expression profiles of lignin-related genes in the leaves of tea plant cultivar 'Longjing 43'. The result showed that shading treatments reduce lignin content and leaf area of the leaves in tea plant. The transcripts of lignin biosynthesis genes were altered under shading treatments. This study may serve as a guide for further elucidating the molecular mechanisms of shade affecting the lignin biosynthesis pathway of tea plant.

Preparation of plant materials
Two-year-old 'Longjing 43' tea plant seedlings were grown in a controlled-environment growth chamber at the Nanjing Agricultural University (Nanjing, China). The chamber conditions were set at 25 °C temperature, 70% relative humidity, 300 μmol m −2 s −1 light intensity and 16 h light/8 h dark cycle. Shade nettings with the corresponding light transmittance coefficients were used to provide shading treatments with approximately 40% (S1) and 80% (S2) shading rates. Tea plants maintained under no shading condition (0% shading rate) were used as control (S0). After shading treatments, tea shoots containing first and second leaves, were collected at 3, 6, and 9 days, respectively. The collected tea plant samples were quickly immersed in liquid nitrogen and then stored at − 80 °C.

Measurement of lignin content
The lignin of tea plant samples was purified and measured according to the previously described procedure with some modifications (Cervilla et al. 2009;Wang et al. 2016). In brief, approximately 1 g of frozen tea plant samples were ground in liquid nitrogen, then immediately homogenized in 99.5% ethanol and centrifuged for 20 min in a refrigerated centrifuge at 12,000 × g. The sediment was collected and spread in a clean petri dish for 24 h to air dry at room temperature. About 10 mg of dried sediment was transferred to a 2 mL plastic tube and then added with 0.1 mL of thioglycolic acid and 1 mL of 2 M HCl. The mixture sample was incubated at 100 °C for 8 h in a thermostatic water bath, and then cooled on ice. After centrifuged at 14,000 × g for 20 min at 4 °C, the sediment was washed with 1 mL of distilled water, centrifuged again and then resuspended with 1 mL of 1 M NaOH. The mixed solution was incubated at 25 °C for 18 h, and then centrifuged at 14,000 × g for 20 min. The water phase on the top was transferred into a new plastic tube and added with 1 mL of concentrated HCl. The mixture was exposed at 4 °C for 6 h and then centrifugation at 14,000 × g for 20 min. The remaining sediment was redissolved with 1 mL of 1 M NaOH. The sample was determined by spectrophotometer at 280 nm with 1 M NaOH as the control. The extraction and determination of lignin from each sample were conducted with three replicates.

Histochemical staining and UV microscopy
Histochemistry staining and UV microscopy were performed to observe lignin distribution. The leaf specimens were cut from healthy tea plant using a razor blade. The first leaves, including major veins, were cut into small pieces and immediately stored in 2.5% glutaraldehyde at 4 °C. For safranin-O/fast green staining, the leaf sections were first deparaffinized in xylene and then washed and dehydrated with ethanol. Subsequently, the sections were stained in 1% safranin-O for 2 h and then counterstained with 0.5% fast green for 15 s. The sections were washed with ethanol to remove the extra stain and then mounted using neutral balsam. Safranin-O stained the lignified cell walls with red, whereas fast green stained the cellulosic tissues with green.
The lignified cell walls exhibit autofluorescence under UV excitation (Donaldson and Knox, 2012;Wang et al. 2017). For autofluorescence observation, the lignin autofluorescence of the leaf specimens were observed by fluorescence microscopy under UV excitation.

Leaf area calculation
First leaf was selected as materials to calculate the leaf area. Three leaves were counted for each treatment. The formula for leaf area is as follows: leaf area (cm 2 ) = leaf width (cm) × leaf length (cm) × 0.7 (Luo 2008).

Selection and analysis of gene involved in lignin biosynthesis
The genes involved in lignin biosynthesis were selected from the Tea Plant Information Archive (TPIA) (http://tpia.teapl ant.org/), a genomic database for tea plant (Wei et al. 2018). A total of 12 genes related to lignin pathway of tea plant were selected, namely CsPAL, CsC4H,Cs4CL,CsHCT,CsC3′H,CsCCoAOMT,CsF5H,CsCOMT,CsCCR ,CsCAD,CsPER and CsLAC. The functional interaction networks of genes involved in the lignin biosynthesis pathway were constructed using STRING software. The upstream 2000 bp regions of these 12 genes involved in the lignin biosynthesis were analyzed to search for putative cis-acting elements using the plant database PlantCARE (http://bioin forma tics.psb.ugent .be/ webto ols/plant care/html/) (Magali et al. 2002).

RNA isolation, reverse transcription, and qRT-PCR detection
Total RNA was extracted from tea leaves by using an RNA extraction kit (Huayueyang Biotech Co., Ltd., Beijing, China) following the manufacturer's instruction. The RNA concentration was assessed using a Nanodrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE). One microgram of each RNA sample was reverse transcribed into cDNAs using the PrimeScript RT reagent kit (TaKaRa, Dalian, China) in accordance with the manufacturer's instructions.
qRT-PCR was carried out using a Bio-Rad CFX96 fluorescence quantitative PCR platform with TaKaRa SYBR Premix Ex Taq (TaKaRa, Dalian, China). The primers used in this study were designed using Primer Premier 5.0 software and listed at Table 1. The Actin gene of tea plant was chosen to normalize the expression levels of lignin related genes (Wu et al. 2016). The reaction conditions were set as follows: 95 °C for 3 min, 40 cycles of 95 °C for 5 s, 58 °C for 20 s, and followed with a melting curve analysis ranging from 65 to 95 °C. The reaction of each sample was conducted with three biological replicates. The relative gene expression was calculated using the 2 −ΔΔCt method (Pfaffl 2001).

Statistical analysis
Statistical analysis was assessed by One-way ANOVA using SPSS18.0 software. Significant differences were detected by Duncan's multiple-range test at significance levels P < 0.05. A Pearson correlation coefficient (PCC) analysis was performed to evaluate the correlations between lignin contents and expression levels of genes in the lignin pathway using SPSS software at significance levels p < 0.01 and p < 0.05.

Phenotypic changes of tea plant leaves
Tea shoots of 'Longjing 43' at 3, 6, and 9 days after shading treatments (S0, S1, and S2) were harvested and investigated. Under shading treatments, the color of tea leaves seems greener, and the leaves are softer, especially in the S2 treatment. Leaf area increased during leaf development, and leaf areas of S0 are obvious larger than that in shading treatments, S1 and S2 (Fig. 1). The leaf areas at 3, 6, and 9 days of S0 are 1.2-, 1.5-, and 1.5-folds of S1, respectively, and 1.5-, 2.2-, and 2.5-folds of S2.

Lignin content levels in tea plant leaves under shading treatments
Tea plant leaves with shading treatments were collected and analyzed for lignin content (Fig. 2). With the growth and development of tea plant, lignin accumulation of the leaves showed an upward trend in S0 and S1, while, the change was not significant in S2. The lignin contents in S0 were obvious higher than that in S1 and S2 at 9 days.

3
The lignin content decreased with increasing shading (S0 > S1 > S2). The results showed that shading might have a negative influence on lignin accumulation in tea plant leaves.

Anatomical structure analysis of tea plant leaves under shading treatments
To further understand the effect of shading on lignin levels of tea plant, the anatomical structure of tea plant leaves was investigated. The transverse sections of leaves were obtained and stained with safranin-O and fast green to highlight the basic anatomical structure of tea plant leaves (Fig. 3). The lignin in the tea plant leaves was mainly distributed in the secondary walls of the xylem region. Compared with control, the lignification of the xylem of leaves under 80% shading treatment appeared to be declining at 9 d. The transverse sections were placed under UV excitation to determine the effect of shading treatments on tea plant leaves (Fig. 4). Lignin was also found in the xylem region of tea plant leaves, which was consistent with the observation in Fig. 3.

Analysis of the promoter regions of the genes involved in the lignin pathway in tea plant
The promoters of 2000 bp upstream of the transcription start site of CsPAL, CsC4H, Cs4CL, CsHCT, CsC3′H, CsCCoAOMT, CsF5H, CsCOMT, CsCCR , CsCAD, CsPER, and CsLAC were analyzed to understand the regulatory mechanisms that control the expression of these genes. As shown in Table 2 and Supplementary Table 1, each gene involved in the lignin pathway contains several lightresponsive elements. For example, Box 4 elements appear at the promoters of all lignin pathway genes. G-box, GT1motif, and TCT-motif are also light-responsive elements, which exist in most lignin pathway genes. Those results suggested that the genes involved in the lignin pathway might be regulated by light.
Other cis-regulatory elements, such as the TGA-element, CGTCA-motif, ABRE, ERE, STRE, W box, ARE, are present in most genes of the lignin pathway (Supplementary Table 1). These elements are related to the signal pathways of methyl jasmonic acid (MeJA), ethylene (Eth), abscisic acid (ABA), anaerobic, and other biotic and abiotic stresses. The results indicated that the expression of these genes in Fig. 3 Safranin-O/fast green staining of transverse sections of tea plant leaves with shading treatments. S0, no shading; S1, approximately 40% shading rate; S2, approximately 80% shading rate. Tea plant leaves were harvested at 3 (a, d, g), 6 (b, e, h), and 9 (c, f, i) day, respectively. Ue upper epidermis, De lower epidermis, Cu cuticula, P phloem, X xylem. Scale bars are equivalent to 50 μm in length Fig. 4 Fluorescence micrographs of transverse sections of tea plant leaves with shading treatments. S0, no shading; S1, approximately 40% shading rate; S2, approximately 80% shading rate. Tea plant leaves were harvested at 3 (a, d, g), 6 (b, e, h), and 9 (c, f, i) day, respectively. Cu cuticula, X xylem. Scale bars are equivalent to 50 μm in length lignin pathway may also be involved in the response to hormones, biotic and abiotic stresses.

Expression patterns of lignin pathway related genes under shading treatments
To further investigate the effect of shading treatments on lignin pathway, the expression profiles of 12 lignin biosynthesis related genes were selected for qRT-qPCR. CsPAL, CsC4H and Cs4CL participate in the general phenylpropanoid pathway. CsHCT, CsC3′H, CsCCoAOMT, CsF5H, CsCOMT, CsCCR , CsCAD, CsPER and CsLAC participate in lignin-specific pathway. General phenylpropanoid pathway During the growth of tea plant, the expression levels of CsPAL, CsC4H and Cs4CL in leaves were gradually increased under S0 and S1 treatments (Fig. 6). With the increase of shade degree, the expression of Cs4CL decreased gradually (S0 > S1 > S2). Compared with S0 (control), the expression profiles of CsPAL and CsC4H were obvious decreased in S2, while they were increased at 9 d in S1.
Specific lignin pathway With the growth and development of tea leaves, the expression profiles of all specific lignin related genes in S0 and S1 showed an upward trend, whereas CsCCoAOMT, CsCOMT and CsLAC significantly decreased under S2 treatment (Fig. 7). The expression level of most genes in S0 was slightly higher than that in S1 treatment. The expression profiles of CsHCT, CsF5H, CsCCR , CsPER and CsLAC were obvious decreased in S2 compared with that in S0. CsC3′H, and CsCAD, showed a similar trend, which is, declining in 3 days and 6 days, while increasing in 9 days with increasing shading.

Correlation analysis of lignin content and expression levels of related genes
The correlation coefficients were calculated by correlating lignin content and gene expression level in lignin pathway by Pearson analysis (Fig. 8 and Table 3). The correlations shading rate. Error bars represent standard deviation among three independent replicates. Different lowercase letters indicate significant differences at P < 0.05 Fig. 7 Expression profiles of genes involved in the specific lignin pathway under shading treatments in tea plant leaves. S0, no shading; S1, approximately 40% shading rate; S2, approximately 80% shading rate. Error bars represent standard deviation among three independent replicates. Different lowercase letters indicate significant differences at P < 0.05 were calculated from the 40% shading treatment (S0&S1), 80% shading treatment (S0&S2), and the whole shading treatments (S0&S1&S2). The expression levels of all lignin related genes were positively correlated with the lignin content of S0&S1, S0&S2, and S0&S1&S2.
The expression profiles of CsPAL, Cs4CL, CsF5H, and CsLAC exhibited significant positively correlation with lignin content in S0&S1, S0&S2, and S0&S1&S2. The expression profile of CsHCT was significant correlated with lignin content in S0&S2 and S0&S1&S2; while, CsCOMT was significant correlated with lignin content in S0&S1 and S0&S1&S2. In addition, the gene expression profile of CsC-CoAOMT was significantly and positively correlated with the lignin content in S0&S1, whereas Cs4CL was significant correlated with lignin content in S0&S2, and CsCCR and CsPER were significant correlated with lignin content in S0&S1&S2.

Discussion
Light is the basis of plant photosynthesis and plays an important role in plant growth and morphogenesis (Yu et al. 2018). A large number of studies have shown that shading conditions can significantly change the plant height stem thickness, leaf area, leaf structure and other biological character, but also affect the change of photosynthetic efficiency and secondary metabolites content (Zhang et al. 2015;Fan et al. 2018). Shading treatment in tea garden can effectively reduce photoinhibition and improve the quality of tea. The present study showed that leaf area decreased with the increase of shade degree. The leaf area of 80% shading treatment (S2) less than that of control (S0). Shading also change the leaf color. With the increase of shade, the tea plant leaves appear to be greener. That's mainly due to shading treatment improved the chlorophyll concentration (Wang et al. 2012a;Dolatkhahi 2013). Shading in tea garden can improve the tenderness of tea plant buds and leaves. The tender age of tea plant leaves is an important basis for judging the quality of tea. Lignin accumulation was negatively correlated with shoot tenderness. The degree of lignification reflects the tenderness of the tea plant leaves and determines the quality of tea. The lignin content of tea plant leaves reduced under shading treatment, indicating that shading might have a negative effect on lignin accumulation in tea plant leaves. Similarly, shading led to a decrease of acid detergent lignin in perennial herbaceous Texas legumes (Muir et al. 2009).
Lignin distribution was qualitatively measured by histochemistry and autofluorescence microscopy. In this study, lignin is mainly distributed in the xylem of tea plant leaves. Our previous research has shown that lignin is mainly distributed in the cell wall of xylem and vascular bundle sheath of main leaf vein of tea plant . That is mainly due to the growth and development stages, and lignin may preferentially deposit in xylem and second vascular bundle sheath, or it is mainly determined by the characteristics of the tea plant cultivar 'Longjing 43'. Histochemistry and autofluorescence microscopy indicated that 80% shading reduced the degree of lignification of xylem. This was consistent with that lignin accumulation was decreased with increasing shading.
Previous studies have shown that changes in lignin content are accompanied with changes of gene expression of involved in lignin biosynthesis (Ali and McNear, 2014;Liu et al. 2018a;Que et al. 2018). Here, 12 genes involved in lignin synthesis (CsPAL,CsC4H,Cs4CL,CsHCT,Fig. 8 Correlation analyses between lignin content and expression levels of related genes. The genes from left to right in abscissa were followed by CsPAL, CsC4H,Cs4CL,CsHCT,CsC3′H,CsCCoAOMT,CsF5H,CsCOMT,CsCCR ,CsCAD,CsPER,and CsLAC ,CsCCoAOMT,CsF5H,CsCOMT,CsCCR ,CsCAD,CsPER and CsLAC) were selected to investigate the regulation mechanism under shading treatment at the transcript level. Promoter analysis indicated that all of these genes contain multiple light responsive elements, indicating that the expression of these genes might be regulated by light. Plants can regulate physiology in response to various environmental stimuli. Tea plants adapt to shade in their natural habitat, according to the photosynthetic characteristics of tea plants, shading can reduce the harm of photoinhibition. Shading treatment was effective way to reduce the biosynthesis of lignin. In control (S0), with the growth and development of tea plant leaves, lignin biosynthetic related genes showed an upward trend, indicating that these genes play an important role in the development of lignin in tea plant (Fig. 9). Compared with no shading treatment, the expression of most lignin biosynthetic related genes in 80% shading treatment was obviously decreased, which was consistent with the decrease of lignin content in 80% shading treatment. PAL is the first enzyme in the lignin biosynthesis pathway, catalyzing the conversion of phenylalanine to cinnamic acid (Xu et al. 2014;de Jong et al. 2015). Previous research has shown that PAL was notably reduced in the shade-treated leaves of tea plant, 'Shuchazao' (Wang et al. 2012a). 4CL is another enzyme in lignin pathway and is also thought to catalyzes the third step of phenylpropanoid pathway (Mo et al. 2010). The lignin content in plants was significantly decreased by the down-regulation of 4CL (Hu et al. 1999;Bin et al. 2011). In the present study, CsPAL and Cs4CL showed a strong positive correlation with lignin concentration, and reduced in shading treatments. PAL, C4H, and 4CL are involved in the general phenylpropanoid pathway and also shared by the formation of other important secondary metabolites, such as flavonoids and coumarin (Riboulet et al. 2009;Wang et al. 2012b;Saito et al. 2013). Research has revealed that the expression of PAL and C4H is consistent with the catechin contents (Eungwanichayapant and Popluechai 2009). The expression of these genes may not only affect the lignin content, but also affect other secondary metabolites.
CsHCT, CsC3′H, CsCCoAOMT, CsF5H, CsCOMT, CsCCR , CsCAD, CsPER, and CsLAC were involved in the specific lignin pathway. Wang and his colleagues found that lignin content was increased compared to darkness in light-induced calli of tea plant, and several lignin-related genes (CCoAOMT, HCT and CCR ) were identified in the light-induced SSH library. The results showed that light can promote the synthesis and accumulation of lignin in tea plant cells during cell differentiation (Wang et al. Fig. 9 Simplified diagram of lignin biosynthesis pathways. Expression levels of genes involved in lignin biosynthesis pathways were marked with colored boxes. Relative expression levels of related genes at 3 d, 6 d, 9 d were showed in the left, center, and right columns, respectively. Relative expression levels of related genes at the first, second and third lines were represented S0, S1, and S2 stages. The enzymes are as follows: phenylalanine ammonia lyase (PAL); cinnamate 4-hydroxylase (C4H); 4-coumarate-CoA ligase (4CL); cinnamoyl-CoA reductase (CCR); cinnamyl alcohol dehydrogenase (CAD); hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT); p-coumaroyl shikimate/ quinate 3′ -hydroxylase (C3′H); caffeoyl-CoA O-methyltransferase (CCoAOMT); ferulate 5-hydroxylase (F5H); caffeic acid O-methyltransferase (COMT); peroxidase (PER), and laccase (LAC) 2012b). Previous studies have shown that changes in lignin content are related to the expression levels of lignin metabolic genes . Here, CsF5H, and CsLAC exhibited significant positive correlation both in the 40% and 80% shading treatments, indicating the importance of these two genes for lignin synthesis under shading conditions. Some genes (CsHCT, CsCCoAOMT and CsCOMT) are highly correlated with 40% or 80% shading treatments. Among them, CsCCoAOMT and CsCOMT have similar expression patterns, suggesting that these genes might have similar functions in lignin metabolism.
Lignin is the main component of the plant secondary cell walls that providing mechanical support and water transport for plants (Boudet 2000;Hui et al. 2009). The lignification of leaf tissues is closely related to the quality and stress resistance of tea plant. Suitable lignification is essential for tea quality. Shading had a certain effect on the growth of tea plants, whereas it also increased the satisfactory amino acid contents. Strong shading seriously affected the development of tea plant leaves, and reduce tea yield. Tea plant leaves grow rapidly in summer, they are usually not processed and utilized due to the deterioration of quality, resulting in the waste of tea raw materials. Shading treatment can significantly improve tea tenderness and tea quality. Tea flavonoids (especially catechins) are the main biologically active components in tea plant, which share the phenylalanine metabolism branch pathway with lignin. Shading reduced the flavonoids, and also lignin content (Wang et al. 2012a). This may be mainly due to shading reduced the carbon source upstream of phenylpropane.
In this study, we evaluated the effect of shading on leaves growth, xylem development, and lignin accumulation in tea plant. Shading treatment inhibited leaves growth and lignin content. Safranin-O/fast green staining and autofluorescence showed that lignin was mainly deposited in the xylem and reduced under strong shading. Shading may affect the lignification of tea plant leaves by regulating the transcript levels of lignin biosynthesis related genes. This study provided potentially useful information for understanding the mechanism of lignin biosynthesis under shading treatments in tea plant, and also provided scientific basis for reducing lignin content and improving tea quality through shading treatment in field operation.