Light-Induced Flavonoid Biosynthesis in Sinopodophyllum hexandrum with High-Altitude Adaptation

Sinopodophyllum hexandrum is a perennial alpine herb producing the anti-cancer metabolite podophyllotoxin (PPT). Although the adaptation of S. hexandrum to high altitudes has been demonstrated and the effects of temperature, precipitation, and UV-B light on plant growth and metabolite accumulation have been studied, knowledge on the role of flavonoid biosynthesis in adapting to high altitudes is limited. In this study, light intensity, amount and type of flavonoids, and differentially expressed proteins (DEPs) and genes (DEGs) at 2300 and 3300 m were analyzed by HPLC, proteomic, transcriptomic, and qRT-PCR analysis. We found that higher light intensity correlated with greater flavonoid, flavonol, and anthocyanin content as well as higher anthocyanin to total flavonoid and flavonol ratios observed at the higher altitude. Based on proteomic and transcriptomic analyses, nine DEPs and 41 DEGs were identified to be involved in flavonoid biosynthesis and light response at 3300 m. The relative expression of nine genes (PAL, CHS1, IFRL, ANS, MYB4, BHLH137, CYP6, PPO1, and ABCB19) involved in flavonoid biosynthesis and seven genes (HSP18.1, HSP70, UBC4, ERF5, ERF9, APX3, and EX2) involved in light stress were observed to be up-regulated at 3300 m compared with 2300 m. These findings indicate that light intensity may play a regulatory role in enhancing flavonoid accumulation that allows S. hexandrum to adapt to elevated-altitude coupled with high light intensity.


Introduction
Sinopodophyllum hexandrum Royle (family Berberidaceae) is a perennial rhizomatous species that is native to the alpine Himalayan region at altitudes of 2000 to 4500 m above sea level [1][2][3]. The dried fruit is referred to as "xiaoyelian" in China and is used widely as a traditional Tibetan medicine to treat gynecological diseases [4]. The rhizomes are the major source of PPT, which can effectively mitigate specific cancers and heal certain skin lesions [5]. Constituents from dried fruit and rhizomes include lignans, flavonoids, and alkaloids [4][5][6]. The species is currently endangered due to over exploitation of wild plants and limited large-scale artificial cultivation [7,8].
To protect the wild S. hexandrum and to satisfy the commercial demand, preliminary preparations for large-scale cultivation have been performed, including breaking seed dormancy [9], promoting seed germination [10,11], accelerating vegetative propagation by dividing the rhizomes [9], and providing the sustainable cultivation-collection model for optimized PPT production [3]. Currently, large-scale cultivation has yet to be realized, largely because of a failure to identify suitable growth conditions in the field.
Geographical modeling for S. hexandrum predicts that altitude determines plant distribution, with optimal altitudes of 2800 to 3600 m for artificial planting [2]. There is a

Ratio Differences for Anthocyanins and Total Flavonoids
The ratio of anthocyanins to total flavonoid content was 1.91-fold higher ( Figure  2A) and the ratio of anthocyanins to four flavonols' contents was 1.99-fold higher at 3300 m than 2300 m ( Figure 2B).

Ratio Differences for Anthocyanins and Total Flavonoids
The ratio of anthocyanins to total flavonoid content was 1.91-fold higher ( Figure  2A) and the ratio of anthocyanins to four flavonols' contents was 1.99-fold higher at 3300 m than 2300 m ( Figure 2B). The ratios of anthocyanins to total flavonoids (A) and flavonol contents (B) at 2300 and 3300 m. The * represents a significant difference (p < 0.05) between 2300 and 3300 m (mean ± SD, n = 3).

Differentially Expressed Proteins at Higher Elevation
A total of 65 proteins were differentially expressed at 3300 m vs. 2300 m, with 46 over-expressed and 19 down-expressed ( Figure S1); among the 46 over-expressed proteins, 30 proteins had known function, while the other 16 proteins had no known function (Table 1). Proteins were classified based on biological function, including primary and secondary metabolism (6), photosynthesis and energy (10), cell morphogenesis (2), transcription (4), and translation (8). Among 30 proteins grouped by biological function, 5 proteins were involved in flavonoid biosynthesis (ANS, CYP6, CYP71BE30, PPO1, and MYB4) and 4 proteins were involved in photosynthesis (ycf3, rbc, rbcL, and petL). The ratios of anthocyanins to total flavonoids (A) and flavonol contents (B) at 2300 and 3300 m. The * represents a significant difference (p < 0.05) between 2300 and 3300 m (mean ± SD, n = 3).

Expression Level of Genes Involved in Flavonoid Biosynthesis
Of the five DEPs (Table 1) and 22 DEGs (Table 2) involved in flavonoid biosynthesis, eight genes (PAL, CHS1, GT6, IFRL, BHLH137, ABCB19, CYP6, and PPO1) and four proteins (ANS, MYB4, CYP6, and PPO1) were identified with CYP6 and PPO1, exhibiting overlap for both gene and protein differential expression at the higher altitude. These subsets of differentially expressed genes/proteins were selected to measure relative expression levels (RELs) by qRT-PCR. A 3.3-(ABCB19) to 12.3-fold (PAL) up-regulation and 0.5-fold (GT6) down-regulation were observed at the higher altitude ( Figure 3). These RELs were consistent with the fold change (FC) at the two altitudes, except for the CHS1 (Table 2). proteins (ANS, MYB4, CYP6, and PPO1) were identified with CYP6 and PPO1, exhibiting overlap for both gene and protein differential expression at the higher altitude. These subsets of differentially expressed genes/proteins were selected to measure relative expression levels (RELs) by qRT-PCR. A 3.3-(ABCB19) to 12.3-fold (PAL) up-regulation and 0.5-fold (GT6) down-regulation were observed at the higher altitude ( Figure 3). These RELs were consistent with the fold change (FC) at the two altitudes, except for the CHS1 (Table 2).

Expression Level of Genes Involved in Light Stress
To further examine the biological function of genes involved in light stress [36], seven genes (HSP18.1, HSP70, UBC4, ERF5, ERF9, APX3, and EX2) were monitored for the REL by qRT-PCR (Table 3). A 2.03-(UBC4) to 15.47-fold (ERF5) up-regulation was observed at 3300 vs. 2300 m (Figure 4), which was consistent with the FC at the two altitudes, except for the UBC4 (Table 3).

Expression Level of Genes Involved in Light Stress
To further examine the biological function of genes involved in light stress [36], seven genes (HSP18.1, HSP70, UBC4, ERF5, ERF9, APX3, and EX2) were monitored for the REL by qRT-PCR (Table 3). A 2.03-(UBC4) to 15.47-fold (ERF5) up-regulation was observed at 3300 vs. 2300 m (Figure 4), which was consistent with the FC at the two altitudes, except for the UBC4 (Table 3). except for the CHS1 (Table 2).

Expression Level of Genes Involved in Light Stress
To further examine the biological function of genes involved in light str seven genes (HSP18.1, HSP70, UBC4, ERF5, ERF9, APX3, and EX2) were moni the REL by qRT-PCR (Table 3). A 2.03-(UBC4) to 15.47-fold (ERF5) up-regula observed at 3300 vs. 2300 m (Figure 4), which was consistent with the FC at the tudes, except for the UBC4 (Table 3).

Discussion
The alpine medicinal plant S. hexandrum has been demonstrated in previous studies to be adapted to high-altitude conditions [1][2][3]. Although anthocyanin dark spots on the leaf surface were proposed to function as UV protection at high altitudes [34,35], the induction of flavonoid biosynthesis by high altitude conditions had not been investigated. Here, we report that higher light intensity is associated with greater flavonoid, flavonol, and anthocyanin content and that higher ratios of anthocyanins to total flavonoids and flavonols are observed for plants grown at 3300 m. Indeed, nine proteins and 41 genes directly involved in flavonoid biosynthesis and light response are differentially expressed in plants grown at the higher altitude.
Previous studies found that climatic factors (e.g., temperature, precipitation, and light) contributed more to elevated lignan and flavonoid content in S. hexandrum than soil elements (e.g., pH, organic matter, and potassium). Specifically, low temperatures (4-15 • C) could enhance plant biomass and accumulation of secondary metabolites, especially in podophyllotoxin (PPT), in S. hexandrum plants by the up-regulation of genes involved in plant growth, PPT biosynthesis, and stress response [16][17][18]; a moderate water deficit could improve PPT accumulation [19]; a moderately shaded habitat could enhance plant growth [20]; UV-B radiation could inhibit plant growth and PPT biosynthesis while improving flavonoid accumulation [21]. Among other effects, the lower temperatures expected in the higher altitude site could probably negatively affect the dark phase of the photosynthesis, therefore exacerbating the photo damage and ROS production caused by high light intensity.
Previous studies have found that higher altitudes (Shangri-La of Yunnan Province and Nyingchi of Tibet) seem to be essential for the accumulation of flavonoids (e.g., quercetin and kaempferol) [14]. Initially, in situ climatic factors including lower annual air temperature, lower annual light duration, and higher annual precipitation at higher elevation growing sites were considered as drivers for robust S. hexandrum growth [3]. However, an alternative driver for growth is significantly higher light intensity at the higher elevation. Indeed, correlation analysis showed a positive correlation between annual light duration and kaempferol and quercetin content [14]. In this study, greater total flavonoids, flavonols, and anthocyanins were observed and correlated with higher light intensity measured at 3300 m than the lower elevation. Light quality and intensity have previously been shown to be a driver of flavonoid biosynthesis in plants [23][24][25][26][27]. In fact, the UV-B radiation enhancing total flavonoid accumulation in S. hexandrum seedlings has been observed [21].
Other climatic factors such as temperature, precipitation, and air pressure may also contribute to flavonoid biosynthesis and/or accumulation at high altitudes [1][2][3]. Several genes involved in upstream (PAL, C4H, and 4CL) and downstream (MTs and UGTs) flavonoid biosynthesis, as well as TFs (MYB, bHLH, and WRKY) and CYPs under low-temperature and water deficit treatments [16,19], have been published; climatic measurements to directly link temperature and/or precipitation with enhanced flavonoid accumulation have yet to be reported.
In this study, five proteins (ANS, CYP6, CYP71BE30, PPO1, and MYB4) and 22 genes (PAL, C4H, CHS1, IFRL, GT6, BHLH137, DTX41, ABCB19, nine CYPs, PPO1, and four MTs) may be involved in flavonoid biosynthesis. Extensive studies have demonstrated that PAL, C4H, CHS1, ANS, and GT6 directly participate in flavonoid biosynthesis, the ABCB19 participates in flavonoid transport, and the MYB4 and BHLH13 participate in the regulation of flavonoid biosynthesis [29,30,[37][38][39][40][41][42]. In addition, nine CYPs and four MTs may also participate in flavonoid biosynthesis [31,33,43]. In addition, the PPO1 and IFRL are involved in the biosynthesis of anthocyanins and isoflavanones, respectively [44,45], and the DTX41 acts as a flavonoid/H + -antiporter controlling the flavonoid transport [46]. It is noteworthy that two TFs, MYB4 and BHLH137, may play critical roles in flavonoid biosynthesis. As is known, the R2R3-MYBs participate in plant growth and development, metabolism (e.g., flavonoid biosynthesis), and stress responses [47]. Previous studies have found that MYB4 serves as a repressor for flavonoid biosynthesis and that MYB4 expression is down-regulated and can enhance sinapate ester levels in Arabidospis leaves with exposure to UV-B light [48]. In buckwheat, in response to UV-B, MYB4R1 regulates flavonoid and anthocyanin biosynthesis by binding to L box motifs in the promoter of CHS, FLS, and UFGT [49]. Moreover, the MYB-bHLH-WD40 complex can regulate genes that encode late step enzymes in the pathway, leading to increased flavonoid biosynthesis [50]. Based on the biological function, the roles of identified proteins and genes were mapped in the flavonoid biosynthetic pathway ( Figure 5). roles in flavonoid biosynthesis. As is known, the R2R3-MYBs participate in plant growth and development, metabolism (e.g., flavonoid biosynthesis), and stress responses [47]. Previous studies have found that MYB4 serves as a repressor for flavonoid biosynthesis and that MYB4 expression is down-regulated and can enhance sinapate ester levels in Arabidospis leaves with exposure to UV-B light [48]. In buckwheat, in response to UV-B, MYB4R1 regulates flavonoid and anthocyanin biosynthesis by binding to L box motifs in the promoter of CHS, FLS, and UFGT [49]. Moreover, the MYB-bHLH-WD40 complex can regulate genes that encode late step enzymes in the pathway, leading to increased flavonoid biosynthesis [50]. Based on the biological function, the roles of identified proteins and genes were mapped in the flavonoid biosynthetic pathway ( Figure 5). The difference in CHS1 gene expression levels based on transcriptomic and qRT-PCR analysis may result from the annotation of the reference transcriptome, amplification efficiency, and/or individual features. Previous studies have found that there are about 85% genes of RNA sequencing consistent with qRT-PCR data [51]. For the down-regulation of the GT6 gene, previous studies on strawberry found that GT6 might be associated with the glucosylation of flavonols [52]; thus, its down-regulation may inhibit the glucosylation of flavonols, which is in accordance with the results of greater contents of flavonols (rutin, quercetin, and kaempferol) at 3300 m than 2300 m.
Initially, flavonoid biosynthesis is regulated by biotic and abiotic stresses that include light stress [53,54]. Here, 10 DEGs (HSP18.1, HSP90-1, HSP22.0, HSP70, UBC4, ERF5, ERF9, TLP, APX3, and EX2) were identified to be involved in light stress. Generally, the HSPs function to prevent damage under heat, high light, and free radicals [55,56]; meanwhile, the up-regulation of HSPs can increase flavonoid content [57]. UBC4 can enhance the flavonoid accumulation by regulating the expression of the CHS gene [58,59]. For other genes, ERFs are involved in the regulation of gene expression by stress factors [60]; TLP can improve plant tolerance [61]; APX3 may be involved in the detoxification of H2O2 [62]; EX2 together with EX1 enables plants to perceive stress signal [63]. In this study, the up-regulation of selected genes (HSP18.1, HSP70, UBC4, ERF5, ERF9, APX3, and EX2) may play important roles in enhancing flavonoid biosynthesis and protecting plants from abiotic stress (e.g., high light) at high altitudes. In addition, elevated expression CAB8, CAB21, and PSBR, as well as proteins ycf3, rbc, rbcL, and petL, at 3300 m versus 2300 m (Tables 1 and 3) suggest that flavonoid accumulation can protect PSII and PSI from stronger UV radiation, which could explain greater biomass that is observed for plants grown at the higher elevation [3].
Based on the above results, a model of high-altitude-induced flavonoid biosynthesis in S. hexdanrum is proposed ( Figure 6). When plants are exposed to high altitudes and The difference in CHS1 gene expression levels based on transcriptomic and qRT-PCR analysis may result from the annotation of the reference transcriptome, amplification efficiency, and/or individual features. Previous studies have found that there are about 85% genes of RNA sequencing consistent with qRT-PCR data [51]. For the down-regulation of the GT6 gene, previous studies on strawberry found that GT6 might be associated with the glucosylation of flavonols [52]; thus, its down-regulation may inhibit the glucosylation of flavonols, which is in accordance with the results of greater contents of flavonols (rutin, quercetin, and kaempferol) at 3300 m than 2300 m.
Initially, flavonoid biosynthesis is regulated by biotic and abiotic stresses that include light stress [53,54]. Here, 10 DEGs (HSP18.1, HSP90-1, HSP22.0, HSP70, UBC4, ERF5, ERF9, TLP, APX3, and EX2) were identified to be involved in light stress. Generally, the HSPs function to prevent damage under heat, high light, and free radicals [55,56]; meanwhile, the up-regulation of HSPs can increase flavonoid content [57]. UBC4 can enhance the flavonoid accumulation by regulating the expression of the CHS gene [58,59]. For other genes, ERFs are involved in the regulation of gene expression by stress factors [60]; TLP can improve plant tolerance [61]; APX3 may be involved in the detoxification of H 2 O 2 [62]; EX2 together with EX1 enables plants to perceive stress signal [63]. In this study, the up-regulation of selected genes (HSP18.1, HSP70, UBC4, ERF5, ERF9, APX3, and EX2) may play important roles in enhancing flavonoid biosynthesis and protecting plants from abiotic stress (e.g., high light) at high altitudes. In addition, elevated expression CAB8, CAB21, and PSBR, as well as proteins ycf3, rbc, rbcL, and petL, at 3300 m versus 2300 m (Tables 1 and 3) suggest that flavonoid accumulation can protect PSII and PSI from stronger UV radiation, which could explain greater biomass that is observed for plants grown at the higher elevation [3].
Based on the above results, a model of high-altitude-induced flavonoid biosynthesis in S. hexdanrum is proposed ( Figure 6). When plants are exposed to high altitudes and high light intensity, genes involved in light stress (e.g., HSPs, ERFs, and TLP) are up-regulated. Photosynthesis will initially be inhibited, as observed with the down-regulation of genes (e.g., CABs, RBCs, and TPT), and subsequently, flavonoids (e.g., flavonols and anthocyanins) will be synthesized and accumulated with the up-regulation of genes (e.g., PAL, CHS1, and CYPs). Additionally, the ratio of anthocyanins will be increased with the up-regulation of genes (e.g., ANS and MYB) and, finally, the anthocyanin pigments in leaves will play critical roles in absorbing the high light radiation (e.g., UV-B) and removing the free radicals (e.g., H 2 O 2 ), which confers the ability of the plants to adapt to the high altitude environmental conditions. down-regulation of genes (e.g., CABs, RBCs, and TPT), and subsequently, flavonoids (e.g., flavonols and anthocyanins) will be synthesized and accumulated with the up-regulation of genes (e.g., PAL, CHS1, and CYPs). Additionally, the ratio of anthocyanins will be increased with the up-regulation of genes (e.g., ANS and MYB) and, finally, the anthocyanin pigments in leaves will play critical roles in absorbing the high light radiation (e.g., UV-B) and removing the free radicals (e.g., H2O2), which confers the ability of the plants to adapt to the high altitude environmental conditions.
S. hexandrum leaves from 3-year-old plants were collected at 12:00 to 14:00 pm on a sunny day from the 2300 and 3300 m sites in July 2016, with 30 leaves at each altitude pooled (×3) and divided into two parts after mixing; one part was rapidly frozen in liquid nitrogen for proteomic partly and the other part was air dried at room temperature for flavonoid analysis (Figure 7). The light intensities at 2300 and 3300 m were measured at 13:00 pm during sunny days (in early July 2016) using the agricultural meteorology monitor (TNHY-7; Zhejiang Tuopu Instrument Co., Ltd., Hangzhou, China).   [3]. The environmental parameters and soil characteristics were shown in Tables S1 and S2, respectively.

Plant Materials
S. hexandrum leaves from 3-year-old plants were collected at 12:00 to 14:00 pm on a sunny day from the 2300 and 3300 m sites in July 2016, with 30 leaves at each altitude pooled (×3) and divided into two parts after mixing; one part was rapidly frozen in liquid nitrogen for proteomic partly and the other part was air dried at room temperature for flavonoid analysis (Figure 7). The light intensities at 2300 and 3300 m were measured at 13:00 pm during sunny days (in early July 2016) using the agricultural meteorology monitor (TNHY-7; Zhejiang Tuopu Instrument Co., Ltd., Hangzhou, China).

Extract Preparation
Air-dried leaf powder (0.3 g) was soaked in ethanol (20.0 mL, 95% v 72 h and then centrifuged at 6000 rpm at 4 °C for 10 min. Following exh

Extract Preparation
Air-dried leaf powder (0.3 g) was soaked in ethanol (20.0 mL, 95% v/v) at 22 • C for 72 h and then centrifuged at 6000 rpm at 4 • C for 10 min. Following exhaustive extraction (×3), the supernatant was increased to 20 mL with 95% ethanol and concentrated in a rotary evaporator at 55 • C. The concentrated residue was re-dissolved with 9 mL methanol [17,18].

Determination of Total Flavonoid Content
Extracts (150 µL) were added into ddH 2 O (2 mL) and 5% NaNO 2 (0.3 mL). After agitating for 5 min, 10% AlCl 3 (0.3 mL) was added and reacted for 1 min and then 1.0 mol/L NaOH (2 mL) was added to stop the reaction. Absorbance was taken at 510 nm [18]. The total flavonoid content was calculated based on the standard curve ( Figure S2).

Determination of Anthocyanin Content
Air-dried leaf powder (0.5 g) was soaked in methanol (5.0 mL, 0.1% HCL v/v) at 22 • C for 72 h, then centrifuged at 5000 rpm at 4 • C for 30 s. Following exhaustive extraction (×3), the supernatant was increased to 20 mL with methanol (0.1% HCL v/v). Absorbance was taken at 530 nm and the anthocyanin content was calculated based on a relative level compared with the blank control [33,65].

Transcriptomic Analysis
Transcriptomic analysis was performed as previously described [36]. For the methodology of RNA-seq, total RNA samples were isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and purified using RNase-free DNase I (TakaRa, Dalian, China); poly-A mRNA was enriched and then used to prepare the paired-end cDNA library (2 × 126 nt) (Illumina, San Diego, CA, USA). Sequences were trimmed with 5 and 3 prime ends to remove poor-quality reads; paired-end sequencing was performed using an Illumina Hiseq 2000 platform. De novo assembly was carried out using Trinity software (version 2.0.6) [66]. Unigenes were searched against a NR, Swiss-Prot, TrEMBL, Pfam, and KOG using a BLASTx procedure with an e-value ≤ 10 −5 [36,67]; DEGs were identified with |log 2 (fold-change) ≥ 1 and p ≤ 0.05 by DESeq2 software and the edgeR package [68,69].

Protein Extraction
Proteins were extracted according to a previously described method [70]. Firstly, frozen leaves were pulverized to a fine powder in liquid nitrogen. Aliquots (200 µL) were then mixed with TCA-2ME-acetone solution (1.8 mL of 10% TCA (w/v), 0.07% 2ME (v/v) in cold acetone) and stored at −20 • C for 1 h. The homogenate was then centrifuged at 10,000× g and 4 • C for 10 min and the precipitate was re-suspended in cold acetone (1.8 mL containing 0.07% 2ME (v/v)). The mixture was then stored at −20 • C for 1 h and then centrifuged at 10,000× g and 4 • C for 15 min. This step was repeated at least three times until the supernatant became colorless. The precipitate was vacuum dried, dissolved in a protein solubilization buffer (1.8 mL of 5 M urea, 2 M thiourea, 2% CHAPS (w/v) with 2% N-decyl-N,N-dimethyl-3-ammonio-1-propane sulfate (w/v), 20 mM DTT, 5 mM phosphine, 0.5% pharmalyte pH 4-6.5 (v/v), and 0.25% pharmalyte pH 3-10 (v/v) in ddH 2 O) and then vortexed for 1 min. Finally, the solubilized mixture was centrifuged at 10,000× g and 25 • C for 15 min and then the supernatant was centrifuged to remove any cellular debris. Protein amount was quantified using a 2-D Quant kit (GE Healthcare, Wauwatosa, WI, USA) and the protein extracts were stored at −80 • C.

Gel Scanning and Image Analysis
The stained gels were scanned using an ImageScanner III (GE Healthcare, Wauwatosa, WI, USA) and images were analyzed using ImageMaster 2-D Platinum v7.0 software (GE Healthcare, Wauwatosa, WI, USA), with three independent biological replicates for the 2300 or 3300 m samples. Spots were automatically detected and matched; mismatched and unmatched spots were artificially modified through manual editing. Spot densities were expressed as mean normalized volumes, and fold changes were calculated between 2300 and 3300 m. Only spots with intensity ratios over 1.5-fold were selected as differentially expressed proteins (DEPs) and the over-expressed proteins at 3300 m vs. 2300 m were subsequently identified.

Protein Identification
The selected DEP spots were excised from CBB-stained gels and digested with trypsin. Peptides were identified using an AB SCIEX 5800 MALDI-TOF/TOF TM system (AB SCIEX, CA, USA) according to a previously described protocol [71]. The obtained peptide masses were used to search the NCBI database using a MASCOT engine (http://www.matrixscience. com (accessed on 1 June 2020)). Biological functions of identified proteins were classified using the Swiss-Prot database (http://www.uniprot.org (accessed on 6 November 2021)). After removing the repeat proteins by selecting the longer sequencing of amino acids for the same protein, the DEPs involved in light response and flavonoid biosynthesis were further screened and validated in this study.

qRT-PCR Validation of Genes Involved in Light Stress and Flavonoids Biosynthesis
Based on the biological functions of the DEGs and DEPs, 17 representative genes were selected to validate their expression level by qRT-PCR. The validation of genes was performed according to our previous protocol [21]. The cycle threshold (Ct) values and standard curves of the reference gene actin (ACT) at different concentrations (0.25, 0.5, 1.0, 1.5, 2.0, and 3.0 µL) were performed to correct expression level of selected genes ( Figures S4 and S5). The primer sequence (Table S3) was designed using the primer-blast tool in NCBI and synthesized by Sangon Biotech (Shanghai, China). Total RNA samples at 2300 and 3300 m were extracted from the leaves using a Plant RNA Kit (R6827; Omega Bio-Tek, Norcross, GA, USA). cDNA was synthesized using a FastKing RT Kit (KR116; Tiangen, Beijing, China) with 42 • C for 15 min and 95 • C for 3 min for one cycle. The qRT-PCR was carried out using a SuperReal PreMix (FP205; Tiangen, Beijing, China) at 95 • C (15 min) for one cycle, followed by 95 • C (10 s), 60 • C (20 s), and 72 • C (30 s) for 40 cycles. The melting curve was analyzed at 72 • C for 34 s. TheREL was calculated using a 2 −∆∆Ct method [72].

Statistical Analysis
All measurements were performed using three biological replicates. Statistical analysis was conducted via a t-test for independent samples. SPSS 22.0 was the software package used, with p < 0.05 as the basis for statistical differences.

Conclusions
From the above observations, greater contents of total flavonoids, flavonols, and anthocyanins in S. hexandrum were observed at higher altitudes, which may be regulated by the up-regulated genes or over-expressed proteins involved in flavonoid biosynthesis and light stress. These findings indicate that light stress may play a determining role in enhancing flavonoid accumulation at high altitudes. In order to exclude the differences in temperature, water, and other factors, the role of light in affecting plant growth and flavonoid biosynthesis will be further studied under controlled laboratory conditions such as shaded habitats.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12030575/s1, Figure S1: 2-DE gels of proteins extracted from leaves of S. hexandrum grown at 2300 and 3300 m; Figure S2: Standard curve of catechin solutions with a range from 25 to 125 µg; Figure S3: Representative HPLC chromatogram of reference standards and samples; Figure S4: The cycle threshold (Ct) values of ACT gene at different volumes (0.25, 0.5, 1.0, 2.0, and 3.0 µL) via PCR amplification with three replications; Figure S5: The standard curve of ACT gene; Table S1: Environmental parameters at the two altitudes over three years; Table  S2: Soil characteristics at the two altitudes; Table S3: Primer sequences of selected genes used for qRT-PCR validation.
Author Contributions: Q.Z., formal analysis, investigation and validation; M.D., investigation and writing-original draft; M.L., conceptualization, project administration, supervision and writingoriginal draft; L.J., resources; P.W.P., writing-review and editing. All authors have read and agreed to the published version of the manuscript.