Expression response of chalcone synthase gene to inducing conditions and its effect on flavonoids accumulation in two medicinal species of Anoectochilus

Anoectochilus roxburghii and Anoectochilus formasanus are the major species of genus Anoectochilus used in traditional Chinese medicine for their abundant content of flavonoids and some other medicinal constituents. In recent years, their wild resources are gradually exhausted due to over-collection and ecological deterioration. Artificial cultivation and tissue culture are employed to increase production. In this study, the open reading frame, promoter and genomic sequences of the chalcone synthase (CHS) gene were cloned from these two species according to their transcriptome information, and used for expression analysis in response to the induction of phenylalanine, ultraviolet light and NaCl, and its effect investigation on accumulation of flavonoids. The results showed that the expression of the CHS genes was upregulated in response to these inductions and resulted in increasing accumulation of total flavonoids. However, the increased flavonoids induced by phenylalanine and ultraviolet light were mainly allocated into the anthocyanidin branch of flavonoids biosynthesis. Not only did it improved the medicinal value, but might have inhibitory effect on plant growth because of the increased malondialdehyde accumulation. Under the induction of appropriate concentration of NaCl, the medicinal constituents of flavonoids were increased without inhibition to plant growth.


Results
The CHS genes and their putative proteins. 8.76 and 10.42 Gb raw bases, and 29213781 and 34742855 raw reads were obtained by RNA-seq of the A. roxburghii and A. formosanus samples. Through quality inspection and filtration, 8.28 and 9.85 Gb clean bases, and 27597990 and 32836885 clean reads were retained, respectively, with Q20 (sequence error rate below 1%) greater than 96% and Q30 (sequence error rate below 0.1%) greater than 90% (Fig. S2). From these clean reads, 130024 and 116423 transcripts were assembled, 30265 and 24425 of them were annotated by SWSSPROT, and 35176 and 28691 of them were annotated by KGO, respectivly. One of them was annotated as the CHS gene for A. roxburghii and A. formosanus, respectively. These two transcripts shared 94.80% similarity (Fig. S3).
Function evaluation of the CHS genes. The green fluorescence signal was observed in the nucleus of the inner epidermis cell of onion transiently expressing vectors pCAMBIA2300-35S-CHS-eGFP, whereas it distributed in the whole cell infiltrated by the negative control vector pCAMBIA2300-35S-eGFP (Fig. 2). This result indicated the subcellular localization of the CHS protein in nucleus in A. roxburghii and A. formosanu.
By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), an obvious additional band about 44.3 kDa was separated from the Escherichia coli lines transformed by the CHS genes of these two medicinal species, and induced by isopropyl β-D-thiogalactopyranoside (IPTG) (Fig. S7). The in vivo CHS enzyme activities of thetransformed E. colilines of the A. roxburghii CHS gene was significantly higher than that transformed by the A. Formosanus CHS gene, while the in vitro activities showed no significant difference (Figs. 3A,B and S8).
From the rice calli transformed by the CHS genes, three T 1 lines (two of A. roxburghiiand one of A. formosanus) were identified as positive by PCR amplification of the CHS gene fragment (Fig. S9). Their contents of total flavonoids were 1.40, 1.68 and 1.26 times higher than that of the untransformed acceptor line, respectively (Fig. 3C).
All these results confirmed that the two amplified ORF sequences were the CHS genes of A. roxburghii and A. formosanus, respectively, and registered at GenBank with accession numbers MK370742 and MK370743.
Differential expression in response to inducing conditions. Under the induction of Phe and UV, the expression of the CHS genes was upregulated significantly in A. roxburghii and A. formosanus, and reached its peak values (81.53 and 7.49 times of the 0 h control, respectively) at 4 and 8 h of the Phe induction, and (21994.7 and 3654.7 times of the 0 h control, respectively) at 8 and 12 h of the UV induction (Fig. 4A,B). In response to the NaCl induction, the expression of the CHS genes was continually upregulated and reached about three times of the 0 h control in A. roxburghii, while the upregulated expression reached 36.2 and 19.5 times of the 0 h control at 2 and 12 h of the NaCl induction in A. formosanus (Fig. 4C). This result indicated the strong expression response of these two CHS genes.
From the products of the second and the third rounds of thermal asymmetric interlaced PCR (TAIL-PCR) amplification from the genomic DNA samples, specific fragments were separated and sequenced for prediction of promoters and cis-acting elements of the CHS genes by Plant CARE software (Fig. S10). The promoter sequence of the CHS gene of A. roxburghii was 342 bp long containing enhance elements TATA-box (4), 5′-UTR Py-rich stretch (1) and CAAT-motif (6), light responsive elements G-box (2), sp1 (18) and I-box (1), and anaerobic inducing element ARE (1). The promoter sequence of the CHS gene of A. formasanus was 314 bp long containing enhance elementsTATA-box (1), AT-rich sequence (1) and CAAT-box (5), light responsive elements G-box (2), CATT-motif (1), CGT-motif (5), GA-motif (1) and sp1 (2), MeJA-responsive elementsCGTCA-motif (1) and TGACG-motif (1), and anaerobic inducing element ARE (1) (Fig. S11). This result might partially explain the expression response of the CHS genes to the UV and NaCl induction. In response to Phe induction, the accumulation of total flavonoids and FRSA in A. roxburghii and A.formoasnus was both increased and reached their peak values (1.38, 1.51, 1.30, and 2.06 times of the 0 d control) on the 5th, 3rd, and 4th day, respectively (Fig. 6A,B). The difference of total flavonoids content was not significant between these two species, but the increased range of FRSA in A. roxburghii was significantly lower than that in A. formoasnus. The accumulation of rutin, quercetin and kaempferol was decreased during the first three days of the induction but rebounded after that with different ranges between the two species ( Fig. 6C-E).The accumulation of anthocyanidin and MDA was increased continually with different ranges between the two species (Fig. 6F,G). The asterisks (*), the black dots (•), the hollow dots (○), the hollow triangles (△) and the plus (+) represent the activation sites, the catalytic residues, the conserved residues, the malonyl-coenzyme A binding motif and the nuclear localization signal, respectively. (B) Phylogenetic tree among the putative proteins of gene CHS of 19 relative species.
Under UV induction, the accumulation of total flavonoids and FRSA in the two species was increased and reached their peak values (2.09, 2.01, 1.47, and 1.82 times of the 0 h control) on the 5th, 4th, 3rd, and 2nd day, respectively (Fig. 7A,B). The accumulation of rutin, quercetin and kaempferol was decreased and rapidly descend to their valley values on the 1st to 5th day ( Fig. 7C-E). The accumulation of anthocyanidin and MAD was increased continually (Fig. 7F,G).
In response to NaCl induction, the accumulation of total flavonoids and FRSA was increased continually with different ranges between A. roxburghii and A. formasanus (Fig. 8A,B). The rutin accumulation in A. roxburghiiwas increased sharply on the 5th and 6th day and reached as high as 21.28 times of the 0 h control, whereas that in A. formasanus did not changed obviously (Fig. 8C). The accumulation of quercetin, kaempferol and anthocyanidin was increased with different ranges between the two species ( Fig. 8D-F). The MAD content was only increased slightly (1.23 and 1.26 times of the 0 h control, Fig. 8G).

Discussion
Numerous studies showed that the biosynthesis of flavonoids, as well as flavonols, took place exclusively in endoplasmic reticulum, cytosol, and other cytoplasmic organelles 19,[36][37][38] . In this study, the subcellular localization of the CHS proteins of A. roxburghii and A. formasanuswas targeted to the nucleus (Fig. 2). This result was also evidenced by the bioinformatics prediction of a nuclear localization signal (Arg 66 -Lys 67 -Arg 68 -His 69 ) at the N-terminal of their primary structure (Fig. 1A). Some recent reports from a number of different plant species www.nature.com/scientificreports www.nature.com/scientificreports/ not only documented the presence of flavonoids in nuclei 39,40 , but also localized at least two of their biosynthetic enzymes to nuclei in several cell types in Arabidopsis 41 . It was speculated that the Anoectochilus CHS proteins might catalyze flavonoids synthesis of the phenylpropane metabolism pathway in nucleus, and their products might be involved not only in the basal metabolism, stress response, reproductive development and many other growth and development processes [30][31][32][33][34][35] , but also in the transcriptional regulation of related genes.
Under Phe, UV and NaCl induction, the expression of the CHS genes of A. roxburghii and A. formasanus were upregulated with different ranges (Fig. 4). The cis-acting elements related to light and anaerobic response were predicted in the promoter sequences of the two CHS genes (Fig. S11) [42][43][44] . These results not only explain the increased accumulation of total flavonoids in the two species under these inducing conditions, but also confirm the rate-limiting role of the CHS enzyme for flavonoids biosynthesis of phenylpropanoid pathway (Fig. S1) 16,[20][21][22][23][24][25][26][27] .    (Fig. S1). This was not conducive to improving medicinal value of these two species. Even more, the increased MDA accumulation revealed the inhibitory effect of Phe and UV on plant growth (Figs. 6G and 7G). Similar effect was also found in cell suspension culture of strawberry and calli of Hydrocotyle bonariensis 28,29 . In response to NaCl induction, the increased flavonols (Fig. 8C-E), especially the sharp peak content of rutin on the 5th and 6th day of induction in A. roxburghii, indicated that the increased accumulation of total flavonoids (Fig. 8A) was mostly allocated into the flavonol branch (Fig. S1), while the MAD content was only increased slightly (Fig. 8G). Along with the higher accumulation of total flavonoids, FRSA, and the accumulation of flavonolsin A. roxburghiiunder non-inducing conditions (Fig. 5), it was suggested that NaCl induction of appreciate concentration (100 mmol/L) in artificial cultivation or tissue culture of A. roxburghii could efficiently increase the accumulation of total flavonoids and flavonols, and improved the medicinal value.

Materials and Methods
Sample preparation. The seedlings of A. roxburghii and A. formosanus were cultured on MS medium for 18 weeks. The uniform seedlings were divided into three groups. Two groups were transplanted into a plastic mesh grid for aquaculture. On the fifth day, Phe and NaCl were added into the nutrient solution with final concentration of 4 mg/L and 100 mmol/L, respectively. The other group was transplanted into plastic pots (five seedlings per pot) with nutritional soil and vermiculite (3:1), and submitted to UV induction of 253.7 nm after recovering. One leaf of each sample of 0 to 24 h of the induction was pulverized to fine powder in liquid nitrogen, and used for RNA extraction with RNeasy Plant Mini Kit (Qiagen, China). After release of probable DNA contamination by RNase-free DNase I (Qiagen, China), detection for concentration, purity and integrity on spectrophotometer (NanoDrop One, Thermo Fisher Scientific, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, USA) respectively, part of each RNA samples was reverse transcribed to cDNA by using PrimeScript RT Reagent Kit (TaKaRa Japan). Part of pulverized leaf samples of the control (0 h) were mixed and used for extraction of genomic DNA with the method of cetyl trimethylammonium bromide (CTAB).
Transcriptome sequencing and CHS gene cloning. Part of the RNA samples of the control (0 h) were mixed and sequenced by Illumina HiseqXten platform at MajorbioBioTech Co., Ltd. The clean reads were assembled by Trinity v2.4.0 (https://github.com/trinityrnaseq/trinityrnaseq/wiki), and used to search for transcript sequences of the CHS genes by SWSSPROT (https://swissmodel.expasy.org/) and KOG (https://genome.jgi.doe. gov/Tutorial/tutorial/kog.html). According to the transcript sequences, a pair of specific primers (Table S1) was synthesized and used to amplify the ORF and genomic sequences of the CHS genes from the cDNA and the genomic DNA samples, respectively, by using Prime STAR HS DNA Polymerase (TaKaRa, China) with proof reading activity. The amplified products were purified by using Universal DNA Purification Kit (Tiangen, China), added dATP at the 3′ ends by using Taq TM (TaKaRa, China), cloned into pMD19-T vector (TaKaRa, Japan), and sequenced at Sangon Biotech Co., Ltd (Shanghai, China). The sequencing results were aligned for gene structure on NCBI website (http://www.ncbi.nl-m.nih.gov), and predicted for putative proteins by using online tool SWISS-MODEL (https://swissmodel.expasy.org/). Phylogenetic analysis was conducted among the putative proteins by using MEGA7.0 software (https://www.megasoftware.net/).

Function evaluation of the CHS genes.
Three pairs of specific primers with appropriate recognition sites (Table S1) were used to amplify the ORF sequences without or with the termination codons from the harbored pMD19-T vectors by using Prime STAR HS DNA Polymerase (TaKaRa, Japan). The amplified products were purified as above, and inserted into transient expression vector pCAMBIA2300, prokaryotic expression vector pET-28a (+) and moncotyledonous expression vector pZZ00026, respectively, by using CloneExpress One Step Cloning Kit (Vazyme, China, Fig. S13). www.nature.com/scientificreports www.nature.com/scientificreports/ The transient expression vectors pCAMBIA2300-35S-CHS-eGFP harboring the CHS genes of the two species, as well as the empty plasmid pCAMBIA2300-35S-eGFP (negative control), was infiltrated into the inner epidermis of onion. After incubation at 28 °C under dark for 24 h, the green fluorescence signal for subcellular localization was observed and photographed under fluorescence microscope (Olympus BX63, Japan) 45 .
The prokaryotic expression vectors pET-28a (+)−CHS were transformed into E. colis train BL21 with freeze-thaw method. Until OD 600 ≈ 0.6, the transformant cultures were added with IPTG to a final concentration of 0.5 mmol L −1 , and incubated at 37 °C for 2 h. The heterologous expression of the CHS genes was detected by SDS-PAGE. The proteins were purified by using Ni-NTA Sefinose TM Resin Kit (Sangon China), and determined for concentration by using NanoDrop ™ One/OneC B-50Q (Thermo, USA). According to the manual (Table S2) of CHS enzyme activity kit (Genmed Scientifics Inc. USA), the purified protein and the IPTG-induced E. coli cells were reacted with 4-coumaric-CoA and 3 untis malonyl-CoA in the presence or absence of luteolin (a sensitive inhibitor of CHS), respectively. Along with the synthesis of chalcone, the released 4 units of CoA-SH with Ellman reagent 5,5-dithiobis (2-nitrobenzoic acid, DTNB) to produced yellow 5-thio-2-nitrobenzoic acid (TNB). The activity of chalcone synthase was quantitatively analyzed by the change of its absorption peak (412 nm wavelength). The in vitro and in vivo CHS enzyme activities were analyzed by the change of the absorption peak at 412 nm.
The moncotyledonous expression vector pZZ00026-Ubi-CHS-T-nos was mobilized into Agrobacterium tumefaciens strain EHA105, and used to transform rice calli of variety ZH11 46 . The transformed lines were identified by PCR amplification of a 1205 bp fragment of the CHS genes, and detected for content of total flavonoids by spectrophotography (described later). expression analysis under inducing conditions. Two pairs of RT-qPCR primers (Table S1)  As described by Liu et al. 49 , six arbitrary degenerate primers (AD) and three nested primers (Table S1) complementary to the coding sequences of the CHS genes were synthesized, and used to amplify the promoter sequences of the CHS genes from the genomic DNA samples by TAIL-PCR. The products of the second and third rounds of amplification were separated by 1.2% argarose gel electrophoresis, purified and sequenced as above. The sequenced results were used for prediction of cis-affecting elements by Plant CARE software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Quantification of total flavonoids and free radical scavenging activity. The other leaf of each sample of 0 to 6 d of the induction was dried at 55 °C, ground to fine powder (extration quality), extracted in 95% alcohol in an ultrasonic instrument at 25 °C for 30 min. The extracts were filtered through Whatman No. 1 paper filter under reduced pressure (extraction volume). Referring to China national standardization for determination of total flavonoids in propolis 50 , 1 mL of each of the filtrates was added with 0.4 ml of 5% NaNO 2 , kept for 5 min, added with 0.4 ml of 10% Al(NO 3 ) 3 , kept for 5 min, added with 4 ml of 4% NaOH for coloration, incubated at room temperature for 20 min, and determined for absorbency at 420 nm in a UV-1800 spectrophotometer (Shimadzu, Japan). The content of total flavonoids was calculated as: where, A 420 were the absorbance at 420 nm, V were total volume of the extract, m were the extration quality from the leaf of each sample (1 g), d were the dilution multiple.
As described by Sharma and Bhat 51 , 2 mL of each of the filtrates, as well as two milliliters of ethanol (blank control), were added with 2 ml of 0.04 mmol/L ethanol solution of 1-diphenyl-2-picrylhydrazyl (DPPH), incubated in dark for 20 min, and detected the absorbance at 517 nm in an UV-1800 spectrophotometer. The free radical scavenging activities were calculated as: where, A i , A b and A n were the absorbance of the induced samples, the non-inducing samples and the blank control.
Quantification of flavonalconstituents and anthocyanidin. Ten  www.nature.com/scientificreports www.nature.com/scientificreports/ As described by Tanaka et al. 52 , one leaf of each sample of 0 (control), 1, 2, 3, 4, 5 and 6 d of the induction, was pulverized to fine powder in liquid nitrogen, extracted with acidified (1% HCl) methanol in dark with shaking for 48 h, and centrifuged at 4000 g for 10 min. The supernatant was used to determine for absorbance at 535 nm in an UV-1800 spectrophotometer. The anthocyanidin content was indicated by absorption value. Determination of MDA content. As described by Yu et al. 53 , the other leaf of the each sample of 0 (control), 1, 2, 3, 4, 5 and 6 d of the induction was pulverized with 10% trichloroacetic acid (TCA) and centrifuged at 4000 g for 10 min. Two milliliters of the supernatant was added with 2 mL of 0.06% thiobarbiuricacid (TBA), and bathed in boiling waterfor 10 min. After cooled, the samples were centrifuged at 7000 g for 5 min. The supernatant was determined for absorbance at 450, 532, and 600 nm, respectively, in an UV-1800 spectrophotometer. The MDA content was calculated as: where, A 450 , A 532 , and A 600 were absorbance at 450, 532, and 600 nm, respectively. V,V' , and W were total volume of the extract, the determined volume (2 mL), and the fresh weight of the sample.