Identification Of Hydrogen Peroxide Responsive ESTs Involved in Phenylethanoid Glycoside Biosynthesis in Cistanche Salsa Cell Culture

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

J. CHEN et al

Abstract
Hydrogen peroxide is an effective abiotic elicitor that can induce secondary metabolite biosynthesis in plants. We show that in the cell suspension culture of a salt-tolerant medicinal plant Cistanche salsa, the production of bioactive components phenylethanoid glycosides (PeGs) was increased after H2O2 treatment. To identify genes related to PeGs (phenylethanoid glycosides) biosynthesis affected by H2O2, we constructed a suppression subtractive hybridization library of H2O2 responsive genes using a Cistanche salsa cell line and identified 105 expressed sequence tags (ESTs) and 85 genes. EST library functional annotation and gene ontology analyses showed genes related to various stress responses, biosynthesis of secondary metabolites, and transcriptional regulation. Among them, we identified two genes related to the PeGs (phenylethanoid glycosides) biosynthesis pathway (4-coumarate coenzyme A ligase and cinnamate 4-hydroxylase), and two WRKY type transcription factors. The expressions of selected genes after the H2O2 treatment were analyzed by RT-qPCR. Early increased transcription of PeG (phenylethanoid glycosides) biosynthesis pathway genes after the treatment revealed that H2O2 induced PeGs (phenylethanoid glycosides) biosynthesis via up-regulation of its key genes.
Additional keywords: cinnamate-4-hydroxylase, 4-coumarate coenzyme A ligase, suppression subtractive hybridization. Cistanche salsa. phenylethanoid glycosides.

Introduction

Cistanche salsa is a perennial plant parasite on the root of Haloxylos ammodendrun, and it grows in the desert area of western China. For centuries, Cistanche species has been used as traditional herbal medicine (Wang et al. 2012). Bioactive components are phenylethanoid glycosides (PeGs) which have a remarkable effect on reactive oxygen species (ROS) clearance, radiation protection, immunity enhancement, and anti-aging (Nan et al. 2013). PeGs (phenylethanoid glycosides) biosynthesis in Cistanche is initiated from phenylalanine (Fig. 1 Suppl.) and synthesized caffeic acid is further glycosylated to form various PeGs (phenylethanoid glycosides) (Wang et al. 2012). Three main PeGs (phenylethanoid glycosides), echinacoside, acteoside, and 2'-acetylacteoside, are considered to be the most active components in Cistanche (Kobayashi et al. 1984). In order to enhance their content in Cistanche cell culture, various elicitors have been tested including chitosan, putrescine, Ag+, and osmotic stress (Ouyang et al. 2005a,b, Cheng et al. 2005, 2006, Liu et al. 2007, Liu and Cheng 2008). However, genes involved in PeGs (phenylethanoid glycosides) biosynthesis in Cistanche, mainly phenylalanine metabolism pathway genes, have been rarely identified. Only a gene encoding phenylalanine ammonia-lyase (PAL) was recently identified in Cistanche deserticola (Hu et al. 2011). Other genes encoding, for example, cinnamate-4-hydroxylase (C4H), which could catalyze the conversion of cinnamate to cumaric acid, and 4-coumarate coenzyme A ligase (4CL), an essential gene for lignin and flavanoids biosyntheses, have not been found in Cistanche.
We used the cell culture of Cistanche salsa to study PeGs (phenylethanoid glycosides) biosynthesis and related genes expression. We also investigated the effect of an effective elicitor H2O2 on PeGs (phenylethanoid glycosides) biosynthesis. Furthermore, to find early responsive genes involved in PeGs (phenylethanoid glycosides) biosynthesis, we carried out suppression subtractive hybridization (SSH) to construct a cDNA library of H2O2-responsive genes.

Fig. 1. Relative gene expression determined by RT-qPCR after H2O2 treatment. CsWRKY1 - WRKY transcription factor (Contig07), CsC4H1 - cinnamate 4-hydroxylase (Contig09), Cs4CL1 - 4-coumarate: ligase (Contig03), CsHSP1 - heat shock protein 70 kDa (Contig 01), and CsSOD1 - superoxide dismutase (Contig16). Cells were treated with 80 μg dm-3 H2O2 in a liquid medium for 2 h and expressions were compared with a control treated with water. Means +- SD of three biological repetitions; * - P < 0.05, ** - P < 0.01.

Materials and methods

The CS2001 cell line was established from petals of Cistanche salsa (C.A. Mey.) Beck collected at Neimenggu province and subcultured. The culture method was similar to a protocol published previously (Liu et al. 2007). Generally, a solid Murashige and Skoog (1962; MS) medium was used for subculture and a liquid medium for PeGs (phenylethanoid glycosides) biosynthesis (it was the modified MS medium supplemented with 30 g dm-3 glucose and 2.0 mg dm-3 indole-3-acetic acid). For H2O2 treatment, 5.0 ± 0.2 g of cells in a fast growth period, usually 20 to 30 d after they were subcultured in the solid medium, were inoculated into a 50 cm3 liquid medium in a 250 cm3 Erlenmeyer flask and cultured in the dark with or without different H2O2 concentrations. The PeGs (phenylethanoid glycosides) content after 10 d induction by H2O2 was analyzed according to a method published previously (Liu et al. 2007) based on reverse-phase high-performance liquid chromatography with methanol gradient elution.

The cells 2 h after the H2O2 treatment were collected from the medium by filter paper using a Büchner funnel, and then they were frozen in liquid nitrogen and ground to powder. Every 2 g of cell powder was lysed using a mixture of 1 cm3 of 2-mercaptoethanol, 1 cm3 of 200 g dm-3 sarcosyl, and 8 cm3 of a GTC buffer (5 M guanidinium isothiocyanate, 62.5 mM Tris-HCl, pH 8.0, 6.25 mM EDTA, pH 8.0, 5 mM thiourea, and 10 mM dithiothreitol). The cells were then incubated on ice for 15 min, then 1/3 volume of 8.5 M CH3COOK, pH 6.0, was added, and the cells were incubated on ice for 15 min and then centrifuged at 15 000 g for 15 min. The supernatant was added to an equal volume of isopropanol to precipitate total RNA. The total RNA was further purified by an RNeasy mini kit (Qiagen, Hilden, USA) according to the manufacturer’s instructions. Messenger RNA was isolated from total RNA using an Oligotex mRNA midi kit (Qiagen).

phenylethanoid glycosides in Cistanche Salsa

An SSH library was generated from control and 30 % H2O2-elicited cell samples 2 h after the elicitation using a PCR-select cDNA subtraction kit (Clontech, Mountain View, USA). For library construction, the products were purified separately using a gel purification kit (Qiagen) and cloned into a pGEM vector using a TA cloning kit (Tiangen, Beijing, China). Plasmids from independent clones were isolated and sequenced using T7 primer. All expressed sequence tag (EST) sequences from the library (with the vector sequences removed) were used for contig assembly with CAP3 (http://doua.prabi.fr/software/cap3). The ESTs were grouped into contigs and singletons; they were used for homology search using the BLASTx program at NCBI (http://blast.ncbi. nlm.nih.gov). Sequences were then annotated, analyzed, and classified according to gene ontology (GO) terms using Blast2GO (http://www. blast2go.com).
Real-time quantitative polymerase chain reaction (RT-qPCR) was performed according to Bustin et al. (2009). The cDNA samples were synthesized using a PrimeScript RT reagent kit (Takara, Dalian, China) from 0.5 μg of total RNA. RT-qPCR was carried out using SYBR Premix Ex Taq II and ROX plus kits (Takara) on an ABI 7300 real-time PCR system (Applied Biosystems, Foster City, USA) with three technical replicates. Primers were designed using the online Primer3 software according to the instruction supplied by Takara; primers used in this study are listed in Table 1 Suppl. The gene expressions were normalized to 18S RNA, the expression of which was stable in all the samples.

The results were analyzed using R version 3.1.1, the significance of differences was verified by the Student's t-test.

Results

Hydrogen peroxide-induced the biosynthesis of all three main PeGs (phenylethanoid glycosides) in Cistanche salsa suspension cells (Table 1) but with no obvious effect on cell growth (Fig. S2). The content of echinacoside increased already after the 10 μg dm-3 H2O2 treatment (P < 0.05), a higher increase was recorded after the 20 μg dm-3 H2O2 treatment (P < 0.01), and the highest increase was after the 40, 80, and 160 μg dm-3 H2O2 treatments. Similar results are shown for acteoside, but its content peaked after the 80 μg dm-3 H2O2 treatment. The biosynthesis of 2'-acetylacteoside was less sensitive to H2O2 treatment; a significant increase of its content (P < 0.05) was at H2O2 concentrations over 40 μg dm-3. After the 80 μg dm-3 H2O2 treatment, the amount of all the three main PeGs (phenylethanoid glycosides) significantly increased (P < 0.01) over the control. Therefore, this concentration was used in further experiments.
To generate a library enriched for early responsive genes that are up-regulated by H2O2, we used cDNA from Cistanche salsa suspension cells treated with 80 μg dm-3 H2O2 for 2 h. Single-pass sequencing 129 recombinant clones in the SSH library yielded 105 clean ESTs after vector sequence exclusion. These clear ESTs were used for assembly by CAP3, and 18 contigs and 67 singletons were generated (Table 2). Thus, 85 genes were isolated in this library. The mean length of the clean 105 ESTs was 527 bp, which is adequate for a sequence similarity-based functional classification.

phenylethanoid glycosides in Cistanche Salsa

All 85 genes obtained in the library were retrieved by BLASTx against the GenBank non-redundant protein database for sequence similarity (E-value ≤ 0.001). Twenty-one genes showed no hits in the database, they might be specific to Cistanche salsa. The results with BLASThits were imported to the Blast2GO software for further GO and KEGG pathway enrichment analyses. Of the 64 genes with BLAST hits, 50 were annotated according to the description of homologous genes from other species. The annotated genes were named according to the description of BLAST hits. The GO analysis (Fig. 3 Suppl.) shows that among these 50 genes, there were 14 genes involved in responses to stimuli, and over 30 genes related to metabolic processes (Tables 1 and 2). Among these genes, two PeG biosynthesis pathway gene homologs were isolated as expected: 4-coumarate coenzyme A ligase (named Cs4CL1: Contig03) and cinnamate 4-hydroxylase (named CsC4H1: Contig14). Further, RT-qPCR was performed to confirm induced expressions of genes from the library after the H2O2 treatment.

To confirm that essential genes identified from the SSH library were responsive to the H2O2 treatment, the expressions of two PeGs (phenylethanoid glycosides) biosynthesis pathway-related genes (Cs4CL1, CsC4H1), two ROS induced genes (CsSOD1: Contig 16, CsHSP1: CISTH099), and one WRKY family transcription factor-like gene (CsWRKY1: Contig 07) were measured by RT-qPCR after the treatment with H2O2 (Fig. 1). CsC4H1, Cs4CL1, CsSOD1, and CsWRKY1 expressions were up-regulated more than 15-fold, 3-fold, 45-fold, and 4-fold, respectively 2 h after the H2O2 treatment. The expression of CsHSP1 was altered only 1.8 fold after the H2O2 treatment.
The time-course of the expressions of CsWRKY1, CsC4H1, and Cs4CL1 after the H2O2 treatment was further analyzed by RT-qPCR (Fig. 2). The CsC4H1 and Cs4CL1 expressions were highly increased (more than 80-fold and 50-fold, respectively) after the 16-h treatment and then down-regulated after 32 h. The expression of CsWRKY1 was up-regulated more than 20-fold at 4 h and then decreased from 8 h afterward (Fig. 2).

Fig. 2. The relative expression of CsWRKY1 (A), CsC4H1 (B), and Cs4CL1 (C) at different times after treatment with 80 μg dm-3 H2O2. The expressions were normalized to a control; (treated with water). Means +- SD of three biological repetitions; * - P < 0.05, ** - P < 0.01.

Discussion

Hydrogen peroxide is a well-known plant elicitor that shows various effects on plants. Like ROS, it can induce the ROS scavenging system composed of superoxide dismutase, catalase, peroxidases, etc., and it serves as the downstream signal of salt, osmotic, and dehydration

stresses (Wi et al. 2006, Kar 2011, Furlan et al. 2013, Sathiyaraj et al. 2014). It was suggested that H2O2 generation in plants is accompanied by induced secondary-metabolite biosynthesis in the Cistanche salsa cell culture. As expected, our results reveal that PeGs (phenylethanoid glycosides) biosynthesis in the Cistanche salsa cell culture was induced after the H2O2 treatment (Table 1). The content of three main PeGs (phenylethanoid glycosides) increased after the H2O2 treatment indicating its pivotal role in PeGs biosynthesis. Genes enriched in the SSH library further suggested its effect on PeGs (phenylethanoid glycosides) biosynthesis induction in Cistanche salsa.

Many known H2O2-responsive genes, such as ascorbate peroxidase (CISTH069, Karyotou and Donaldson 2005), h-quinone oxidoreductase (CISTH125, Bello, et al. 2001), CsSOD1, CsHSP1 (Baruah et al. 2014), and disease resistance response genes (Contig10, Nanda, et al. 2010), were enriched in the SSH library (Table 3, Table 2 Suppl.). Some of these typical ROS responsive genes with an induced expression after the H2O2 treatment were further confirmed by RT-qPCR (Figs. 1 and 2) indicating that a qualified SSH library was constructed here. Two PeG biosynthesis pathway genes, CsC4H1 and Cs4CL1, were also found in this library and these two gene expressions were induced by the H2O2 treatment in a time-dependent manner (Fig. 2). It suggests that the H2O2 treatment induced PeGs (phenylethanoid glycosides) biosynthesis at the genetic level via the regulation of PAL pathway genes. However, PAL and other downstream PeGs biosynthesis genes (Wang et al. 2012) were not identified in the SSH library; it might be because the library has not been sequenced sufficiently. A relation between CsC4H1 and Cs4CL1 induced expressions and an increased PeGs biosynthesis after the H2O2 treatment should be clarified by further analysis.

In addition to the PAL pathway genes induced by the H2O2 treatment, two WRKY family transcription factors (CsWRKY1, CsWRKY2) were found in the SSH library (Table 3 and Table 2 Suppl.), and a CsWRKY1 induced expression was confirmed by RT-qPCR. Its expression was responsive to the H2O2 treatment earlier than the expression of two PAL pathway genes, Cs4CL1 and CsC4H1 (Fig. 2). From previous studies, co-expression of some WRKY transcription factors and PAL pathway genes has been observed in rice (Gupta et al. 2012). Furthermore, PAL gene expression is up-regulated in an OsWRKY03 over-expression mutant (Liu et al. 2005). The above-mentioned two WRKY family transcription factors might be also involved in PeGs (phenylethanoid glycosides) biosynthesis in Cistanche salsa. Interestingly, dehydration-related ESTs (CISTH067, CISTH064) were found in the SSH library, which may suggest that the H2O2 treatment was related to dehydration responsive genes expression in Cistanche salsa as observed in other plants (Schmidt et al. 2013, Zhou et al. 2013). One gibberellin (GA) signaling-related gene, gibberellin receptor gid1b-like (CISTH092), was identified in the SSH library implying the connection of GA signaling and H2O2 treatment in Cistanche salsa, which was also reported in barley (Bahin et al. 2011). However, more studies are needed to confirm whether these genes are related to PeGs (phenylethanoid glycosides) biosynthesis. Other than the genes mentioned above, the SSH library constructed here also presents genes with or without BLAST hits. It improves our understanding of the H2O2 effect on C. salsa and firstly supplies the information of its genes involved in PeGs biosynthesis.

In conclusion, we assessed the effect of H2O2 on PeGs (phenylethanoid glycosides) induction in the Cistanche salsa cell culture. The SSH library was constructed after the H2O2 treatment, and the expression of selected genes was confirmed by RT-qPCR. Two PeG biosynthesis pathway-related genes and other H2O2-responsive genes were identified. These findings will extend our understanding of H2O2-induced PeGs (phenylethanoid glycosides) biosynthesis in Cistanche salsa to the molecular level, providing the information for gene manipulation of Cistanche salsa cell cultures to improve PeGs (phenylethanoid glycosides) production.

From: ' Identification of hydrogen peroxide responsive ESTs involved in phenylethanoid glycoside biosynthesis in Cistanche salsa cell culture' by J. CHEN et al.

--- BIOLOGIA PLANTARUM 59 (4): 695-700, 2015 J. CHEN et al. DOI: 10.1007/s10535-015-0541-y

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