Introduction
Tree peony (Paeonia suffruticosa Andr.), a deciduous bush, is known as one of the top 10 well-known traditional flowers in China for its high ornamental and commercial value. It has been proposed as pot plant and cut flower production and is also widely used as an outdoor ornamental plant (Beruto et al. 2004). Currently, tree peony is primarily propagated by tuft division and grafting under a commercial point of view (Cheng 2001; Gao et al. 2001). However, The traditional propagation approaches already cannot satisfy the needs of the market development because of lower propagation efficiency and high production costs (Bouza et al. 1994; Orlikowska et al. 1998). Taking into consideration the problems w ith conv entional p ropagation m ethod, t he propagation techniques of tree peony can benefit greatly from the plant tissue culture. Since Partanen (1965) released the reports on tissue culture of tree peony, scholars both domestic and abroad have done a lot of researches in this field (Beruto and Curir 2007; Chen 2005; Demois and Partanen 1969; He et al. 2011; Luo 2010; Shen et al. 2014; de Silva et al. 2012; Wang et al. 2008; Wang et al. 2012; Wang et al. 2014). Tree peony tissue culture currently has suffered from several difficulties; serious browning and contamination, difficulty in rooting and transplanting, in which rooting is the most difficult challenge (de Silva et al. 2012).
Thus far, most studies on the rooting of tree peony plantlets in vitro are mainly related to several factors, including the basal medium, plant growth regulators, the phenol compound, temperature, and activate carbon (AC) (Fu et al. 2011; He et al. 2011; Li et al. 2007; Luo 2010) and have made progress to a certain extent. A report shows that Planteck in Canada is the only company in the world offering large quantities of Itoh peonies from the tissue culture to the horticultural market in China Flower & Gardening News (2012). Itoh peonies, which flowers like the tree peony’s and the habit of growth similar to the herbaceous peony’s, are the herbaceous and tree peonies hybrids and are relatively easier to gain success than the tree peony (wood plant) in tissue culture (Shen 1992). Beruto et al. (2004), Li (2004), and Wang (2008) obtained the rooting plantlets of tree peony in vitro successfully, but the technique was not applied to a quite large-scale production. Wang (2008) found a phenomenon that the vascular bundles of root weren’t connected to the stem’s in the rooting plantlets of tree peony in vitro, and it could result in the low survival rate of transplanting. Proteins plays essential roles in all aspects of plant growth and development cellular processes, such as blooming and root formation (Li 2002). However, the research on the rooting protein of the plantlets of tree peony in vitro has not been published to date.
Proteomics, the large-scale analysis of proteins, has become the center of the science research in the post-genomic era. Two-dimensional gel electrophoresis (2-DE), combined with mass spectrometry technology, can be able to quickly identify the quantity of protein expression with good repeatability and high credibility (Eschen et al. 2004). At present, the applications of the 2-DE proteomic are mainly concentrated in model plants (Caruso et al. 2008), crop plants (Caruso et al. 2008; Porubleva et al. 2001; Zhang et al. 2015), medicinal plants (Kumari et al. 2015), and some forestry trees (Hua et al. 2014; Simova-Stoilova et al. 2015) to reveal the molecular basis of development and stress response. However, there are almost no researches of 2-DE proteomic on ornamental plants, especially in tree peony.
The adventitious root induction of plantlets in vitro of tree peony, one of the national flowers for China, is a key problem of its regeneration system. The present study on the proteins associated with adventitious root induction of tree peony aimed to sift the proteins associated with adv entitious root induction and further conquer the difficult rooting problem of tree peony plantlets in vitro.
Materials and Methods
Plant materials and culture
With the characteristics of strong growth potential and high rooting rate, ‘Wu Long Peng Sheng’ was selected as the stock plant in the experiment (Wang et al. 2012). The plantlets in vitro, induced from the sterilized axillary buds of P. suffruticosa ‘Wu Long Peng Sheng’ at the key laboratory of Garden Plant Biotechnology in Henan Agricultural Univ ersity, were uniform in size and growth state as laboratory materials in the experiment. The sterilized axillary buds were cultured in solid medium, containing MS + 6-BA 0.5 mg·L-1 + NAA 0.5 mg·L-1 + sucrose 30 g·L-1 + agar 7 g·L-1 (pH 5.8), under normal conditions (at a temperature of 24 ± 1°C, the light intensity of 36 μmol·m-2·s-1, and the illumination time of 12 h·d-1). After 35 d, the rootless plantlets in vitro were transferred to the rooting medium, containing WPM + IBA 4 mg·L-1 + sugar 30 g·L-1 + Gellan gum 2 g·L-1 (pH 5.8), and were cultured under normal conditions above. The previous studies in our lab showed that root primordia of tree peony was induced on the 3rd day and formed on the 5th day after rooting culture (He et al. 2011). Samples were taken respectively at 0, 1, 2, 3, 5 and 7 d during the rooting culture. 30 test-tube plantlets were selected in each treatment randomly and rinsed with pure water. The caudexes were cut after drying water and ground to a fine powder in liquid nitrogen quickly. The powders were stored at the temperature of -70°C for subsequent experiment.
Protein extraction and quantification
The previous screening experiment of our laboratory indicated that TCA/acetone precipitation was the most appropriate protocol for protein extraction of tree peony among the three methods: TCA/acetone precipitation, phenol extraction and SDS-based extraction (Wang et al. 2010). TCA/acetone precipitation was based on the work of Damerval et al. (1986) with some modifications. Approximately 2 g powder stored was resuspended with 3.5 volumes of ice-cold acetone containing 10% TCA (w/v) and 0.07% B ME ( w/v ) . The m ixture was homogenized and proteins were precipitated at 20°C for 2 - 3 hrs before centrifuging at 20,000 g for 20 min at 4°C. The supernatant was discarded and the pellet was washed by suspending in acetone/water (90 : 10 v/v) until a clear supernatant was obtained. The pellet was resuspended with ice-cold acetone and kept at -20°C for 1 hr, then centrifuged. Centrifugation was repeated as above, and then the pellet dried by vacuum was kept at -20°C until use. The dried pellet kept at -20°C was dissolved in IEF buffer (7 mmol·L-1 urea, 2 mmol·L-1 thiourea, 4% CHAPS, 1% DTT, 0.5% IPG buffer pH 3 - 10) by vortexing for 1 hr at 30°C. The supernatant was collected, and the residue was re-extracted with IEF buffer after centrifuging at 25°C for 30 min at 30,000 g. The combined supernatants were centrifuged and used for proteins estimation and 2-DE analysis. The protein concentration was estimated according to the protocol of Bradford (1976) with bovine serum albumin as a standard.
Two-dimensional electrophoresis
Isoelectric focusing (IEF) was carried out on an Ettan IPGpor II system (GE Healthcare Life Science) with immobilized pH gradient (IPG) strips (pH 3.0 - 10.0, linear gradient, 24 cm), according to the method of Görg et al. (1999) 1999) with several modifications. The IPG strips were rehydrated for 12 h at 50 V with 450 μL rehydration buffer (7 mol·L-1 urea, 2 mol·L-1 thiourea, 4% CHAPS, 65 mmol·L-1 DTT, 0.5% pharmalyte, 0.002% bromophenol blue) containing a certain amount of proteins. The voltage settings for IEF were 250 V for 1 hr, 500 V for 1 hr, 1000 V for 1 hr and 10,000 V for 10.5 hrs. The temperature was maintained at 18°C and the current limit was 50 μA per strip. Prior to the second dimension analysis, the strips were equilibrated in 10 mL equilibration solutionⅠ (50 mmol·L-1 Tris-HCl, pH 8.8, 6 mol·L-1 urea, 20% glycerol, 4% SDS, 1.5% DTT) for 15 min and re-equilibrated in 10 mL equilibration solutionⅡ (50 mmol·L-1 Tris-HC1, pH 8.8, 6 mol·L-1 urea, 20% glycerol, 4% SDS, 2.5% iodoacetamide, 0.002% bromophenol blue) for 15 min. The separation in the second dimension was performed on the Ettan Dalt II System (Amersham Biosciences) with lab cast 1 mm SDS polyacrylamide gels (12%): 2 w/gel (45 min) and 10 w/gel (6 hrs).
Protein visualization, image analysis
Proteins were visualized by colloidal Coomassie brilliant blue (CBB) staining (Candiano et al. 2004). Gels were fixed for 40 min in a fixing solution (10% glacial acetic acid, 40% methanol) and stained 8 hrs or overnight in a staining solution (20% methanol, 0.12% w/v CBB G-250, 10% phosphoric acid, 10% w/v ((NH4)2SO4 ). Stained gels were washed in ultrapure water, and then were scanned with alpha gel imaging analysis system. Image analysis was performed with Image Master 2-D platinum Software version 5.0.
Mass spectrometry and statistical analysis
The differentially expressed protein spots were selected and analyzed by using Electron Spray Ionization (ESI) and Tandem Mass Spectrometry (MS/MS). Database searches were performed with using Genbank (http:// www.ncbi. nlm. nih.gov/) and Swiss-Prot (http://www. expasy.ch).
Results and Discussion
The extraction of tree peony caudexes protein
Protein extraction was the first step of two-dimensional electrophoresis, and was also one of the key factors to determine the repeatability and resolution of the 2-DE map (Carpentier et al. 2005; Gevaert 2000; Rabilloud 2002) Rabilloud 2002). At recent, the standard protocol of protein extraction applied to all kinds of samples proteome analysis has not been reported, the specific protocols of a new plant sample still need to explore according to its interfering substance and the characteristics. TCA/acetone precipitation has broadly been used for plant protein extraction as a general method for its remarkable value for better inhibition on proteases activity and removal of phenols interfering compounds (Damerval et al. 1986; Jorge et al. 2005; Rodrigues et al. 2009). In the study, The 2-DE maps (Fig. 1) of the tree peony caudexes protein has proved the TCA/acetone precipitation method were suitable for 2-DE system of tree peony possibly because TCA could eliminate high protease activity of tree peony tissues instantly to reduce protein degradation effectively.
The 2-DE maps of tree peony caudexes protein at the root induction period
In order to investigate the changes of caudexes proteome in response to rooting induction, 2-DE analysis of the total proteins in the caudexes of tree peony in vitro was carried out during the root induction period (0 – 7 d). The results of 2-DE maps (Fig. 1) showed that most of the proteins in the caudexes of tree peony in vitro were concentrated in the acidity gel region (pH 4 - 7), only few proteins were in the alkaline area. The 2-DE maps for different periods had a good similarity, but they still revealed some differences on the quantity of protein spots and the abundance of same protein spots. The results showed that the number of protein spots ranged from 373 to 462 on 2-DE maps during the root induction period (0 – 7 d) and the maximum was presented to the 2nd day (Fig. 1C). And we found that the abundance of 8 protein spots was obviously changed with the extension of the time of rooting induction. The abundance of a few protein spots had a small fluctuation at the first two days, but no changes generally (Figs. 1B and 1C). The abundance of No. 1, 4, 5, 6 and 7 protein spots were sharply reduced, and that of No. 2 and 3 spot proteins had an opposite tendency on the 3rd day (Fig. 1D). A new spot (No. 8) appeared in the 2-DE map on the 5th day and the abundance of most protein spots had shown an upward trend after day 5 (Figs. 1E and 1F).
Identification of tree peony caudexes differential proteins at the root induction period
Eight differential expression protein spots were eluted from representative 2-D gels, digested with trypsin, and analyzed with a mass spectrometer (LC/ESI/MS/MS). The 8 proteins were identified by comparing the results of mass spectrometry and the amino acid rank in Genbank database and Swiss-Prot database (Table 1). The molecular weight (MW) and isoelectric point (pI) of the 8 proteins spots were identified, and the proteins were categorized into four types of ATP synthase β-subunit belonged to Paeonia. The protein spots 1 - 4 and 8 were similar to the ATP synthase β-subunit of P. suffruticosa. The peptide information of protein spots 5, 6 and 7 were consistent with the ATP synthase β-subunit of P. tenuifolia, P. californica and P. brownie respectively. The protein spots 1 - 4 and 8 were identified as a single protein and distributed in different locations in the 2-DE gel probably because of the presence of dimeric and monomeric forms of the ATP synthase beta subunit, the different post-translational modification and processing, and the protein chemical modification (Porubleva et al. 2001; Zhu et al. 2006).
ATP synthase β-subunit
ATP synthase is a key enzyme of energy metabolism in higher plants, which is widely distributed in the inner membrane of mitochondria and chloroplast thylakoid membrane. It synthesizes ATP driven by the transmembrane proton motive force and involves in oxidative phosphorylation and photosynthetic phosphorylation reaction (Boyer 1997). Some researchers hav e indicated that ATP and i ts related proteins were associated with the adventitious root formation. Haissig (1990) has found that ATP concentrations in the base of auxin-treated cuttings are about twice as great as in controls at the fourth day of propagation, and then has speculated that spatiotemporal changes in ATP concentrations are positively related to normal and auxin-induced callusing-rooting in Pinus banksiana. Sukumar et al. (2013) has indicated that the localized synthesis of ATP-binding cassette B19 (ABCB19) protein after hypocotyl excision leads to enhanced IAA transport and local IAA accumulation driving adventitious root formation in Arabidopsis thaliana. Furthermore, Bellamine and Gaspar (1998) 1998) have detected that the ATPase activity of the microsomal vesicles of poplar shoots is increased after 7 h induction on rooting medium, and they have indicated strongly that the ATPase activ ity may be necessary for the induction and expression of rooting.
The ATP synthase is composed of the membrane-bound F0 portions and the soluble F1. The soluble F1 has five subunits (α3, β3, γ, δ, ε) for catalyzing ATP hydrolysis and synthesis (Stock et al. 1999). The β-subunit, as catalytic active center, plays an important role in the process of ATP synthase catalysis by binding ADP and converting it to ATP in the presence of transmembrane proton gradient (Ni and Wei 2002; Lai et al. 2010). However, even with relatively strong nucleotide binding site, isolated α-, β- subunits can still not play a role, ATP hydrolysis and synthesis only can be catalyzed by mixtures of some or all F1 and F0 subunits (Müller and Grüber 2003). The experimental results showed that the obviously decreased period of t he m ajority ATP synthase β-subunit (the 3rd day) were consistent with the induction period of adventitious root primordial of tree peony plantlet in vitro (He et al. 2011). The decreased phenomenon was possibly because the ATP synthase β-subunit was consumed for assembling ATP synthase, and ATP synthase catalyzed ATP synthesis in order to meet the energy needs of cellular proliferation and differentiation in the adventitious root primordia formation process.
The experimental had built the 2-DE system of tree peony to explore the peony proteome, laying a basis on tree peony proteomic study. Meanwhile, we inferred that ATP synthase β-subunit was probably associated with adventitious root initiation of tree peony plantlets in vitro from the change trend of ATP synthase β-subunit on the 2-DE map. It would explore a new path for conquering the difficult rooting problem of tree peony plantlets in vitro.
