Polyphenol Oxidase ( PPO ) in Early Stage of Browning of Phalaenopsis Leaf Explants

Explant browning is one of the most common problems during the early stage of tissue culture. The aim of this study was to explore the roles of polyphenol oxidase (PPO, EC 1.10.3.2) in the browning process of leaf explants of Phalaenopsis (Doritaenopsis Queen Bee “Red Sky”) under a routine culture condition. Along with the observation of the time course of browning and PPO distribution, PPO gene expression in leaf explants were investigated. In addition, the expression and activity of PPO were also examined. This work provides some insights into further understanding the relationship between PPO activation and explant browning.


Introduction
Explant browning can occur in laboratory and in nature (Martini et al., 2013).It is one of the major problems in the tissue culture process and generally considered to be associated with the enzymatic oxidation of phenolic compounds.Polyphenol oxidase (PPO), peroxidase (POD), and L-phenylalanine ammonialyase (PAL) are three major proteases involved in plant tissue browning (Mikal, 2000;Xu & Li, 2006).PPO is an enzyme widely distributed in plants and catalyzes two types of reactions: (a) hydroxylation of phenols (such as tyrosine) to an o-diphenol (cresolase activity) (EC1.14.18.1) and (b) oxidation of diphenols to o-quinones (catecholase activity) (EC 1.10.3.1).PPO is a copper-containing enzyme which uses oxygen molecule as a co-substrate (Mayer, 2006).
Enzymatic browning in plants is mainly related to the oxidation of phenolic compounds to unstable o-quinones which are highly electrophilic molecules and their polymerization can lead to the appearance of brown, red or black pigmentation (Mayer & Harel, 1979;Holderbaum et al., 2010).The degree of browning depends on the nature and amount of phenolic compounds, the presence of oxygen, reducing substances, metal ions, pH, temperature, and the activity of PPO as well (Yoruk & Marshall, 2003).
At present, plant tissue culture is mainly used for the rapid propagation of Phalaenopsis.However, explant browning can impair the success of the rapid propagation.A recent in vitro study utilizing the variety 'R4' of Phalaenopsis demonstrated that the browning of the explants could be affected by pH and temperature (Zhao et al., 2006).The tissue browning of Phalaenopsis is also closely related to the phenol concentration of explant.After Phalaenopsis explant browning, the vascular bundle is filled with tannin and the upper epiderm becomes shriveled (Xu et al., 2005).
Enzyme activity of protease PPO and POD is closely related to explant browning of Phalaenopsis.It has been reported that the POD enzyme activity gradually increase at the beginning of culture and then begin to decrease while the PPO activity rises during the first 6 days then gradually declines (Xu & Li, 2006).In the browning explants of Phalaenopsis, the content of total phenol compounds and the activity of PAL can also increase in culture conditions (Xu et al., 2005).protein-like protein (Chen et al., 2012).In this study, we further investigated the expression pattern of PPO during the browning of Phalaenopsis leaf explants in in vitro culture.The tissue-specific distribution of PPO protein in the leaf explant of Phalaenopsis were examined and the pattern of PPO protein synthesis and PPO enzyme activity were assayed in order to provide some insights into the relationship between the PPO and Phalaenopsis leaf explant browning.

Preparation of Plant Materials
Phalaenopsis (Doritaenopsis Queen Bee "Red Sky") seedlings (from Botanical Garden of South China Normal University) were grown on MS medium containing active carbon (1 g/L).Light intensity was maintained at 35 μmol•m -2 •s -1 with 16h-day/8h-night cycle and temperature controlled at 24±2 o C. Leaves of Phalaenopsis seedlings were cut into 0.5 cm × 0.5 cm segments and immediately transferred to fresh MS medium containing 6-benzylaminopurine (3 mg/L).All cultures were maintained under cool white fluorescent light of approximately 35 μmol•m -2 •s -1 photon flux density and 16h-day/8h-night cycle at 24±2 o C. Leaf explants were collected at 0, 6, 12, 24, 48 and 72 h, respectively and frozen immediately in liquid nitrogen and stored at -80 o C.

Evaluation of Browning
The browning of the cut edges of the lettuce segments was evaluated visually and the number of explants showing brown pigment was counted.

Analysis of PPO Gene Expression
Semi-quantitative RT-PCR was used to analyze PPO gene transcription.Total RNA extracts of leaf samples obtained at 0, 6, 12, 24 and 72 h were used to synthesize the first strand of cDNA by TaKaRa PrimeScript RT reagent Kit.cDNA was used as a template for amplifying a partial sequence of the PPO gene.The full length of cDNA of PhPPO is 1851 bp with an open read frame of 1647 bp (Xu et al., 2009).The PPO primers were designed as PPO-F (5'-AATATGTGGCGAAGTACGCC-3') and PPO-R (5'-GATCCCGTTCGCCTCCAATA-3').β-Actin was used as the internal control to verify the RT-PCR reaction.The primers of the internal control gene were designed as ACT-F (5'-ATTGTAAGGGACGTGAAGGAGAAGC-3') and ACT-R (5'-AAGTAGTCTCATGGATTCCCGCTGC-3'), respectively.The PCR reaction mixture (20 μL) contained 2 μL 10×PCR buffer, 1.6 μL dNTPs (2.5 mM), 0.4 μL (10 mM) of each primers, 1 μL DNA template and 0.1 μL Taq (5 U, Fermentas), 14.5 μL H 2 O.The cycling conditions were 94 o C, 5 min for initial denaturation and a total of 27 cycles of 94 o C for 30 s, 55 o C for 30 s and 72 o C for 30 s, followed by a final extension at 72 o C for 10 min.

PhPPO Antibody Preparation
The PPO gene was cloned from Phalaenopsis and PhPPO protein was generated from BL21 (DE3) cells (Xu et al., 2009).Purified PhPPO protein was submitted to Invitrogen and used to raise polyclonal antibodies against PPO from the rabbit.The crude antiserum was purified by affinity purification and used as anti-PhPPO polyclonal antibodies in immunostaining and western blot assays.

In Situ Immunostaining of PPO
Leaf samples were fixed at 4 o C in 2% (w/v) EDC, 3% (w/v) paraformaldehyde, 05% (v/v) glutaraldehyde and 4% (w/v) sugar in 10 mmol/L PBS buffer (pH 7.2).The samples were dehydrated with 30% (w/v) sugar.Leaf cross-sections in 20 μm thickness were cut with a microtome (Sorvall MT-6000 ultramicrotome) and dried on object glass slides.Immunoenzyme detection of extensins in the sections was performed using the streptavidin method.The sections were blocked with 5% BSA in PBS, then incubated with anti-PhPPO polyclonal antibodies (1:200 in 5% BSA/10 mM PBS, pH 7.2) at 4 °C overnight.Slides were rinsed three times with PBS and incubated with a HRP-labeled goat anti-rabbit IgG antibody (1:200) (Bio Rad) at 37 °C for 20 min.Following triple washing with PBS, the sections were treated with DAB at room temperature for 10 min and then washed with distilled water.The localization of PPO was examined under a microscope (Olympus IX-70, Japan).Sections treated with PBS/BSA solution without primary antibody were used as negative controls.

Analysis of PPO Synthesis by Western Blotting
Frozen leaf explants (0.5 g) were ground in liquid nitrogen to a fine powder and then homogenized in 1 mL pre-cooled lysis buffer (Keygen Biotech, Guangzhou, China) containing 1 μL protease inhibitor, 10 μL DTT (1M) and 10 μL PMSF (100 mM).The homogenate was centrifuged at 14,000 g for 10 min at 4 o C. The supernatant, referred to as the crude enzyme extract, was used in western blot analysis.For western blots and protein analyses, soluble proteins (50 μg total protein) were separated on SDS-PAGE (12% acrylamide) gels and either visualized by Coomassie Blue staining or transferred to NC membranes (Millipore corporation, Billerica, USA) to react with anti-PhPPO polyclonal antibodies (1:2000 in 5% dried nonfat milk).Immunocom-plexes were detected with HRP-labeled goat anti-rabbit IgG antibody.The protein content was determined by the Bradford method using bovine serum albumin (BSA) as the standard.

PPO Activity Assay
Frozen leaf explants of Phalaenopsis (200 mg) were mixed with 2 ml pre-cooled 0.2 M sodium phosphate buffer (pH 8.0) containing 0.1% TritonX-100.The homogenate was centrifuged at 12,000 g for 10 min at 4 o C. The supernatant, referred as the crude enzyme extract, was collected and stored at -20 o C. For PPO activity assay, 0.2 ml crude enzyme extract was mixed with 3 ml 2 M phosphate buffer (pH 8.0) and 0.2 ml 2% catechol.The reaction was carried out at 32 o C. The changes in absorbance at 310 nm were recorded by a spectrophotometer (UV6000PC, Shanghai Metash Instruments Co.) every 20 s for 2 min.PPO activity was expressed as units mg −1 protein.One unit of PPO activity was defined as the change in absorbance of 0.001 min -1 mg -1 of enzyme.The protein content was determined as described above.

Statistical Analyzes
Browning percentage and enzymatic activity were expressed as the mean values of three independent experiments.Statistical analysis was carried out using the Student's t-test by SPSS statistical package (SPSS 17.0 version).Each time point had two replicates, and experiments were repeated three times.

Browning of Phalaenopsis Leaf Explants Under Routine Culture Condition
The browning areas in the cut sections of Phalaenopsis leaf explants collected at Day 1, 2, 3 and 6 were visually examined, respectively.The browning percentages were calculated using the fresh cut (0 h) of Phalaenopsis leaf explants as a control.As shown in Figure 1, the leaf explants gradually turned brown under routine culture condition.The browning spots could hardly be observed at Day 1, however, there were 25.0±3.0%and 42.0±2.6%sections of cut leaf explants that turned brown at Day 2 and 3, respectively.

Three Phases of PPO Gene Expression in Phalaenopsis Leaf Explants
The PPO homolog, designated as PhPPO, was cloned from Phalaenopsis leaf explants by RT-PCR and RACE.The time course of PPO gene expression pattern is shown in Figure 2. No PPO gene expression could be observed in the fresh cut (i.e.0 h) but a strong expression was seen at 6 h.There were a noticeable expression of PPO gene at 12 and 24 h but the expression level was much lower compared to 6 h.Then the PPO expression level raised up again at 72 h but was still lower than 6 h.Based on these results, it could be suggested that for the first 3 days the PPO gene expression of Phalaenopsis leaf explants in the culture condition could be divided into three phases: the rapid response phase (6 h), the fatigue phase (12 to 24 h) and second response phase (72 h).

Tissue-Specific Distribution of PPO Protein in Phalaenopsis Leaf Explants
The distribution of PPO protein in leaf tissues was examined using an in situ immunostaining method (Figure 3).At 6 h, the PPO protein was mainly located in the vascular tissues enriched in the vascular wall (Figure 3B and 3F) and deep in the epidermis compared to the control (Figure 3C and 3D).At 72 h, the location of PPO protein rich vascular tissues was deepened compared to 6 h (Figure 4), indicating that more PPO proteins were synthesized at 72 h than at 6 h.

PPO Protein Synthesis and Activity
Early study has shown that PhPPO encodes a protein of 549 amino acids with a molecular mass of 60.43 kD and shares the characteristics of tyrosinase family (Xu et al., 2009).To confirm the synthesis pattern of PPO protein in Phalaenopsis leaf, western blot was performed to evaluate the PPO protein levels.As shown in Figure 5, the PPO protein presented in samples collected at every time point.Compared to the fresh cut leafs (0 h) there was no significant increase at 6, 12 or 24 h.However, there was a significant increase at 72 h, nearly two times higher than 0 h.These results indicated that intrinsic PPO existed in Phalaenopsis leaf explants before being subjected to culture and its level gradually increased during tissue browning and maximized at Day 3.This was consistent with the results of PPO activity assay.As shown in Figure 6, the PPO activity was relativity stable and maintained at the baseline level (0 h) at least up to the first 24 h in culture.The enzyme activity increased significantly at 72 h, which was 0.9 times higher than that of the baseline level. Figure Figure 3. L B: vertical