Subcellular Localization Studies of Three Phenylalanine Ammonia-Lyases and Cinnamate 4-Hydroxylase from Scutellaria Baicalensis Using GFP Fusion Proteins

1Department of Plant Science, Gangeung-Wonju National University, 7 Jukheon-gil, Gangneung-si, Gangwon-do 210-702, Korea 2College of Life Science, Inner Mongolia University for Nationalities, Inner Mongolia, China 3Department of Botany and Microbiology, Addiriyah Chair for Environmental Studies, College of Science, King Saud University, P. O. Box 2455, Riyadh 11451, Saudi Arabia Department of Crop Science, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon, 305-764, Korea 5 Visiting Professor Program (VPP), King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia


Introduction
Scutellaria baicalensis Georgi, golden root, is a species of flowering plant in the Lamiaceae family. It is one of the 50 fundamental herbs used in traditional Chinese medicine and is prescribed for fever, cold and hematemesis. In Western medicine, it has been used to treat inflammation, respiratory tract infections, diarrhea, dysentery, jaundice/liver disorders, hypertension, hemorrhaging and insomnia (Li et al., 2000;Nishikawa et al., 1999). Root of S. baicalensis is rich in flavones, a class of flavonoids produced by plants. These compounds exhibit biological and pharmacological properties, including antioxidative activity, cancer prevention and the treatment and prevention of coronary heart disease (Martens and Mithöfer, 2005).
Baicalin, baicalein and wogonin are the most well studied flavone constituents of S. baicalensis (Gao et al., 2000;Horvath et al., 2005). They provide a variety of health benefits; wogonin, especially, is a natural neuroprotective compound that inhibits the inflammatory response of microglia (Lee et al., 2003). S. baicalensis contains high concentrations of melatonin, a potent antioxidant (Murch et al., 2004). The main flavone compounds of S. baicalensis represent important sources for the development of anti-cancer, anti-inflammatory and neuroprotective drugs.
Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the first key enzyme of the phenylpropanoid pathway, catalyzes the conversion of L -phenylalanine to transcinnamic acid. PAL has been extensively studied in plants because of its important role in the biosynthesis of various secondary metabolites. PAL is thought to be responsible for many essential functions, including the establishment of mechanical support, the generation of pigments such as anthocyanins and signaling during flavonoid nodulation (Weisshaar and Jenkins, 1998). PAL also induces phenylpropanoid biosynthesis in response to biotic and abiotic stressors, such as pathogen attacks, UV irradiation, mechanical wounding and light (Dixon and Paiva, 1995).
Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11), the second key enzyme of the phenylpropanoid pathway, catalyzes the hydroxylation of trans-cinnamic acid to pcoumaric acid (Russel, 1971). C4H constitutes the CYP73 family of the cytochrome P450 monooxygenases that catalyze monooxygenase reactions in plants that are often involved in the biosynthesis of diverse metabolites (Chapple, 1998). C4H controls the carbon flux required for the synthesis of many phytoalexins when plants are challenged by pathogens (Teutsch et al., 1993). Therefore, PAL and C4H are important compounds involved in the regulation of biosynthesis and flux control in the S. baicalensis flavone pathway (Fig. 1a).

Gene Cloning and Construction
Full-length S. baicalensis PALs (SbPAL1, SbPAL2 and SbPAL3) and C4H (SbC4H) (GenBank accession numbers HM062775, HM062776, HM062777 and HM062778, respectively) were cloned by using rapid amplification of cDNA ends (RACE)-Polymerase Chain Reaction (PCR). SbPAL1, -2, -3 and SbC4H constructs fused with GFP for transient expression were generated by cloning Reverse Transcription (RT)-PCR products corresponding to the coding sequence for each gene into the pENTR/D-TOPO vector (Invitrogen). These were then recombined into a pK7FWG2 destination vector (obtained from the Functional Genomics Unit of the Department of Plant System Biology, VIB-Ghent University, The Netherlands) using Gateway LR Clonase II Plus Enzyme Mix (Invitrogen).

GFP Analysis
The GFP fusion proteins were transiently expressed following the biolistic transformation of onion (Allium cepa) epidermal cells as previously described (Park and Muench, 2007). Briefly, 5 µg of DNA was vigorously mixed with tungsten particles in a solution containing 1 M CaCl 2 and 16 mM spermidine. After being washed with 70% and 95% ethanol, the DNA-coated particles were loaded onto macrocarriers and bombarded into onion epidermal cells using a particle gun (PDS-1000; Bio-Rad). Peeled epidermal cell layers were mounted on microscope slides in MS medium, covered with a cover glass and observed using a UPlanFL N 40 × objective lens attached to a fluorescence microscope (BX51; Olympus, Tokyo, Japan). Images were captured using a DP71 microscope digital camera (Olympus).

Results
Green Fluorescent Protein (GFP) imaging has been widely used to study plant cells despite early problems with the use of the jellyfish GFP gene due to the production and distribution of modified, thermostable, highly fluorescent GFP molecules suitable for expression in plant cells (Haseloff and Siemering, 1998). GFP coding sequences have been fused at either the 5' or 3' end of the coding region of a DNA sequence of interest, leading to the production of Nor C-terminal GFP fusion proteins, respectively.  (Scott et al., 1999). The transient expression of GFP-fused SbPAL2, SbPAL3 and SbC4H resulted in a reticulate distribution of fluorescence in the examined cells; however, SbPAL1 localization was entirely cytosolic (Fig. 1b). Fluorescence from GFP only was observed throughout the cytoplasm and nucleus (data not shown).

Discussion
PAL and C4H participate in the first and second steps of the phenylpropanoid pathway, respectively. C4H anchors the enzyme complex, formed by the general phenylpropanoid enzymes, to the ER membrane (Winkel-Shirley, 1999). The ER localization of C4H has previously been shown in the hybrid poplar (Ro et al., 2001). In addition, tobacco PAL-and C4H-GFP fusion proteins localize to the ER and their co-localization was confirmed by using dual-labeling immunofluorescence and fluorescence resonance energy transfer studies (Achnine et al., 2004). Recent evidence that the other flavonoid enzymes, such as Chalcone Synthase (CHS) and Chalcone Isomerase (CHI), are localized in A. thaliana in the nucleus, suggests that flavonoid regulation of transcription is developmentally regulated at the subcellular level (Saslowsky et al., 2005).
Although our experiment did not show direct evidence that PALs co-localized with ER, the transient expression of GFP-fused SbPAL2, SbPAL3 and SbC4H resulted in a reticulate distribution of fluorescence in the examined cells. Unlike SbPAL2 and SbPAL3, however, SbPAL1 localization was entirely cytosolic. This result may be related that SbPAL1 formed a distinct group with SbPAL2 and SbPAL3 in a phylogenetic tree constructed from the deduced amino acid sequences of plant PALs (Xu et al., 2010).

Conclusion
Using in vivo localization technique, the results of this study indicate that SbPAL2, SbPAL3 and SbC4H proteins possible localize to the ER; however, SbPAL1 was a cytosolic protein. Unlike SbPAL2 and SbPAL3, SbPAL1 may be expected to have a different function in the flavone biosynthetic pathway.