Molecular Characterization of Ferulate 5-Hydroxylase Gene from Kenaf (Hibiscus cannabinus L.)

The purpose of this study is to clone and characterize the expression pattern of a F5H gene encoding ferulate 5-hydroxylase in the phenylpropanoid pathway from kenaf (Hibiscus cannabinus L.). Kenaf is a fast-growing dicotyledonous plant valued for its biomass. F5H, a cytochrome P450-dependent monooxygenase (CYP84), is a key enzyme for syringyl lignin biosynthesis. The full length of the F5H ortholog was cloned and characterized. The full-length F5H ortholog consists of a 1,557-bp open reading frame (ORF) encoding 518 amino acids (GenBank Accession number JX524278). The deduced amino acid sequence showed that kenaf F5H had the highest similarity (78%) with that of Populus trichocarpa. Transcriptional analysis of F5H ortholog was conducted using quantitative real-time PCR during the developmental stages of various tissues and in response to various abiotic stresses. The highest transcript level of the F5H ortholog was observed in immature flower tissues and in early stage (6 week-old) of stem tissues, with a certain level of expression in all tissues tested. The highest transcript level of F5H ortholog was observed at the late time points after treatments with NaCl (48 h), wounding (24 h), cold (24 h), abscisic acid (24 h), and methyl jasmonate (24 h).


Introduction
Kenaf (Hibiscus cannabinus L.) is an annual dicotyledonous and a fast-growing herbaceous plant, spanning a wide ecological habitat from temperate to tropical areas [1]. Kenaf is regarded as the third largest fiber crop following cotton and jute and is indispensible for pulp and paper industries [2,3]. Since kenaf has large ranging habitats and fast growth rate, it has great potential for use in biomass production [4][5][6]. Lignin is an aromatic heteropolymer that is predominantly found in the secondary plant cell walls with cellulose and hemicellulose and is the second most abundant biopolymer on earth after cellulose [7][8][9]. It provides rigidity to the plant cell wall and hydrophobicity to the vascular system of plants. In addition, lignin is highly associated with the plant defense mechanisms against biotic and abiotic stresses [7][8][9]. However, lignin is one of the major obstructions in the conversion of plant biomass to pulps, papers, or biofuels [8,10]. Lignin protects polysaccharides in plant cell walls from microbial degradation. As lignin is strongly bound to polysaccharides, cellulose and hemicellulose polymers are able to get only a limited access to microbial hydrolytic enzymes such as cellulases. For the efficient use of polysaccharides in pulps or biofuels, lignin should be reduced or removed. In addition, the residual products generated during the removal of lignin can inhibit the subsequent processes of saccharification and fermentation. The removal of lignin from plant biomass is a costly process and generates environmental pollutants [10][11][12]. These shortcomings of the presence of lignin prompted many scientists to develop 2 The Scientific World Journal genetically modified plants with less lignin content or an altered lignin component for its easy degradation [7,8,13]. Lignin is synthesized through the phenylpropanoid pathway [14]. There are two major steps of lignin biosynthesis in plants.
In the first step, three hydroxycinnamyl alcohol monomers (p-coumaryl, coniferyl, and sinapyl alcohols) are synthesized through the phenylpropanoid pathway. In the second step, these alcohol monomers are integrated into the lignin polymers by peroxidase and laccase during polymerization to form -hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units, respectively [15,16]. Among the three units, the S unit is relatively unbranched and has a higher degree of degradability [14]. The biomass of kenaf fibers is favorable for delignification because these fibers contain less amount of lignin and have a high S/G ratio with small amounts of H unit (S 83.3%, G 15.4%, and H 1.3%) [17,18]. Many attempts have been made to modify the content and composition of lignin in order to increase pulping efficiency. Upregulation of F5H increases the S/G ratio [10,13]. F5H, a cytochrome P450-dependent monooxygenase (CYP84), is specifically required for the synthesis of S lignin [14]. It catalyzes the 5hydroxylation of coniferaldehyde and/or coniferyl alcohol in the phenylpropanoid pathway. Upregulation of F5H in poplar tree was found to increase the S/G ratio that subsequently led to increased pulping efficiency [19]. Conversely, downregulation of F5H in alfalfa caused a decrease in the S/G ratio [13]. Arabidopsis transgenic lines overexpressing F5H showed that it contained S lignin in a larger proportion (92%) [20]. Therefore, F5H is a key enzyme involved in synthesizing monolignol sinapyl alcohol and, ultimately, S lignin moieties. In this study, we cloned and characterized F5H ortholog in kenaf to investigate the expression patterns of F5H ortholog in various developmental stages, in different tissues, and under various abiotic stresses such as NaCl, wounding, cold, ABA, MeJA, and drought using quantitative real-time PCR (QPCR).

Plant Materials.
Dr. Si-Yong Kang (Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup, Korea) generously provided kenaf seeds (Hibiscus cannabinus L., C-9) that were originally from Russia (GenBank of Korea Rural Development Administration IT number 202789). Seeds were germinated in small pots filled with a sterile nonsoil mixture (Tobietec, Chungbuk, Korea). Kenaf seedlings were grown for up to 4 weeks in a growth room, watered twice a week, and maintained under the following growth condition: 16-h light/8-h dark, 22 ∘ C, and 100 mol m −2 s −1 light intensity. After 4 weeks in the growth room, kenaf seedlings were transplanted into larger pots (20 cm diameter) filled with nonsoil mixture and were grown in a greenhouse for up to 20 weeks with watering twice a week. Various tissues (roots, stems, leaves, petioles, and flowers) were harvested from 16-week-old kenaf plants during midday of light length. Harvested samples were immediately frozen in liquid nitrogen and stored at −80 ∘ C. Leaf tissues were harvested at three development stages: (1) young leaf (YL), <2 cm long; (2) immature leaf (IL), 3-5 cm long; and (3) mature leaf (ML), >9 cm long. In addition, flower samples were harvested at three development stages: (1) young flower (YF), unopened green flower, <2 cm long with green sepal; (2) immature flower (IF), unopened white flower, >3 cm long with green sepal; and (3) mature flower, open white flower.

Stress Treatments.
Three-week-old plants, grown in the growth room as previously described, were treated with diverse abiotic stresses. Stem tissues were harvested for RNA extraction. For the wounding treatment, stem tissues were cut twice longitudinally with scissors. For cold treatment, plants were put into a cold room under following growth conditions: 16 h light/8 h dark, 10 ∘ C, and 100 mol m −2 s −1 light intensity. Plants were treated with ABA (100 M) or NaCl (200 mM) [21,22]. Plants treated without ABA and NaCl were used as control samples. Plants were sprayed with 100 M MeJA dissolved in 0.004% ethanol and then covered with a plastic bag. Plants sprayed with 0.004% ethanol were used as control samples. Stem tissues were harvested 1, 6, 12, 24, and 48 h after treatments. For drought treatment, watering was stopped for 4, 7, 10, and 14 days. For control samples, plants were grown in normal growth conditions with watering once in every 3 days. The harvested samples were frozen in liquid nitrogen and stored at −80 ∘ C.

Cloning of a Full-Length F5H Ortholog in Kenaf.
The full length of kenaf F5H ortholog was cloned using degenerate primers and rapid amplification of cDNA ends (RACE) method. Kenaf F5H ortholog (GenBank Accession number JX524278) consists of a 1,557-bp open reading frame (ORF) that encodes 518 amino acids ( Figure 1). The predicted molecular weight of the deduced protein was 58.62 kDa with an isoelectric point (pI) of 5.84 as calculated by the ExPASy Proteomic Server. BlastP analysis revealed that the deduced amino acid sequence of kenaf F5H ortholog had high identities with other F5H sequences ( Figure 2). The deduced kenaf F5H ortholog shared 78, 78, 77, 77, and 77% identities with F5H sequences from Camptotheca acuminate (AAT39511), Populus trichocarpa (CAB65335), Eucalyptus globulus (ACU45738), Arabidopsis thaliana (CAC26936), and Arabidopsis lyrata (XP002867029), respectively (Figures 2 and 3). SignalP 4.0 and TargetP V1.1 analyses showed that no signal peptide was detected for subcellular localization in kenaf F5H ortholog, which implies cytoplasmic localization. Motif Finder and PROSITE analyses suggested that kenaf F5H ortholog may contain a cytochrome P450 cysteine heme-iron ligand signature (FGSGRRSCPG, amino acid position 448-457; Figures 1 and 2), which exactly matched with the previously reported heme-binding ligand in Arabidopsis [25]. A phylogenetic tree was constructed from 10 F5H amino acid sequences using Mega 5 program ( Figure 3). The phylogenetic tree showed that kenaf F5H ortholog had the closest relationship with Populus trichocarpa (CAB65335) and was grouped into a subcluster of 4 proteins: P. trichocarpa, Camptotheca acuminate (AAT39511), and Solanum lycopersicum × Solanum peruvianum (AAD37433). Cumulatively, all these results emphatically suggest that kenaf F5H ortholog is a F5H enzyme. Therefore, kenaf F5H ortholog was designated as HcF5H.

Expression of HcF5H
Ortholog in Tissues. The expression patterns of HcF5H transcript were analyzed in various tissues and organs including roots, stems, leaves, petioles, and flowers using QPCR. HcF5H transcript was found to be ubiquitous in all the tested samples ( Figure 4(a)). A high level of HcF5H transcript expression was observed in root, stem, and petiole tissues of 16-week-old kenaf plants. HcF5H was highly expressed in young stems (6 week old, 4.54% relative ACTIN; Figure 4(b)) and immature flowers (5.61% relative ACTIN; Figure 4(c)), while the transcript level was not significantly induced during leaf development (Figure 4(d)). During stem development, HcF5H transcript showed a significant increase at 6 weeks and then sharply decreased for up to 20 weeks. During flower development, the highest transcript level of HcF5H was observed in immature flowers, while low level of the transcript was found in young and mature flowers. The transcript values during developmental stages in all tissues were significant ( ≤ 0.05), except for the transcripts in leaf development ( > 0.05).

Expression of HcF5H Ortholog in Response to Various
Abiotic Stresses. HcF5H expression patterns were analyzed using 3-week-old stem tissues in their response to various abiotic stresses (NaCl, wounding, cold, ABA, MeJA, and drought) using QPCR. HcF5H transcript was induced by all treatments with the highest responses to NaCl and wounding treatments ( Figure 5). The highest induction of HcF5H was observed at 24-48 h after NaCl treatment. In wounding treatment, a biphasic expression was observed. HcF5H was induced at 6 h after the treatment, declining marginally by 12 h. The secondary induction was observed at 24 h after treatment, which was the highest in wounding treatment. The transcript was sharply downregulated later on. Biphasic expression was also observed in cold treatment, in which HcF5H transcript was upregulated at 6 h after treatment and then sharply downregulated at 12 h. The secondary induction was observed at 24 h, when the highest transcript level was occurred. When kenaf was treated with ABA and MeJA, HcF5H transcript reached its maximum level at 24 h after treatment. In ABA treatment, the transcript level increased up to 24 h after treatment and showed a decrease at 48 h. In the case of MeJA, the transcript level was sharply increased at 12-24 h after treatment and then decreased at 48 h. HcF5H transcript level was maximum 7 days after watering was stopped and then dramatically decreased. All transcript values in response to various abiotic stresses were statistically significant based on Duncan's multiple range test ( ≤ 0.05).

Characteristics of HcF5H
. F5H enzyme is a cytochrome P450-dependent monooxygenase (P450s) that catalyzes the 5-hydroxylation of coniferaldehyde and/or coniferyl alcohol in the phenylpropanoid pathway [15]. P450s are a large group of heme-containing enzymes that generally catalyze NADPH-and O 2 -dependent hydroxylation reactions. In plants, two P450s catalyze reactions of the general phenylpropanoid pathway: cinnamate 4-hydroxylase (C4H) and ferulate 5-hydroxylase (F5H). While C4H belongs to P450 CYP73 family, F5H is a member of the CYP84 family of P450s [25]. Arabidopsis F5H was first defined as CYP84 and two F5H homologs (AtF5H1 and AtF5H2) were identified in the Arabidopsis genome [25]. AtF5H1 is now well characterized in many plants including Arabidopsis, Liquidambar styraciflua, and Brassica napus [25][26][27]. AtF5H2 is a more divergent member of the CYP84 family, and no genes that closely resemble AtF5H2 are found in other plants [28]. Three F5H homologs were identified in Brassica napus (BnF5H1, BnF5H2, and BnF5H3) [27]. BnF5H1 and BnF5H2 are very similar to each other (98% identity at nucleotide sequence  and 99% identity at amino acid sequence) and show high identity with AtF5Hs (90% identity at amino acid sequence and 93% identity at amino acid sequence). Therefore, BnF5H1 and BnF5H2 are thought to belong to one group. BnF5H3 also shows the similar level of identity to the corresponding portion but lacks a codon for Proline at position 39, which is present in other F5H genes [27]. These two groups of BnF5H therefore must have been formed before the divergence. Also, the divergence between either BnF5H1 or BnF5H2 and BnF5H3 suggests that the duplication may have occurred very    early in the lineage of the Brassica spp., whose ancestor has not been identified yet [29]. Only one putative HcF5H gene was isolated in this study, but we suspect there might be more than one in kenaf. The deduced amino acid sequence of HcF5H showed 74-78% identity with F5H sequences of other plants (Figure 2). Phylogenic tree showed that HcF5H shared amino acid sequence identity with other plant F5Hs (Figure 3). HcF5H may not contain a significant signal peptide, and so it is thought to be a cytoplasmic protein. In previous studies, however, AtF5H1 and AtF5H2 were found to contain fully conserved membrane anchor regions, and AtF5H2 was expected to have an ER-targeting peptide [28]. In our study, we identified that HcF5H contains a cytochrome P450 cysteine heme-iron ligand signature (FGSGRRSCPG), which is homologous with the previously reported hemebinding domain that is well conserved in the CYPs of higher plants [30]. In addition, alignment of seven proteins of the CYP73 gene family showed similar results with the previous study [31]. Therefore, we conclude that HcF5H may function as an F5H enzyme.

Significant Difference in HcF5H Expression in Different
Tissues and Developmental Stages. QPCR analysis revealed that HcF5H was expressed in all tissues tested (Figure 4(a)). HcF5H was not regulated during leaf development (Figure 4(b)). The highest expression of HcF5H was detected in immature flowers and young stems (Figures 4(c) and 4(d)). These results resemble the expression profile of AtF5H in Arabidopsis [28]. In Arabidopsis, both AtF5H1 and AtF5H2 were present in all tissues, with stem tissues expressing them at a high level. Moreover, AtF5H2 had the highest expression in the early stage of stem development. Most abundant transcript level of BnF5H was also detected in the stem tissues of B. napus [27]. In kenaf, the highest expression level of HcF5H was detected in young stem during stem development. The F5H-deficient mutant (fah1) accumulated only guaiacyl lignin, while F5H-overexpressing transgenic plant produced lignin with mostly syringyl propane units [20]. These results imply that F5H expression is essential for S lignin synthesis during stem development. In contrast to the previous studies, HcF5H showed the highest expression level in immature flower tissues in our study. In flowers and seeds, F5H is required for the production of sinapate esters, which are intermediates of syringyl lignin biosynthesis in higher plants, and some of them serve as precursors for important secondary metabolites [32]. For example, phenylpropanoidderived compounds such as sinapate and flavonoids were detected at high levels in Arabidopsis flowers [33]. In addition, disruption of AtF5H led to a limited production of lignin, which resulted in a dwarf floral architecture in the f5 h mutant [34]. Therefore, F5H is an important enzyme in flower development. In this study, HcF5H expression was found to be significantly induced during flower development, reaching its maximum level in the immature flower. Overexpression of AtF5H in AtCOMT-deficient Arabidopsis caused the accumulation of 5H monomers [35]. These 5H monomers were either directly or indirectly detrimental to pollen wallforming sporopollenin biosynthesis, which is thought to protect pollens from UV radiation and desiccation. The overexpression of HcF5H during flower development could be explained by the pollen wall formation. Overall, expression patterns of HcF5H strongly point toward the possibility that this putative enzyme may function as F5H in kenaf.

Significant Induction of HcF5H in Response to Various
Abiotic Stresses. Plants are subjected to various stress conditions during their life time. They have a generic signal transduction pathway that starts with signal perception, followed by the generation of second messengers such as calcium, reactive oxygen species (ROS), and inositol phosphates [36]. These biotic and abiotic stresses can turn on a plant's defense mechanisms, which include the production of various defensive enzymes and proteins as well as cell wall reinforcement through lignin deposition [23,[37][38][39]. Lignin induction has been correlated with cold, drought, or light stresses as well as mechanical injuries in plants [23]. Although lignin biosynthesis itself is well characterized, the mechanism regulating ectopic deposition of lignin in response to environmental stimuli is not well understood. Environmental stresses can activate the synthesis of phytohormones in plants [40,41]. These phytohormones can produce the initial signals for the gene regulation in lignin biosynthesis and others. The initial signals subsequently produce a second-round signaling pathway. In previous studies, we showed that kenaf genes involved in lignin biosynthesis were controlled by various environmental stresses and phytohormones [42][43][44][45][46]. However, this is the first report that showed the upregulation of HcF5H as a result of various abiotic stresses in kenaf.
HcF5H is found to be induced in response to treatments that include wounding, cold, drought, and additions of NaCl, ABA, and MeJA ( Figure 5).
NaCl. Soil salinity poses a severe environmental restriction limiting the availability of land for sustainable agriculture [47]. Many scientists have identified the genes that cause salt tolerance, so that the effects of salinity in agriculture can be overcome. According to previous reports, increased lignification or altered monolignol composition in plant cell walls provides an effective way to overcome salinity stress [48]. In this study, HcF5H was highly induced by NaCl ( Figure 5). Similar result was reported in Brassica juncea [49]. Moreover, NaCl treatment also induced the genes encoding S-adenosyl-l-methionine synthase (SAMS) in tomato, Omethyltransferase (COMT) in Tamarix hispida, and phenylalanine ammonia-lyase (PAL), -coumarate 3-hydroxylase (C3H), and caffeoyl-coenzyme A 3-O-methyltransferase (CCoAMT) in kenaf [42,44,45,50,51].
Wounding. Wounding has proved to be one of the most effective ways by which control of phenylpropanoid biosynthesis can be understood [23,52,53]. For example, wounding induces the genes involved in lignin biosynthesis, such as PAL (phenylalanine ammonia-lyase), C4H (cinnamate 4hydroxylase), F5H, CAD (cinnamyl alcohol dehydrogenase), CCR (cinnamoyl-CoA reductase), and 4CL (4-coumarate-CoA ligase), which caused accumulation of lignin compounds surrounding the wounding sites [23,52,53]. Wounding also induced the synthesis of lignin precursors and ferulate esters in Solanum tuberosum [54]. QPCR analysis suggests that HcF5H is highly induced by wounding ( Figure 5). These results point to the fact that plants enhance lignin production in order to protect them from wounding through the regulation of the genes involved in lignin biosynthesis such as F5H.
Cold. Low temperature causes an alteration in plant lignin contents [23]. The adaptation of plants to low temperature  is thought to involve a genetic response controlling the physical and biochemical changes. It was reported that poplar seedlings grown at 10 ∘ C showed an increase in lignin content [55]. Cold treatment induced the accumulation of phenylpropanoid lignin precursors such as -coumaric acid, ferulic acid, and synaptic acid in Brassica napus [56]. A total 3,379 genes showed altered expression patterns during cold acclimation in Arabidopsis [57]. As a key enzyme in lignin synthesis, F5H induction by cold might lead to lignification in kenaf stem tissues. It has been reported that cold treatment can upregulate the genes involved in lignin synthesis such as PAL, CCoAOMT, C3H, HCT, and COMT1 in kenaf stem tissues [42][43][44][45][46].
ABA. The universal stress hormone ABA is substantially induced during a plant's response to abiotic stress [58]. When the salt stress signal is received by plants, ABA synthesis is induced, which triggers other signaling processes such as the accumulation of calcium ions [59]. ABA is an important phytohormone and plays a critical role in response to stresses including drought, cold, and osmotic stress [60,61]. Studies suggest that two types of ABA signal transduction pathways are present in plants: one is ABA-independent, and the other is ABA-dependent [60]. The genes involved in ABA biosynthesis were induced by drought and cold treatments, and they were not responsive to exogenous ABA treatment [60]. When detached leaves were treated with ABA, F5H was found to be induced in Camptotheca acuminata [62], suggesting that F5H might belong to the ABA-dependent pathway. Similar response pattern was observed when HcF5H was treated with ABA, indicating that HcF5H can be categorized into an ABA-dependent pathway. Similar induction patterns were observed for treatments with ABA and NaCl.
MeJA. Exogenous application of MeJA caused the accumulation of lignin, phenolic compounds, and defense enzymes in eggplant roots [63]. Jasmonic acid (JA) or its methyl ester jasmonate acts as an important regulator of responses to wounding and environmental stresses [64]. CaF5H transcript was induced by MeJA treatment in Camptotheca acuminate The Scientific World Journal 9 [62]. In our study, we found the upregulation of F5H by MeJA in kenaf. This result strongly suggests that MeJA-dependent pathway regulates wound-induced F5H activation similar to other F5H orthologs. However, MeJA treatment only weakly induced HcF5H compared to other treatments. Similar result was observed in the expression of CaF5H [62]. It is thought that exogenous MeJA alone might not be effective in mediating the expression of F5H. Other factors such as ABA and H 2 O 2 are also required for stronger expression.
Drought. In this study, drought induced HcF5H expression at early time point after watering was stopped (7 days). HcF5H sharply dropped to initial levels later. The level of lignin deposition and gene expression may be dependent on plant tissues and drought period. In a previous study, there was a reduction in the amount of ferulic acid and an induction of -coumaric and caffeic acids in maize xylem sap 12 days after watering was stopped [65]. Previous studies reported the induction of genes involved in lignin biosynthesis [23,[42][43][44][45]. During the drought period, plant cell wall becomes impermeable to water due to the accumulation of lignin, which causes the reduction in water transpiration [66]. Therefore, it is thought that lignification is one of the primary responses to drought stress. In summary, a full length of the gene F5H encoding ferulate 5-hydroxylase was cloned from kenaf (Hibiscus cannabinus L.). Transcriptional and phylogenetic analyses indicated that HcF5H ortholog belonged to known F5H genes of plants. On the transcript level, HcF5H was regulated by the developmental stages in various tissues and in response to various abiotic stresses. These results suggest that HcF5H may play important roles in kenaf plant for better adaptation to environmental stresses.