ACTA BOT. CROAT. 77 (1), 2018 97 Acta Bot. Croat. 77 (1), 97–101, 2018 CODEN: ABCRA 25 DOI: 10.1515/botcro-2017-0022 ISSN 0365-0588 eISSN 1847-8476 Short communication Identification and expression profiling of flax (Linum usitatissimum L.) polyamine oxidase genes in response to stimuli Seung Hee Eom1, Jae Kook Lee1, Dong-Ho Kim2, Heekyu Kim3, Keum-Il Jang2, Hojin Ryu4, Tae Kyung Hyun1 1 Department of Industrial Plant Science and Technology, College of Agricultural, Life and Environmental Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea 2 Department of Food Science and Biotechnology, College of Agricultural, Life and Environmental Sciences, Chungbuk National University, Cheongju 28644, Republic of Korea 3 Nature Environment Research Park of Gangwon Province, Hongcheon 250-884, Republic of Korea 4 Department of Biology, College of Natural Science, Chungbuk National University, Cheongju 28644, Republic of Korea Abstract – Polyamine oxidases (PAOs) are known to be involved in either the terminal catabolism or the back conv– ersion of polyamines, which affect a range of physiological processes, including growth, development, and stress responses. In this study, based on genome-wide analysis, we identified five putative PAO genes (LuPAO1 to LuPAO5) in flax (Linum usitatissimum L.) that contain the amino-oxidase domain and FAD-binding-domain. The expression analysis using quantitative real-time PCR revealed spatial variations in the expression of LuPAOs in different organs. In addition, the expression level of LuPAOs in the flax cell suspension culture was increased by treatment with met- hyl-jasmonate (MeJA) or pectin, but not with salicylic acid or chitosan. This indicates that LuPAOs might be involved in the MeJA-mediated biological activities. Taken together, our genome-wide analysis of PAO genes and expression profiling of these genes provide the first step toward the functional dissection of LuPAOs. Keywords: cell suspension culture, flax, methyl-jasmonate, pectin, polyamine oxidase * Corresponding author, e-mail: taekyung7708@chungbuk.ac.kr Introduction Polyamines (PAs), including spermine (Spm), spermi- dine (Spd), and putrescine (Put), are low-molecular-mass aliphatic polycations that are ubiquitously distributed in organisms. Due to the cationic nature of PAs, they bind to macromolecules, such as DNA, RNA, and proteins, through electrostatic linkages that can cause either stabilization or destabilization (Kusano et al. 2008). Thus, they have been implicated in a range of fundamental cellular processes, in- cluding the regulation of gene expression, translation, cell proliferation, cell growth, differentiation, modulation of cell signaling, membrane stabilization, and modulation of ion-channel function and stability (Kusano et al. 2008, Ji- ménez-Bremont et al. 2014, Minocha et al. 2014, Tiburcio et al. 2014). Endogenous PA contents depend upon the reg- ulation of biosynthesis, transport, and catabolism in both prokaryotes and eukaryotes, including plants (Kusano et al. 2008, Takahashi et al. 2010). PAs are oxidatively deam- inated by two types of amine oxidases: copper-containing amine oxidases (CuAOs, EC 1.4.3.6) and FAD-dependent polyamine oxidases (PAOs, EC 1.5.3.6) (Cona et al. 2006). The extracellular PAOs, such as the PAOs from monocoty- ledonous plants oxidize the carbon on the endo-side of the N4-nitrogen of Spd and Spm to produce 4-aminobutanal and N-(3-aminopropyl)-4-aminobutanal, respectively, along with 1,3-diaminopropane and H2O2, and are thus consid- ered involved in the terminal catabolism of PAs. Differently, intracellular (cytosolic and peroxisomal) PAOs oxidize the carbon at the exo-side of the N4-nitrogen of Spd and Spm with the production of Spd from Spm and Put from Spd, 3-aminopropanal, and H2O2, and are considered involved in a polyamine back-conversion pathway (Planas-Portell et al. 2013, Ahou et al. 2014). Although it has been explained that PAOs in monocotyledonous plants are involved in the terminal catabolism of PAs, four rice PAOs were found to mailto:taekyung7708@chungbuk.ac.kr EOM S. H., LEE J. K., KIM D.-H., KIM H., JANG K.-I., RYU H., HYUN T. K. 98 ACTA BOT. CROAT. 77 (1), 2018 be involved in the PA back-conversion pathway (Ono et al. 2012). This suggests that the PA back-conversion pathway also exists in monocotyledonous plants. Plant PAOs have been suggested to play an important role in physiological processes, including growth, develop- ment, and responses to abiotic and biotic stresses (Angelini et al. 2010). The physiological role of PAO proteins is medi- ated by the regulation of cellular PA levels, but also by H2O2 (an important signaling molecule in the promotion of plant cell death and biotic or abiotic stress response) synthesis via the terminal catabolism and back conversion of PAs (Mino- cha et al. 2014, Tiburcio et al. 2014). Although accumulat- ing evidence has shown that PAOs play roles in modulating a range of physiological processes, most PAO family mem- bers in higher plants, except rice and Arabidopsis PAOs, are poorly understood. Therefore, in this study, we identified genes potential- ly encoding PAOs in flax (Linum usitatissimum L.), which is a medicinally important oil seed crop. Based on in-silico analysis, gene structures, sequence homology, intron phase, and cis-elements in the promoter regions of five PAO genes were investigated. In addition, the expression patterns of flax PAOs in elicitor-treated flax cell suspensions were examined. Our systematic analysis provides new insights into the un- derstanding of the potential roles of flax PAOs in response to stimuli. Materials and methods Identification and sequence analysis of LuPAO genes and promoters Protein sequences of Arabidopsis and rice PAOs were used as queries in a search against the flax genome sequence (Phytozome v9.1; http://www.phytozome.net /search. php?method=Org_Lusitatissimum). The information on LuPAO gene features, including introns and exons, was ob- tained from Phytozome v9.1. The intron phases of different introns were analyzed using Wise 2.0 (http://www.ebi.ac.uk/ Tools/Wise2). In addition, the molecular weight (MW) and the theoretical isoelectric point (pI) were calculated using the Compute pI/Mw tool available on the Expert Protein Analysis System site (http://web.expasy.org/compute_pi/), and the amino acid sequences of putative LuPAO were ana- lyzed to predict subcellular localization using HybridGO- Loc web services (http://bioinfo.eie.polyu.edu.hk/Hybrid- GoServer/) and WoLF PSORT (https://wolfpsort.hgc. jp/). The program MEME (http://meme.sdsc.edu/meme4_6_1/ cgi-bin/meme.cgi) was used for the recognition of motifs in LuPAOs. The phylogenetic analysis was performed with the use of the Phylogeny.fr server (http://www.phylogeny.fr) in the “one-click” mode, as described by Hyun et al. (2014). For the cis-element analysis, all 1000-bp upstream se- quences of LuPAO genes, except LuPAO1 (437-bp up- stream), were compared with known cis-regulatory elements in the collection of the PLACE database (http://www.dna.af- frc.go.jp/PLACE/). Plant growth Flax seeds (golden variety) were obtained from Danong Co. Ltd in South Korea. The seeds were germinated and grown in soil at 22 °C±2 °C /16±2 °C, at a light intensity of 180 μmol m–2 s–1 and a 16-h-light/8-h-dark cycle. The seeds, cotyledons and young leaves were harvested for tissue spe- cific PAO gene expression analysis. Cell culture treatment The cell suspension culture of flax (golden variety), de- scribed previously by Hano et al. (2006), was used as the experimental system. For the stress treatment, cell suspen- sion cultures were sub-cultured every two weeks and incu- bated on a rotary shaker set to 120 rpm in darkness at 25 °C. For elicitor treatment, suspension-cultured cells were treat- ed with 50 µM methyl-jasmonate (MeJA), 1 mM salicylic acid (SA), 50 mg L–1 chitosan, or 50 mg L–1 pectin. The cells were harvested at different time points (5 h, 24 h, and 48 h after treatment) by centrifugation and stored at –80 °C un- til analysis. Quantitative real-time PCR analysis Total RNA was extracted using the FavorPrep Plant Total RNA Purification Mini Kit (FAVORGEN, Ping-Tung, Tai- wan) according to the manufacturer’s instructions and was reverse-transcribed into cDNA using the QuantiTect® Re- verse Transcription Kit (QIAGEN) in accordance with the manufacturer’s recommendations. Quantitative real-time PCR (qRT-PCR) was performed using the AmpiGene qP- CR Green Mix (Enzo Life Sciences Inc., Lausen, Switzer- land) in the ECOTM Real-time PCR system (Illumina) with default parameters. The expression levels of different genes were normalized to the constitutive expression level of flax actin. Specific primer pairs are listed in On-line Suppl. Tab. 1. Tab. 1. Gene catalog and nomenclature of polyamine oxidases (PAOs) in Linum usitatissimum. The subcellular locations of polyamine oxidases were predicted by HybridGO-Loc web services (a) and WoLF PSORT (b). Name Gene ID Location CDS (bp) AA Intron Nr. pI kDa Subcellular localization LuPAO1 Lus10020726 scaffold 303:384767 – 390953 1473 490 9 5.43 54.27 Peroxisome a LuPAO2 Lus10005021 scaffold 637:175302 – 177892 1395 464 9 5.67 50.82 Peroxisome a LuPAO3 Lus10039599 scaffold 15:686508 – 691160 1491 496 9 4.95 55.52 Plastid a LuPAO4 Lus10029495 scaffold 55:372940 – 377831 1419 472 9 4.75 52.29 Extracellular b LuPAO5 Lus10019725 scaffold 420:540284 – 543964 1446 481 6 6.78 53.69 Extracellular b http://www.phytozome.net https://wolfpsort.hgc POLYAMINE OXIDASE GENES IN FLAX ACTA BOT. CROAT. 77 (1), 2018 99 Determination of PAO activity PAO activity was determined according to Han et al. (2014) with slight modifications. Soluble proteins were ex- tracted by grinding cultured cells in 0.1 M sodium phosphate buffer (pH 6.5). After centrifugation (10 min, 10,000 g) at 4°C, the supernatant was used in the assays. Reaction solu- tions (1.5 mL) contained 0.9 ml of 0.1 M sodium phosphate buffer (pH 6.5), 0.45 mL of crude enzyme extracts, 0.05 ml of peroxidase (200 U mL–1), 0.1 mL of 4-aminoantipyrine and N, N’-dimethylaniline solution. The reaction was initiated by the addition of 7.5 μL Spd (20 mM) for the determina- tion of PAO activity. The reaction mixture was incubated at 25 °C for 20 min, and then terminated with the addition of 0.25 mL of 10% trichloroacetic acid. A 0.001 change in the absorbance value at 550 nm was regarded as one enzyme ac- tivity unit. Protein concentration was determined according to the method described by Bradford (1976) with bovine se- rum albumin as the standard. Statistical analysis Statistical differences were analyzed using ANOVA based on Duncan's multiple range tests (p < 0.05). All experiments were repeated at least three times, and all data were expressed as means ± standard error. Results and discussion The availability of the flax genome sequence (Phytozome v9.1) has made it possible to identify the putative PAO fam- ily in this plant species for the first time. In order to iden- tify PAO genes, sequences of PAOs from Arabidopsis and rice were analyzed using BLASTp against all scaffold se- quences of flax. The redundant sequences were removed ac- cording to the self-BLAST of sequences, resulting in a to- tal of five putative PAO genes from flax (Tab. 1). Then, to further support the hypothesis that the five computation- ally predicted LuPAO proteins belong to the PAO family, the presence of the amino-oxidase domain (PF01593) and FAD-binding- domain, which are conserved in PAOs (Se- bela et al. 2001, Gaweska and Fitzpatrick 2011), was ana- lyzed using SMART (http://smart.embl-heidelberg.de/) and Pfam. Based on the phylogenetic analysis of the PAO pro- teins from different plants, the PAOs were classified into four major classes (I, II, III, and IV). Class I contained LuPAO1 and LuPAO2, whereas LuPAO3 and LuPAO4 were clustered into class III. In addition, LuPAO5 belonged to class IV (On- line Suppl. Fig. 1).It was not clear whether flax lacked class II of the PAO family or whether class II of LuPAOs might be not sequenced. Multiple-sequence alignments of putative LuPAOs showed that two PAOs (LuPAO 1 and LuPAO2) contained a putative peroxisomal targeting signal (On-line Suppl. Fig. 2), which was defined as a tripeptide of the C-terminus ([SA] [RK][LM]) (Reumann 2004). In addition, these LuPAO pro- teins were predicted to be peroxisomal proteins, whereas Lu- PAO 4 and 5 localize to the extracellular (Table 1). Arabi- dopsis (AtPAO2, AtPAO3 and AtPAO4) and rice (OsPAO3, OsPAO4, and OsPAO5) polyamine oxidases, clustered into class I (On-line Suppl. Fig. 1), are known as peroxisomal proteins like LuPAO1 and LuPAO2 (Ono et al. 2012; Planas- Portell et al. 2013). This indicates that class I PAOs are per- oxisomal proteins (On-line Suppl. Fig. 1) and are involved in a PA back-conversion pathway. Furthermore, LuPAO3 was predicted as a plastid-associated PAO (Tab. 1). The occur- rence of PAs at all stage of plastid development suggested that PAs serve as a nitrogen source for proteins and chlo- rophyll synthesis, which play a role in plastid differentia- tion (Sobieszczuk-Nowicka and Legocka 2014). PA content depends not only on biosynthesis, but also on the catabo- lism (Sobieszczuk-Nowicka and Legocka 2014), suggesting that plastid-associated PAOs including LuPAO3 should be involved in the plastid differentiation via controlling PA ca- tabolism. Conserved gene structures, including the same num- ber of nucleotides in the exons and the conserved intron phases, indicate the similarities between the studied genes (von Schantz et al. 2006). As shown in Fig. 1, LuPAO1 and 2 shared eight exons, with the same number of nucleotides and the same intron phase, whereas LuPAO3 and 4 shared six exons. In addition, we used the MEME program to iden- tify the conserved motifs in LuPAOs. As shown in On-line Suppl. Fig. 3, we found a total of three conserved motifs with low E values. Three motifs were shared by LuPAO3, LuPAO4, and LuPAO5 proteins, and motif 3 was not found in LuPAO1 and LuPAO2. These differences represent the evolutionary and functional relationship between LuPAOs. To investigate the spatial organization of transcripts for LuPAOs, the expression patterns of LuPAO genes in differ- Fig. 1. Phylogenetic analysis and intron-exon structures of PAO gene family in flax. Default values were used except for 100 bootstraps. Numbers in boxes are nucleotide length of each exon, and the connecting thin boxes indicate the positions of the introns. The numbers above the introns indicate the phase of the intron. EOM S. H., LEE J. K., KIM D.-H., KIM H., JANG K.-I., RYU H., HYUN T. K. 100 ACTA BOT. CROAT. 77 (1), 2018 ent tissues and cultured cells were analyzed by qRT-PCR. As shown in Fig. 2, the transcription levels of all LuPAOs, except LuPAO3, were detected in all the tested tissues with high ex- pression level compared to the cultured cells. Plant PAOs have been reported to be involved in plant responses to abiotic and biotic stresses (Angelini et al. 2010). Therefore, we analyzed the expression patterns of LuPAOs in response to external stimuli by subjecting suspension flax cell cultures to different treatments, including MeJA, SA, chitosan, and pectin. When flax-cultured cells were treated with MeJA or pectin, increased expression levels of all Lu- PAOs were observed, whereas the expression of no LuPAOs was significantly affected by SA and chitosan treatments (Fig. 3A). In addition, LuPAOs exhibited different expres- sion patterns during response to MeJA or pectin, indicating the divergent functions of LuPAOs in response to stimuli. Although copper amine oxidases are also able to oxidize Put and Spd, with the subsequent release of H2O2 (Planas-Portell et al. 2013), the increased transcription level of LuPAOs by MeJA or pectin treatment resulted in the induction of enzy- matic activity for oxidizing Spd (Fig. 3B). In addition, the enzymatic activity was not changed by treatment with SA or chitosan (Fig. 3B). However, the cis-elements like T/GBOX- ATPIN2 for jasmonate signaling were not found in LuPAOs (On-line Suppl. Fig. 4), indicating the presence of a nov- el jasmonate-responsive element in the LuPAO promoters. PA accumulation depends on de novo synthesis and catabo- lism under stress conditions (Kusano et al. 2008, Takahashi et al. 2010), suggesting that the expression of PAOs under stress conditions is required for the induction of a PA-medi- ated response. In fact, stress-induced PAO expressions have been observed in higher plants (Planas-Portell et al. 2013, Wang and Liu 2015). In addition, several stress-responsive elements were found in the LuPAO gene promoters, includ- ing the W box (WBOXNTCHN48), ELRECOREPCRP1 mo- tif (elicitor responsive element), MYB1AT (dehydration-re- sponsive), GT1CONSENSUS (Consensus GT-1 binding site in many light-regulated genes), and BIHD1OS (BELL home- odomain transcription factor in disease resistance respons- es) (On-line Suppl. Fig. 4). The presence of the aforemen- tioned putative cis-elements in LuPAO promoters indicates the contribution of PAO to stress defense responses. In conclusion, based on genome-wide analysis, we iden- tified five flax PAO genes, which belong to three groups. Plant PAOs are known to be responsible for either the ter- minal catabolism or the back conversion of PAs. Therefore, a further motivating challenge would be to investigate the Fig. 2. Tissue-specific expression of Linum usitatissimum poly- amine oxidase (LuPAO) genes. The expression levels for each gene in different tissue samples were calculated relative to its expression in the cultured cells. The Y-axis represents the normalized relative expression values (Log2). Data represent the means ± SE of three independent experiments. Values with different superscript letters are significantly different (p < 0.05). N.D = not detected. Fig. 3. Effects of elicitation on the expression of Linum usitatissimum polyamine oxidase (LuPAO) genes and enzymatic activity in sus- pension-cultured cells. (A) The expression analysis of LuPAO genes. Transcript levels of LuPAO1-5 were normalized to the constitutive expression level of flax actin, and were expressed relative to the values at 0 hour. The Y-axis represents the normalized relative expression values (Log2). (B) The variation of LuPAO enzymatic activity. Flax suspension-cultured cells were treated with methyl-jasmonate (MeJA), salicylic acid (SA), pectin, or chitosan. Mock indicates the treated control (mock-treated control). Data represent the means ± SE of three independent experiments. Values in the same column with different superscripted letters are significantly different (p < 0.05). POLYAMINE OXIDASE GENES IN FLAX ACTA BOT. CROAT. 77 (1), 2018 101 References Ahou, A., Martignago, D., Alabdallah, O., Tavazza, R., Stano, P., Macone, A., Pivato, M., Masi, A., Rambla, J. L., Vera-Sirera, F., Angelini, R., Federico, R., Tavladoraki, P., 2014: A plant spermine oxidase/dehydrogenase regulated by the protea- some and polyamines. Journal of Experimental Botany 65, 585–1603. Angelini, R., Cona, A., Federico, R., Fincato, P., Tavladoraki, P., Tisi, A., 2010: Plant amine oxidases "on the move": an update. Plant Physiology and Biochemistry 48, 560–564. Bradford, M.M., 1976: A rapid and sensitive method for the quan- titation of microgram quantities of protein utilizing the prin- ciple of protein-dye binding. Analytical Biochemistry 72, 248– 254. Cona, A., Rea, G., Angelini, R., Federico, R., Tavladoraki, P., 2006: Functions of amine oxidases in plant development and de- fence. Trends in Plant Science 11, 80–88. Gaweska, H., Fitzpatrick, P.F., 2011: Structures and mechanism of the monoamine oxidase family. Biomolecular Concepts 2, 360–377. Han, B., Yang, Z., Xie, Y., Nie, L., Cui, J., Shen, W., 2014: Arabi- dopsis HY1 confers cadmium tolerance by decreasing nitric oxide production and improving iron homeostasis. Molecu- lar Plant 7, 388–403. Hano, C., Addi, M., Bensaddek, L., Crônier, D., Baltora-Ros- set, S., Doussot, J., Maury, S., Mesnard, F., Chabbert, B., Hawkins, S., Lainé, E., Lamblin, F., 2006: Differential ac- cumulation of monolignol-derived compounds in elicited flax (Linum usitatissimum) cell suspension cultures. Planta 223, 975–989. Hyun, T. K., Rim, Y., Kim, E., Kim, J. S., 2014: Genome-wide and molecular evolution analyses ofthe KT/HAK/KUP family in tomato (Solanum lycopersicum L.). Genes & Genomics 36, 365–374. Jiménez-Bremont, J. F., Marina, M., Guerrero-González Mde, L., Rossi, F. R., Sánchez-Rangel, D., Rodríguez-Kessler, M., Ruiz, O. A., Gárriz, A., 2014: Physiological and molecular implica- tions of plant polyamine metabolism during biotic interac- tions. Frontiers in Plant Science 5, 95. Kusano, T., Berberich, T., Tateda, C., Takahashi, Y., 2008: Poly- amines: essential factors for growth and survival. Planta 228, 367–381. Minocha, R., Majumdar, R., Minocha, S. C., 2014: Polyamines and abiotic stress in plants: a complex relationship. Frontiers in Plant Science 5, 175. Ono, Y., Kim, D. W., Watanabe, K., Sasaki, A., Niitsu, M., Berb- erich, T., Kusano, T., Takahashi, Y., 2012: Constitutively and highly expressed Oryza sativa polyamine oxidases localize in peroxisomes and catalyze polyamine back conversion. Amino Acids 42, 867–876. Planas-Portell, J., Gallart, M., Tiburcio, A. F., Altabella, T., 2013: Copper-containing amine oxidases contribute to terminal polyamine oxidation in peroxisomes and apoplast of Arabi- dopsis thaliana. BMC Plant Biology 13, 109. Reumann, S., 2004: Specification of the peroxisome targeting sig- nals type 1 and type 2 of plant peroxisomes by bioinformatics analyses. Plant Physiology 135, 783–800. Sebela, M., Radová, A., Angelini, R., Tavladoraki, P., Frébort, I., Pec, P., 2001: FAD-containing polyamine oxidases: a timely challenge for researchers in biochemistry and physiology of plants. Plant Science 160, 197–207. Sobieszczuk-Nowicka, E., Legocka, J., 2014: Plastid-associated polyamines: their role in differentiation, structure, func- tioning, stress response and senescence. Plant Biology 16, 297–305. Takahashi, Y., Cong, R., Sagor, G. H., Niitsu, M., Berberich, T., Ku- sano, T., 2010: Characterization of five polyamine oxidase iso- forms in Arabidopsis thaliana. Plant Cell Reports 29, 955–965. Tiburcio, A. F., Altabella, T., Bitrián, M., Alcázar, R., 2014: The roles of polyamines during the lifespan of plants: from devel- opment to stress. Planta 240, 1–18. von Schantz, M., Jenkins, A., Archer, S. N., 2006: Evolutionary history of the vertebrate period genes. Journal of Molecular Evolution 62, 701–707. Wang, W., Liu, J. H., 2015: Genome-wide identification and ex- pression analysis of the polyamine oxidase gene family in sweet orange (Citrus sinensis). Gene 555, 421–429. specific roles of each LuPAO in metabolism. An in-depth analysis of LuPAO gene expression patterns under different stress conditions suggested that LuPAO should be involved in the MeJA-mediated biological activities. Taken together, our genome-wide analysis and expression analysis provide a solid foundation for developing further understanding of the potential function of PAOs. Acknowledgements This research was supported by Basic Science Resear- ch Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF- 2015R1A4A1041869)