JXB Advance Access originally published online on November 22, 2006
Journal of Experimental Botany 2006 57(15):4155-4169; doi:10.1093/jxb/erl193
RESEARCH PAPER |
Cloning and characterization of Arabidopsis and Brassica juncea flavin-containing amine oxidases
Department of Biological Sciences and Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117543
* To whom correspondence should be addressed. E-mail: dbsyuhao{at}nus.edu.sg or yuhao{at}tll.org.sg
Received 18 July 2006; Accepted 7 September 2006
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Abstract |
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Polyamines (PAs) are low molecular weight metabolites involved in various physiological and developmental processes in eukaryotic and prokaryotic cells. The cellular PA level is regulated in part by the action of amine oxidases (AOs) including copper diamine oxidases (DAOs) and flavoprotein polyamine oxidases (PAOs). In this study, the isolation and characterization of flavin amine oxidases (FAOs) from Brassica juncea (BJFAO) and Arabidopsis (ATFAO1) are reported that were clustered in the same group as polyamine oxidases from maize (MPAO) and barley (BPAO1) and monoamine oxidases from mammalian species. ATFAO1 was temporally and spatially regulated in Arabidopsis and showed distinct expression patterns in response to different stress treatments. To investigate the in vivo function of FAO, transgenic Arabidopsis plants expressing sense, antisense, and double-stranded BJFAO RNAs were generated and those with altered activity of FAOs were selected for further characterization. It was found that the shoot regeneration response in transgenic plants was significantly affected by the modulated PA levels corresponding to FAO activities. Tissues that originated from transgenic plants with down-regulated FAO activity were highly regenerative, while those from transgenic plants with upregulated FAO activity were poorly regenerative. The shoot regeneration capacity in these transgenic plants was related to the levels of individual PAs, suggesting that FAO affects shoot regeneration by regulating cellular PAs. Furthermore, it was found that the effect of FAO activity on shoot regeneration was exerted downstream of the Enhancer of Shoot Regeneration (ESR1) gene, which may function in a branch of the cytokinin signalling pathway.
Key words: Arabidopsis thaliana, cytokinin, flavin-containing amine oxidase, polyamine, shoot regeneration
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Polyamines (PAs) are polycations of low molecular mass that are found ubiquitously in all living organisms. They play an important role in growth and development of prokaryotes and eukaryotes. In plants, the most common aliphatic PAs are putrescine (Put), the tri-amine spermidine (Spd), and the tetra-amine spermine (Spm) (Galston and Sawhney, 1990). Many other di- and polyamines are present in plants and other organisms, such as 1,3-diaminopropane and cadaverine (1,5-diaminopentane). Unusual PAs have also been identified in bacteria, algae, fungi, animals, and higher plants (Niitsu and Samejima, 1993).
Plant PAs often occur in free form as soluble PAs. However, they can be conjugated to low molecular mass molecules by the formation of an amide linkage or bound to macromolecules such as proteins. Because of their polycationic nature, PAs can interact with anionic macromolecules such as DNA, RNA, phospholipids, and certain proteins (Tiburcio et al., 1997). Therefore, they can play important roles in stabilization and protection of these molecules to maintain cell homeostasis, growth, and tumorigenesis (Wallace et al., 2003).
In plants, PAs have been shown to play important roles in a myriad range of physiological processes during growth and development, including embryogenesis, dormancy breaking of tubers and seed germination, stimulation and development of flower buds, flower and leaf senescence, fruit set, development and ripening, and in vitro organogenesis (Evans and Malmberg, 1989). Apart from plant growth and development, PAs are also involved in the response to environmental stimuli such as osmotic stress, mineral deficiency, salinity (Bouchereau et al., 1999), and pathogen infection (Walters, 2003). Plants usually respond to stress by increasing their endogenous PA content, accompanied by an increase in the activity of enzymes involved in PA biosynthesis. It was therefore suggested that PAs might play a role in plant survival under stress (Bouchereau et al., 1999).
PAs are oxidatively deaminated by the action of amine oxidases (AOs), which can be divided into copper-containing AOs (Cu-AOs; EC 1.4.3.6
[EC]
) and FAD-dependent AOs (FAD-AOs; EC 1.4.3.4
[EC]
). In plants, Cu-AO, also known as DAO, catabolizes Put to produce 4-aminobutyraldehyde concomitantly with hydrogen peroxide (H2O2) and ammonia, while Spd and Spm are degraded primarily by FAD-AO, also known as PAO. Together with H2O2 and diaminopropane that is converted to ß-alanine, 4-aminobutyraldehyde and aminopropylpyrroline are produced from the oxidation of Spd and Spm, respectively. 4-Aminobutyraldehyde cyclizes spontaneously to yield 
1-pyrroline that can be further oxidized to form 
-aminobutyric acid (GABA) by the action of pyrroline dehydrogenase (Flores and Filner, 1985). GABA can be transaminated and oxidized to form succinic acid, which is subsequently incorporated into the Krebs cycle. This degradation pathway can thus ensure the recycling of carbon and nitrogen from PAs.
It is important to underline that the enzymes involved in PA catabolism and the products derived from their oxidation have been implicated in some essential physiological processes (Martin-Tanguy, 1997; 
ebela et al., 2001). For example, several lines of evidence indicate that H2O2, a product of PA oxidation, can drive the cross-linking of cell wall proteins and serve as a secondary messenger to trigger the plant hypersensitive response (Levine et al., 1994) as well as increase disease resistance (Kachroo et al., 2003) and somatic embryogenesis (Luo et al., 2001). PAOs are also involved in the production of uncommon PAs, such as norspermidine, norspermine, cardopentamine, caldohexamine, homocaldopentamine, and monocaldohecamine, that may be involved in co-ordinating the growth response of various organisms under extreme environmental conditions (Philips and Kuehn, 1991).
In recent years, major efforts have been dedicated to perturbation of PA production in transgenic plants through genes that overexpress or down-regulate enzymes involved in the PA metabolic pathways (Bastola and Minocha, 1995; Bhatnagar et al., 2001; Lepri et al., 2001). The majority of these studies have focused on the PA biosynthetic pathways and only a few on the catabolic pathways (Wisniewski and Brewin, 2000; Rea et al., 2004). In the latter scenario, PAOs have been isolated mostly from monocotyledonous species, especially cereals belonging to the Graminae (ebela et al., 2001), where maize PAO (MPAO) (Tavladoraki et al., 1998) and barley PAOs (BPAO1 and BPAO2) (Cervelli et al., 2001) are the most studied members of this class. Hitherto, evidence for the occurrence of PAO in dicotyledonous species has been reported only once from Medicago sativa L. (alfalfa) (Bagga et al., 1991), where only the enzyme activity of PAO was examined.
In this study, the isolation and functional characterization of putative PAOs from Brassica juncea (BJFAO) and Arabidopsis (ATFAO1) are presented. The results show that the ATFAO1 gene is temporally and spatially regulated and is responsive to stress. Alteration of endogenous FAO activity in Arabidopsis can change the levels of aliphatic PAs and their relative ratios, resulting in different shoot regeneration responses.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Plant materials
Arabidopsis thaliana (ecotype Columbia) seeds were surface-sterilized by sequential washes in 70% ethanol (v/v) for 1 min and 15% Clorox® (1% sodium hypochlorite) solution for 10 min, and rinsed with sterile distilled water before being sown onto agar plates. The MS agar medium contained Murashige and Skoog salts (Murashige and Skoog, 1962), 3% sucrose, and 0.8% agar, pH 5.7. The plates were incubated at 4 °C for 2 d before being placed at room temperature under white light for germination and growth. Two-week-old seedlings were used for the experiments. For growth on soil, seeds were sown on commercial potting compost and stratified at 4 °C in a cold room for 4 d before being transferred to a growth chamber under constant illumination at a light intensity of 50–100 mE m–2 s–1 and a temperature of 22 °C. The humidity was maintained at 60%. Three-week-old plants were used for the experiments. FAO transgenic lines were grown on selection media consisting of MS agar medium and kanamycin (50 mg l–1), while FAO transgenic lines crossed with pER8-ESR1 lines were grown on selection media consisting of MS agar medium, kanamycin (50 mg l–1), and hygromycin (30 mg l–1) that select for S-BJFAO2 and AS-BJFAO8, and pER8:ESR1 constructs, respectively.
Genomic DNA isolation and Southern analysis
Genomic DNA was isolated using the DNAzol ES kit according to the manufacturer's protocol (Molecular Research Centre, Inc., Cincinnati, OH, USA). For Southern analysis, 10 µg of genomic DNA was digested with different restriction endonucleases and separated on a 1% agarose gel by electrophoresis in 1x TRIS-borate-EDTA buffer. The gel was treated with 0.25 N HCl for 10 min, denatured twice in a solution of 0.5 N NaOH/1.5 M NaCl, each for 15 min with shaking, rinsed with sterile water, and transferred to the neutralization solution [0.5 M TRIS–HCl (pH 7.5) and 3 M NaCl] twice, each for 15 min with shaking. The DNA was transferred overnight onto a positively charged membrane (Roche Diagnostics GmbH, Mannheim, Germany) by capillary action in 10x SSC [3 M NaCl and 0.3 M sodium citrate (pH 7.0)]. The membrane was cross-linked, prehybridized at 42 °C for 2 h in DIGTM Easy Hybridization buffer (Roche Diagnostics GmbH), and hybridized overnight at 42 °C in the same buffer containing 25 ng ml–1 of a digoxigenein (DIG)-labelled double-stranded DNA probe. The membrane was washed in 2x SSC/0.1% SDS twice, each for 5 min at room temperature, followed by 0.5x SSC/0.1% SDS twice, each for 15 min at 68 °C, and 0.1x SSC/0.1% SDS for 15 min at 68 °C. Detection of the hybridized signal was carried out by capturing the chemiluminescent signal on an X-ray film after CDP-StarTM (Roche Diagnostics GmbH) was applied.
Stress and chemical treatments
Dehydration was induced by removing plants from the medium and placing them on a dry filter paper, which was placed in a flow hood for 30 min. Osmotic stress was imposed by NaCl or polyethylene glycol (PEG) (average molecular mass 6000 Da) treatment. Seedlings were pulled out of agar and placed on filter papers soaked with different concentrations of NaCl or PEG solutions. For abscisic acid (ABA), 1-aminocyclopropane-1-carboxylate (ACC), silver nitrate (AgNO3), 2-aminoethoxyvinyl glycine (AVG), benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), gibberellic acid (GA3), H2O2, methyl jasmonic acid (MeJA), Put, salicylic acid (SA), Spd, and Spm treatment, different concentrations of the chemicals in appropriate solvent solutions were sprayed on the seedlings. Seedlings were immersed in water of different pHs for pH treatment. For temperature treatment, 2-week-old seedlings in agar plates were incubated at 0 °C or 4 °C in the dark, and 22, 28, 30, or 37 °C under light, respectively. For cold treatment, seedlings were thawed at room temperature for 2 h. Control treatments with seedlings soaked or sprayed with solvent solutions or on agar plates under room temperature were also conducted simultaneously. All treatments conducted on Petri dishes were sealed and conducted under light at room temperature for 6 h unless otherwise stated. For phenotypic analysis of stress treatments of transgenic plants, 2-week-old wild-type and transgenic seedlings on the same MS agar plates were subjected to optimum conditions or different concentrations of stress and chemical treatments.
Shoot regeneration
Shoots were regenerated from Arabidopsis root culture as described by Banno et al. (2001). Seven-day-old seedlings were transferred to B5 liquid medium and incubated for 15 d with shaking at 125 rpm. The roots were cut into ~1 cm segments and six such segments were incubated on callus-inducing medium (CIM) consisting of MS medium (MS salts, Gamborg's B5 vitamins, 1% sucrose, and 0.25% Phytagel as gelling agent) supplemented with 2 µM 2,4-D. After 4 d on CIM, the root segments were transferred to shoot-inducing medium (SIM) comprising MS medium supplemented with 0.8 µM indole-3-acetic acid (IAA) and 12.5 µM N6-2-isopentyladenine. For shoot regeneration of pER8-ESR1 and crossed lines, SIM was supplemented with 10 µM 17-ß-oestradiol for induction of ESR1 activity. For each genotype, shoot regeneration experiments were repeated three times with 48 root segments tested in each independent experiment.
RNA isolation and real-time PCR
Total RNA from plant tissues was isolated using the RNeasy Kit (Qiagen, CA, USA) and reverse-transcribed by using the ThermoscriptTM real-time PCR System (Invitrogen Life Technologies, Carlsbad, CA, USA). Real-time PCR assays were performed in triplicate on a Bio-Rad iCycler iQ Real-Time Detection System (Bio-Rad Laboratories Inc., Baltimore, MD, USA) using tubulin (TUB2) as an internal standard. From the diluted cDNA, 1 µl (15 ng) was used as template in a 20 µl PCR containing 1x SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and 0.25 µM primer (each). The PCR thermal cycling parameters were 95 °C for 3 min, 95 °C for 20 s, followed by 55 cycles of 58 °C for 20 s, 72 °C for 20 s, 95 °C for 1 min, 55 °C for 1 min, and 80 cycles of 55 °C for 10 s. The difference between the cycle threshold (Ct) of the target gene and the Ct of tubulin, Ct=Cttarget gene–Cttubulin, was used to obtain the normalized expression of target genes, which corresponds to 2–Ct. Each experiment was replicated at least three times using samples collected separately. Primers designed for real-time PCR were as follows: SBJPRT1 (5'-TTCACCGTACCATACGCA-3') and SBJPRT2 (5'-GGCAGTGTAAAGCTTGTTCG-3') for BJFAO, ATP1RT1 (5'-CAACACCTTCGAGCTCTCAGT-3') and ATP1RT2 (5'-AACAGGGCTTCCACCGATTCC-3') for ATFAO1, and TUB2-P1 (5'-ATCCGTGAAGAGTACCCAGAT-3') and TUB2-P2 (5'-AAGAACCATGCACTCATCAGC-3') for TUB2.
Sequence analysis
The nucleotide sequence of cDNA was determined using the ABI PRISMTM Big Dye and dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, Boston, MA, USA). Sequence data were compared with the relevant sequences retrieved from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) database. Phylogenetic analyses were carried out using the program PAUP version 4.0b4 (Swofford, 2000) after alignment using ClustalW (Thompson et al., 1994).
Plasmid construction
The FAO fragments that were amplified from mustard and the Arabidopsis cDNA library were cloned into pGEM®-T Easy Vector (Promega, Madison, WI, USA) prior to preparation of sense, antisense, and double-stranded cDNA fragments. The sense BJFAO cDNA of 1764 bp was amplified using Sense Fwd PAO (5'-GCTCTAGAGCTTCACCGTACCATACG-3'; XbaI site underlined) and SP6 (5'-TATTTAGGTGACACTATAG-3') primers, respectively. After digestion, the XbaI–SacI fragment was inserted in a sense orientation between the cauliflower mosaic virus (CaMV) 35S promoter and the nopaline synthase (NOS) terminator in the pBI121 vector pre-digested with the same enzymes. The sense ATFAO cDNA of 1772 bp was amplified using ATSF (5'-CCCTTGGGAACACCGACAAGGAGATTCC-3'; HindIII site underlined) and ATSR (5'-TCCCCCGGGGGAGCTTCTCTAAACACATTACCC-3'; SmaI site underlined). After digestion with HindIII and SmaI, the cDNA was inserted in a sense orientation between two CaMV 35S promoters and the CaMV terminator in pGreen 0229 IT vector pre-digested with the same enzymes. For the antisense BJFAO plasmid, the 967 bp cDNA was amplified by PCR using AS Fwd PAO (5'-GCTCTAGAGCGGAGGACTAAACAAC-3'; XbaI site underlined) and AS Rev PAO (5'-CGAGCTCGTTCACCGTACCATACG-3'; SacI underlined). The resulting XbaI–SacI fragment was ligated into the pBII121 vector predigested with the same enzymes. The double-stranded construct of BJFAO was produced as described by Chuang and Meyerowitz (2000). Generally, the construct consisted of one ß-glucuronidase (GUS) segment (nucleotides 787–1809) flanked by two BJFAO fragments with the same sequence but in reverse directions. The BJFAO fragment used for the double-stranded construct was 610 bp (–1 to 610 bp) in length.
The sense, antisense, and double-stranded constructs were designated as S-BJFAO, AS-BJFAO, and DS-BJFAO, respectively. All constructs were introduced into Agrobacterium tumefaciens strain (LBA4404) and used to transform wild-type (Col-0) Arabidopsis thaliana by the floral dip method as described previously (Clough and Bent, 1998).
Enzyme activity
The level of PAO activity in plant tissues was determined by following the formation of a pink adduct (
515=2.6x104 m–1 cm–1), resulting from the oxidation and condensation of 0.1 mM 4-aminoantipyrine (4-AAP) and of 1 mM 3,5-dichloro-2-hydroxybenzenzenesulphonic acid (DCHBS) catalysed by 0.08 mg ml–1 of horseradish peroxidase in 0.2 M sodium phosphate buffer, pH 6.5, at 25 °C (Smith and Barker, 1988). Plant materials were homogenized in 0.2 M sodium phosphate buffer [tissue/buffer ratio 1:5 (w/v)]. The homogenate was centrifuged at 12 000 g for 20 min. A 20 µl aliquot of the supernatant was used for measurement of protein concentration by Bradford assay (Bradford, 1976) and 40 µl was used for enzyme assay. The reaction mixture comprised horseradish peroxidase (1 mg ml–1), sodium phosphate buffer (pH 6.5, 0.2 M), 4-AAP (0.1 mM), and DCHBS (1 mM) as well as 2 mM Spm as substrate. Enzyme activity was expressed as U g–1FW.
Polyamine measurement
The levels of PAs, i.e. Put, Spd, and Spm, in cultured Arabidopsis explants during shoot regeneration were determined based on the method described previously (Flores and Galston, 1982). Tissue (200 mg) was extracted by homogenization with 1 ml of 5% (v/v) cold perchloric acid. The extract was incubated on ice for 1 h and centrifuged at 14 000 rpm. for 20 min at 4 °C. Free PAs were dansylated overnight by mixing 200 µl of supernatant with 400 µl of dansyl chloride (5 mg ml–1 acetone) and 200 µl of saturated sodium carbonate. After overnight incubation, 100 µl of proline (100 mg ml–1) was added and the mixture was allowed to stand for 30 min at room temperature to remove excess dansyl reagent. This was followed by the addition of 0.5 ml of benzene to the mixture and vortexing for 60 s, after which the organic phase was retained in a glass vial and stored at –20 °C until use. The dansylated PAs were separated on silica gel thin-layer chromatography plates (Whatman LK6D, Middlesex, UK) and developed for 70 min in chloroform:triethylamine (25:2, v/v). The plates were dried for 5 min at 100 °C, and the dansyl-PA bands were identified under UV light, scraped, and eluted with 2 ml of ethyl acetate. PAs in the eluant were quantified using a fluorescent spectrophotometer (Shimadzu RF-1501, Shimadzu Incorporation, Tokyo, Japan) with excitation at 350 nm and emission at 495 nm. The standard curve for each PA was plotted by quantifying known concentrations of Put dihydrochloride, Spd trihydrochloride, and Spm tetrahydrochloride (Sigma).
Ethylene measurement
The level of ethylene produced by cultured explants at different intervals was measured as described previously (Chi et al., 1991). Six root segments were cultured in a 50 ml Erlenmeyer flask with 20 ml of CIM in the first 4 d and transferred to SIM on the fourth day. Prior to ethylene measurement, the flasks were aerated for 1 h in the laminar flow hood, sealed with airtight rubber stoppers, and allowed to stand for an additional 2 h. A 1 ml gas sample was withdrawn from each flask with a 1 ml hypodermic syringe. The ethylene content in the gas sample was determined by gas chromatography (Chi et al., 1991).
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Results |
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Cloning and sequence analysis of BJFAO and ATFAO1
A partial 750 bp fragment homologous to Arabidopsis AO was obtained from a specific cDNA pool that was created from B. juncea leaf discs grown on shoot regeneration medium for 5 d. Based on the above fragment, a 1747 bp full-length cDNA was cloned using rapid amplification of cDNA ends (accession no. AY188087). This cDNA consisted of a 90 bp 5'-untranslated region, a 31 bp 3'-untranslated region, and a 1626 bp open reading frame that encoded a putative polypeptide of 542 amino acid residues with an estimated mol. wt. of 59 390 Da and a theoretical pI of 5.32. This mustard polypeptide (BJFAO) shared 81% sequence identity with an Arabidopsis AO family protein designated ATFAO1 (At4g29720). Conserved domain analysis revealed that both FAOs possessed a flavin-containing amine oxidoreductase, which is a fingerprint motif in the family consisting of various AOs, including MPAO and other flavin-containing monoamine oxidases (MAOs) (Fraaije et al., 2000). ATFAO1 and BJFAO shared 21% and 17% amino acid identity with MPAO (Tavladoraki et al., 1998), and 20% and 18% with BPAO1 (Cervelli et al., 2001), respectively (Fig. 1).
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Phylogenetic analyses demonstrated that BJFAO and ATFAO1 were clustered closely with AOs from other species (Fig. 2). In addition, they revealed the presence of at least eight other Arabidopsis AOs that were grouped together with both ATFAO1 and BJFAO in the same cluster (Fig. 2). These putative PAOs were clustered closely with MPAO (ZM 064411; Tavladoraki et al., 1998), BPAO1 (HV1 AJ298131 [GenBank] ; Cervelli et al., 2001), and some animal MAOs, which demonstrated AO activities in previous studies (Hsu et al., 1988; Powell et al., 1989; Grimsby et al., 1991; Hashizume et al., 2003; Chen et al., 2004; Okauchi et al., 2004; Setini et al., 2005). Although MAO and PAO differed in their specificity towards substrates, they could be grouped in the same structural and functional class of flavin-dependent oxidases (Tavladoraki et al., 1998).
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DNA gel blot analyses of mustard and Arabidopsis genomic DNA were performed to determine the relative copy number of FAO genes (Fig. 3A). The full-length BJFAO probe was hybridized to several distinct bands (Fig. 3A), suggesting that BJFAO is encoded by a small gene family, while the full-length ATFAO1 probe was hybridized to single distinct bands, suggesting that ATFAO1 is encoded by a single gene (Fig. 3A). In particular, BJFAO and ATFAO1 shared low homology (19–25% amino acid identity) with eight Arabidopsis FAOs that were clustered together in the same subgroup.
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Spatial and temporal FAO expression in Arabidopsis organs and in response to treatments
To investigate whether FAO transcripts were spatially regulated, expression of ATFAO1 was examined by real-time PCR from total RNA isolated from various organs of wild-type Arabidopsis plants. Transcripts were detected in all organs, among which the expression levels varied greatly (Fig. 3B). While the gene expression was highest in cauline leaves and stems, it was lower in flower buds, roots, rosette leaves, and siliques, and least abundant in flowers.
In addition, ATFAO1 expression was detected in plants from 1 week old up to 7 weeks old (Fig. 3C). The gene expression was significantly up-regulated in the second week when the plants were at the floral transitional stage.
ATFAO1 expression was also investigated in response to external stimuli by subjecting Arabidopsis seedlings to various treatments such as exogenous phytohormones (Fig. 4A), temperature (Fig. 4B), osmotic stress (Fig. 4C), PAs (Fig. 4D), ethylene inhibitors (Fig. 4E), SA, H2O2, pH, and dehydration (Fig. 4F). After responsive treatments, the ATFAO1 transcripts were up-regulated in response to cold temperature (Fig. 4B, 0 °C and 4 °C), NaCl (Fig. 4C, 50–400 mM), PEG (Fig. 4C, 10–40%), and SA (Fig. 4F, 0.1–1 mM). In contrast, the transcripts were down-regulated in response to ABA (Fig. 4A, 50–400 µM), ACC (Fig. 4A, 10–500 µM), 2,4-D (Fig. 4A, 0.01–2.5 mg l–1), GA3 (Fig. 4A, 0.01–2.5 mg l–1), MeJA (Fig. 4A, 10–500 µM), high temperature (Fig. 4B, 28–42 °C), Put (Fig. 4D, 1–50 mM), Spd (Fig. 4D, 1–50 mM), Spm (Fig. 4D, 1–50 mM), and AVG (Fig. 4E, 5–50 µM). Dehydration (Fig. 4F) treatment did not significantly affect ATFAO1 expression. In some cases, the expression fluctuated upon treatment with different concentrations of chemicals, such as BA (Fig. 4A), AgNO3 (Fig. 4E), H2O2 (Fig. 4F, 10–100 mM), and pH (Fig. 4F, 5.5–8).
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Molecular characterization of transgenic Arabidopsis plants
To investigate the in vivo role of FAO in plants, FAO expression was modulated by expressing sense, antisense, and double-stranded BJFAO cDNAs under the control of the CaMV 35S promoter in transgenic Arabidopsis plants (Fig. 5A). The BJFAO cDNA used for antisense and double-stranded constructs shared 85% and 84% identity, respectively, with the corresponding sequences of Arabidopsis ATFAO1. The copy number of transgenes was determined in these transgenic plants by investigation of genetic segregation in their progeny and Southern blot analysis (see supplementary Fig. 1 at JXB online). The T3 homozygous lines were isolated for transformants with a single insertion, while the T1 generation line was maintained by tissue culture for transformants with multiple insertions, because isolation of their homozyous progeny was difficult due to complicated genetic segregation. In addition, an Arabidopsis line containing a putative T-DNA insert in At4g29720 (SALK_009671, designated atfao1) was obtained from the SALK Institute's SIGnAL collection (Alonso et al., 2003; Fig. 5B), and was confirmed by PCR analysis (data not shown).
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Real-time PCR analysis revealed that the BJFAO transcripts were highly accumulated in two sense transformants (Fig. 5C; and supplementary Fig. S1 at JXB online), while the endogenous ATFAO1 expression was not affected in these sense transformants (data not shown). The ATFAO1 transcripts were reduced to various degrees in all antisense (Fig. 5C, AS1–AS12) and double-stranded transformants (Fig. 5C, DS2–DS4), and atfao1 mutants (Fig. 5C, Salk). The mRNA expression of the other nine Arabidopsis FAOs that were closely clustered with BJFAO and ATFAO1 was also checked, and it was found that their expression was not significantly affected in these sense, antisense, and double-stranded transformants (data not shown).
Enzyme activity assays of selected transgenic plants were performed for every construct (Fig. 5D). It was observed that most of the transgenic plants showed enzyme activity relevant to the expression of the FAO mRNAs. However, the enzyme activities of two antisense lines (AS4 and AS12) and the atfao1 Salk line were higher than those in wild-type plants (Fig. 5D). In view of the fact that the expression of the other nine Arabidopsis FAOs was not significantly affected, it was deduced that ATFAO1 activity in these lines was either post-transcriptionally regulated or regulated via some unknown feedback mechanisms.
FAO activity does not affect plant development and stress tolerance
Transgenic plants with different levels of FAO activity grew normally and were phenotypically indistinguishable from wild-type plants throughout plant growth and development. Since plants usually respond to stress by increasing endogenous PA content, an investigation was carried out to determine whether the changes in PA content affected the stress response of transgenic plants with modulated PA levels (Bouchereau et al., 1999). These transgenic plants were treated under different external stimuli, and transgenic seedlings subjected to responsive stress treatments displayed similar phenotypes to wild-type plants (data not shown).
Effect of FAO activity on shoot regeneration in relation to ESR1, polyamines, and ethylene
To investigate whether shoot regeneration from cultured explants was affected by the altered endogenous levels of FAO activity, the regeneration capacity of root explants excised from wild-type and transgenic plants was compared. An obvious difference in shoot regeneration between wild-type and transgenic plants was observed after 20 d in culture (Fig. 6A). Transgenic lines with down-regulated FAO activity (AS-BJFAO6, AS-BJFAO8, DS-BJFAO2, DS-BJFAO3, and DS-BJFAO4) were more regenerative (79–89%) in culture compared with wild-type (65%) and pBI121 control explants (59%), while the transgenic lines with upregulated FAO activity (S-BJFAO2 and S-BJFAO5) were poorly regenerative, with only 24% and 34% explants forming shoots (Fig. 6A). In another independent experiment, Arabidopsis transgenic lines overexpressing the sense ATFAO1 cDNA were also generated (see supplementary Fig. S2 at JXB online). Two transgenic lines, named S-ATFAO1 and S-ATFAO4, respectively, were selected for further shoot regeneration analyses. They exhibited comparable low capacity of shoot regeneration with S-BJFAO2 and S-BJFAO5 (see supplementary Fig. 3 at JXB online).
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There is increasing evidence showing that shoot regeneration is genetically regulated. One typical example is the involvement of an ESR1 gene, which encodes a putative transcription factor with an AP2/EREBP domain (Banno et al., 2001). Expression of ESR1 could be up-regulated by exogenous cytokinin. Overexpression of ESR1 in transgenic plants conferred cytokinin-independent shoot formation from root explants and increased shoot regeneration efficiency in the presence of cytokinin (Banno et al., 2001). To elucidate further the molecular basis of FAO function in shoot regeneration, two representative T3 homozygous transgenic lines, S-BJFAO2 and AS-BJFAO8, that had a single insertion of a transgene, were chosen for further crossing with an oestrogen receptor-based inducible ESR1 overexpression line, designated pER8-ESR1 (Zuo et al., 2000; Banno et al., 2001). The crossed plants were grown on selection media that selected for S-BJFAO2, AS-BJFAO8, as well as the pER8:ESR1 construct, and F3 homozygous hybrid plants were used for shoot regeneration studies. The induction of ESR1 in the crossed plants was also confirmed as previously published (Banno et al., 2001). In accordance with the previous study (Banno et al., 2001), induced pER8-ESR1 explants (80%) were more regenerative than non-induced pER8-ESR1 explants (63%) (Fig. 6B). Interestingly, when induced, pER8-ESR1 AS-BJFAO8 (83%) showed a similar regeneration rate to AS-BJFAO8 (85%) and pER8-ESR1 (80%), whereas pER8-ESR1 S-BJFAO2 (23%) exhibited s similar regeneration rate to S-BJFAO2 (26%). Thus, the effect of FAO activity on shoot regeneration was clearly downstream of ESR1 activity.
The altered FAO activity may be associated with different concentrations of PAs, which is a possible factor causing the change of shoot regeneration responses in generated transgenic plants, as shown in some previous studies (Monteiro et al., 2002; Hunter and Burritt, 2005). To verify the above possibility, the content of PAs in cultured tissues was analysed during shoot organogenesis. In general, all transgenic lines possessed higher levels of Put than the wild type during culture (Fig. 7A). The Put content in wild-type explants was shown to increase up to 5 d and decreased thereafter during the remaining culture period (Fig. 7A). Unlike wild-type plants, the Put content in S-BJFAO2 and AS-BJFAO8 decreased upon transfer to SIM (day 5) and subsequently increased up to 8 d and 10 d, respectively (Fig. 7A). With respect to Spd, there was a marked difference between wild-type and transgenic explants after 4 d of culture in that Spd levels in the wild type surged markedly (Fig. 7A). Wild-type explants were shown to possess a significantly higher level of Spd than all transgenic lines on day 5 (Fig. 7A), while all the transgenic lines exhibited similar Spd levels throughout the culture period (Fig. 7A). Like Spd, the Spm levels in the wild type also increased greatly after 4 d of culture compared with those in transgenic lines (Fig. 7A). Moreover, AS-BJFAO8 contained higher levels of Spm than S-BJFAO2 throughout the culture period (Fig. 7A). In general, the individual concentrations of endogenous PAs were significantly different between wild-type and FAO transgenic explants after they were transferred from CIM (day 4) to SIM (day 5).
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Since it has been suggested that high ratios of Put/Spd and Put/Spd+Spm may cause low morphogenic capacity (Bajaj and Rajam, 1996; Hunter and Burritt, 2005), the ratios of different PAs in wild-type and transgenic explants were compared further (Fig. 7B). It was found that S-BJFAO2 possessed the highest Put/Spd and Put/Spd+Spm throughout the entire culture period. The common trend of all these plants after transfer to SIM (day 5) is the decreased ratio of Put/Spd and Put/Spd+Spm (Fig. 7B), suggesting that low ratios of Put/Spd and Put/Spd+Spm are markers of shoot induction.
As evidence from previous studies showed that shoot regeneration was associated with ethylene (Pua and Chi, 1993; Pua and Lee, 1995), the effect of BJFAO expression on ethylene production in cultured explants of wild-type and transgenic plants was also examined in this study. Surprisingly, the patterns of ethylene production in both wild-type and transgenic plants were similar during 20 d of culture (Fig. 7C).
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Discussion |
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Amine oxidation is important in a number of basic biological processes ranging from lysyl oxidation in the cross-linking of collagen to the catabolism of neurotransmitters and PAs. The oxidation of biogenic amines is catalysed by either the quinoprotein class of enzymes (usually primary amines) (Hartmann and McIntire, 1997) or the flavin-containing AOs (primary, secondary, or tertiary amines) (Massey, 2000).
AOs represent a class of enzymes heterogeneous in structure, catalytic mechanism, and mode of substrate oxidation. They are found widespread in bacteria, fungi, animals, and plants, and are classified into Cu-AO and FAD-AO. PAOs belong to the latter subclass and they occur in high specific activity in the Leguminosae and Graminae. To date, only one study of dicotyledenous PAO has been reported in alfalfa (Bagga et al., 1991). Although several putative dicotyledenous PAO sequences can be retrieved from the Arabidopsis database, their concrete functions are unknown. In this study, ATFAO1 and Brassica BJFAO were characterized, both of which show close sequence similarity to maize MPAO and barley BPAO1.
MPAO is expressed constitutively in all organs tested, with higher levels of expression in leaves and coleoptiles than in roots, mesocotyl outer tissue, and stele (Cervelli et al., 2000). In barley, BPAO1 was expressed only in the ear while BPAO2 could be detected in all tissues examined (Cervelli et al., 2001). In the present study, it was found that ATFAO1 transcripts were detectable in all organs, with the most abundant transcripts in cauline leaves. The variable expression of ATFAO1 observed in whole seedlings from 1–7-weeks-old indicates that ATFAO1 expression may be temporally regulated. In particular, the significantly increased expression of ATFAO1 in the second week after seed germination implies the involvement of the PA metabolic pathway in floral transition of Arabidopsis. This is consistent with the previous suggestion that optimum levels of endogenous PA may be required for flowering (Applewhite et al., 2000).
PAs have been suggested to play a protective role against stress in plants because PA biosynthesis enzymes and cellular PAs usually increase in response to stress (Bouchereau et al., 1999). The present study showed that the ATFAO1 gene was up-regulated in response to cold temperature, NaCl, PEG, and SA, while it was down-regulated in response to ABA, ACC, 2,4-D, GA3, MeJA, high temperature, Put, Spd, Spm, and AVG. The down-regulation of ATFAO1 by synthetic auxin (2,4-D) is in agreement with the finding that MPAO1 expression in outer tissues of maize mesocotyl was reduced by auxin treatment (Cona et al., 2003). Some of the above changes of ATFAO1 expression may reflect the direct response of ATFAO1 in defence against stress. However, some stress treatments may indirectly affect ATFAO1 expression by regulating the substrates or catabolic products of PAOs, because the inhibitory effects of Put, Spd, Spm, and H2O2 on ATFAO1 could be clearly observed (Fig. 4). In addition, ATFAO1 expression was variable under the treatment with different levels of ethylene (AgNO3) and BA (Fig. 4), indicating that ATFAO1 expression may involve complex feedback regulatory mechanisms.
For the last 15 years, there has been increasing evidence, derived mainly from physiological studies, showing that PAs are implicated in plant morphogenesis in vitro. Increased regeneration has been correlated with endogenous PA accumulation (Pua et al., 1999) or exogenous application of PAs (Chi et al., 1994). Furthermore, the inhibition of PA synthesis has been shown to prevent regeneration (Pua et al., 1996). The regulatory role of PAs in plant morphogenesis in vitro has also been supported by transgenic studies, in which carrot cells expressing mouse ODC cDNA showed increased Put biosynthesis and promoted somatic embryogenesis (Bastola and Minocha, 1995). The similar promoting effect of increased PA titres on the plant regeneration potential has been reported in rice callus expressing an ODC cDNA (Kumria and Rajam, 2002). These findings have shown an essential role for PAs in somatic embryogenesis and implicated the importance of regulating the cellular PA levels.
The results of this study have provided direct evidence showing that FAO is an important enzyme in the PA catabolic pathway, and the activity of FAO can affect the levels of individual PAs, thus regulating shoot regeneration. Overexpression of BJFAO markedly decreased the shoot regeneration capacity of the cultured tissue, while down-regulation of ATFAO1 expression promoted regeneration. Moreover, transgenic plants overexpressing the Arabidopsis homologue ATFAO1 under the control of the 35S promoter showed similar shoot regeneration results to S-BJFAO2 plants. This demonstrated that the results obtained by the heterologous system are comparable with those obtained by the homologous system.
In the transgenic plants, the changes of the individual concentrations of endogenous PAs were significantly different from those of wild-type plants upon transfer from CIM to SIM, indicating that the levels of PAs are closely related to shoot regeneration capacity. In addition, the demonstration of higher Put/Spd and Put/Spd+Spm ratios in S-BJFAO2 throughout the culture period is in line with previous findings that cultured tissues with a higher Put/Spd ratio displayed a low morphogenic capacity (Bajaj and Rajam, 1996; Hunter and Burritt, 2005). It is noteworthy that FAO may affect shoot regeneration downstream of the ESR1 gene, which has been suggested to function in a branch of the cytokinin signalling pathway that directs shoot regeneration (Banno et al., 2001). This is supported by the genetic crossing data showing that the effect of FAO activity on shoot regeneration capacity was independent of overexpression of ESR1 (Fig. 6). This result, together with the observation that BA could up-regulate ATFAO1 expression (Fig. 4A), suggests that the cytokinin signalling pathway may be involved in the regulation of PA levels in plants.
Enhancement of shoot regeneration and somatic embryogenesis can also be achieved by inhibition of ethylene synthesis or action using ethylene inhibitors (Pua and Chi, 1993), or down-regulation of genes of ethylene biosynthetic enzymes (Pua and Lee, 1995). Although the mechanism of ethylene action in plant morphogenesis is not clear, it has been suggested that enhanced shoot regeneration by ethylene inhibition may be attributed to increased PA synthesis, in view of the scenario that PAs and ethylene may compete for the same precursor S-adenosylmethionine for their synthesis (Pua et al., 1996). In the present study, modulation of FAO activity regulated shoot regeneration, but did not affect ethylene levels, indicating that altered PA levels rather than decreased ethylene levels are directly responsible for shoot regeneration.
Although the evidence thus far indicates the role of PAs in shoot morphogenesis, the mechanism of PA action is not clear. The results of several studies in recent years indicated the possible implication of the PA oxidative product, H2O2, in somatic embryogenesis. This assumption is in accordance with the results of several recent studies showing that stress induces the occurrence of several morphogenic events in vitro, including shoot formation in flax (Mundhara and Rashid, 2001), somatic embryogenesis of Astragalus adsurgens (Luo et al., 2001), cotton (Kumria et al., 2003), and Arabidopsis (Ikeda-Iwai et al., 2003), and androgenesis and subsequent plant regeneration in rye (Immonen and Anttila, 1999) and triticale (Immonen and Robinson, 2000). On the other hand, it was shown that overexpression of glutathione-S-transferase (GST, EC 2.5.1.18 [EC] ), an enzyme associated with limiting oxidative damage, enhanced the capability of shoot regeneration from Arabidopsis explants (Gong et al., 2005). Similarly, activation of the reactive oxygen species-scavenging mechanism resulted in increased regeneration of plant protoplasts (Papadakis and Roubelakis-Angelakis, 2002). Therefore, it seems that H2O2 can play a bivalent role in shoot regeneration, and its exact relevance to the effect of PAs needs to be further investigated.
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Supplementary data |
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Supplementary data can be found at JXB online.
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Acknowledgements |
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This work was supported by the National University of Singapore (research grants R-154-000-209-112 and R-154-000-232-101) and intramural research funds from Temasek Life Sciences Laboratory. TSL and PH were supported by postgraduate scholarships from the National University of Singapore. The authors would like to thank the SALK Institute Genomic Analysis Laboratory and the Arabidopsis Biological Research Centre for providing the T-DNA insertion line, Dr Nam Hai Chua from the Rockefeller University for the XVE-ESR1 stock/pER8-ESR1 line, Dr Banno Hiroharu of Chubu University for useful advice on pER8-ESR1 line, and Drs Toshiro Ito and Wenwei Hu for critical reading of the manuscript.
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Abbreviations |
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2,4-D, 2,4-dichlorophenoxyacetic acid; ABA, abscisic acid; ACC, 1-aminocyclopropane-1-carboxylate; AO, amine oxidase; AVG, 2-aminoethoxyvinyl glycine; BA, benzyladenine; CaMV, cauliflower mosaic virus; CIM, callus-inducing medium; Cu-AO, copper amine oxidase; DAO, diamine oxidase; FAD-AO, flavin adenine dinucleotide amine oxidase; FAO, flavin-amine oxidase; GA, gibberellic acid; GABA,
-aminobutyric acid; H2O2, hydrogen peroxide; MAO, monoaminase oxidase; MeJA, methyl jasmonic acid; PA, polyamine; PAO, polyamine oxidase; PEG, polyethylene glycol; Put, putrescine; SA, salicylic acid; SIM, shoot-inducing medium; Spd, spermidine; Spm, spermine; UTR, untranslated region. |
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