Isolation and characterization of three polyamine oxidase genes from Zea mays§
Introduction
Polyamine oxidase (PAO), a flavin adenine dinucleotide (FAD) containing enzyme, catalyses the oxidation of polyamines at the secondary amino group, giving different products in different taxa. In particular, vertebrate PAOs (EC 1.5.3.11) efficiently transform N1-acetyl derivatives of spermidine and spermine into putrescine and spermidine, respectively, plus acetamidopropanal and H2O2, and participate in the inter-conversion of polyamines [6], [17], [19]. PAOs with similar characteristics occur in fungi [6]. On the other hand, plant [6], bacterial [19] and protozoa [11] PAOs oxidize spermidine and spermine to 4-aminobutyral or 3-aminopropyl-4-aminobutyral, respectively, plus 1,3-diaminopropane and H2O2. As these compounds cannot be converted directly to other polyamines, this class of PAO is considered to be involved in terminal catabolism of polyamines. Plant PAOs, which apparently occur mainly in the cell wall of monocots, have been purified and partially characterized from few species [6], [9]. PAO from maize (MPAO), the most studied member of this enzyme class, is a monomeric glycoprotein with a molecular mass of 53 kDa, as determined by SDS-PAGE, containing one molecule of FAD [10], [18]. Recently, its complete amino acid sequence has been determined by a combined approach of protein and cDNA sequencing [21], and its crystal structure has been solved to a resolution of 0.19 nm [5]. Growing evidence suggests that the production of H2O2 in the cell wall is a mediator of several physiological events such as programmed cell death, lignification, wall stiffening and cellular defence [12], [15]. It has been suggested that PAO is important in producing H2O2 in vivo for peroxidase-catalysed wall-stiffening reactions during cell growth and differentiation [13]. Moreover, H2O2 produced by PAO is an essential cofactor in lignification of xylem tissues and a general trigger of programmed cell death [13].
In order to understand the regulatory mechanism of PAO synthesis, it is necessary to isolate the corresponding genes from a given plant. In this paper, we have isolated in maize, by PCR amplification, three genes encoding for polyamine oxidase, here designated MPAO1, MPAO2 and MPAO3. They show a conserved gene organization and almost identical amino acid sequences, indicating that they have originated from an ancestral gene by duplication events. Long inverted repeat sequences, of unknown origin and also present in other maize genes, have been localized within the second intron. The 5’-flanking regions of MPAO1 and MPAO2 genes have been cloned and analysed searching for putative cis-acting promoter motifs. The results from genomic Southern analysis support the existence of at least three members in the PAO gene family in maize and also confirm the presence of a PAO gene family in other monocots. On the contrary, three dicots examined did not show any detectable hybridization signal. A northern blot analysis revealed a unique hybridizing band of about 1 900 nt and a tissue-specific accumulation of the MPAO mRNA, which is higher in leaves and coleoptiles compared to cortex, roots and stele. A similar tissue-specific accumulation is observed for the MPAO protein, indicating that the expression of this enzyme is most probably controlled at the transcriptional level.
Section snippets
MPAO gene cloning and analysis
Using specific primers designed on the reported MPAO cDNA sequence [21], three genomic fragments of approximately 3.8, 3.6 and 3.3 kb resulted from direct PCR amplification of total DNA extracted from maize. The cloning and sequencing of these PCR products revealed that they correspond to three different copies of the MPAO gene, designated MPAO1, MPAO2 and MPAO3. In order to test the fidelity of these sequences, several independent gene specific clones and PCR products were sequenced and
MPAO gene sequences
Three maize MPAO genes have been cloned by direct PCR; their sequencing has shown a conserved mosaic structure consisting of eight exons and seven introns and an almost identical protein coding sequence, indicating that they arose by gene duplication. At first glance, the high degree of similarity among the three sequences raises the question of whether MPAO1, MPAO2 and MPAO3 may in fact be allelic. However, this is not the case, since the gene sequences are substantially divergent in the
Plant material and growth conditions
Commercially available seeds of maize (Zea mays, cultivar Paolo, Dekalb) were soaked for 12 h in running tap water and germinated at 26 °C in moistened vermiculite and in the dark. Three-day-old etiolated seedlings were exposed to continuous white light from fluorescent lamps (9 W·m–2) for 24 h. For expression experiments roots, leaves, coleoptiles, cortical plus epidermal tissues of mesocotyl (outer tissues) and mesocotyl stele were harvested, frozen immediately in liquid nitrogen and stored
Acknowledgements
This research was supported by grants from the ‘Ministero dell’Università e della Ricerca Scientifica e Tecnologica’ (MURST 60 %) and from the ‘Consiglio Nazionale delle Ricerche’ (‘Progetto Finalizzato Biotecnologie’, contract No. 97.01017.PF.49). The authors wish to thank Dr Elena Nebuloso for technical assistance.
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§ The nucleotide sequences of MPAO1, MPAO2 and MPAO3 genes have been submitted to the EMBL nucleotide sequences databases under accession numbers AJ251568, AJ251018 and AJ251019 respectively.