Barley polyamine oxidase isoforms 1 and 2, a peculiar case of gene duplication.
Polyamine oxidases (PAOs, EC 1.5.3.11) are key enzymes responsible for the terminal catabolism of polyamines in plants, bacteria and protozoa. In barley, two PAO isoforms (HvPAO1 and HvPAO2) have been previously analyzed as regards their tissue expression and subcellular localization. Only the major isoform HvPAO2 has been biochemically characterized up to now. In order to study the ear-specific expression of the HvPAO1 isoform in detail, RT-PCR analysis was performed in barley on the whole ear and on various ear tissues. Moreover, HvPAO1promoter::GUS transient expression was examined in barley developing caryopses at 30-day postfertilization. Results from these analyses have demonstrated that the HvPAO1 gene is specifically expressed in all the ear organs analyzed (i.e. basal lemma, rachis, awn, embryo-deprived caryopsis, embryo and sterile spikelets), at variance with the HvPAO2 gene, which is expressed at high levels in sterile spikelets and at very low levels in embryos. We purified HvPAO1 from barley immature caryopses and characterized its catalytic properties. Furthermore, we carried out in vitro synthesis of HvPAO1 protein in a cell-free translation system. The HvPAO1 enzymes purified from immature caryopses and in vitro synthesized showed the same catalytic properties, in particular, an optimum at pH 7.0 for Spd and Spm oxidation and comparable Km values for both substrates, i.e. 0.89x10(-5) M and 0.5x10(-5) M for Spd and Spm, respectively. It has been found that HvPAO1 enzyme activity significantly differs in substrate specificity and pH optimum when compared with the major isoform HvPAO2. As a whole, these data strongly suggest that, in barley, the two PAO genes evolved separately, after a duplication event, to code for two distinct tissue-specific enzymes, and they are likely to play different physiological roles.
Cervelli
Manuela
M
Dipartimento di Biologia, Università Roma Tre, Rome, Italy.
Bianchi
Marzia
M
Cona
Alessandra
A
Crosatti
Cristina
C
Stanca
Michele
M
Angelini
Riccardo
R
Federico
Rodolfo
R
Mariottini
Paolo
P
D001483:Base Sequence; D019143:Evolution, Molecular; D020440:Gene Duplication; D020131:Genes, Duplicate; D001467:Hordeum / Q000033:anatomy & histology / Q000201:enzymology* / Q000235:genetics; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D014018:Tissue Distribution / Q000235:genetics; D000097667:Polyamine Oxidase
D007527:Isoenzymes; D000587:Oxidoreductases Acting on CH-NH Group Donors; D010940:Plant Proteins; D012333:RNA, Messenger; D000097667:Polyamine Oxidase