Transgenic cell lines as a useful tool to study the biochemistry of down-regulation of an endogenous rice gene using a heterologous diamine-oxidase cDNA

Transgenic rice (Oryza sativa L.) cell lines engineered with the pea diamin e oxidase cDNA in antisense orientation under the control of two different promoters were recovered using particle bombardment. Plasmids p35Sdaoa and pEdaoa contained the pea diamine oxidase cDNA driven by the CaMV35S and the pea ENOD12 nodulin promoter, respectively. Molecular analyses confirmed th e stable integration of the transgene and active transcription (mRNA) with both promoters driving the gene of interest. We observed a a-fold decrease in diamine oxidase activity (P < 0.01) in transformed callus lines expressi ng p35Sdaoa compared to wild-type or lines expressing the selectable marker gene alone (hpt). In cell lines transformed with pEdaoa, a 2.5-fold reduct ion in enzyme activity was measured (P < 0.01). Biochemical analysis of cel lular polyamines from p35Sdaoa transformants indicated up to a B-fold incre ase in putrescine levels (P < 0.001). Concentrations of higher polyamines a lso increased, with spermidine and spermine levels increasing 8- (P < 0.05) and 3.5-fold (P < 0.01) respectively. A maximum of 2.5-fuld increase in pu trescine (P < 0.001) and spermidine (P < 0.001) was measured in lines trans formed with pEdaoa. No significant variation (P > 0.05) was observed in spe rmine levels in pEdaoa transformants. We demonstrate for the first time the down-regulation of an endogenous enzyme involved in polyamine metabolism i n plants accompanied by a concomitant increase in the concentration of putr escine and spermidine. (C) 2000 Editions scientifiques et medicales Elsevie r SAS.