Promoter strength influences polyamine metabolism and morphogenic capacityin transgenic rice tissues expressing the oat adc cDNA constitutively

We analyzed molecularly and biochemically a series of transgenic rice lines expressing the oat ade (arginine decarboxylase) cDNA under the control of the constitutive maize ubiquitin 1 promoter. We established baseline bioche mical parameters to elucidate the role of polyamines (PAs) during morphogen esis. We measured mRNA levels, ADC enzyme activity and cellular PAs in dedi fferentiated callus. Polyamine levels were also quantified in two subsequen t developmental stages - regenerating tissue and differentiated shoots. We observed significant (P < 0.05) differences in the levels of individual PAs at the three developmental stages. The amounts of putrescine (Put) and spe rmidine (Spd) in dedifferentiated transgenic callus were lower than those i n the wild type or in hpt (hygromycin resistant)-controls, whereas the amou nt of spermine (Spm) was increased up to two-fold. In regenerating tissue, this trend was reversed, with significantly higher levels of Put and Spd (P < 0.05), and lower levels of Spm (P < 0.05) compared to non-transformed or hpt-control tissues at the same developmental stage. In differentiated sho ots, there was a general increase in PA levels, with significant increases in Put, Spd, and Spm (P < 0.05); on occasion reaching six times the level o bserved in wild type and hpt-control tissues. These results contrast those we reported previously using the weaker CaMV 35S promoter driving adc expre ssion. mRNA measurements and ADC enzyme activity were consistently higher ( P < 0.01) in all tissues expressing pUbiadcs compared to equivalent tissues engineered with 35Sadc. Our findings are consistent with a threshold model which postulates that high ade expression leading to production of Put abo ve a basal level is necessary to generate a big enough metabolic pool to tr igger PA flux through the pathway leading to an increase in the concentrati on of Spd and Spm. This can be best accomplished by a strong constitutive p romoter driving adc. We discuss our results in the context of flux through the PA pathway and its impact on morphogenesis.