The Pea Nodulation Mutant R50 (sym16) Displays Altered Activity and Expression Profiles for Cytokinin Dehydrogenase

Abstract

R50 (sym16) is a pleiotropic mutant of pea (Pisum sativum L.) which develops few, pale nodules and has pale young leaves. This phenotype coincides with elevated cytokinin content in vegetative organs, especially mature shoots. Because cytokinin content is known to be tightly regulated by the catabolic action of cytokinin dehydrogenase (CKX), this study focuses on whether CKX-mediated regulation of cytokinin content is involved in the R50 phenotype. Thus, we analyzed the biochemical activity of this enzyme in vitro and found that R50 displayed an aberrant activity profile. During development, PsCKX activity was significantly reduced when compared to wild-type (WT); this was observed in many tissues, specifically in mature shoots and nodules where decrease in activity correlated with elevated cytokinin content. To further address this issue, a full-length cDNA corresponding to CKX1 from pea (PsCKX1) was obtained via RACE-PCR. Although sequencing the entire PsCKX1 cDNA from R50 did not reveal any significant mutations that could have linked PsCKX1 to the sym16 mutation, relative transcript levels of PsCKX1 and of another PsCKX homolog (PsCKX2) were compared between R50 and WT using semiquantitative reverse transcriptase PCR. Interestingly, transcription of these homologs was upregulated in the tissues of R50 displaying the most aberrant phenotype, namely, the mature shoots and nodules. We propose that the R50 phenotype is linked to elevated cytokinin content as a result of deficient PsCKX activity and that transcription of two PsCKX homologs is upregulated as a means to compensate for the biochemical deficiency of this enzyme in R50 mutants.