Functional analysis of the isopentenyltransferase gene MdIPT3a from apple (Malus pumila Mill.)

Plant isopentenyltransferases (IPTs) catalyse the first and rate-limiting step in the synthesis of cytokinin phytohormones. To understand the function of IPTs in apple (Malus pumila Mill.), a gene encoding an IPT (MdIPT3a; GenBank Accession No. HQ585947) was cloned from shoot tips of 'Fuji' apple using the reverse transcription-polymerase chain reaction (RT-PCR) method. Semi-quantitative RT-PCR showed that expression of MdIPT3a was detected in shoot-tips, young leaves, bark tissue, leaf buds, floral buds, flowers, and young fruit. Transformation of tobacco plants with the MdIPT3a gene, driven by the cauliflower mosaic virus (CaMV) 35S promoter, showed that over-expression of MdIPT3a led to the inhibition of root development, enhanced outgrowth of axillary buds, and a delay in flowering. Cytokinin-specific organogenesis of shoots in transformed tobacco was observed by in vitro culture of Md/PT3a-transformed leaf explants that over-expressed MdIPT3a and generated adventitious shoots on plant growth regulator (PGR)-free Murashige and Skoog (MS) medium. The endogenous concentrations of isopentenyl adenosine, zeatin riboside, and dihydrozeatin were significantly higher in Md/PT3a transformed T1 tobacco plants with more axillary buds than in non-transformed control plants. These results indicate that expression of MdIPT3a is involved in plant growth and development. Expression of MdIPT3a from the CaMV 35S promoter appears to enhance the morphogenic potential of transformed plants by increasing endogenous cytokinin levels.