Modified expression of cysteine protease affects seed germination, vegetative growth and nodule development in transgenic lines of Medicago truncatula

Transgenic lines of Medicago truncatula (R108-1) were constructed to analyse Cyp15a (cysteine protease) gene functioning and localisation. The promoter region of PsCyp15a was fused to the coding sequence of uidA. GUS-analysis of transgenic plants containing this construct revealed strong expression from the Cyp15 promoter in cotyledonary leaves, senescent leaves, and root nodules. A seven-fold increase in GUS activity was observed following treatment of seedlings with 0.6 M mannitol and a five-fold increase with 75 mM NaCl. This confirmed previous reports that Cyp15a is a stress-inducible gene. Immunolabelling of nodule sections showed localization of CYP15A antigen in large vacuolar bodies and to a smaller extent in cytoplasmic vesicles. In seeds, antigenicity was located in the protein storage vacuoles. In a separate series of experiments, part of the coding sequence for MsCyp15a was fused in antisense orientation to the promoter region of early nodulin gene MtENOD12 and to the promoter region of the Medicago leghaemoglobin gene Lb1 (a late nodulin gene). Analysis of both sets of antisense lines revealed unexpected developmental effects in vegetative tissues as well as changes in nodule development. Seed germination was significantly impaired compared to untransformed lines, although treatment with gibberellin (1 μM GA3) could overcome this inhibition. Transformed plants ranged from slow growing and fatal phenotypes to leafy and late-senescing types. Cytological analysis of nodules revealed effects ranging from mild (infected cells with larger vacuoles and a low number of bacteroids) to severe (degenerate nodules with poor colonization of host cells). These results indicate a role of Cyp15a cysteine protease in germination and stress adaptation and also in nodule organogenesis and function.