Cloning and characterisation of three groups of cysteine protease genes expressed in the senescing zone of white clover (Trifolium repens) nodules

In the present study, we have cloned and characterised three full-length and two partial cysteine protease-encoding genes from a white clover (Trifolium repens) genomic library. The five genes could be assigned to three groups based on their nucleotide and deduced amino acid sequences and they belong to the C1 family of cysteine proteases. Semi-quantitative RT-PCR analysis revealed that the three groups of genes were expressed in nodules but not in root, leaves, petioles and internodes. Transcription of two of the gene groups was initiated in the nodules 24 days after inoculation while the third group initiated transcription at a slightly later stage. In situ hybridisation analysis assigned the transcriptional activity to the senescing zone of the nodules. Comparative semi-quantitative RT-PCR analyses of two T. repens genotypes revealed no differences with respect to timing, specificity and quantity of gene transcription between the two genotypes. Southern blot analysis suggested the presence of two to three loci for each of the three groups with some degree of polymorphism between the two genotypes. The identified T repens cysteine protease genes showed high homology to cysteine proteases from Astragalus sinicus, Alnus glutinosa and Medicago truncatula. These cysteine proteases all show enhanced/specific expression in the senescing zone of nodules. (C) 2004 Elsevier Ireland Ltd. All rights reserved.