The white clover enod40 gene family. Expression patterns of two types of genes indicate a role in vascular function

Enod40 is one of the genes associated with legume nodule development and has a putative role in general plant organogenesis. We have isolated a small enod40 gene family from white clover (Trifolium repens), with three genes designated Trenod40-1, Trenod40-2, and Trenod40-3, all containing the conserved enod40 regions I and II. Trenod40-1 and Trenod40-2 share over 90% homology in the transcribed regions and high levels of similarity in their upstream regulatory sequences. Trenod40-1 and Trenod40-2 are similar to the enod40 genes of legumes forming indeterminate nodules (group II) and are predominantly expressed in nodules. Trenod40-3 shares only 32.8% identity with Trenod40-1 and Trenod40-2 within the transcribed region. Trenod40-3 is similar to the enod40 genes of legumes with determinate nodules (group I) and is not predominantly expressed in nodules. To our knowledge, this is the first report of both group I- and group II-type enod40 genes being expressed in a single legume species. In situ hybridization studies revealed that Trenod40 genes were highly expressed in non-symbiotic tissues, particularly in stolon nodes during nodal root and lateral shoot development. High levels of Trenod40 transcripts were also present in the vascular bundles of mature plant organs, mainly at sites of intensive lateral transport, suggesting a role in vascular tissue function. The expression pattern of Trenod40 genes was analyzed further using Trenod40 promoter-gus fusions in transgenic white clover and tobacco (Nicotiana tabacum), indicating that white clover and tobacco share the regulatory mechanisms for Trenod40-1/2 promoters and some aspects of Trenod40-3 regulation.