Comparative studies on ENOD40 in legumes and non-legumes

Plants, unlike animals, continue to form organs after the completion of embryogenesis. This continuous formation of new organs allows plants to be flexible in a constantly changing environment. A unique example of adaptation to an environmental signal can be found among members of the family of Leguminosae . Legumes can enter a symbiosis with Rhizobium bacteria that leads to the formation of a complete new organ, the root nodule. Inside these nodules, the hosted bacteria fix atmospheric nitrogen into ammonia that can be used by the plant. Root nodule formation is considered to be acquired by legumes in the course of evolution (Gualtieri and Bisseling, 2000). Several studies show that evolution of new traits involves changes of transcription factors, signalling molecules and structural proteins (Carroll et al ., 2001). Homologues of nodulin genes (genes that are highly induced during nodule formation) can be found in non-legumes. This indicates that at least some of the genes and maybe even processes necessary for nodule development must be functioning in non-legumes, suggesting that rhizobia have recruited genes involved in general plant development for nodule formation. The presence of nodulin homologues in non-legumes opens the possibility to compare the regulation and function of nodulins and nodulin homologues in legumes and non-legumes. From such studies one might learn how the regulation or function of these genes was adjusted to establish a symbiosis with Rhizobium .

We studied the regulation and function of the legume ENOD40 gene and its tomato homologue LeENOD40 . ENOD40 homologues have been found in legumes as well as non-legumes such as rice (Kouchi et al . 1999), maize, citrus and tobacco (Van de Sande et al . 1996). In the legume Medicago truncatula , ENOD40 was shown to be required for proper nodule development (Charon et al . 1999). Ectopic expression of soybean ENOD40 in the non-legume tobacco leads to reduced apical dominance (Van de Sande et al. 1996). This indicates that ENOD40 plays an important role in both nodule formation and non-symbiotic plant development.

In chapter 1 an introduction on root nodule development in legumes is described and the current knowledge on the role of ENOD40 in this process is summarised.

In chapter 2 the isolation of the LeENOD40 gene from tomato and its mapping position on the tomato genome is described. To enable detailed expression studies of ENOD40 , LeENOD40::GUS is introduced in tomato. Expression of LeENOD40::GUS is analysed throughout the plant life cycle. LeENOD40::GUS expression strikingly co-localises with sites of increased ethylene production in plant development such as in the seed after germination and in flowers before the onset of and during flower senescence. Furthermore, the expression studies show that LeENOD40 is negatively regulated during initiation of lateral root formation, suggesting that the gene plays a role in lateral root formation.

In chapter 3 we compare regulation of LeENOD40::GUS and GmENOD40::GUS expression in a legume and a non-legume background. Our studies show that LeENOD40::GUS and GmENOD40::GUS expression are similarly regulated in non-legumes. We also show that LeENOD40::GUS is expressed in similar nodular tissues as the endogenous ENOD40 .

In chapter 4 we investigate the effect of ectopic expression of GmENOD40 ( 35S::GmENOD40 ) on tomato development in transgenic tomato plants. Preliminary studies show that ectopic expression of GmENOD40 causes an increase in flower and leaf size in 2 transgenic tomato plants. The leaves and flowers of these plants contain cells larger than wild-type cells in the epidermis.

In chapter 5 we investigate whether we can assign a function to the peptide encoded by ENOD40 by searching for protein binding partners for the peptide. For this we used the yeast Two-Hybrid system to screen a cDNA library of young pea nodules. This resulted in the isolation of Ps-p40, a pea homologue of the ribosomal protein p40. In situ hybridisation studies show that the expression sites of p40 and ENOD40 partly overlap in nodules. Further investigation is necessary to confirm or disprove the interaction between p40 and ENOD40.

In the concluding remarks of this thesis (chapter 6) we discuss a possible function of ENOD40 and we discuss whether ENOD40 represents a gene that is recruited during evolution for nodule formation.