Progress on the Regulation and Function of Enod8, an Early Nodulin Gene from Medicago that May Encode a Lipolytic Enzyme

Abstract

Enod8 is an early nodule-specific gene isolated from Medicago truncatula and alfalfa (M. sativa). Its expression patterns indicate that it has a likely role in nodule organogenesis. The characterization of Enod8 expression, by Northern blot, indicates that Enod8 is expressed as early as the nodule is visible on the root, at 5 days post-inoculation. Its sequence predicts a glycosylated 41.7 kD protein (Dickstein et al. 1993; Peng, Dickstein 1994). Enod8 is a member of an emerging plant lipolytic gene family (Brick et al. 1995). Two stratégies are being used to determine if Enod8 encodes a lipase. First, the pSBET expression system (Schenk et al. 1995) was used to obtain Enod8 expression in Escherichia coli. So far, Enod8 expressing strains do not show an increase in lipase activity. The second strategy is to purify Enod8 from nodules. Anti-Enod8 peptide antibodies recognize two nodule-specific polypeptides of approximately 54 and 56 kD. Partial purification of these two proteins has resulted in an increase in lipase specific activity (D. Pringle, R. Dickstein, unpublished). A sensitive RT-PCR Enod8 expression assay shows that low levels of Enod8 expression are found in young roots, expecially root tips. Thus, Enod8 is not strictly nodule-specific. Spot inoculation of R. meliloti on roots in the root hair emergent area resulted in increased Enod8 expression levels 24 hours post inoculation. Increased Enod8 expression was not observed in mock-inoculated controls, nor in roots spot inoculated with SL44 (Fisher et al. 1988), a Nod⁻ R. meliloti strain (M. Konduri, R. Dickstein, unpublished), suggesting that Nod factor participates directly or indirectly in the induction of Enod8 expression. The results point to Enod8 having a role in an aspect of lipid metabolism early in the nitrogen-fixing root nodule development and a similar role in root growth and development.

We acknowledge USDA NRICGP grants #91-37305-6584 and #96-35305-3413.