In vivo fluorescence correlation microscopy (FCM) reveals accumulation andimmobilization of Nod factors in root hair cell walls

Fluorescence correlation microscopy (FCM) is a new single-molecule detectio n technique based on the confocal principle to quantify molecular diffusion and concentration of fluorescent molecules (particles) with sub-micron res olution. In this study, FCM is applied to examine the diffusional behaviour of fluorescent Nod factor analogues on living Vicia sativa root hairs. Thr ee recently described Nod factors with a fluorescent acyl chain (Goedhart e t al. Biochemistry 1999, 38, 10898-10907) were used. Plasmolysis of fluores cently labelled root hairs showed that the Nod factors are predominantly lo cated in the cell wall, as hardly any fluorescence could be detected in the plasma membrane. After Nod factor-induced root hair deformation, the new o utgrowth was not labelled, indicating a lack of migration of Nod factors to the newly synthesized cell wall. In agreement, FCM showed a > 1000-fold re duction of molecular mobility of the fluorescence Nod factors upon binding to the cell wall. In addition, FCM demonstrated that Nod factors, when exog enously applied in aqueous solution at 10 nM, markedly concentrate in the c ell wall of root hairs (up to 50-fold). The feasibility of applying FCM for the study of living plant cells as well as the implications of our results for the perception of Nod factors are discussed.