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A strong GUS signal was found in the dividing outer cortical cells during the nodule primordium formation between 2 and 5 dpi [22]. During primordia development, blue staining was present in the nodule cortex and in the root stele in direct contact with the primordium. In the nodules examined at 21 dpi, GUS expression was mainly present in the nodule vascular bundles and occasionally, a weak diffused signal was found in the nodule cortex [22]. Similar results were obtained in work by Takanashi et al.

[26] described detailed patterns of auxin accumulation during early nodule development in stable transformants of L. japonicus bearing green fluorescent protein (GFP) under the control of a highly active synthetic auxin-responsive promoter DR5 . In that study, auxin accumulated in pre-primordial cortical cells adjoining curled root hair with infection thread and in the divided cortical cells at the site of infection [26]. A strong GFP signal was maintained until the primordia “emerged” above the root surface. In young nodules formed at 12 dpi, i.e., after differentiation of bacteroid-containing tissue, GFP signal remained in the nodule cortex, including vascular bundles [26]. All those results show a striking spatial coincidence existing between distribution of auxin maxima and LjPIN genes expression pattern presented in this work. However, it should be noted that auxin distribution pattern was obtained using a construct bearing a GH3 promoter from soybean and that four different GH3 genes are expressed during the development of L. japonicus nodules ( LjGH3–3 , LjGH3–4 , LjGH3–5 and LjGH3–18 ) [50].

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