Vacuolar SNAREs function in the formation of the leaf vascular network by regulating auxin distribution

In normal leaf development, a two-dimensional pattern of leaf veins is known to form by differentiation of vascular cells from ground meristem cells in a manner that is regulated by the polar flow of auxin. However, the mechanisms regulating the distribution of auxin in the leaf primordium are largely unknown. Here we show that vacuolar SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), VAM3 and VTI11, are required for the formation of the leaf vascular network in a dosage-dependent manner. This is the first report to show that the pre-vacuolar compartment (PVC)-vacuole traffic pathway is required for the formation of the leaf vascular network. vam3-4, a VAM3-defective mutant, was found to have an immature vascular network. An analysis of the DR5 reporter in vam3-4 indicated that VAM3 is involved in the proper pattern formation of auxin maxima in the leaf primordium. This suggests that the immature vascular network in vam3-4 was mainly determined at the stage of procambium formation in the leaf primordium. The abnormal distribution of auxin maxima was caused by the non-polarized localization of the auxin efflux carrier PIN1 (PIN-FORMED 1) in leaf primordium cells. VAM3 is the first key protein which is required for the proper localization of PIN1 in leaf cells. Finally, we found that PIN1 proteins were constitutively transported to vacuoles in leaf and roots cells. Our findings demonstrate that the PVC-vacuole pathway is required for the formation of auxin maxima, which regulates the polar localization of PIN1, which, in turn, is required for the formation of the leaf vascular network.