Auxin-responsive DR5 promoter coupled with transport assays suggest separate but linked routes of auxin transport during woody stem development in Populus.

Polar auxin transport (PAT) is a major determinant of plant morphology and internal anatomy with important roles in vascular patterning, tropic growth responses, apical dominance and phyllotactic arrangement. Woody plants present a highly complex system of vascular development in which isolated bundles of xylem and phloem gradually unite to form concentric rings of conductive tissue. We generated several transgenic lines of hybrid poplar (Populus tremula x alba) with the auxin-responsive DR5 promoter driving GUS expression in order to visualize an auxin response during the establishment of secondary growth. Distinct GUS expression in the cambial zone and developing xylem-side derivatives supports the current view of this tissue as a major stream of basipetal PAT. However, we also found novel sites of GUS expression in the primary xylem parenchyma lining the outer perimeter of the pith. Strands of primary xylem parenchyma depart the stem as a leaf trace, and showed GUS expression as long as the leaves to which they were connected remained attached (i.e., until just prior to leaf abscission). Tissue composed of primary xylem parenchyma strands contained measurable levels of free indole-3-acetic acid (IAA) and showed basipetal transport of radiolabeled auxin ((3)H-IAA) that was both significantly faster than diffusion and highly sensitive to the PAT inhibitor NPA. Radiolabeled auxin was also able to move between the primary xylem parenchyma in the interior of the stem and the basipetal stream in the cambial zone, an exchange that was likely mediated by ray parenchyma cells. Our results suggest that (a) channeling of leaf-derived IAA first delineates isolated strands of pre-procambial tissue but then later shifts to include basipetal transport through the rapidly expanding xylem elements, and (b) the transition from primary to secondary vascular development is gradual, with an auxin response preceding the appearance of a unified and radially-organized vascular cambium.