A Whole-plant Approach to Identifying Sites of Auxin Biosynthesis in Populus

The plant hormone auxin regulates many processes throughout plant growth and development. Aboveground auxin biosynthesis is thought to occur exclusively in the shoot apex, where it then moves via polar auxin transport (PAT) down through developing vasculature in the stem. In contrast to the canonical view of the shoot apex as the sole site of auxin production, there is some evidence to suggest that other tissues contribute auxin to the stem. Woody plants in particular may require additional sources of auxin to support the extensive vascular development in the woody stem, but very little is known about whole-plant auxin dynamics outside of herbaceous (i.e., non-woody) plants. A series of experiments were conducted using the model woody plant hybrid poplar (Populus tremula x alba) in order to better understand the sources of auxin that supply the developing stem. First, shoot apices were removed and the auxin content of developing xylem at three positions along the stem was determined after 48 hours. Removal of the apex led to a 40% reduction in the auxin content of developing xylem located 30 cm beneath the apex but had no effect further down, suggesting either that PAT is extremely slow, or that mature leaves contribute auxin directly to the developing xylem. Plants were then defoliated along 75% of the lowermost stem. Interestingly, defoliation reduced auxin levels in the apex by about 50% but had no effect on auxin levels in the developing xylem further down the stem. However, analysis of a transformant expressing an auxin-responsive reporter construct (DR5:GUS) showed that cells associated with the vasculature (primary xylem parenchyma; PXP) in the petioles of mature leaves were indeed auxin-responsive. Application of the auxin transport inhibitor NPA to petioles confirmed that PAT from leaf to stem is occurring, as auxin levels “downstream” from the site of NPA application were reduced by about 60%. In addition, qRTPCR analysis showed that the auxin biosynthetic gene YUCCA1 is expressed in mature leaves, albeit it at low levels. Despite this evidence for the production of auxin and its export by mature leaves, more extensive application of NPA to petioles had no effect on the auxin content of PXP or developing xylem in the stem, suggesting that the auxin moving through the petiole may not enter the stem. If all of the auxin in the stem is indeed derived from the shoot apex, transport through the stem must be exceptionally slow, as the levels of auxin in the apex are much lower than in the developing xylem. Transport rates this slow would imply that auxin is remarkably long-lived, given the distance it must travel in a large woody stem. These findings underscore the need for a better understanding of the rate and mechanism of auxin turnover, in addition to its transport and biosynthesis.