Localized gene expression changes during adventitious root formation in black walnut (Juglans nigra L.)

Cutting propagation plays a large role in the forestry and horticulture industries where superior genotypes need to be clonally multiplied. Integral to this process is the ability of cuttings to form adventitious roots. Recalcitrance to adventitious root development is a serious hurdle for many woody plant propagation systems including black walnut (Juglans nigra L.), an economically valuable species. The inability of black walnut to reliably form adventitious roots limits propagation of superior genotypes. Adventitious roots originate from different locations, and root induction is controlled by many environmental and endogenous factors. At the molecular level, however, the regulation of adventitious root formation is still poorly understood. In order to elucidate the transcriptional changes during adventitious root development in black walnut, we used quantitative real-time polymerase chain reaction to measure the expression of nine key genes regulating root formation in other species. Using our previously developed spatially explicit timeline of adventitious root development in black walnut softwood cuttings, we optimized a laser capture microdissection protocol to isolate RNA from cortical, phloem fiber and phloem parenchyma cells throughout adventitious root formation. Laser capture microdissection permitted high-resolution, site-specific analysis of gene expression that differentiated between participatory and non-participatory root progenitor cells. Results indicated mRNA abundance was altered in all nine rooting-related genes in response to auxin treatment in both juvenile and mature cuttings. SCARECROW LIKE-1 (SCL) had the greatest change in expression in juvenile rooting-competent cells at days 16 and 18, with a 24- and 23-fold increase relative to day 0, respectively. Tissues not linked to root organogenesis had little change in SCL expression at similar time points. AUXIN RESPONSE FACTOR (ARF)6 and ARF8 as well as SHORTROOT expression also increased 2- to 4-fold in rooting-competent tissue. The greatest transcript abundance in rooting-competent cuttings was restricted to root progenitor cells, while recalcitrant cuttings had a diffuse mRNA signal among tissue types.