title: New insights into pioneer root xylem development: evidence obtained from Populus trichocarpa plants grown under field conditions.
abstract: OBJECTIVE
  Effective programmed xylogenesis is critical to the structural framework of the plant root system and its central role in the acquisition and long-distance transport of water and nutrients. The process of xylem differentiation in pioneer roots under field conditions is poorly understood. In this study it is hypothesized that xylogenesis, an example of developmental programmed cell death (PCD), in the roots of woody plants demonstrates a clearly defined sequence of events resulting in cell death. A comprehensive analysis was therefore undertaken to identify the stages of xylogenesis in pioneer roots from procambial cells to fully functional vessels with lignified cell walls and secondary cell wall thickenings.
  
  
  METHODS
  Xylem differentiation was monitored in the pioneer roots of Populus trichocarpa at the cytological level using rhizotrons under field conditions. Detection and localization of the signalling molecule nitric oxide (NO) and hydrogen peroxide (H2O2) was undertaken and a detailed examination of nuclear changes during xylogenesis was conducted. In addition, analyses of the expression of genes involved in secondary cell wall synthesis were performed in situ.
  
  
  RESULTS
  The primary event in initially differentiating tracheary elements (TEs) was a burst of NO in thin-walled cells, followed by H2O2 synthesis and the appearance of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling)-positive nuclei. The first changes in nuclear structure were observed in the early stages of xylogenesis of pioneer roots, prior to lignification; however, the nucleus was detectable under transmission electron microscopy in differentiating cells until the stage at which vacuole integrity was maintained, indicating that their degradation was slow and prolonged. The subsequent sequence of events involved secondary cell wall formation and autophagy. Potential gene markers from the cinnamyl alcohol dehydrogenase (CAD) gene family that were related to secondary wall synthesis were associated with primary xylogenesis, showing clear expression in cells that undergo differentiation into TEs and in the thin-walled cells adjacent to the xylem pole.
  
  
  CONCLUSIONS
  The early events of TE formation during pioneer root development are described, together with the timing of xylogenesis from signalling via NO, through secondary cell wall synthesis and autophagy events that are initiated long before lignification. This is the first work describing experiments conducted in planta on roots under field conditions demonstrating that the process of xylogenesis in vivo might be gradual and complex.
authors.affiliation: Department of General Botany agabag@amu.edu.pl.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Bagniewska-Zadworna
authors.affiliation: Department of Plant Ecophysiology, Institute of Experimental Biology, Faculty of Biology, Adam Mickiewicz University, Umultowska 89, 61-614 Poznań, Poland.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Arasimowicz-Jelonek
authors.affiliation: Department of Cell Biology, Institute of General and Molecular Biology, Nicolaus Copernicus University, Lwowska 1, 87-100 Toruń, Poland.
authors.forename: Dariusz J
authors.initials: DJ
authors.lastname: Smoliński
authors.affiliation: Department of General Botany.
authors.forename: Agnieszka
authors.initials: A
authors.lastname: Stelmasik
mesh_terms: D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D015870:Gene Expression; D006861:Hydrogen Peroxide / Q000378:metabolism; D017404:In Situ Hybridization, Fluorescence; D020287:In Situ Nick-End Labeling; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D009569:Nitric Oxide / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D018517:Plant Roots / Q000166:cytology / Q000235:genetics / Q000254:growth & development* / Q000502:physiology / Q000648:ultrastructure; D032107:Populus / Q000166:cytology / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D015398:Signal Transduction; D052584:Xylem / Q000166:cytology / Q000235:genetics / Q000254:growth & development / Q000502:physiology*
chemical_list: D010940:Plant Proteins; D009569:Nitric Oxide; D006861:Hydrogen Peroxide; D000429:Alcohol Oxidoreductases; C018656:cinnamyl alcohol dehydrogenase
keywords: PCD; Populus trichocarpa; black cottonwood; cell wall synthesis; nitric oxide signalling; pioneer root development; programmed cell death; tracheary elements; xylogenesis