Tyloses and Phenolic Deposits in Xylem Vessels Impede Water Transport in Low-Lignin Transgenic Poplars: A Study by Cryo-Fluorescence Microscopy    

Abstract

Of 14 transgenic poplar genotypes (Populus tremula 3 Populus alba) with antisense 4-coumarate:coenzyme A ligase that were grown in the field for 2 years, five that had substantial lignin reductions also had greatly reduced xylem-specific conductivity compared with that of control trees and those transgenic events with small reductions in lignin. For the two events with the lowest xylem lignin contents (greater than 40% reduction), we used light microscopy methods and acid fuchsin dye ascent studies to clarify what caused their reduced transport efficiency. A novel protocol involving dye stabilization and cryofluorescence microscopy enabled us to visualize the dye at the cellular level and to identify water-conducting pathways in the xylem. Cryo-fixed branch segments were planed in the frozen state on a sliding cryo-microtome and observed with an epifluorescence microscope equipped with a cryo-stage. We could then distinguish clearly between phenolic-occluded vessels, conductive (stain-filled) vessels, and nonconductive (water-or gas-filled) vessels. Low-lignin trees contained areas of nonconductive, brown xylem with patches of collapsed cells and patches of noncollapsed cells filled with phenolics. In contrast, phenolics and nonconductive vessels were rarely observed in normal colored wood of the low-lignin events. The results of cryo-fluorescence light microscopy were supported by observations with a confocal microscope after freeze drying of cryo-planed samples. Moreover, after extraction of the phenolics, confocal microscopy revealed that many of the vessels in the nonconductive xylem were blocked with tyloses. We conclude that reduced transport efficiency of the transgenic low-lignin xylem was largely caused by blockages from tyloses and phenolic deposits within vessels rather than by xylem collapse.

Improved saccharification and ethanol yield from field-grown transgenic poplar deficient in cinnamoyl-CoA reductase

“…S7), as was previously noticed in greenhouse-grown CCR-downregulated poplars (13). In 4CL-down-regulated poplar, the reduced growth has been attributed to collapsed xylem and the formation of tyloses in the vascular system, and it was shown that vascular tissue in the red areas is defective in its transport capacity (22,28). One option to circumvent the negative consequences of lignin down-regulation on plant performance while still achieving strong down-regulation is to specifically target the down-regulation to fibers but leaving the vessels intact.…”

RELATIONSHIP BETWEEN LIGNIN CONTENT AND QUALITY OF Pinus taeda SEEDLINGS1

“…Lignin in stems and root cell walls promote greater resistance to embolism, a phenomenon associated with death of woody plants under severe conditions of water deficit and winds capable of raising water pressure in xylem vessels resulting in increased transpiration rate (RETUERTO; WOODWARD, 1993;MARTÍNEZ-VILALTA;PIÑOL, 2003;KITIN et al, 2010). Therefore, the value of the partial correlation coefficient between lignin content of seedling root system with survival (r = 0.98) can also be associated with the maintenance of hydraulic conductivity in the xylem.…”

Antisense Down-Regulation of 4CL Expression Alters Lignification, Tree Growth, and Saccharification Potential of Field-Grown Poplar

“…2, F and L). Extensive anatomical investigations of lowlignin poplar wood (Kitin et al, 2010) suggest that this partial collapse can be caused by slide preparation or partial recovery of cells that were almost completely collapsed before preparation. In either case, the cell wall material was apparently weaker.…”