Kitin, Peter , Voelker, Steven L. , Lachenbruch, Barbara , Beeckman, Hans , Meinzer, Frederick , Strauss, Steven H. .
Transgenic poplars with very low lignin in their cell walls produce tyloses and phenolic deposits in xylem vessels that impede water transport: a study by cryo-fluorescence microscopy.
Of 14 transgenic poplar genotypes with low-lignin contents (downregulated 4-coumarate: coenzyme A ligase (4CL)) that were grown in the field, two showed greatly reduced wood specific conductivity (ks) compared to that of normal trees. To clarify what causes the reduced transport efficiency of xylem, dye-flow experiments with acid-fuchsin were performed on transgenic and control trees. It has been previously recognized that dye-tracks must be interpreted with caution because pressure gradients in the plant tissues can be easily altered during sample preparation, and hence flow directions. A novel protocol of dye stabilization and cryo-fluorescence microscopy enabled us to visualize the dye at the single cellular level and to identify active conductive paths of the xylem. As soon as dye uptake reached leaf petioles, branches were snap-frozen in liquid Nitrogen, while the cut ends were still immersed in the dye solution. Cryo-fixed branch segments were planed in the frozen state on a sliding cryo-microtome and observed with an epi-fluorescence microscope equipped with a cryo-stage. Alternatively, the cryo-planed samples were freeze-dried and observed at room temperature with the epi-fluorescence microscope or a confocal microscope.
The cryo-fluorescence microscope provided lower resolution compared to cryo-scanning electron microscopy but enabled us to clearly distinguish between phenolic-occluded vessels, actively conducting (stained), and not actively conducting (unstained) water-filled vessels. All vessels within brown areas of the xylem of the extreme low-lignin lines, as well as the fibers and ray cells, were filled with phenolics. Within “normally” colored wood of the modified trees, not all of the water-filled vessels were actively conducting and deposition of phenolics was also observed. In contrast, all vessels in the control trees were traced with the dye and no phenolics were observed. The reduced transport efficiency of the transgenic low-lignin xylem is caused by ectopic deposition of tyloses and phenolics within vessels.
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1 - Oregon State University, Dept. of Wood Science and Engineering, 119 Richardson Hall, Corvallis, OR, 97331, USA
2 - Dept. of Wood Science and Engineering, Oregon State University, Corvallis, OR, 97331, USA
3 - Royal Museum for Central Africa, Laboratory for Wood Biology and Xylarium, Leuvensesteenweg 13, Tervuren, 3080, Belgium
4 - USDA Forest Service, Forestry Sciences Laboratory, 3200 SW Jefferson Way, Corvallis, Oregon, 97331, USA
5 - Oregon State University, Dept. of Forests, Ecosystems, and Society, Corvallis, OR, 97331, USA
Presentation Type: Poster:Posters for BSA Sections
Location: Event Tent/Cliff Lodge
Date: Monday, July 27th, 2009
Time: 5:30 PM