X-ray micro-computed tomography in willow reveals tissue patterning of reaction wood and delay in programmed cell death

Abstract

Background: Variation in the reaction wood (RW) response has been shown to be a principle component driving differences in lignocellulosic sugar yield from the bioenergy crop willow. The phenotypic cause(s) behind these differences in sugar yield, beyond their common elicitor, however, remain unclear. Here we use X-ray micro-computed tomography (μCT) to investigate RW-associated alterations in secondary xylem tissue patterning in three dimensions (3D).

Results: Major architectural alterations were successfully quantified in 3D and attributed to RW induction. Whilst the frequency of vessels was reduced in tension wood tissue (TW), the total vessel volume was significantly increased. Interestingly, a delay in programmed-cell-death (PCD) associated with TW was also clearly observed and readily quantified by μCT.

Conclusions: The surprising degree to which the volume of vessels was increased illustrates the substantial xylem tissue remodelling involved in reaction wood formation. The remodelling suggests an important physiological compromise between structural and hydraulic architecture necessary for extensive alteration of biomass and helps to demonstrate the power of improving our perspective of cell and tissue architecture. The precise observation of xylem tissue development and quantification of the extent of delay in PCD provides a valuable and exciting insight into this bioenergy crop trait.

Figure 1

Reaction wood impact on basic density and 2D xylem architecture. A Basic density of debarked willow cultivar Resolution after 3 months of growth either unperturbed or including 6 weeks of RW induction (tipping at 45° from vertical). n = 3 trees. B Transverse middle stem section (25 μm) of a RW induced tree stained with safranin O (red – nonspecific staining the cell wall) and chlorazol black (black – specifically staining the g-layer of g-fibres). Panels: OW (left) and TW (right) are included with scale bar = 100 μm. *p < 0.05 (Students t-test).

Figure 2

2D transverse X-Ray CT scans. A single representative image from the stack reconstructed from the X-ray CT scanning of each stem segment. Each tree is either RW induced (T1, T2 and T3) or a control grown without induction (C1, C2 and C3). Regions of interest assessed for voxel intensity and distribution, TW, OW and NW are highlighted in red. High voxel intensity, and therefore 554 great X-Ray attenuation, is visible as lighter regions whereas regions of low voxel intensity are visible as darker regions. Scale bar = 4mm.

Figure 3

Voxel distribution. A Matlab histograms of voxel intensity distribution for each ROI, TW, OW and NW and 3D render of each ROI, units are not included as the number of voxels varied (histograms are to compare intensity distribution). Each tree, RW induced (T1, T2 and T3) or controls (C1, C2 and C3) were scanned including a common internal standard – allowing comparison of average voxel intensity. B Average ROI voxel intensity. Error bars = standard error of tissue type across 3 trees. * p < 0.05 (one-way ANOVA).

Figure 4

3D xylem architecture. A 3D render of each ROI (TW, OW or NW) from X-ray CT scans of RW induced trees (tipped T1, T2 and T3) or controls (C1, C2 and C3). The 3D ROI render on the right after the common vessel specific transfer function was applied in silico. B Total volume of vessels as a percentage of each ROI was averaged for each tissue. C Vessel surface area:volume ratio of each ROI was averaged for each tissue. Error bars = standard error of tissue type across 3 trees. * p < 0.05 (one-way ANOVA).

Figure 5

Tension wood delay in programmed-cell-death. A Top, single representative images from the stack reconstructed from the X-ray CT scanning of each RW induced stem segment. Bottom, Transverse middle stem section (25 μm) of a RW induced tree stained with safranin O (red – non-specific staining the cell wall) and chlorazol black (black – specifically staining the g-layer of g-fibres). Scale bar = 4 mm. B Confocal micrograph of coomassie stained OW (top) and TW (bottom), autofluorescence is shown in red (excitation and emission wavelengths were 488nm and 500-700nm respectively). Panes highlight the difference between OW fibre and TW g-fibre development in terms of individual cell structure and greater tissue architecture in relation to the whole stem. Blue scale bar = 500 μm.