Suppression of CCR impacts metabolite profile and cell wall composition in Pinus radiata tracheary elements.

Suppression of the lignin-related gene cinnamoyl-CoA reductase (CCR) in the Pinus radiata tracheary element (TE) system impacted both the metabolite profile and the cell wall matrix in CCR-RNAi lines. UPLC-MS/MS-based metabolite profiling identified elevated levels of p-coumaroyl hexose, caffeic acid hexoside and ferulic acid hexoside in CCR-RNAi lines, indicating a redirection of metabolite flow within phenylpropanoid metabolism. Dilignols derived from coniferyl alcohol such as G(8-5)G, G(8-O-4)G and isodihydrodehydrodiconiferyl alcohol (IDDDC) were substantially depleted, providing evidence for CCR's involvement in coniferyl alcohol biosynthesis. Severe CCR suppression almost halved lignin content in TEs based on a depletion of both H-type and G-type lignin, providing evidence for CCR's involvement in the biosynthesis of both lignin types. 2D-NMR studies revealed minor changes in the H:G-ratio and consequently a largely unchanged interunit linkage distribution in the lignin polymer. However, unusual cell wall components including ferulate and unsaturated fatty acids were identified in TEs by thioacidolysis, pyrolysis-GC/MS and/or 2D-NMR in CCR-RNAi lines, providing new insights into the consequences of CCR suppression in pine. Interestingly, CCR suppression substantially promoted pyrolytic breakdown of cell wall polysaccharides, a phenotype most likely caused by the incorporation of acidic compounds into the cell wall matrix in CCR-RNAi lines.

Suppression of 4-coumarate-CoA ligase in the coniferous gymnosperm Pinus radiata.

Severe suppression of 4-coumarate-coenzyme A ligase (4CL) in the coniferous gymnosperm Pinus radiata substantially affected plant phenotype and resulted in dwarfed plants with a "bonsai tree-like" appearance. Microscopic analyses of stem sections from 2-year-old plants revealed substantial morphological changes in both wood and bark tissues. This included the formation of weakly lignified tracheids that displayed signs of collapse and the development of circumferential bands of axial parenchyma. Acetyl bromide-soluble lignin assays and proton nuclear magnetic resonance studies revealed lignin reductions of 36% to 50% in the most severely affected transgenic plants. Two-dimensional nuclear magnetic resonance and pyrolysis-gas chromatography-mass spectrometry studies indicated that lignin reductions were mainly due to depletion of guaiacyl but not p-hydroxyphenyl lignin. 4CL silencing also caused modifications in the lignin interunit linkage distribution, including elevated beta-aryl ether (beta-O-4 unit) and spirodienone (beta-1) levels, accompanied by lower phenylcoumaran (beta-5), resinol (beta-beta), and dibenzodioxocin (5-5/beta-O-4) levels. A sharp depletion in the level of saturated (dihydroconiferyl alcohol) end groups was also observed. Severe suppression of 4CL also affected carbohydrate metabolism. Most obvious was an up to approximately 2-fold increase in galactose content in wood from transgenic plants due to increased compression wood formation. The molecular, anatomical, and analytical data verified that the isolated 4CL clone is associated with lignin biosynthesis and illustrated that 4CL silencing leads to complex, often surprising, physiological and morphological changes in P. radiata.

Chitosan-mediated changes in cell wall composition, morphology and ultrastructure in two wood-inhabiting fungi.

The effect of chitosan on cell wall deposition was investigated in the two wood-inhabiting fungal species Trichoderma harzianum (CBS 597.91) and Sphaeropsis sapinea (NZFS 2725). The study used three independent analytical techniques to quantify chitin in the fungal mycelium. A colorimetric method for the detection of D-glucosamine was compared with two gas chromatography-mass spectroscopy (GC-MS) methods employing alditol acetates analysis and pyrolysis. The latter used a stable-isotope-labelled internal standard, d(3)-N-acetyl glucosamine. At least in the case of S. sapinea, the study provided evidence of an increase in the chitin content in the mycelium due to chitosan treatment, indicating that chitosan treatment affected cell wall deposition. Electron microscopy techniques showed alteration in surface morphology and cell wall texture due to chitosan treatment. The implications of these results are discussed with a view to analysing possible mechanisms for growth inhibitory effects of chitosan on fungal hyphae.

Gene silencing of cinnamyl alcohol dehydrogenase in Pinus radiata callus cultures.

Xylem-derived Pinus radiata cell cultures, which can be induced to differentiate tracheary elements (TEs), were transformed with an RNAi construct designed to silence cinnamyl alcohol dehydrogenase (CAD), an enzyme involved in the biosynthesis of monolignols. Quantitative enzymatic CAD measurements revealed reduced CAD activity levels in most transclones generated. TEs from transclones with approximately 20% residual CAD activity did not release elevated levels of vanillin, which was derived from coniferyl-aldehyde through a mild alkali treatment. However, the activation of the phenylpropanoid pathway in transclones with approximately 20% residual CAD activity through the application of non-physiological concentrations of sucrose and l-phenylalanine produced phenotypic changes. The accumulation of metabolites such as dihydroconiferyl-alcohol (DHCA), which also accumulates in the P. taeda CAD mutant cad-n1, was observed. These results indicate that a substantial reduction in CAD activity is necessary for this enzyme to become a rate-limiting step in lignin biosynthesis in conifers such as P. radiata and confirm that transformable P. radiata callus cultures can be useful to investigate the function of xylogenesis-related genes in conifers.