Syringyl lignin production in conifers: Proof of concept in a Pine tracheary element system

Significance This study shows that metabolic engineering can be used to imbue pine tracheary elements with an ability to synthesize sinapyl alcohol, a lignin monomer not normally used for lignification in conifers such as pine. The dynamic nature of the lignification process enables pines to incorporate this monolignol, allowing them to produce hardwood-like lignins that are known to facilitate refining processes such as biofuel production and chemical pulping. The potential to improve the refining of conifer-derived biomass through lignin manipulations is important, as even small improvements in yield can lead to significant environmental and economic benefits in such processes.


PtreSAD f -5′CATGGACTAGTTCCGGTTACGTAGCTAGCA and
PtreSAD r -5′AGCTTGCTAGCTACGTAACCGGAACTAGTC and after Nco I/Spe I digest inserted into pWVC50 digested with Nco I/Spe I. The integrity of the generated PrUbiquitin pro -PtreSAD-nos ter construct was con firmed by sequencing. A schematic diagram of lignin-related constructs used in this study is shown in Fig. S2. S2. Schematic diagram of the constructs used for transformation of nondifferentiated Pinus radiata TE cultures. Vector pWVC50 contains the P. taeda Ubiq uitin (PtUbq) promoter and the ferulate 5-hydroxylase (coniferaldehyde 5-hydroxylase) gene from Liquidambar styraciflua (LsF5H). Vector pWVC55 contains the P. taeda 4-coumarate-CoA ligase (Pt4CL) promoter and the O-methyltransferase gene from L. styraciflua (LsCOMT). Vector pHF21 contains the P. taeda Ubq promoter and the sinapyl alcohol dehydrogenase gene from P. tremuloides (PtreSAD). All constructs contain the P. taeda Ubq 3'UTR followed by the NOS termina tor.
Tissue culture, transformation and molecular monitoring procedures P. radiata callus cultures were co-transformed with vector(s) pWVC50, pWVC55 and pHF21 as described earlier (2). The expression of transformed and endogenous genes in transgenic lines was monitored by quantitative RT-PCR as described by (3)    to differentiate tracheary elements (TEs) as described earlier (4). Cultures freshly induced to form TEs were stained with acriflavin (0.0025% in water) for 10 min and mounted in 50% glycerol. Tissue was imaged using a Leica TCS NT confocal microscope using 488/568 nm excitation, and 530/600 nm emission.

Pyrolysis-GC/MS
Pyrolysis-GC/MS was on powdered, freeze-dried pine callus cultures and purified TEs as described previously (5). Thermal breakdown products of pyrolysed materials were identified by using mass spectra of lignin and polysaccharide-derived pyrolysis products (6-9).  ) and incompletely methylated pathway intermediates (5-hydroxy-vinyl-guaiacol (m/z 166)) in transgenic TE cultures. None of these pyrolysis products was detectable in wild-type controls.

Preparation of enzyme lignins from purified TEs
Preparation of enzyme lignin (EL) samples for NMR was performed essentially as described previously (10)(11)(12)  (HMBC) experiments ("hmbcgplpndqf") for the acetylated ELs had the para meters described previously (16,17). Processing to a final matrix of 2 k by 1 k datapoints used typical matched Gaussian apodization in F2 (LB, −45; GB, 0.15), squared sine-bell in F1, and one level of linear prediction in F1 (32 coefficients). Table S1. NMR-derived syringyl/guaiacyl (S/G) data and interunit linkage data for lignin from a wild-type control and transgenic F5H/COMT/SAD Line 6 and Line 19 (see also  Table S2. Raw pyrolysis data for initial selection of best lines, and for estimating relative S/G A Student's t-test showed a statistically significant difference in the S/G-ratios (P < 0.05, highlighted in yellow) between the F5H lines and the F5H/COMT and F5H/COMT/SAD lines, but no significant difference between the F5H/COMT and F5H/COMT/SAD, suggesting that SAD does not significantly contribute to S-lignin creation in this system. Nevertheless, the two lines, Line 6 and Line 19, with the highest S/G were F5H/COMT/SAD lines, i.e., with all three constructs.  Darker shading: data used for Figure 2.

Raw Pyrolysis Data, and Significance
The average and SD of the three ratios were used for this Figure. No biological reps. The three ratios are considered technical reps.