Cell Wall Chemotyping for Functional Genomics Applications of Pyrolysis–Gas Chromatography / Mass Spectrometry

The interest in lignocellulose as a sustainable resource for energy and materials has fueled research on biotechnology applications in tree breeding to improve biomass production and wood properties. An important aspect of this research is the basic understanding of gene function in wood formation, where analysis of wood chemistry and wood structure is of utmost importance. Current research strategies often involve large-scale screening of plant material, and therefore there is a need for rapid chemical characterisation and classification (chemotyping) of samples. Pyrolysis-Gas Chromatography/Mass Spectrometry (Py-GC/MS) provides a chemical fingerprint with more than hundred peaks representing each sample. Despite the fact that the technique is very informative, the wealth of data resulting from each sample has prohibited its use as a high– throughput method in large scale projects. For this reason, a novel application of Py-GC/MS was developed. An automated data processing pipeline was created and implemented, which together with a new simplified rule-set to analyse Py-GC/MS data overcame the bottleneck of time-consuming data handling. The added value of the method was demonstrated by fingerprinting and classifying the wood of 736 transgenic hybrid aspen (Populus tremula x tremuloides) trees representing 44 different genotypes. Among these genes were fructokinases (FRK1 and 2) and sucrose synthase (SUS) isoforms, both involved in primary carbon metabolism. A battery of chemotyping tools demonstrated that the downregulation of FRK resulted more specifically in a decrease in cellulose biosynthesis, whereas the repression of SUS affected the amount of all of the major wood polymers. It was further demonstrated that the wood of transgenic SUS trees was affected in structure and mechanical properties. In another study, it was shown that down-regulating a lignin biosynthetic gene, cinnamate 4-hydroxylase, resulted in a modified wood structure in hybrid aspen. Interestingly, the large reduction in lignin content resulted in only minor effects on the ultimate tensile strength of the wood. Finally, the battery of chemotyping tools was used in a study to show that the transcription factor MYB103 is required for ferrulate-5-hydroxylase (F5H ) expression and S-lignin biosynthesis in Arabidopsis thaliana.