Breaking Down the Complex Regulatory Web Underlying Lignin Biosynthesis

Abstract

Vascular plants would not dominate the terrestrial ecosystem today if they had not evolved the ability to synthesize lignin. Lignin, the second most abundant biological material on earth (after cellulose), helps vascular plants stand upright and serves as a scaffold for water and nutrient transport systems, enabling vascular plants to tower over their bryophyte relatives. Lignin is one of the most difficult biopolymers to degrade, making it an excellent barrier to pests and pathogens. Unfortunately, lignin also interferes with efforts to extract sugars from plant cellulose to convert them to biofuels, and it represents the major barrier to efficient extraction of cellulose fibers for pulp and paper production (Novaes et al., 2010). Thus, while lignin is indispensible to vascular plants, much time and money is spent trying to get rid of it. Lignin is a complex material primarily comprising so-called monolignol subunits, mainly derived from coniferyl alcohol (which forms guaiacyl units after polymerization), and sinapyl alcohol (which forms syringyl units after polymerization), as well as smaller amounts of phenylpropanoid aldehydes, acetates, esters, and other compounds. While some lignin biosynthetic enzymes and associated transcription factors have been identified and manipulated to alter lignin content and/or composition, and genomics-based descriptions of lignin pathway genes are available, the genetic pathway regulating lignin formation remains to be fully elucidated. Powerful techniques have now been developed to elucidate the regulatory pathways underlying lignin formation in the energy crop switchgrass (Panicum virgatum) and wood formation (in general) in poplar (Populus trichocarpa). Shen et al. (pages 4342–4361) employed two systems to identify candidate genes for lignin biosynthesis in the complex switchgrass genome: the fourth internode region of mature tillers, which contains a wide range of lignification and lignified tissue types, and suspension cells capable of undergoing lignification (see figure). They compared all available putative switchgrass unique transcript sequences to known Arabidopsis thaliana sequences involved in lignin biosynthesis and tested their expression in internodes and suspension cells using microarray analysis. Protein sequence alignment and phylogenetic analysis were used to identify candidate genes, which were tested using RNA silencing, culminating in the identification of candidate genes encoding enzymes involved in the early steps of the currently accepted monolignol biosynthesis pathway and the construction of a more detailed putative pathway to monolignols in switchgrass. Induced (+) and noninduced (−) switchgrass suspension cells harvested at days 1 and 7 and stained with phloroglucinol. The red color indicates the presence of lignin. (Reprinted from Shen et al. [2013], Figure 2B.) Lin et al. (pages 4324–4341) set out to identify a regulatory hierarchy underlying wood formation, specifically, the network of genes targeted by Secondary Wall-Associated NAC Domain-B1 (SND1-B1), a transcription factor affecting wood formation in poplar. First, they developed a differentiating xylem protoplast system from poplar stems, which is no small feat, since wood-forming cells are generally resistant to protoplast isolation. Protoplasts were transfected with SND1-B1 and differentially expressed genes identified using RNA sequencing. The authors inferred direct interactions between SND1-B1 and the identified genes by integrating time-course RNA sequencing data and top-down graphical Gaussian modeling–based algorithms. To verify these inferred interactions in vivo, they developed an antibody-based chromatin immunoprecipitation method for wood-forming cells, which is also a difficult task, as these cells are highly recalcitrant to nuclear and chromatin isolation. Chromatin immunoprecipitation analysis of differentiating xylem and the production of stably transgenic P. trichocarpa helped confirm the functions of the identified genes. By combining robust genetic and cell culture techniques, much can be learned about the material that helps vascular plants stand tall and interferes with their utilization.