Manipulation of Lignin Monomer Composition Combined with the Introduction of Monolignol Conjugate Biosynthesis Leads to Synergistic Changes in Lignin Structure.

The complexity of lignin structure impedes efficient cell wall digestibility. Native lignin is composed of a mixture of three dominant monomers, coupled together through a variety of linkages. Work over the past few decades has demonstrated that lignin composition can be altered through a variety of mutational and transgenic approaches such that the polymer is derived almost entirely from a single monomer. In this study, we investigated changes to lignin structure and digestibility in Arabidopsis thaliana in near-single-monolignol transgenics and mutants and determined whether novel monolignol conjugates, produced by a FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) or a p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (PMT), could be integrated into these novel polymers to further improve saccharification efficiency. Monolignol conjugates, including a new conjugate of interest, p-coumaryl p-coumarate, were successfully integrated into high-H, high-G and high-S lignins in A. thaliana. Regardless of lignin composition, FMT- and PMT-expressing plants produced monolignol ferulates and monolignol p-coumarates, respectively, and incorporated them into their lignin. Through the production and incorporation of monolignol conjugates into near-single-monolignol lignins, we demonstrated that substrate availability, rather than monolignol transferase substrate preference, is the most important determining factor in the production of monolignol conjugates, and lignin composition helps dictate cell wall digestibility.

The impact of alterations in lignin deposition on cellulose organization of the plant cell wall.

BACKGROUND: Coordination of synthesis and assembly of the polymeric components of cell walls is essential for plant growth and development. Given the degree of co-mingling and cross-linking among cell wall components, cellulose organization must be dependent on the organization of other polymers such as lignin. Here we seek to identify aspects of that codependency by studying the structural organization of cellulose fibrils in stems from Arabidopsis plants harboring mutations in genes encoding enzymes involved in lignin biosynthesis. Plants containing high levels of G-lignin, S-lignin, H-lignin, aldehyde-rich lignin, and ferulic acid-containing lignin, along with plants with very low lignin content were grown and harvested and longitudinal sections of stem were prepared and dried. Scanning X-ray microdiffraction was carried out using a 5-micron beam that moved across the sections in 5-micron steps and complete diffraction patterns were collected at each raster point. Approximately, 16,000 diffraction patterns were analyzed to determine cellulose fibril orientation and order within the tissues making up the stems. RESULTS: Several mutations-most notably those exhibiting (1) down-regulation of cinnamoyl CoA reductase which leads to cell walls deficient in lignin and (2) defect of cinnamic acid 4-hydroxylase which greatly reduces lignin content-exhibited significant decrease in the proportion of oriented cellulose fibrils in the cell wall. Distinctions between tissues were maintained in all variants and even in plants exhibiting dramatic changes in cellulosic order the trends between tissues (where apparent) were generally maintained. The resilience of cellulose to degradative processes was investigated by carrying out the same analysis on samples stored in water for 30 days prior to data collection. This treatment led to significant loss of cellulosic order in plants rich in aldehyde or H-lignin, less change in wild type, and essentially no change in samples with high levels of G- or S-lignin. CONCLUSIONS: These studies demonstrate that changes in lignin biosynthesis lead to significant disruption in the orientation and order of cellulose fibrils in all tissues of the stem. These dramatic phenotypic changes, in mutants with lignin rich in aldehyde or H-units, correlate with the impact the mutations have on the enzymatic degradation of the plant cell wall.

Tissue specific specialization of the nanoscale architecture of Arabidopsis.

The Arabidopsis stem is composed of five tissues - the pith, xylem, phloem, cortex and epidermis - each of which fulfills specific roles in support of the growth and survival of the organism. The lignocellulosic scaffolding of cell walls is specialized to provide optimal support for the diverse functional roles of these layers, but little is known about this specialization. X-ray scattering can be used to study this tissue-specific diversity because the cellulosic components of the cell walls give rise to recognizable scattering features interpretable in terms of the underlying molecular architecture and distinct from the largely unoriented scatter from other constituents. Here we use scanning X-ray microdiffraction from thin sections to characterize the diversity of molecular architecture in the Arabidopsis stem and correlate that diversity to the functional roles the distinct tissues of the stem play in the growth and survival of the organism.

The Arabidopsis REF8 gene encodes the 3-hydroxylase of phenylpropanoid metabolism.

The activity of p-coumarate 3-hydroxylase (C3H) is thought to be essential for the biosynthesis of lignin and many other phenylpropanoid pathway products in plants; however, no conditions suitable for the unambiguous assay of the enzyme are known. As a result, all attempts to purify the protein and clone its corresponding gene have failed. By screening for plants that accumulate reduced levels of soluble fluorescent phenylpropanoid secondary metabolites, we have identified a number of Arabidopsis mutants that display a reduced epidermal fluorescence (ref) phenotype. Using radiotracer-feeding experiments, we have determined that the ref8 mutant is unable to synthesize caffeic acid, suggesting that the mutant is defective in a gene required for the activity or expression of C3H. We have isolated the REF8 gene using positional cloning methods, and have verified that it encodes C3H by expression of the wild-type gene in yeast. Although many previous reports in the literature have suggested that C3H is a phenolase, the isolation of the REF8 gene demonstrates that the enzyme is actually a cytochrome P450-dependent monooxygenase. Although the enzyme accepts p-coumarate as a substrate, it also exhibits significant activity towards other p-hydroxylated substrates. These data may explain the previous difficulties in identifying C3H activity in plant extracts and they indicate that the currently accepted version of the lignin biosynthetic pathway is likely to be incorrect.