The fields of lignin chemistry, biochemistry, molecular biology, and plant cell wall biomechanics/anatomy are of greatly renewed interest as the twenty-first century unfolds. This contrasts with the preliminary studies of the mid-twentieth century on lignin consti­tution, which more or less relegated this important class ofmacromolecules to the scientific curiosity of being randomly assembled. This, in turn, was largely as a result of the severe technological barriers that existed during the 1950s and 1960s, and the speculations of the time. Yet, even today, there remains spirited discussion as regards both lignin structure and how they are formed.

From the standpoint of the authors, the recent advances made largely in the last decade or so now beckon a different viewpoint of the lignins — particularly as regards their potential to help facilitate either cellulose-to-ethanol or related biofuels/bioproducts technologies. This is thus a very exciting time for studying lignins, as there is still much to be learned about both lignins and the associated cell wall assembly processes. In addition, such problems need to be solved to meet humanity’s needs for future generations.

Several areas that seem to be of particular promise for future study are summarized below:

1 Monolignol Transport to the Cell Wall: Establishing the biochemical basis of how monolig — nols 1,3, and 5 (or derivatives/homologues thereof) are targeted to precise regions within the cell walls, and how this is regulated, are important questions to resolve; while (ABC) transporters (351) have been postulated as involved, this has not yet been proven.

2 Lignin Initiation Sites: Lignin deposition begins at so-called “initiation sites” in the cell corners and then progresses to eventually encapsulate the entire cell wall prior to apoptosis (see Figure 7.5). In this regard, establishing the nature and function of each of the proteins, enzymes and genes involved at these initiation sites appears to be a particularly attractive line of enquiry. Resolution to this should substantially clarify any remaining questions on how lignins are formed.

3 Lignin Primary Sequences/New Chemistries: A related need is to develop methodologies to establish the primary sequences of the lignins being formed initially, as well as developing new technological means and new chemistries to both identify and quantify all interunit linkages. This will also include establishing unambiguously whether lignins contain either no branches, or short or long branches, and how lignin-carbohydrate linkages are both formed and regulated.

4 Re-oxidation of the Growing Lignin Chains: Another urgently needed emphasis is in es­tablishing biochemically how monolignols 1, 3, and 5 and the growing lignin chains are re-oxidized in the cell wall following initial coupling, i. e., thereby enabling radical/radical generation to reoccur. This is a problem that has long been recognized, namely, how does one-electron re-oxidation of lignin continue at sites presumably distant from the one-electron oxidative enzymes. Whether this occurs, for example, via some form of elec­tron transfer through the lignin matrix (e. g., originating via oxidation by the presumed peroxidase/H2O2) or whether a diffusible oxidant, such as has been suggested for Mn3+ is involved (352), needs to be established.

5 Monolignol Radical and Lignin Primary Chain Interactions, Proposed Template Polymeriza­tion, and Lignin Association: Unambiguously determining whether the monolignol radi­cals being generated are never “free,” but instead are transiently immobilized as a result of strong л… л interactions between the substrates and a pre-existing lignin macromolec­ular template is also another important goal, i. e., following on the previously proposed template polymerization for replication of lignin primary chains (353). Recently, Sarkanen and Chen modeled non-covalent interactions between a monolignol (coniferyl alcohol, 3) radical and a representative monomer residue (= veratryl alcohol) for a lignin macro­molecule. Mo5-2x/6J1 + G (d, p) density functional theory calculations led to a gas phase stabilization energy of 13.4 kcal/mole for a cofacial complex with one strong and one weak intermolecular bond, versus that of 8.6 kcal/mole due to dynamic electron cor­relations in the interacting л-constituents alone. These researchers also suggested that head-to-tail orientations of the interacting species were preferred, and additionally pro­posed that an antiparallel double-stranded lignin template was responsible for obtaining macromolecular lignin domains lacking either crystallinity or optical activity. According to these researchers, the intermolecular interactions between monolignol radicals and the substructures in lignins may be considerably stronger than Watson-Crick A-T or G-C nucleobase pairs in DNA: in the latter, a weak hydrogen bond in an oligonucleotide has been estimated to only amount to an increment of ~0.4-2.0 kcal/mole to the stabilization energy. While the gas-phase calculations favor the concept of template polymerization, the potential ramifications of this clearly need to be more fully investigated. It will thus be most instructive to establish how many lignin-forming templates there possibly are, and to what extent each would be able to display limited substrate degeneracy. In addition to the question of proposed template replication, another important related emphasis is to unambiguously define the precise molecular basis for the strong associative forces observed between adjacent lignin chains/lignin preparations.

6 Transcriptional Control of Individual Cell Wall Formation Processes, Biomechanics, and Biodegradation of Plant Cell Walls: With the recent demonstration of specific transcrip­tion factors required for fiber formation, this now offers the opportunity to identify how the various cell wall types are differentially generated. This, in turn, may also permit investigation of lignification in specific cell types, and thus as to how they are individu — ally/differentially formed. In any event, this gives another new direction to the possibility of systematically modulating overall plant structure and plant properties for humanity’s use — one cell type at a time.

The areas of biomechanics, biodegradation, and factors affecting disease resistance are other most important emphases that need to be enhanced in terms of current levels of sci­entific investigation, i. e., in order to understand fully the potential of lignin modification. For example, to what extent can lignin contents and composition be manipulated without adversely affecting cell wall properties/vascular integrity for growth/development, harvest­ing, storage, and further processing? Furthermore, what effects do such manipulations have on biodegradation feasibility (e. g., with different lignin compositions/contents), as well as on plant defense.

In short, there is much still to do in determining how Nature’s second most abundant vascular plant biopolymers are formed and their potential (through manipulation) for

humanity’s varied needs. The next 5-10 years should be exciting ones for both lignin and plant cell wall research, and promise to be an exciting challenge for the twenty-first century.

Acknowledgments

The authors thank the National Science Foundation (MCB-0417291), the United States Department of Energy (DE FG03-97ER20259), the National Institute of General Medical Sciences (5 R01 GM066173-02), McIntire Stennis, and the G. Thomas and Anita Hargrove Center for Plant Genomic Research for generous financial support.