Manipulating Monocot (Grass) Lignin-forming Dirigent Protein Complexes for Optimal Bioenergy/Bioproducts
EMSL Project ID
51912
Abstract
The overall objective of this proposal is to establish the (bio)chemical basis as to why monocot (grass) lignocellulosic tissues are significantly less recalcitrant to (bio)degradation than their (woody) dicot counterparts. Resolution to this question, we hypothesize, will provide much needed new insight into developing effective universal strategies for overcoming woody dicot lignocellulosic recalcitrance.A major difference between monocot (grass) and dicot lignins is the presence of hydroxycinnamic acid-derived moieties, either ester- or ether-linked, in grass lignins versus essentially only monolignol-derived constituents that are ether or C-C inter-unit linked in (woody) dicot lignins. Based on current knowledge, this difference is the most important feature in grass lignins being more (bio)degradable, as compared to dicots.
To approach resolving this question, we focus here on the strategically important, DOE BER-approved, bio-feedstock Brachypodium distachyon, in order to delineate the biochemical basis for the reduced lignocellulosic recalcitrance.
A class of proteins we discovered, and that we trivially named dirigent proteins (DPs), apparently evolved during transition of aquatic plants to land. This transition was also associated with emergence of lignification. One DP subfamily has been correlated with lignification in dicots. In addition to this, in grasses, other unique DP sub-family members are apparently associated with lignification in its stems, strongly suggesting their possible involvement in grass lignin formation. (All known DPs provide entry into distinct plant phenol metabolic classes).
Through an existing JGI-supported project we have, JGI is providing constructs for all 46 Brachypodium DPs, in order that we express them in recombinant form and establish their biochemical functions. In this specific project, our focus is on 9 of the grass-unique DPs, known to be expressed in lignifying stem tissue, as well as 3 others. In addition, in our ongoing work, we are successfully utilizing CRISPR-Cas9 gene editing approaches to target all 9 of these grass species unique DPs, and 3 Dir-e DPs. Currently, our DP gene-edited Brachypodium has successfully been done with 2 of the 9 target DP genes, with the remainder under construction.
In this project, there are 3 Specific Aims, all of which critically depend upon EMSL capabilities. In Specific Aim 1, we will utilize EMSL laser micro-dissection capabilities in order to individually study lignifying stem cell types (fiber and/or vascular bundles) of wild type (WT) and DP gene edited Brachypodium lines. In addition to investigating their lignin compositions and contents in our lab, the EMSL capabilities allow for us to use NanoPots (nano-proteomics), HPLC MS/MS (for metabolomics) and MALDI-TOF metabolite imaging, in order to gain needed insight into effects of DP gene-editing on both lignification and associated secondary metabolism at the whole tissue and individual lignifying cell type level.
The second Specific Aim will study the 9 recombinant DPs and the Dir-e homologs, in terms of establishing their biochemical functions. EMSL capabilities will allow for their structural characterization by cryo-electron microscopy, NMR, and native MS, as appropriate.
Specific Aim3 will utilize the recombinant DPs in pull-down experiments to probe for interacting proteins associated with the DPs, including those proteins that are membrane-bound.
With the unique EMSL capabilities, we consider that our study will provide a new paradigm for lignification, and in strategies for overcoming woody dicot lignocellulosic recalcitrance.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2021-10-01
End Date
2024-01-31
Status
Closed
Released Data Link
Team
Principal Investigator
Co-Investigator(s)
Team Members