BdCESA7, BdCESA8, and BdPMT Utility Promoter Constructs for Targeted Expression to Secondary Cell-Wall-Forming Cells of Grasses.

Deborah L Petrik,Steven D Karlen,John Ralph,Cynthia L Cass,Dharshana Padmakshan,Cliff E Foster,John P Vogel,John C Sedbrook

doi:10.3389/fpls.2016.00055

Abstract

Utility vectors with promoters that confer desired spatial and temporal expression patterns are useful tools for studying gene and cellular function and for industrial applications. To target the expression of DNA sequences of interest to cells forming plant secondary cell walls, which generate most of the vegetative biomass, upstream regulatory sequences of the Brachypodium distachyon lignin biosynthetic gene BdPMT and the cellulose synthase genes BdCESA7 and BdCESA8 were isolated and cloned into binary vectors designed for Agrobacterium-mediated transformation of monocots. Expression patterns were assessed using the β-glucuronidase gene GUSPlus and X-glucuronide staining. All three promoters showed strong expression levels in stem tissue at the base of internodes where cell wall deposition is most active, in both vascular bundle xylem vessels and tracheids, and in interfascicular tissues, with expression less pronounced in developmentally older tissues. In leaves, BdCESA7 and BdCESA8 promoter-driven expression was strongest in leaf veins, leaf margins, and trichomes; relatively weaker and patchy expression was observed in the epidermis. BdPMT promoter-driven expression was similar to the BdCESA promoters expression patterns, including strong expression in trichomes. The intensity and extent of GUS staining varied considerably between transgenic lines, suggesting that positional effects influenced promoter activity. Introducing the BdPMT and BdCESA8 Open Reading Frames into BdPMT and BdCESA8 utility promoter binary vectors, respectively, and transforming those constructs into Brachypodium pmt and cesa8 loss-of-function mutants resulted in rescue of the corresponding mutant phenotypes. This work therefore validates the functionality of these utility promoter binary vectors for use in Brachypodium and likely other grass species. The identification, in Bdcesa8-1 T-DNA mutant stems, of an 80% reduction in crystalline cellulose levels confirms that the BdCESA8 gene is a secondary-cell-wall-forming cellulose synthase.

Highlights

Efforts are underway to engineer plant vegetative biomass, such as stems and leaves of grasses, to be more deconstructed and converted to liquid biofuels such as ethanol (Carroll and Somerville, 2009)
We showed that the Brachypodium Brachypodium p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (BdPMT) gene catalyzes the formation of monolignol p-coumarate (ML-p-coumaric acid (pCA)) ester conjugates that become polymerized into lignin within secondary cell walls (Petrik et al, 2014)
We reasoned that the promoter and cis-acting regulatory sequences of the BdPMT gene would be useful for driving the expression of gene knockdown constructs or genes of interest, with spatial and temporal patterns associated with lignin deposition

Summary

Introduction

Efforts are underway to engineer plant vegetative biomass, such as stems and leaves of grasses, to be more deconstructed and converted to liquid biofuels such as ethanol (Carroll and Somerville, 2009). One approach to improving biomass deconstructability is to engineer genetic changes that alter flux through the lignin biosynthetic pathway, by knocking down or overexpressing key monolignol biosynthetic pathway genes resulting in lower lignin quantity, altered lignin composition, and/or altered cell wall polymer crosslinking (Vanholme et al, 2012; Eudes et al, 2014; Wilkerson et al, 2014). Another approach to improve plant biomass yields and deconstruction properties is to introduce genetic changes that either increase deposition of secondary cell wall polysaccharides or result in the deposition of less tightly packed (amorphous) polysaccharides. The creation of a toolbox of directed gene promoters that drive expression of transgenes in cells involved in secondary cell wall formation would be of benefit to researchers aiming to improve plant biomass properties for biofuels generation while minimizing plant fitness costs

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