Overexpression of microRNA397 and its effects on the expression of laccases involved in lignin biosynthesis

Nguyen DQ1, Furbank R2, Eamens A1 and Grof C1

  1. Centre for Plant Science, School of Environmental and Life Science, University of Newcastle, Callaghan, NSW, 2308, Australia.
  2. Research School of Biology, Australian National University, ACT, 2600, Australia.

Lignocellulosic biomass derived from C4 grasses and other energy feedstocks is currently viewed as an attractive, alternative energy source. However, hydrolysis of lignocellulosic biomass to obtain fermentable sugars for the production of bioethanol is an inefficient process due to the presence of a complex network of lignin. Lignin, the third most abundance component in plant cell walls, is a recalcitrant biopolymer that consists of 3 monolignols (p-coumaryl, coniferyl and sinapyl alcohols), the precursors of 3 monomeric lignin subunits, p-hydroxyphenyl (H), guaicyl (G) and syringyl (S), respectively. Monolignols are initially synthesised in cytosol and transported to the apoplasm before they are oxidised and incorporated into the plant cell wall. The incorporation of lignin subunits into the plant cell wall has been reported to be controlled by a class of laccase enzymes. The genetic manipulation of LACCASE (LAC) gene function, or the activity of the post-transcriptional regulator of LAC gene expression, microRNA397 (miR397), is a potentially viable approach to modify plant cell wall composition and structure. Such an approach may maximise the efficiency of hydrolysis, and therefore, enhance the yield of bioethanol, while reducing input costs. The initial goal of this study was to identify putative Setaria viridis LACs (SvLACs), and a targeting small RNA (sRNA), and to profile the expression domains of both the miRNA and its targeted LAC genes across 13 developmentally distinct tissues of S. viridis. Secondly, to transform wild-type Arabidopsis thaliana (Col-0) plants with a miR397 overexpression construct to allow the study of the efficacy of the sRNA in knocking down the expression of Arabidopsis LAC target genes. Finally, the cell walls of the miR397 overexpression plant line will be anatomically and chemically analysed to compare their lignin content to that of non-transformed, wild-type Arabidopsis cell walls.