Lignocellulose in woody biomass is one of the most important sources of renewable energy around the word. Many countries are promoting biofuels from lignocellulose as a substitute for oil to replace nonrenewable fossil fuels. However, the conversion of lignocellulosic feedstocks to fermentable sugar for biofuel production is inefficient, and most strategies to enhance efficiency directly target lignin biosynthesis^{1, 2}, with associated negative growth impacts^3-5.

In the collaborative research by Prof. Richard A. Dixon and Prof. Jinxing Lin from the University of North Texas and Beijing Forestry University, we demonstrated the potential of manipulating non-coding RNA to achieve both enhanced biomass and reduced cell wall recalcitrance, critical for the development of an environmentally friendly lignocellulosic biofuels industry. 

We found that overexpression miR408 in poplar can promote plant height and stem diameter, and enhance saccharification significantly, with no requirement for acid-pretreatment, for both laboratory- and field-grown plants (Figure 1a-d). To investigate the mechanism that miR408 promotes the saccharification efficiency in poplar, we first utilized green fluorescent protein (GFP)-tagged CBM1/3 (CtCBM3-GFP and TrCBM1-GFP) and green dye-labeled cellulase enzyme to identify exposed cellulose surfaces. The results showed significantly increased accessibility of cellulase to the cell walls of miR408_OX plants (Figure 1e-i). Furthermore, in the cross sections of natural dried one-year-old poplar stems, the miR408_OX plants showed many more collapsed cells, probably due to water loss, whereas the cells were not collapsed in the cross sections of fresh material of the same lines.

Figure 1. Overexpression of miR408 enhances biomass yield, cell wall accessibility and saccharification efficiency in poplar.

 To understand the relationship between altered cell wall morphology and the changes in bulk lignin levels, we employed various techniques such as confocal Raman microspectroscopy (CRM), stimulated Raman scattering (SRS) and two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance (2D-HSQC NMR) spectroscopy and other techniques. These results showed overexpression of miR408 alters lignin deposition in poplar by reduced in lignin content and decreased molecular weights compared with WT.

To investigate the target(s) of miR408 that influence cell wall structure and composition, we employed 5’ RACE and found miR408 targets LAC19，LAC25 and LAC32. We also generated the LACCASE loss of function mutants and the overexpression poplars. It is very interesting that the LACCASE loss of function mutants exhibit significantly increased growth and saccharification efficiency in xylem, similar with miR408 overexpression poplars (Figure 2a-d).

Laccases are considered to function in the polymerization of lignin monomers, potentially at the stage of polymer initiation, and subsequently in concert with peroxidases⁶. Given that reduced degree of lignin polymerization is associated with improved lignin extractability and reduced biomass recalcitrance⁷, we speculate that the changes in lignin distribution and composition observed in the present miR408_OX plants result largely from the post-transcriptional regulation of three target LACCASES.

Lignification and growth are opposing processes competing for cellular resources. Our finding demonstrates that it is possible to engineer directionally opposite changes in these two processes with a single transgene. In the model in Figure 2h, we suggest that the decreased recalcitrance phenotypes and enhanced growth of the miR408_OX biomass result from repressed polymerization of monolignols, leading to less polymer–polymer cross-linking of lignin, increased wall porosity and reduced wall cohesiveness, which together increase enzyme access to biomass during saccharification. On the other hand, the looser microfibril structure may be easier to expand when the cell is growing under turgor pressure.

Figure 2. The lac19 lac25 lc32 triple mutant shows enhanced saccharification efficiency.

In conclusion, down-regulating multiple laccases through targeting of specific miRNAs may be a promising way to enhance biomass saccharification through reducing lignin polymerization. These findings, which are translatable to the field, can facilitate generation of improved tree feedstocks coupling enhanced saccharification efficiency with high total biomass, providing a promising and effective approach to the production of lignocellulosic bioenergy.

References

1. Fu C, et al. Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass. Natl. Acad. Sci. U S A 108, 3803-3808 (2011).
2. Saleme MLS, et al. Silencing CAFFEOYL SHIKIMATE ESTERASE affects lignification and improves saccharification in poplar. Plant Physiol. 175, 1040-1057 (2017).
3. Ruben V, et al. Caffeoyl shikimate esterase (CSE) is an enzyme in the lignin biosynthetic pathway in Arabidopsis. Science 341, 1103-1106 (2013).
4. Bonawitz ND, et al. Disruption of Mediator rescues the stunted growth of a lignin-deficient Arabidopsis Nature 509, 376-380 (2014).
5. Ha CM, et al. Ectopic defense gene expression is associated with growth defects in Medicago truncatula lignin pathway mutants. Plant Physiol. 181, 63-84 (2019).
6. Zhao Q, et al. Laccase is necessary and nonredundant with peroxidase for lignin polymerization during vascular development in Arabidopsis. Plant Cell 25, 3976-3987 (2013).
7. Ziebell A, et al. Increase in 4-coumaryl alcohol units during lignification in alfalfa (Medicago sativa) alters the extractability and molecular weight of lignin. Biol. Chem. 285, 38961-38968 (2010).