Knockdown of a laccase in Populus deltoides confers altered cell wall chemistry and increased sugar release.

Plant laccases are thought to function in the oxidation of monolignols which leads to higher order lignin formation. Only a hand-full of laccases in plants have been functionally evaluated, and as such little is known about the breadth of their impact on cell wall chemistry or structure. Here, we describe a previously uncharacterized laccase from Populus, encoded by locus Potri.008G064000, whose reduced expression resulted in transgenic Populus trees with changes in syringyl/guaiacyl ratios as well as altered sugar release phenotypes. These phenotypes are consistent with plant biomass exhibiting reduced recalcitrance. Interestingly, the transgene effect on recalcitrance is dependent on a mild pretreatment prior to chemical extraction of sugars. Metabolite profiling suggests the transgene modulates phenolics that are associated with the cell wall structure. We propose that this particular laccase has a range of functions related to oxidation of phenolics and conjugation of flavonoids that interact with lignin in the cell wall.

Control of pollen-mediated gene flow in transgenic trees.

Pollen elimination provides an effective containment method to reduce direct gene flow from transgenic trees to their wild relatives. Until now, only limited success has been achieved in controlling pollen production in trees. A pine (Pinus radiata) male cone-specific promoter, PrMC2, was used to drive modified barnase coding sequences (barnaseH102E, barnaseK27A, and barnaseE73G) in order to determine their effectiveness in pollen ablation. The expression cassette PrMC2-barnaseH102E was found to efficiently ablate pollen in tobacco (Nicotiana tabacum), pine, and Eucalyptus (spp.). Large-scale and multiple-year field tests demonstrated that complete prevention of pollen production was achieved in greater than 95% of independently transformed lines of pine and Eucalyptus (spp.) that contained the PrMC2-barnaseH102E expression cassette. A complete pollen control phenotype was achieved in transgenic lines and expressed stably over multiple years, multiple test locations, and when the PrMC2-barnaseH102E cassette was flanked by different genes. The PrMC2-barnaseH102E transgenic pine and Eucalyptus (spp.) trees grew similarly to control trees in all observed attributes except the pollenless phenotype. The ability to achieve the complete control of pollen production in field-grown trees is likely the result of a unique combination of three factors: the male cone/anther specificity of the PrMC2 promoter, the reduced RNase activity of barnaseH102E, and unique features associated with a polyploid tapetum. The field performance of the PrMC2-barnaseH102E in representative angiosperm and gymnosperm trees indicates that this gene can be used to mitigate pollen-mediated gene flow associated with large-scale deployment of transgenic trees.

Overexpression of a Domain of Unknown Function 231-containing protein increases O-xylan acetylation and cellulose biosynthesis in Populus.

BACKGROUND: Domain of Unknown Function 231-containing proteins (DUF231) are plant specific and their function is largely unknown. Studies in the model plants Arabidopsis and rice suggested that some DUF231 proteins act in the process of O-acetyl substitution of hemicellulose and esterification of pectin. However, little is known about the function of DUF231 proteins in woody plant species. RESULTS: This study provides evidence supporting that one member of DUF231 family proteins in the woody perennial plant Populus deltoides (genotype WV94), PdDUF231A, has a role in the acetylation of xylan and affects cellulose biosynthesis. A total of 52 DUF231-containing proteins were identified in the Populus genome. In P. deltoides transgenic lines overexpressing PdDUF231A (OXPdDUF231A), glucose and cellulose contents were increased. Consistent with these results, the transcript levels of cellulose biosynthesis-related genes were increased in the OXPdDUF231A transgenic lines. Furthermore, the relative content of total acetylated xylan was increased in the OXPdDUF231A transgenic lines. Enzymatic saccharification assays revealed that the rate of glucose release increased in OXPdDUF231A transgenic lines. Plant biomass productivity was also increased in OXPdDUF231A transgenic lines. CONCLUSIONS: These results suggest that PdDUF231A affects cellulose biosynthesis and plays a role in the acetylation of xylan. PdDUF231A is a promising target for genetic modification for biofuel production because biomass productivity and compositional quality can be simultaneously improved through overexpression.

Down-regulation of p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H) and cinnamate 4-hydroxylase (C4H) genes in the lignin biosynthetic pathway of Eucalyptus urophylla × E. grandis leads to improved sugar release.

BACKGROUND: Lignocellulosic materials provide an attractive replacement for food-based crops used to produce ethanol. Understanding the interactions within the cell wall is vital to overcome the highly recalcitrant nature of biomass. One factor imparting plant cell wall recalcitrance is lignin, which can be manipulated by making changes in the lignin biosynthetic pathway. In this study, eucalyptus down-regulated in expression of cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) or p-coumaroyl quinate/shikimate 3'-hydroxylase (C3'H, EC 1.14.13.36) were evaluated for cell wall composition and reduced recalcitrance. RESULTS: Eucalyptus trees with down-regulated C4H or C3'H expression displayed lowered overall lignin content. The control samples had an average of 29.6 %, the C3'H reduced lines had an average of 21.7 %, and the C4H reduced lines had an average of 18.9 % lignin from wet chemical analysis. The C3'H and C4H down-regulated lines had different lignin compositions with average S/G/H ratios of 48.5/33.2/18.3 for the C3'H reduced lines and 59.0/39.8/1.2 for the C4H reduced lines, compared to the control with 65.9/33.2/1.0. Both the C4H and C3'H down-regulated lines had reduced recalcitrance as indicated by increased sugar release as determined using enzymatic conversion assays utilizing both no pretreatment and a hot water pretreatment. CONCLUSIONS: Lowering lignin content rather than altering sinapyl alcohol/coniferyl alcohol/4-coumaryl alcohol ratios was found to have the largest impact on reducing recalcitrance of the transgenic eucalyptus variants. The development of lower recalcitrance trees opens up the possibility of using alternative pretreatment strategies in biomass conversion processes that can reduce processing costs.