Search

The presence of aromatic polymer lignin is a bottleneck that hampers the efficient processing of Eucalyptus wood into downstream products such as cellulose pulp and biofuels. However, despite the impact of lignin on the economic uses of Eucalyptus wood, little is known regarding the molecular mechanisms underlying lignin deposition in this genus. Here, we report on the identification of a laccase gene potentially involved in lignin polymerization in Eucalyptus globulus by employing a combination of phylogenetic analysis, tissue-specific expression analysis, and genetic complementation assays. EglLAC48 is phylogenetically close to the lignin-related gene AtLAC15 of Arabidopsis thaliana and showed increased expression upon in vitro lignification-promoting conditions. Reverse transcription polymerase chain reaction analysis showed that the expression of EglLAC48 is higher in stems when compared with leaves and roots. In addition, in situ hybridization experiments revealed that EglLAC48 mRNA was mainly localized in developing xylem cells, an important lignification site. In order to provide genetic evidence to support a role for EglLAC48 in lignification, this gene was expressed in the Arabidopsis lac17 mutant background using both the endogenous AtLAC17 promoter and the constitutive CaMV35S promoter. Both strategies were successful in restoring lignin content, but the levels of guaiacyl units were still significantly reduced when compared with those of wild-type plants. The fact that the expression of EglLAC48 was able to restore the lignin levels but not lignin composition of the lac17 mutant suggests that this laccase is potentially involved in lignin polymerization in E. globulus but show different enzyme specificity when compared with AtLAC17.

The successful development of genetically engineered monocots using Agrobacterium-mediated transformation has created an increasing demand for compatible vectors. We have developed a new expression vector, pGVG, for efficient transformation and expression of different constructs for gene overexpression and silencing in sugarcane. The pCAMBIA2300 binary vector was modified by adding Gateway recombination sites for fast gene transfer between vectors and the maize polyubiquitin promoter Ubi-1 (ZmUbi1), which is known to drive high gene expression levels in monocots. Transformation efficiency using the pGVG vector reached up to 14 transgenic events per gram of transformed callus. Transgenic plants expressing the β-glucuronidase (GUS) reporter gene from pGVG showed high levels of GUS activity. qRT-PCR evaluations demonstrated success for both overexpression and hairpin-based silencing cassettes. Therefore, pGVG is suitable for plant transformation and subsequent applications for high-throughput production of stable transgenic sugarcane. The use of an expression cassette based on the ZmUbi1 promoter opens the possibility of using pGVG in other monocot species.