Wood Architecture and Composition Are Deeply Remodeled in Frost Sensitive Eucalyptus Overexpressing CBF/DREB1 Transcription Factors.

Eucalypts are the most planted trees worldwide, but most of them are frost sensitive. Overexpressing transcription factors for CRT-repeat binding factors (CBFs) in transgenic Eucalyptus confer cold resistance both in leaves and stems. While wood plays crucial roles in trees and is affected by environmental cues, its potential role in adaptation to cold stress has been neglected. Here, we addressed this question by investigating the changes occurring in wood in response to the overexpression of two CBFs, taking advantage of available transgenic Eucalyptus lines. We performed histological, biochemical, and transcriptomic analyses on xylem samples. CBF ectopic expression led to a reduction of both primary and secondary growth, and triggered changes in xylem architecture with smaller and more frequent vessels and fibers exhibiting reduced lumens. In addition, lignin content and syringyl/guaiacyl (S/G) ratio increased. Consistently, many genes of the phenylpropanoid and lignin branch pathway were upregulated. Most of the features of xylem remodeling induced by CBF overexpression are reminiscent of those observed after long exposure of Eucalyptus trees to chilling temperatures. Altogether, these results suggest that CBF plays a central role in the cross-talk between response to cold and wood formation and that the remodeling of wood is part of the adaptive strategies to face cold stress.

Implementing the CRISPR/Cas9 Technology in Eucalyptus Hairy Roots Using Wood-Related Genes.

Eucalypts are the most planted hardwoods worldwide. The availability of the Eucalyptus grandis genome highlighted many genes awaiting functional characterization, lagging behind because of the lack of efficient genetic transformation protocols. In order to efficiently generate knock-out mutants to study the function of eucalypts genes, we implemented the powerful CRISPR/Cas9 gene editing technology with the hairy roots transformation system. As proofs-of-concept, we targeted two wood-related genes: Cinnamoyl-CoA Reductase1 (CCR1), a key lignin biosynthetic gene and IAA9A an auxin dependent transcription factor of Aux/IAA family. Almost all transgenic hairy roots were edited but the allele-editing rates and spectra varied greatly depending on the gene targeted. Most edition events generated truncated proteins, the prevalent edition types were small deletions but large deletions were also quite frequent. By using a combination of FT-IR spectroscopy and multivariate analysis (partial least square analysis (PLS-DA)), we showed that the CCR1-edited lines, which were clearly separated from the controls. The most discriminant wave-numbers were attributed to lignin. Histochemical analyses further confirmed the decreased lignification and the presence of collapsed vessels in CCR1-edited lines, which are characteristics of CCR1 deficiency. Although the efficiency of editing could be improved, the method described here is already a powerful tool to functionally characterize eucalypts genes for both basic research and industry purposes.

Eucalyptus hairy roots, a fast, efficient and versatile tool to explore function and expression of genes involved in wood formation.

Eucalyptus are of tremendous economic importance being the most planted hardwoods worldwide for pulp and paper, timber and bioenergy. The recent release of the Eucalyptus grandis genome sequence pointed out many new candidate genes potentially involved in secondary growth, wood formation or lineage-specific biosynthetic pathways. Their functional characterization is, however, hindered by the tedious, time-consuming and inefficient transformation systems available hitherto for eucalypts. To overcome this limitation, we developed a fast, reliable and efficient protocol to obtain and easily detect co-transformed E. grandis hairy roots using fluorescent markers, with an average efficiency of 62%. We set up conditions both to cultivate excised roots in vitro and to harden composite plants and verified that hairy root morphology and vascular system anatomy were similar to wild-type ones. We further demonstrated that co-transformed hairy roots are suitable for medium-throughput functional studies enabling, for instance, protein subcellular localization, gene expression patterns through RT-qPCR and promoter expression, as well as the modulation of endogenous gene expression. Down-regulation of the Eucalyptus cinnamoyl-CoA reductase1 (EgCCR1) gene, encoding a key enzyme in lignin biosynthesis, led to transgenic roots with reduced lignin levels and thinner cell walls. This gene was used as a proof of concept to demonstrate that the function of genes involved in secondary cell wall biosynthesis and wood formation can be elucidated in transgenic hairy roots using histochemical, transcriptomic and biochemical approaches. The method described here is timely because it will accelerate gene mining of the genome for both basic research and industry purposes.

EgMYB1, an R2R3 MYB transcription factor from eucalyptus negatively regulates secondary cell wall formation in Arabidopsis and poplar.

• The eucalyptus R2R3 transcription factor, EgMYB1 contains an active repressor motif in the regulatory domain of the predicted protein. It is preferentially expressed in differentiating xylem and is capable of repressing the transcription of two key lignin genes in vivo. • In order to investigate in planta the role of this putative transcriptional repressor of the lignin biosynthetic pathway, we overexpressed the EgMYB1 gene in Arabidopsis and poplar. • Expression of EgMYB1 produced similar phenotypes in both species, with stronger effects in transgenic Arabidopsis plants than in poplar. Vascular development was altered in overexpressors showing fewer lignified fibres (in phloem and interfascicular zones in poplar and Arabidopsis, respectively) and reduced secondary wall thickening. Klason lignin content was moderately but significantly reduced in both species. Decreased transcript accumulation was observed for genes involved in the biosynthesis of lignins, cellulose and xylan, the three main polymers of secondary cell walls. Transcriptomic profiles of transgenic poplars were reminiscent of those reported when lignin biosynthetic genes are disrupted. • Together, these results strongly suggest that EgMYB1 is a repressor of secondary wall formation and provide new opportunities to dissect the transcriptional regulation of secondary wall biosynthesis.