We chose some of these candidate transcription factors according to their expression patterns and novelty for functional characterization and transformed Arabidopsis and poplar lines with overexpressing and/or dominant repressor constructs.

As an example, the ectopic expression of a stabilized version of an E. grandis Aux/IAA gene [EgrIAA4m] in Arabidopsis reduced dramatically primary and lateral growth resulting into dwarf plants with very thin stems [Fig. 2].  The development of the interfascicular fibres was strongly inhibited [Fig. 2, Yu et al 2015].

Wood formation is highly plastic and secondary cell walls [SCW] structure and composition respond to environmental constraints. We showed that in response to luxuriant nitrogen fertilization, SCWs are weaker and less lignified [Fig 4 (a,b); Camargo et al., 2014]. Cold temperatures trigger the precocious formation of thick and lignified SCWs unusually close to cambium [Fig 4 (c,d); Ployet et al., 2017). Potassium supply and water availability modify vessel size and density (Ployet et al., in prep). Temperature changes and nutrients supply also modify polysaccharides accessibility (saccharification yield), a key parameter for biofuel generation.

Fig 4: Nutrition and temperature modify secondary cell wall (SCW) structure in Eucalyptus xylem. SEM images of Eucalyptus xylem SCW under limiting (a) and luxuriant (b) nitrogen fertilization conditions [Camargo et al, 2014]. Images of Eucalyptus xylem SCW in plant grown at 25°C (c) and in plants grown at 4°C (d) [Ployet et al, 2017].

In order to get a comprehensive view of the regulatory pathways underlying xylem response to abiotic stresses, we developed system biology approaches integrating ‘omics (transcriptomics, metabolomics, epigenetics and genetics) and phenotypic data. Using multivariate analyses, gene network reconstruction, and large-scale data integration, we were able to correlate SCW-related gene modules to wood properties and to further identify new promising transcription factors regulating xylem formation under stress conditions. Altogether system biology approach and functional characterization of CGs, showed that stress-signalling pathways could co-opt the regulatory pathways controlling SCW formation to modify cell differentiation, secondary metabolites accumulation and SCW deposition in xylem.

Fig 5: Targeted gene correlation network highlights Xylem and Cambium specific transcription factors (TFs) regulated by cold. (a) Gene correlation network based on expression profiles of 250 genes including 150 TFs obtained in >30 conditions and or tissues. A “cambium” module contains MYB and NAC TFs induced by cold stress. A “xylem” module encloses new candidate genes potentially involved in SCW formation, such as EgNAC64 (b), strongly correlated to genes described in the regulation and the deposition of SCW, such as EgNAC61 (AtSND1) in (c) [Ployet et al, 2017].

Fig 6: Drought and nutrition can modulate SCW structure and composition through a network of cell wall related genes. We performed a data integration approach on transcriptomic, metabolomic, phenotypic and eco-physiological datasets. WGCNA modules detection highlighted 12 modules related to cell activity, primary and secondary metabolism, ion transport, cytoskeleton, stress response and cell wall formation. Cell wall related modules (1 to 6) were strongly correlated to wood phenotypic traits and new mass signatures corresponding to flavonoids, terpenes and coumarins [Ployet et al, in prep].

To cope with abiotic stresses using limited resources, plants have evolved diverse mechanisms underlying a trade-off between stress tolerance and plant growth. However, to date, the precise molecular mechanisms involved in this trade-off are yet to be investigated.

Given its opportunistic growth without endodormancy, Eucalyptus represents a suitable model to address this question. On various woody species including Eucalyptus, abundant transcriptomics data have highlighted the DREB1/CBF (“Drought Responsive Element Binding/ CRT-repeat Binding Factor”) as a key transcription factor in the cold-induced regulation (Wisniewski et al, 2014). Moreover, the spectacular increase of CBF family members in Eucalyptus compared to other species, suggests that these transcriptional factors are master regulators of cold tolerance in this species (Cao et al 2015). Together with the comparison of Eucalyptus species (Nguyen et al, 2017), the phenotype of Eucalyptus CBF-overexpressors (CBF-OE) provided strong evidence of the involvement of CBF in the cross-talk between cold tolerance and growth (Navarro et al, 2011; Cao et al, submitted).

Beyond the compromise between primary growth and stress tolerance, a vascular trade-off between conduction efficiency and resistance to cavitation is often described but molecular control is still to be elucidated. Recent investigations on wood properties of Eucalyptus CBF-OE shed a new light on the gene regulation of this trade-off. CBF overexpressors were characterized by modifications of xylem architecture and dynamics of differentiation, both resulting in modulation of lignin content and composition. Interestingly, smaller vessels are associated with lower xylem vulnerability to cavitation which is consistent with improvement of stem frost tolerance observed in CBF-OE (see hypothetic model below). By controlling xylem maturation and architecture under stress, the CBF may be a key in the trade-off between growth and tolerance, representing the balance between efficiency and safety for Eucalyptus as a model tree species.

Hypothetic model of CBF control on the trade-off between growth and cole resistance through modification of xylem architecture.

Because protein-protein interaction is a major mechanism regulating the activity of TFs, we constructed a yeast-two-hybrid library from Eucalyptus xylem and started to seek for the protein partners of EgMYB1, a transcription factor known to repress lignin biosynthesis (Legay et al, 2007, 2010). EgMYB1 interacts specifically with a linker histone variant, EgH1.3. This interaction enhances the repression of EgMYB1’s target genes, strongly limiting the amount of lignin deposited in xylem cell walls. Our results suggest that a complex between EgMYB1 and EgH1.3 integrates developmental signals to prevent premature or inappropriate lignification of secondary cell walls,  roviding a mechanism to fine-tune the differentiation of xylem cells in time and space [Soler et al, 2016a].