Nathalie Ladouce

Reference genes for high-throughput quantitative reverse transcription-PCR analysis of gene expression in organs and tissues of Eucalyptus grown in various environmental conditions.

Interest in the genomics of Eucalyptus has skyrocketed thanks to the recent sequencing of the genome of Eucalyptus grandis and to a growing number of large-scale transcriptomic studies. Quantitative reverse transcription-PCR (RT-PCR) is the method of choice for gene expression analysis and can now also be used as a high-throughput method. The selection of appropriate internal controls is becoming of utmost importance to ensure accurate expression results in Eucalyptus. To this end, we selected 21 candidate reference genes and used high-throughput microfluidic dynamic arrays to assess their expression among a large panel of developmental and environmental conditions with a special focus on wood-forming tissues. We analyzed the expression stability of these genes by using three distinct statistical algorithms (geNorm, NormFinder and ΔCt), and used principal component analysis to compare methods and rankings. We showed that the most stable genes identified depended not only on the panel of biological samples considered but also on the statistical method used. We then developed a comprehensive integration of the rankings generated by the three methods and identified the optimal reference genes for 17 distinct experimental sets covering 13 organs and tissues, as well as various developmental and environmental conditions. The expression patterns of Eucalyptus master genes EgMYB1 and EgMYB2 experimentally validated our selection. Our findings provide an important resource for the selection of appropriate reference genes for accurate and reliable normalization of gene expression data in the organs and tissues of Eucalyptus trees grown in a range of conditions including abiotic stresses.

Pathogen-associated molecular pattern-triggered immunity and resistance to the root pathogen Phytophthora parasitica in Arabidopsis

The cellulose binding elicitor lectin (CBEL) of the genus Phytophthora induces necrosis and immune responses in several plant species, including Arabidopsis thaliana. However, the role of CBEL-induced responses in the outcome of the interaction is still unclear. This study shows that some of CBEL-induced defence responses, but not necrosis, required the receptor-like kinase BAK1, a general regulator of basal immunity in Arabidopsis, and the production of a reactive oxygen burst mediated by respiratory burst oxidases homologues (RBOH). Screening of a core collection of 48 Arabidopsis ecotypes using CBEL uncovered a large variability in CBEL-induced necrotic responses. Analysis of non-responsive CBEL lines Ws-4, Oy-0, and Bla-1 revealed that Ws-4 and Oy-0 were also impaired in the production of the oxidative burst and expression of defence genes, whereas Bla-1 was partially affected in these responses. Infection tests using two Phytophthora parasitica strains, Pp310 and Ppn0, virulent and avirulent, respectively, on the Col-0 line showed that BAK1 and RBOH mutants were susceptible to Ppn0, suggesting that some immune responses controlled by these genes, but not CBEL-induced cell death, are required for Phytophthora parasitica resistance. However, Ws-4, Oy-0, and Bla-1 lines were not affected in Ppn0 resistance, showing that natural variability in CBEL responsiveness is not correlated to Phytophthora susceptibility. Overall, the results uncover a BAK1- and RBOH-dependent CBEL-triggered immunity essential for Phytophthora resistance and suggest that natural quantitative variation of basal immunity triggered by conserved general elicitors such as CBEL does not correlate to Phytophthora susceptibility.

Development and functional annotation of an 11,303-EST collection from Eucalyptus for studies of cold tolerance

A cDNA library was constructed from leaves of a cold-acclimated Eucalyptus gunnii elite clone and 13,056 ESTs were sequenced. When the 5,457 unique sequences were compared with Arabidopsis, rice, and poplar datasets, more than 38% of no-hits were found and the best annotation was provided by the woody perennial poplar database. However, among the 62% of sequences that could be annotated, the vast majority (87.5%) was found to be common to the four compared plant species, which shows how highly conserved this gene pool is across the plant kingdom. When the distribution of the annotated sequences was studied according to the Gene Ontology classification, some typical features of the dehydrative stress transcriptome were observed. In particular, genes from sugar metabolism and, above all, those induced by stress or external stimuli were well represented (6% in total). In addition, the library was found to contain a substantial number of ESTs encoding putative transcription factors including CBF (CRT-Binding Factor). The enrichment of this library with stress-related genes was strongly suggested by the high redundancy (up to 300 ESTs) of several genes known to be involved in cell protection such as the PCP (Pollen Coat Protein), Lti6b (Low temperature induced) or metallothionein. It was further confirmed by demonstrating the cold induction of a set of representative genes through Real Time-PCR. All these characteristics make this Eucalyptus EST collection very suitable for investigating the molecular basis of cold tolerance in a woody perennial and for isolating important or new candidate frost tolerance genes.

Special trends in CBF and DREB2 groups in Eucalyptus gunnii vs Eucalyptus grandis suggest that CBF are master players in the trade-off between growth and stress resistance.

Annotation of the Eucalyptus grandis genome showed a large amplification of the dehydration-responsive element binding 1/C-repeat binding factor (DREB1/CBF) group without recent DREB2 gene duplication compared with other plant species. The present annotation of the CBF and DREB2 genes from a draft of the Eucalyptus gunnii genome sequence reveals at least one additional CBF copy in the E. gunnii genome compared with E. grandis, suggesting that this group is still evolving, unlike the DREB2 group. This study aims to investigate the redundancy/neo- or sub-functionalization of the duplicates and the relative involvement of the two groups in abiotic stress responses in both E. grandis and E. gunnii (lower growth but higher cold resistance). A comprehensive transcriptional analysis using high-throughput quantitative real-time polymerase chain reaction (qRT-PCR) was performed on leaves, stems and roots from the two Eucalyptus species after cold, heat or drought treatment. A large CBF cluster accounted for most of the cold response in all the organs, whereas heat and drought responses mainly involved a small CBF cluster and the DREB2 genes. In addition, CBF putative target genes, known to be involved in plant tolerance and development, were found to be cold-regulated. The higher transcript amounts of both the CBF and target genes in the cold tolerant E. gunnii contrasted with the higher CBF induction rates in the fast growing E. grandis. Altogether, the present results, in agreement with previous data about Eucalyptus transgenic lines over-expressing CBF, suggest that these factors, which promote both stress protection and growth limitation, participate in the trade-off between growth and resistance in this woody species.

Foliar treatments with Gaultheria procumbens essential oil induce defense responses and resistance against a fungal pathogen in Arabidopsis.

Essential oil from Gaultheria procumbens is mainly composed of methylsalicylate (MeSA) (>96%), a compound which can be metabolized in plant tissues to salicylic acid, a phytohormone inducing plant immunity against microbial pathogens. The potential use of G. procumbens essential oil as a biocontrol agent was evaluated on the model plant Arabidopsis thaliana. Expression of a selection of defense genes was detected 1, 6, and 24 h after essential oil treatment (0.1 ml/L) using a high-throughput qPCR-based microfluidic technology. Control treatments included methyl jasmonate and a commercialized salicylic acid (SA) analog, benzo(1,2,3)-thiadiazole-7carbothiolic acid (BTH). Strong induction of defense markers known to be regulated by the SA pathway was observed after the treatment with G. procumbens essential oil. Treatment induced the accumulation of total SA in the wild-type Arabidopsis line Col-0 and analysis of the Arabidopsis line sid2, mutated in a SA biosynthetic gene, revealed that approximately 30% of MeSA sprayed on the leaves penetrated inside plant tissues and was demethylated by endogenous esterases. Induction of plant resistance by G. procumbens essential oil was tested following inoculation with a GFP-expressing strain of the Arabidopsis fungal pathogen Colletotrichum higginsianum. Fluorescence measurement of infected tissues revealed that treatments led to a strong reduction (60%) of pathogen development and that the efficacy of the G. procumbens essential oil was similar to the commercial product BION®. Together, these results show that the G. procubens essential oil is a natural source of MeSA which can be formulated to develop new biocontrol products.

Long cold exposure induces transcriptional and biochemical remodelling of xylem secondary cell wall in Eucalyptus

Although eucalypts are the most planted hardwood trees worldwide, the majority of them are frost sensitive. The recent creation of frost-tolerant hybrids such as Eucalyptus gundal plants (E. gunnii × E. dalrympleana hybrids), now enables the development of industrial plantations in northern countries. Our objective was to evaluate the impact of cold on the wood structure and composition of these hybrids, and on the biosynthetic and regulatory processes controlling their secondary cell-wall (SCW) formation. We used an integrated approach combining histology, biochemical characterization and transcriptomic profiling as well as gene co-expression analyses to investigate xylem tissues from Eucalyptus hybrids exposed to cold conditions. Chilling temperatures triggered the deposition of thicker and more lignified xylem cell walls as well as regulation at the transcriptional level of SCW genes. Most genes involved in lignin biosynthesis, except those specifically dedicated to syringyl unit biosynthesis, were up-regulated. The construction of a co-expression network enabled the identification of both known and potential new SCW transcription factors, induced by cold stress. These regulators at the crossroads between cold signalling and SCW formation are promising candidates for functional studies since they may contribute to the tolerance of E. gunnii × E. dalrympleana hybrids to cold.

Master regulators of wood formation in Eucalyptus

With the current global focus on bioenergy, forest plantations are increasingly becoming important sources for second generation biofuel, where the whole plant lignocellulosic biomass is to be mobilized. The lignocellulosic biomass is mainly composed of secondary walls (SW) possessing unique characteristics (biochemical composition and tridimensional association of polymers), which govern the intrinsic properties of wood. They especially contain high amounts of lignins, hydrophobic phenolic polymers which constitute an obstacle to the optimal utilization of plant species in paper industry and for saccharification prior to bioethanol production. Among perennial species, Eucalyptus species grow very fast and produce high yields of lignocellulosic biomass. They represent the main industrial plantations in the worldand one of the most appealing lignocellulosic feedstock for bioenergy production. Dissection of the molecular switches controlling the coordinated lignin biosynthetic genes is therefore of utmost importance to understand the molecular mechanisms underlying tissue specific deposition of lignin and be able to improve secondary cell wall properties. With the objective of improving Eucalyptus wood quality to better-fit industrial applications, we are focusing our efforts towards the identification and functional characterisation of regulatory genes controlling the biosynthesis of the cell wall polymers (mainly lignins). We performed a precise mapping and functional characterization of the cis-regulatory elements contained in the promoters of two genes encoding key and consecutive steps of the lignin biosynthetic pathway i.e. Cinnamoyl CoA reductase (CCR) and Cinnamyl Alcohol dehydrogenase (CAD) (Rahantamala et al, 2010). Our results supported a major role for the MYB transcription factors (TF) consensus sites in the control of the coordinated expression of these two genes. The functional analysis of two MYB factors (EgMYB1 and EgMYB2) preferentially expressed in Eucalyptus xylem revealed that they are able to bind specifically to these promoters and regulate transcriptionin vivo. EgMYB1 behaves as a repressor whereas EgMYB2 is an activator (Goicoechea et al, 2005, Legay et al, 2010) of the lignin biosynthetic genes but also of the secondary wall biosynthesis. Indeed, both MYBs were shown recently to be master genes regulating the entire secondary wall biosynthetic program including cellulose, xylan and lignin genes (Zhong et al, 2010; Legay et al 2010). The presence of both positive and negative regulators in Eucalyptus xylem offers the possibility of a combinatorial control of gene expression that could provide the necessary flexibility to ensure tight temporal and spatial regulation of lignin biosynthesis or secondary cell wall. To address this question and get a deeper insight the complex regulation of the SW formation in Eucalyptus, we are now studying the regulation of these two MYBs including fine spatial and temporal expression, identification of their direct targets genes and of their protein partners. We have constructed a yeast-two-hybrid library from Eucalyptus xylem that will also be instrumental for deciphering the interactants of landmark genes for the International Eucalyptus community. Thanks to the recent release of the E. grandis genome (Eucalyptus grandis Genome Project 2010, http://www.phytozome.net/eucalyptus), we have performed a genome-wide survey of the large R2R3-MYB superfamily. The phylogenetic comparison of this family with Arabidopsis, rice, poplar and grapevine showed a marked expansion of some clusters putatively involved in wood-related processes. Some R2R3 MYB genes seem to be specific of woody plants. The spatiotemporal expression patterns of members of such clusters are currently being studied. Although Auxin is known as a key regulator of cambium activity and wood formation, the Auxin response mediators [Auxin/Indole-3-Acetic Acid (Aux/IAA) and Auxin Response Factor (ARF) transcription factors] extensively characterized in model plants, are still largely uncharacterized in tree species. We have identified 23 Aux/IAA and 17 ARF in the E. grandis genome. Comparative phylogenetic analysis revealed that several Aux/IAA and ARF subgroups have differentially expanded or contracted amongst the three dicotyledonous plants studied (Arabidopsis, Populus and Eucalyptus). Expression analysis and EST database surveys are currently underway to explore the transcript levels of each member in the different organs and tissues of Eucalyptus at key developmental stages as well as in response to hormonal treatments and to environmental stresses. Further functional genomics studies conducted on new candidate transcription factors, their regulation under the developmental or environmental stimuli will help identifying major factors underpinning the physicochemical properties of cell walls, the recalcitrance of which remains a key scientific challenge for establishing highly efficient, sustainably produced, second-generation biofuels.