Genome-wide analysis of the lignin toolbox of Eucalyptus grandis

Lignin, a major component of secondary cell walls, hinders the optimal processing of wood for industrial uses. The recent availability of the Eucalyptus grandis genome sequence allows comprehensive analysis of the genes encoding the 11 protein families specific to the lignin branch of the phenylpropanoid pathway and identification of those mainly involved in xylem developmental lignification. We performed genome-wide identification of putative members of the lignin gene families, followed by comparative phylogenetic studies focusing on bona fide clades inferred from genes functionally characterized in other species. RNA-seq and microfluid real-time quantitative PCR (RT-qPCR) expression data were used to investigate the developmental and environmental responsive expression patterns of the genes. The phylogenetic analysis revealed that 38 E. grandis genes are located in bona fide lignification clades. Four multigene families (shikimate O-hydroxycinnamoyltransferase (HCT), p-coumarate 3-hydroxylase (C3H), caffeate/5-hydroxyferulate O-methyltransferase (COMT) and phenylalanine ammonia-lyase (PAL)) are expanded by tandem gene duplication compared with other plant species. Seventeen of the 38 genes exhibited strong, preferential expression in highly lignified tissues, probably representing the E. grandis core lignification toolbox. The identification of major genes involved in lignin biosynthesis in E. grandis, the most widely planted hardwood crop world-wide, provides the foundation for the development of biotechnology approaches to develop tree varieties with enhanced processing qualities.This work, part of the LABEX project TULIP (ANR-10-LABX-41), was supported by grants from the Project Tree For Joules (ANR-2010-KBBE-007-01 and FCT-PKBBE/ AGR_GPL/0001/2010), the CNRS, the Toulouse III University (UPS), and the FCT project microEGo (PTDC/AGR-GPL/098179/2008). V.C. was supported by a FCT PhD grant (SFRH/BD/72982/2010). M.S. received a postdoctoral fellowship ‘Beatriu de Pinós’ from the DURSI de la Generalitat de Catalunya. J.A.P.P. acknowledges FCT for the research contract Ciência 2008 program and the postdoctoral fellowship SFRH/BPD/92207/2013. We are grateful to C. Araujo and L. Neves (Altri Florestal, Portugal), C. Marques (RAIZ, Portugal) and L. Harvengt (FCBA, France) for kindly providing and/or allowing collection of Eucalyptus samples, and C. Graça (IBET, IICT), N. Saidi, H. Yu and E. Camargo (all LRSV) for help with sample design/collection and RNA extraction. We also thank N. Ladouce (LRSV) and the Genotoul Genomic platform http://get.genotoul.fr/ for technical assistance with the Biomark Fluidigm RT-qPCR amplifications, and E. Mizrachi for assistance with RNA-seq data analysis. RNA-seq was funded by Mondi and Sappi through the FMG Program, the THRIP Program (UID 80118), the NRF (UID 71255 and 86936) and the DST of South Africa.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1469-8137hb2016Genetic

Comprehensive genetic dissection of wood properties in a widely-grown tropical tree: Eucalyptus

Background: Eucalyptus is an important genus in industrial plantations throughout the world and is grown for use as timber, pulp, paper and charcoal. Several breeding programmes have been launched worldwide to concomitantly improve growth performance and wood properties (WPs). In this study, an interspecific cross between Eucalyptus urophylla and E. grandis was used to identify major genomic regions (Quantitative Trait Loci, QTL) controlling the variability of WPs. Results: Linkage maps were generated for both parent species. A total of 117 QTLs were detected for a series of wood and end-use related traits, including chemical, technological, physical, mechanical and anatomical properties. The QTLs were mainly clustered into five linkage groups. In terms of distribution of QTL effects, our result agrees with the typical L-shape reported in most QTL studies, i.e. most WP QTLs had limited effects and only a few (13) had major effects (phenotypic variance explained > 15%). The co-locations of QTLs for different WPs as well as QTLs and candidate genes are discussed in terms of phenotypic correlations between traits, and of the function of the candidate genes. The major wood property QTL harbours a gene encoding a Cinnamoyl CoA reductase (CCR), a structural enzyme of the monolignol-specific biosynthesis pathway. Conclusions: Given the number of traits analysed, this study provides a comprehensive understanding of the genetic architecture of wood properties in this Eucalyptus full-sib pedigree. At the dawn of Eucalyptus genome sequence, it will provide a framework to identify the nature of genes underlying these important quantitative traits. (Résumé d'auteur

Critical Players and Gene Expression Regulation in Eucalyptus Xylogenesis

Secondary xylem, commonly known as wood, is essentially formed by highly lignified secondary cell walls of both fibres and vessels. The process of formation of secondary xylem is also termed xylogenesis. Secondary xylem derives from the vascular cambium, whose dividing cells undergo irreversible differentiation, under a strict temporal and spatial control.(...

Critical Players and Gene Expression Regulation in Eucalyptus Xylogenesis

Secondary xylem, commonly known as wood, is essentially formed by highly lignified secondary cell walls of both fibres and vessels. The process of formation of secondary xylem is also termed xylogenesis. Secondary xylem derives from the vascular cambium, whose dividing cells undergo irreversible differentiation, under a strict temporal and spatial control.(...

The EucalyptusEucalyptus grandis R2R3‐ MYB transcription factor family: evidence for woody growth‐related evolution and function

International audienceThe R2R3-MYB family, one of the largest transcription factor families in higher plants, controls a wide variety of plant-specific processes including, notably, phenylpropanoid metabolism and secondary cell wall formation. We performed a genome-wide analysis of this superfamily in Eucalyptus, one of the most planted hardwood trees world-wide. A total of 141 predicted R2R3-MYB sequences identified in the Eucalyptus grandis genome sequence were subjected to comparative phylogenetic analyses with Arabidopsis thaliana, Oryza sativa, Populus trichocarpa and Vitis vinifera. We analysed features such as gene structure, conserved motifs and genome location. Transcript abundance patterns were assessed by RNAseq and validated by high-throughput quantitative PCR. We found some R2R3-MYB subgroups with expanded membership in E. grandis, V. vinifera and P. trichocarpa, and others preferentially found in woody species, suggesting diversification of specific functions in woody plants. By contrast, subgroups containing key genes regulating lignin biosynthesis and secondary cell wall formation are more conserved across all of the species analysed. In Eucalyptus, R2R3-MYB tandem gene duplications seem to disproportionately affect woody-preferential and woody-expanded subgroups. Interestingly, some of the genes belonging to woody-preferential subgroups show higher expression in the cambial region, suggesting a putative role in the regulation of secondary growth

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