Marçal Soler

The genome of Eucalyptus grandis

Eucalypts are the world’s most widely planted hardwood trees. Their outstanding diversity, adaptability and growth have made them a global renewable resource of fibre and energy. We sequenced and assembled >94% of the 640-megabase genome of Eucalyptus grandis. Of 36,376 predicted protein-coding genes, 34% occur in tandem duplications, the largest proportion thus far in plant genomes. Eucalyptus also shows the highest diversity of genes for specialized metabolites such as terpenes that act as chemical defence and provide unique pharmaceutical oils. Genome sequencing of the E. grandis sister species E. globulus and a set of inbred E. grandis tree genomes reveals dynamic genome evolution and hotspots of inbreeding depression. The E. grandis genome is the first reference for the eudicot order Myrtales and is placed here sister to the eurosids. This resource expands our understanding of the unique biology of large woody perennials and provides a powerful tool to accelerate comparative biology, breeding and biotechnology.

A Genomic Approach to Suberin Biosynthesis and Cork Differentiation

Cork (phellem) is a multilayered dead tissue protecting plant mature stems and roots and plant healing tissues from water loss and injuries. Cork cells are made impervious by the deposition of suberin onto cell walls. Although suberin deposition and cork formation are essential for survival of land plants, molecular studies have rarely been conducted on this tissue. Here, we address this question by combining suppression subtractive hybridization together with cDNA microarrays, using as a model the external bark of the cork tree (Quercus suber), from which bottle cork is obtained. A suppression subtractive hybridization library from cork tree bark was prepared containing 236 independent sequences; 69% showed significant homology to database sequences and they corresponded to 135 unique genes. Out of these genes, 43.5% were classified as the main pathways needed for cork biosynthesis. Furthermore, 19% could be related to regulatory functions. To identify genes more specifically required for suberin biosynthesis, cork expressed sequence tags were printed on a microarray and subsequently used to compare cork (phellem) to a non-suberin-producing tissue such as wood (xylem). Based on the results, a list of candidate genes relevant for cork was obtained. This list includes genes for the synthesis, transport, and polymerization of suberin monomers such as components of the fatty acid elongase complexes, ATP-binding cassette transporters, and acyltransferases, among others. Moreover, a number of regulatory genes induced in cork have been identified, including MYB, No-Apical-Meristem, and WRKY transcription factors with putative functions in meristem identity and cork differentiation.

CYP86A33-Targeted Gene Silencing in Potato Tuber Alters Suberin Composition, Distorts Suberin Lamellae, and Impairs the Periderm's Water Barrier Function

Suberin is a cell wall lipid polyester found in the cork cells of the periderm offering protection against dehydration and pathogens. Its biosynthesis and assembly, as well as its contribution to the sealing properties of the periderm, are still poorly understood. Here, we report on the isolation of the coding sequence CYP86A33 and the molecular and physiological function of this gene in potato (Solanum tuberosum) tuber periderm. CYP86A33 was down-regulated in potato plants by RNA interference-mediated silencing. Periderm from CYP86A33-silenced plants revealed a 60% decrease in its aliphatic suberin load and greatly reduced levels of C18:1 ω-hydroxyacid (approximately 70%) and α,ω-diacid (approximately 90%) monomers in comparison with wild type. Moreover, the glycerol esterified to suberin was reduced by 60% in the silenced plants. The typical regular ultrastructure of suberin, consisting of dark and light lamellae, disappeared and the thickness of the suberin layer was clearly reduced. In addition, the water permeability of the periderm isolated from CYP86A33-silenced lines was 3.5 times higher than that of the wild type. Thus, our data provide convincing evidence for the involvement of ω-functional fatty acids in establishing suberin structure and function.

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.

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

The 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.

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.

The potato suberin feruloyl transferase FHT which accumulates in the phellogen is induced by wounding and regulated by abscisic and salicylic acids

The present study provides new insights on the role of the potato (Solanum tuberosum) suberin feruloyl transferase FHT in native and wound tissues, leading to conclusions about hitherto unknown properties of the phellogen. In agreement with the enzymatic role of FHT, it is shown that its transcriptional activation and protein accumulation are specific to tissues that undergo suberization such as the root boundary layers of the exodermis and the endodermis, along with the tuber periderm. Remarkably, FHT expression and protein accumulation within the periderm is restricted to the phellogen derivative cells with phellem identity. FHT levels in the periderm are at their peak near harvest during periderm maturation, with the phellogen becoming meristematically inactive and declining thereafter. However, periderm FHT levels remain high for several months after harvest, suggesting that the inactive phellogen retains the capacity to synthesize ferulate esters. Tissue wounding induces FHT expression and the protein accumulates from the first stages of the healing process onwards. FHT is up-regulated by abscisic acid and down-regulated by salicylic acid, emphasizing the complex regulation of suberin synthesis and wound healing. These findings open up new prospects important for the clarification of the suberization process and yield important information with regard to the skin quality of potatoes.

Genome-Wide Characterization and Expression Profiling of the AUXIN RESPONSE FACTOR (ARF) Gene Family in Eucalyptus grandis

Auxin is a central hormone involved in a wide range of developmental processes including the specification of vascular stem cells. Auxin Response Factors (ARF) are important actors of the auxin signalling pathway, regulating the transcription of auxin-responsive genes through direct binding to their promoters. The recent availability of the Eucalyptus grandis genome sequence allowed us to examine the characteristics and evolutionary history of this gene family in a woody plant of high economic importance. With 17 members, the E. grandis ARF gene family is slightly contracted, as compared to those of most angiosperms studied hitherto, lacking traces of duplication events. In silico analysis of alternative transcripts and gene truncation suggested that these two mechanisms were preeminent in shaping the functional diversity of the ARF family in Eucalyptus. Comparative phylogenetic analyses with genomes of other taxonomic lineages revealed the presence of a new ARF clade found preferentially in woody and/or perennial plants. High-throughput expression profiling among different organs and tissues and in response to environmental cues highlighted genes expressed in vascular cambium and/or developing xylem, responding dynamically to various environmental stimuli. Finally, this study allowed identification of three ARF candidates potentially involved in the auxin-regulated transcriptional program underlying wood formation.

A potato skin SSH library yields new candidate genes for suberin biosynthesis and periderm formation

Potato (Solanum tuberosum) tubers are underground storage organs covered by the skin or periderm, a suberized layer that protects inner flesh from dehydration and pathogens. Understanding the molecular processes associated with periderm formation is of great importance for a better knowledge of this protective tissue and for improving the storage life of tubers. Here, to isolate new candidate genes for potato periderm, a suppression subtractive hybridization library from potato skin was performed. This library yielded a comprehensive list of 108 candidate genes that were manually sorted in functional categories according to the main cellular and metabolic processes in periderm. As expected, the list contains Suberin and wax genes, including some genes with a demonstrated role in the biosynthesis of these cell wall aliphatic compounds. Moreover, Regulation and Stress and defence genes are highly abundant in the library in general agreement with previous potato skin proteomic studies. The putative function of the genes in periderm is discussed.

Transcriptional Regulation of the Lignin Biosynthetic Pathway Revisited: New Players and Insights

The discovery that AC elements coordinated the regulation of genes belonging to the entire lignin biosynthetic pathway was the first breakthrough in understanding how lignin biosynthesis is regulated. Since then, tremendous progress has been made in the identification and characterization of many transcription factors (TFs) that regulate the genes of the phenylpropanoid branch pathway leading to lignin. A major breakthrough consisted in the discovery of a hierarchical transcriptional network regulating the biosynthesis of lignified secondary walls (SWs) in Arabidopsis. The NAC TFs (VND/NST/SND) work as the first layer of master switches activating the whole SW biosynthetic network through the regulation of a cascade of downstream TFs. Among these, MYB46/83 act as a second layer of master switches. Recent findings, however, reveal that the regulation of SW formation is far more complex than initially thought, involving both positive and negative regulators, dual function regulators, feedback loops, combinatorial complexes and cross talk between pathways. Finally, because of the great potential that lignocellulosic biomass represents for the production of bioenergy, there is a great interest in further elucidating the molecular mechanisms underlying the regulation of lignified SW and subsequently applying this knowledge to improve their saccharification potential for the generation of biofuels.