Eucalyptus grandis AUX/INDOLE‑3‑ACETIC ACID 13 (EgrIAA13) is a novel transcriptional regulator of xylogenesis

Auxin is a crucial phytohormone regulating multiple aspects of plant growth and diferentiation, including regulation of vascular cambium activity, xylogenesis and its responsiveness towards gravitropic stress. Although the regulation of these biological processes greatly depends on auxin and regulators of the auxin signalling pathway, many of their specifc functions remain unclear. Therefore, the present study aims to functionally characterise Eucalyptus grandis AUX/INDOLE3-ACETIC ACID 13 (EgrIAA13), a member of the auxin signalling pathway. In Eucalyptus and Populus, EgrIAA13 and its orthologs are preferentially expressed in the xylogenic tissues and downregulated in tension wood. Therefore, to further investigate EgrIAA13 and its function during xylogenesis, we conducted subcellular localisation and Induced Somatic Sector Analysis experiments using overexpression and RNAi knockdown constructs of EgrIAA13 to create transgenic tissue sectors on growing stems of Eucalyptus and Populus. Since Aux/IAAs interact with Auxin Responsive Factors (ARFs), in silico predictions of IAA13-ARF interactions were explored and experimentally validated via yeast-2-hybrid experiments. Our results demonstrate that EgrIAA13 localises to the nucleus and that downregulation of EgrIAA13 impedes Eucalyptus xylem fbre and vessel development. We also observed that EgrIAA13 interacts with Eucalyptus ARF2, ARF5, ARF6 and ARF19A. Based on these results, we conclude that EgrIAA13 is a regulator of Eucalyptus xylogenesis and postulate that the observed phenotypes are likely to result from alterations in the auxin-responsive transcriptome via IAA13-ARF modules such as EgrIAA13-EgrARF5. Our results provide the frst insights into the regulatory role of EgrIAA13 during xylogenesis.Open Access funding enabled and organized by CAUL and its Member Institutions. Funding for this project was secured by GB. During this work, NK was supported by a Melbourne Research Scholarship and the Albert Shimmins Fund provided by the University of Melbourne.A Melbourne Research Scholarship and the Albert Shimmins Fund provided by the University of Melbourne. Open Access funding enabled and organized by CAUL and its Member Institutions.http://link.springer.com/journal/11103dm2022BiochemistryForestry and Agricultural Biotechnology Institute (FABI)GeneticsMicrobiology and Plant Patholog

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

La régulation transcriptionnelle dépendante de l’auxine lors du développement du fruit: caractérisation fonctionnelle du gène DR4, un homologue d’Aux/IAA chez la tomate (Lycopersicon esculentum, Mill)

Fruit development is a genetically programmed process under multi-hormonal control. While the role of ethylene in triggering the ripening process is well demonstrated in the case of climacteric fruit like tomato, little is known about the involvement of other hormones. Differential screening allowed the isolation of transcriptional regulators involved in auxin responses that show ripening and ethylene-regulated expression during the late stages of fruit development. The work described here deals with the functional characterization of DR4, an Aux/IAA gene expressed in tomato fruit. Aux/IAA proteins are transcriptional regulators known to mediate many aspects of plant responses to the phytohormone auxin. Though a number of gain-of-function mutations affecting Aux/IAA genes in Arabidopsis have been characterized, strikingly, phenotypes associated with loss-of-function mutations yet have not been described. We report here on the down-regulation of DR4, a tomato Aux/IAA-like gene, which altered auxin-related responses. Under-expressing DR4 through antisense strategy in the tomato resulted in pleiotropic phenotypes with the most dramatic changes affecting leaf morphology and fruit development. The leaves in DR4-inhibited were simple shaped compared to compound leaves in wild type and fruit development was triggered prior to flower fertilization giving rise to parthenocarpy. DR4 down-regulated plants also displayed a wide range of auxin-associated responses including enhanced hypocotyl, root and stem elongation, reduced apical dominance, a multiple organ fusion and a transition from basipetal to acropetal lateral shoot development. Genetic crosses between DR4 antisense lines and plants expressing GUS driven by an auxin responsive promoter resulted in massive increase of the reporter gene expression in the hybrid lines indicating that in planta, DR4 acts as a negative regulator of auxin responsive genes. These data represent the first description of phenotypes associated with down-regulation of an Aux/IAA gene and uncover for the first time the essential role of these factors in leaf morphogenesis and fructification.Le développement des fruits de l’anthèse à la maturation est un processus génétiquement programmé dont le déroulement se trouve sous un contrôle multihormonal. Alors que l’éthylène joue un rôle déterminant au cours de la maturation des fruits climactériques comme la tomate, les modalités d’intervention des autres hormones ne sont toujours pas connues. Une approche de criblage différentiel a permis d’isoler au laboratoire plusieurs gènes codant pour des régulateurs transcriptionnnels impliqués dans la réponse à l’auxine et montrant un profil d’expression qui varie au cours de la maturation et en réponse à l’éthylène. Le travail présenté dans cette thèse est focalisé sur la caractérisation fonctionnelle du gène DR4 apparenté à la famille des Aux/IAA de la tomate dont le rôle en tant que médiateurs de la réponse des plantes à l’auxine est bien connu. Alors que de nombreux mutants gain de fonction touchant les gènes Aux/IAA ont été décrits chez Arabidopsis, de façon remarquable, aucun mutant perte de fonction n’a été caractérisé à ce jour. Le travail présenté ici montre que la sous-expression du gène DR4 chez la tomate affecte plusieurs processus de développement dépendant de l’auxine. Ainsi, les lignées transgéniques exprimant une construction anti-sens du gène DR4 (DR4AS) montrent un phénotype pléïotropique qui touche essentiellement la morphologie des feuilles et le développement du fruit. Les lignées DR4AS possèdent des feuilles simples et non pas composées comme chez le sauvage et le développement des fruits survient en l’absence de toute pollinisation et fécondation ce qui conduit à la production de fruits parthénocarpiques. Les plantes DR4AS montrent également plusieurs phénotypes associés à une réponse exacerbée à l’auxine tel qu’une forte élongation de l’hypocotyle, de la tige et de la racine, une réduction de la dominance apicale, des fusions d’organes et une transition d’un développement basipétal des tiges axillaires à un mode acropétal. Les croisements génétiques des plantes DR4AS avec des lignées exprimant le gène GUS sous contrôle d’un promoteur répondant à l’auxine engendre une augmentation substantielle de l’expression du gène rapporteur dans les lignées hybrides indiquant que la protéine DR4 agit comme régulateur négatif des gènes de réponse à l’auxine. Les données présentées dans cette thèse constituent la première description de phénotypes associés à une sous-expression d’un gène Aux/IAA et révèlent pour la première fois le rôle essentiel joué par un membre de la famille des Aux/IAA dans la morphogenèse des feuilles et le développement du fruit

Overexpression of EgrIAA20 from Eucalyptus grandis, a Non-Canonical Aux/IAA Gene, Specifically Decouples Lignification of the Different Cell-Types in Arabidopsis Secondary Xylem

Wood (secondary xylem) formation is regulated by auxin, which plays a pivotal role as an integrator of developmental and environmental cues. However, our current knowledge of auxin-signaling during wood formation is incomplete. Our previous genome-wide analysis of Aux/IAAs in Eucalyptus grandis showed the presence of the non-canonical paralog member EgrIAA20 that is preferentially expressed in cambium. We analyzed its cellular localization using a GFP fusion protein and its transcriptional activity using transactivation assays, and demonstrated its nuclear localization and strong auxin response repressor activity. In addition, we functionally tested the role of EgrIAA20 by constitutive overexpression in Arabidopsis to investigate for phenotypic changes in secondary xylem formation. Transgenic Arabidopsis plants overexpressing EgrIAA20 were smaller and displayed impaired development of secondary fibers, but not of other wood cell types. The inhibition in fiber development specifically affected their cell wall lignification. We performed yeast-two-hybrid assays to identify EgrIAA20 protein partners during wood formation in Eucalyptus, and identified EgrIAA9A, whose ortholog PtoIAA9 in poplar is also known to be involved in wood formation. Altogether, we showed that EgrIAA20 is an important auxin signaling component specifically involved in controlling the lignification of wood fibers

Comprehensive Genome-Wide Analysis of the Aux/IAA Gene Family in Eucalyptus: Evidence for the Role of EgrIAA4 in Wood Formation

International audienceAuxin plays a pivotal role in various plant growth and development processes, including vascular differentiation.The modulation of auxin responsiveness through the auxin perception and signaling machinery is believed to be a major regulatory mechanism controlling cambium activity and wood formation. To gain more insights into the rolesof key Aux/IAA gene regulators of the auxin response in these processes, we identified and characterized membersof the Aux/IAA family in the genome of Eucalyptus grandis, a tree of worldwide economic importance. We found that the gene family in Eucalyptus is slightly smaller than that in Populus and Arabidopsis, but all phylogenetic groupsare represented. High-throughput expression profiling of different organs and tissues highlighted several Aux/IAAgenes expressed in vascular cambium and/or developing xylem, some showing differential expression in response todevelopmental (juvenile vs. mature) and/or to environmental (tension stress) cues. Based on the expression profiles,we selected a promising candidate gene, EgrIAA4, for functional characterization. We showed that EgrIAA4 protein is localized in the nucleus and functions as an auxin-responsive repressor. Overexpressing a stabilized version of EgrIAA4 in Arabidopsis dramatically impeded plant growth and fertility and induced auxin-insensitive phenotypes such as inhibition of primary root elongation, lateral root emergence and agravitropism. Interestingly, the lignified secondary walls of the interfascicular fibers appeared very late, whereas those of the xylary fibers were virtually undetectable, suggesting that EgrIAA4 may play crucial roles in fiber development and secondary cell wall deposition

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

Explosive Tandem and Segmental Duplications of Multigenic Families in Eucalyptus grandis

International audiencePlant organisms contain a large number of genes belonging to numerous multigenic families whose evolution size reflects some functional constraints. Sequences from eight multigenic families, involved in biotic and abiotic responses, have been analyzed in Eucalyptus grandis and compared with Arabidopsis thaliana. Two transcription factor families APETALA 2 (AP2)/ethylene responsive factor and GRAS, two auxin transporter families PIN-FORMED and AUX/LAX, two oxidoreductase families (ascorbate peroxidases [APx] and Class III peroxidases [CIII Prx]), and two families of protective molecules late embryogenesis abundant (LEA) and DNAj were annotated in expert and exhaustive manner. Many recent tandem duplications leading to the emergence of species-specific gene clusters and the explosion of the gene numbers have been observed for the AP2, GRAS, LEA, PIN, and CIII Prx in E. grandis, while the APx, the AUX/LAX and DNAj are conserved between species. Although no direct evidence has yet demonstrated the roles of these recent duplicated genes observed in E. grandis, this could indicate their putative implications in the morphological and physiological characteristicsofE. grandis, andbethekeyfactorforthesurvivalofthisnondormantspecies.Globalanalysisofkeyfamilieswouldbea good criterion to evaluate the capabilities of some organisms to adapt to environmental variations

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

International audienceEucalypts 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 knockout 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

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

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

International audienceAuxin 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