Search

SummaryTo improve our understanding of genetic mechanisms underlying complex traits in plants, a comprehensive analysis of gene variants is required. Eucalyptus is an important forest plantation genus that is highly outbred. Trait dissection and molecular breeding in eucalypts currently relies on biallelic SNP markers. These markers fail to capture the large amount of haplotype diversity in these species and thus multi-allelic markers are required. We aimed to develop a gene-based haplotype mining panel for Eucalyptus species. We generated 17 999 oligonucleotide probe sets for targeted sequencing of selected regions of 6 293 genes implicated in growth and wood properties, pest and disease resistance and abiotic stress responses. We identified and phased 195 834 SNPs using a read-based phasing approach to reveal SNP-based haplotypes. A total of 8 915 target regions (at 4 637 gene loci) passed tests for Mendelian inheritance. We evaluated the haplotype panel in four Eucalyptus species (E. grandis, E. urophylla, E. dunnii and E. nitens) to determine its ability to capture diversity across eucalypt species. This revealed an average of 3.13 to 4.52 haplotypes per target region in each species and 33.36% of the identified haplotypes were shared by at least two species. This haplotype mining panel will enable the analysis of haplotype diversity within and between species and provide multi-allelic markers that can be used for genome-wide association studies and gene-based breeding approaches.Significance StatementWe developed a haplotype sequencing panel for Eucalyptus targeting 8915 regions at 4637 gene loci associated with growth and wood properties, pest and disease resistance and abiotic stress response providing a genome-wide, multi-allelic, gene centric genotyping resource for eucalypts. We tested the panel in four Eucalyptus species (E. grandis, E. dunnii, E. nitens and E. urophylla) and found an average of 3.65 haplotypes per target region per species, and 9.98 across all four species.

The importance of drought as a constraint to agriculture and forestry is increasing with climate change. Genetic improvement of plants’ resilience is one of the mitigation strategies to curb this threat. Although recovery from drought stress is important to long-term drought adaptation and has been considered as an indicator of dehydration tolerance in annual crops, this has not been well explored in forest trees. Thus, we aimed to investigate the physiological and transcriptional changes during drought stress and rewatering in Eucalyptus grandis W. Hill ex Maiden. We set up a greenhouse experiment where we imposed drought stress on 2-year-old seedlings and rewatered the recovery group after 17 days of drought. Our measurement of leaf stomatal conductance (gs) showed that, while gs was reduced by drought stress, it fully recovered after 5 days of rewatering. The RNA-seq analysis from stem samples revealed that genes related to known stress responses such as phytohormone and reactive oxygen species signaling were upregulated, while genes involved in metabolism and growth were downregulated due to drought stress. We observed reprogramming of signal transduction pathways and metabolic processes at 1 day of rewatering, indicating a quick response to rewatering. Our results suggest that recovery from drought stress may entail alterations in the jasmonic acid, salicylic acid, ethylene and brassinosteroid signaling pathways. Using co-expression network analysis, we identified hub genes, including the putative orthologs of ABI1, ABF2, ABF3, HAI2, BAM1, GolS2 and SIP1 during drought and CAT2, G6PD1, ADG1 and FD-1 during recovery. Taken together, by highlighting the molecular processes and identifying key genes, this study gives an overview of the mechanisms underlying the response of E. grandis to drought stress and recovery that trees may face repeatedly throughout their long life cycle. This provides a useful reference to the identification and further investigation of signaling pathways and target genes for future tree improvement.

A multi-tiered transcriptional network regulates xylem differentiation and secondary cell wall (SCW) formation in plants, with evidence of both conserved and lineage-specific SCW network architecture. We aimed to elucidate the roles of selected R2R3-MYB transcription factors (TFs) linked to Eucalyptus wood formation by identifying genome-wide TF binding sites and direct target genes through an improved DAP-seq protocol combined with machine learning for target gene assignment (DAP-seq-ML). We applied this to five TFs including a well-studied SCW master regulator (EgrMYB2; homolog of AtMYB83), a repressor of lignification (EgrMYB1; homolog of AtMYB4), a TF affecting SCW thickness and vessel density (EgrMYB137; homolog of PtrMYB074) and two TFs with unclear roles in SCW regulation (EgrMYB135 and EgrMYB122). Each DAP-seq TF peak set (average 12,613 peaks) was enriched for canonical R2R3-MYB binding motifs. To improve the reliability of target gene assignment to peaks, a random forest classifier was developed from ArabidopsisDAP-seq, RNA-seq, chromatin, and conserved noncoding sequence data which demonstrated significantly higher precision and recall to the baseline method of assigning genes to proximal peaks. EgrMYB1, EgrMYB2 and EgrMYB137 predicted targets showed clear enrichment for SCW-related biological processes. As validation, EgrMYB137 overexpression in transgenic Eucalyptus hairy roots increased xylem lignification, while its dominant repression in transgenic Arabidopsis and Populus reduced xylem lignification, stunted growth, and caused downregulation of SCW genes. EgrMYB137 targets overlapped significantly with those of EgrMYB2, suggesting partial functional redundancy. Our results show that DAP-seq-ML identified biologically relevant R2R3-MYB targets supported by the finding that EgrMYB137 promotes SCW lignification in planta.

Eucalypts are the most planted trees worldwide, but most of them are frost sensitive. Overexpressing transcription factors for CRT-repeat binding factors (CBFs) in transgenic Eucalyptus confer cold resistance both in leaves and stems. While wood plays crucial roles in trees and is affected by environmental cues, its potential role in adaptation to cold stress has been neglected. Here, we addressed this question by investigating the changes occurring in wood in response to the overexpression of two CBFs, taking advantage of available transgenic Eucalyptus lines. We performed histological, biochemical, and transcriptomic analyses on xylem samples. CBF ectopic expression led to a reduction of both primary and secondary growth, and triggered changes in xylem architecture with smaller and more frequent vessels and fibers exhibiting reduced lumens. In addition, lignin content and syringyl/guaiacyl (S/G) ratio increased. Consistently, many genes of the phenylpropanoid and lignin branch pathway were upregulated. Most of the features of xylem remodeling induced by CBF overexpression are reminiscent of those observed after long exposure of Eucalyptus trees to chilling temperatures. Altogether, these results suggest that CBF plays a central role in the cross-talk between response to cold and wood formation and that the remodeling of wood is part of the adaptive strategies to face cold stress.

Human life intimately depends on plants for food, biomaterials, health, energy, and a sustainable environment. Various plants have been genetically improved mostly through breeding, along with limited modification via genetic engineering, yet they are still not able to meet the ever-increasing needs, in terms of both quantity and quality, resulting from the rapid increase in world population and expected standards of living. A step change that may address these challenges would be to expand the potential of plants using biosystems design approaches. This represents a shift in plant science research from relatively simple trial-and-error approaches to innovative strategies based on predictive models of biological systems. Plant biosystems design seeks to accelerate plant genetic improvement using genome editing and genetic circuit engineering or create novel plant systems through de novo synthesis of plant genomes. From this perspective, we present a comprehensive roadmap of plant biosystems design covering theories, principles, and technical methods, along with potential applications in basic and applied plant biology research. We highlight current challenges, future opportunities, and research priorities, along with a framework for international collaboration, towards rapid advancement of this emerging interdisciplinary area of research. Finally, we discuss the importance of social responsibility in utilizing plant biosystems design and suggest strategies for improving public perception, trust, and acceptance.

International audience; Wood production in fast-growing Eucalyptus grandis trees is highly dependent on both potassium (K) fertilization and water availability but the molecular processes underlying wood formation in response to the combined effects of these two limiting factors remain unknown. E. grandis trees were submitted to four combinations of K-fertilization and water supply. Weighted gene co-expression network analysis and MixOmics-based co-regulation networks were used to integrate xylem transcriptome, metabolome and complex wood traits. Func- tional characterization of a candidate gene was performed in transgenic E. grandis hairy roots. This integrated network-based approach enabled us to identify meaningful biological pro- cesses and regulators impacted by K-fertilization and/or water limitation. It revealed that modules of co-regulated genes and metabolites strongly correlated to wood complex traits are in the heart of a complex trade-off between biomass production and stress responses. Nested in these modules, potential new cell-wall regulators were identified, as further con- firmed by the functional characterization of EgMYB137. These findings provide new insights into the regulatory mechanisms of wood formation under stressful conditions, pointing out both known and new regulators co-opted by K-fertilization and/or water limitation that may potentially promote adaptive wood traits.

Wood, which represents the most abundant lignocellulosic biomass on earth, fulfils key roles in trees, and is also a raw material for multiple end-uses by mankind. The differentiation of this complex vascular tissue starts with cell division in the vascular cambium and is characterized by a massive deposition of lignified secondary cell walls mainly in fibres and vessels. A transcriptional network underlying this differentiation process ensures a tight regulation of the expression of thousands of genes both at the spatial and temporal levels. Most of our current knowledge of this hierarchical network was extrapolated from studies performed in Arabidopsis. Here, we review recent findings on the regulation of wood formation in angiosperm trees species highlighting conserved and distinct mechanisms with Arabidopsis. We provide examples shedding light on the central role of auxin and its cross-talk with other hormones at different stages of secondary xylem differentiation. Functional studies of trees' wood-associated transcription factors revealed diversified functions as compared to their Arabidopsis orthologs. Sophisticated mechanisms of alternative splicing and cross-regulation between the two distinct groups of top level NAC-domain master regulators were uncovered. These findings underlie the high level of complexity of wood formation in trees and provide a framework for future lines of research in this exiting research field.

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.

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.