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Information on public attitudes towards the use of transgenic trees in forest plantations is important in the decision-making process and policy implementation for safe tree development, particularly at the EU level. In Europe, the use of transgenic forest trees is very limited and therefore such information is completely lacking. To address this issue within the FP0905 European COST Action on the Biosafety of Transgenic Forest Trees a pioneer cross-country pilot survey on public attitudes towards the use of transgenic forest trees was conducted using young population as a focus group. This was decided mainly because this focus group represents the future consumers, policy makers or developers. Specifically, the survey aimed to: i) assess the level of young people’s knowledge about transgenic forest trees, ii) identify issues of concern to them regarding the cultivation of transgenic forest trees and iii) explore whether they approve or disapprove of the use of transgenic forest trees in plantations. Purposive sampling was performed and university students of different disciplines were included in the research as sampling subjects. In total, 1868 completed questionnaires from 15 European and non-European countries were analyzed. The young educated people that took part in the survey appeared to approve of the use of transgenic forest trees in plantations and would be willing to buy forest transgenic products. The potential loss of biodiversity due to a risk of gene flow between transgenic and wild trees was seen as the safety issue of most concern when considering the commercial release of transgenic forest trees. However, a serious perceived lack of knowledge about potential benefits and risks of the cultivation of transgenic forest trees was recorded in most of the countries. K-means clustering was implemented on respondents’ positive responses to identify potential country patterns. No differences in patterns of public attitude towards the acceptance of the commercial growing of transgenic forest trees were observed between European and non-European countries. Extended research on public attitude issues towards the use of transgenic forest trees is strongly recommended as a basis for policy implementation on safe tree development.

Myrtle rust poses a significant biosecurity threat to Australia with potential for long‐term damaging impacts on native flora and plant industries. This study describes the disease cycle of Austropuccinia psidii, the myrtle rust pathogen, in Eucalyptus globulus and Eucalyptus obliqua, two commercially and ecologically important species from different subgenera of Eucalyptus. Ontogeny and morphology of infection structures of A. psidii on plants of both Eucalyptus species with different rust response phenotypes, i.e. completely resistant, hypersensitive and highly susceptible, were investigated. Plants were inoculated with single‐uredinium‐derived urediniospores and examined by scanning electron microscopy. No differences between rust response phenotypes were observed in germination of urediniospores, formation of appressoria or length of germ tubes. The growth of germ tubes had no affinity towards stomata of either species. Histological observations indicated direct penetration by infection pegs through the leaf cuticle and no penetration beyond the epidermis on rust‐resistant E. obliqua. Eucalyptus obliqua plants that were identified as susceptible to A. psidii at 3‐ and 6‐months‐old showed no disease when reinoculated with A. psidii at 12‐months‐old; this indicated possible early acquisition of adult plant resistance to A. psidii in this species. In the susceptible phenotype of E. globulus rust inoculation led to rapid colonization of leaf parenchyma cells with the disease cycle completed within 10 days. These findings provide valuable insights into host–pathogen interactions in the Eucalyptus–A. psidii pathosystem, which might be useful for the development of effective rust control strategies across Eucalyptus subgenera. [ABSTRACT FROM AUTHOR]

Trees play a pivotal role in terrestrial ecosystems as well as being an important natural resource. These attributes are primarily associated with the capacity of trees to continuously produce woody tissue from the vascular cambium, a ring of stem cells located just beneath the bark. Long-lived trees are exposed to a myriad of biological and environmental stresses that may result in wounding, leading to a loss of bark and the underlying vascular cambium. This affects both wood formation and the quality of timber arising from the tree. In addition, the exposed wound site is a potential entry point for pathogens that cause disease. In response to wounding, trees have the capacity to regenerate lost or damaged tissues at this site. Investigating gene expression changes associated with different stages of wound healing reveals complex and dynamic changes in the activity of transcription factors, signalling pathways and hormone responses. In this review we summarise these data and discuss how they relate to our current understanding of vascular cambium formation and xylem differentiation during secondary growth. Based on this analysis, a model for wound healing that provides the conceptual foundations for future studies aimed at understanding this intriguing process is proposed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Key Message: Our Induced Somatic Sector Analysis and protein-protein interaction experiments demonstrate that Eucalyptus grandis IAA13 regulates xylem fibre and vessel development, potentially via EgrIAA13 modules involving ARF2, ARF5, ARF6 and ARF19. Auxin is a crucial phytohormone regulating multiple aspects of plant growth and differentiation, 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 specific functions remain unclear. Therefore, the present study aims to functionally characterise Eucalyptus grandis AUX/INDOLE-3-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 fibre 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 first insights into the regulatory role of EgrIAA13 during xylogenesis., (© 2022. Crown.)

Recent advances in our understanding of the molecular control of secondary cell wall (SCW) formation have shed light on molecular mechanisms that underpin domestication traits related to wood formation. One such trait is the cellulose microfibril angle (MFA), an important wood quality determinant that varies along tree developmental phases and in response to gravitational stimulus. The cytoskeleton, mainly composed of microtubules and actin filaments, collectively contribute to plant growth and development by participating in several cellular processes, including cellulose deposition. Studies in Arabidopsis have significantly aided our understanding of the roles of microtubules in xylem cell development during which correct SCW deposition and patterning are essential to provide structural support and allow for water transport. In contrast, studies relating to SCW formation in xylary elements performed in woody trees remain elusive. In combination, the data reviewed here suggest that the cytoskeleton plays important roles in determining the exact sites of cellulose deposition, overall SCW patterning and more specifically, the alignment and orientation of cellulose microfibrils. By relating the reviewed evidence to the process of wood formation, we present a model of microtubule participation in determining MFA in woody trees forming reaction wood (RW)., Competing Interests: The authors declare no conflict of interest.

We used sector analysis to study cambium development and dynamics and to test whether fundamental developmental and functional differences exist between cambial initials as true 'stem cells' and more differentiated mother cells. In many higher plants, a cylindrical lateral meristem, the vascular cambium, forms along the plant axis. Most notably in stems of perennial tree species, this meristem gives rise to xylem (wood) towards the inside of the trunk and phloem (bark) towards the outside. As such, the vascular cambium is responsible for the production of most of the planet's forest biomass, significantly contributing to the global carbon cycle. Using the bacterial uidA reporter gene in Agrobacterium-based in vivo stem transformation experiments in poplar trees, we created 379 cambium sectors that originated from the transformation of individual cells. Results from our analysis of sector frequency and patterns are consistent with the poplar cambium featuring a single layer of true cambial initials (being able to divide both anti- and periclinally). We show that initials are frequently lost from the cambium, that such cell loss rarely occurs at mother cell level, that phloem and xylem differentiation are controlled independently, and that the frequency of mother cell replenishment is not pre-determined.

Secondary stem growth in trees and associated wood formation are significant both from biological and commercial perspectives. However, relatively little is known about the molecular control that governs their development. This is in part due to physical, resource and time limitations often associated with the study of secondary growth processes. A number of in vitro techniques have been used involving either plant part or whole plant system in both woody and non-woody plant species. However, questions about their applicability for the study of secondary stem growth processes, the recalcitrance of certain species and labor intensity are often prohibitive for medium to high throughput applications. Also, when looking at secondary stem development and wood formation the specific traits under investigation might only become measurable late in a tree's lifecycle after several years of growth. In addressing these challenges alternative in vivo protocols have been developed, named Induced Somatic Sector Analysis, which involve the creation of transgenic somatic tissue sectors directly in the plant's secondary stem. The aim of this protocol is to provide an efficient, easy and relatively fast means to create transgenic secondary plant tissue for gene and promoter functional characterization that can be utilized in a range of tree species. Results presented here show that transgenic secondary stem sectors can be created in all live tissues and cell types in secondary stems of a variety of tree species and that wood morphological traits as well as promoter expression patterns in secondary stems can be readily assessed facilitating medium to high throughput functional characterization.

Climate change can negatively affect sensitive tree species, affecting their acclimation and adaptation strategies. A common garden experiment provides an opportunity to test whether responses of trees from different provenances are genetically driven and if this response is related to factors at the site of origin. We hypothesized that antioxidative defence systems and leaf mass area ofAcacia melanoxylonR. Br. samples collected from different provenances will vary depending on local rainfall. Thirteen provenances ofA. melanoxylonoriginating from different rainfall habitats (500-2000 mm) were grown for 5 years in a common garden. For 2 years, phyllode samples were collected during winter and summer, for measurements of leaf mass area and concentrations of glutathione and ascorbic acid. Leaf mass area varied between seasons, years and provenances ofA. melanoxylon, and an increase was associated with decreasing rainfall at the site of origin. Ascorbic acid and glutathione concentrations varied between seasons, years (i.e., environmental factors) and among provenances ofA. melanoxylon In general, glutathione and ascorbic acid concentrations were higher in winter compared with summer. Ascorbic acid and glutathione were different among provenances, but this was not associated with rainfall at the site of origin., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

REALLY INTERESTING NEW GENE (RING) proteins play important roles in the regulation of many processes by recognizing target proteins for ubiquitination. Previously, we have shown that the expression of PtaRHE1, encoding a Populus tremula × Populus alba RING-H2 protein with E3 ubiquitin ligase activity, is associated with tissues undergoing secondary growth. To further elucidate the role of PtaRHE1 in vascular tissues, we have undertaken a reverse genetic analysis in poplar. Within stem secondary vascular tissues, PtaRHE1 and its corresponding protein are expressed predominantly in the phloem. The downregulation of PtaRHE1 in poplar by artificial miRNA triggers alterations in phloem fibre patterning, characterized by an increased portion of secondary phloem fibres that have a reduced cell wall thickness and a change in lignin composition, with lower levels of syringyl units as compared with wild-type plants. Following an RNA-seq analysis, a biological network involving hormone stress signalling, as well as developmental processes, could be delineated. Several candidate genes possibly associated with the altered phloem fibre phenotype observed in amiRPtaRHE1 poplar were identified. Altogether, our data suggest a regulatory role for PtaRHE1 in secondary phloem fibre development., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

We present a phylogenetic analysis and comparison of structural features of chloroplast genomes for 39 species of the eucalypt group (genera Eucalyptus, Corymbia, Angophora, and outgroups Allosyncarpia and Stockwellia). We use 41 complete chloroplast genome sequences, adding 39 finished-quality chloroplast genomes to two previously published genomes. Maximum parsimony and Bayesian analyses, based on >7000 variable nucleotide positions, produced one fully resolved phylogenetic tree (35 supported nodes, 27 with 100% bootstrap support). Eucalyptus and its sister lineage Angophora+Corymbia show a deep divergence. Within Eucalyptus, three lineages are resolved: the 'eudesmid', 'symphyomyrt' and 'monocalypt' groups. Corymbia is paraphyletic with respect to Angophora. Gene content and order do not vary among eucalypt chloroplasts; length mutations, especially frame shifts, are uncommon in protein-coding genes. Some non-synonymous mutations are highly incongruent with the overall phylogenetic signal, notably in rbcL, and may be adaptive. Application of custom informatics pipelines (GYDLE Inc.) enabled direct chloroplast genome assembly, resolving each genome to finished-quality with no need for PCR gap-filling or contig order resolution. Analysis of whole chloroplast genomes resolved major eucalypt clades and revealed variable regions of the genome that will be useful in lower-level genetic studies (including phylogeography and geneflow)., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The increasing focus on plantation forestry as a renewable source of cellulosic biomass has emphasized the need for tools to study the unique biology of woody genera such as Eucalyptus, Populus and Pinus. The domestication of these woody crops is hampered by long generation times, and breeders are now looking to molecular approaches such as marker-assisted breeding and genetic modification to accelerate tree improvement. Much of what is known about genes involved in the growth and development of plants has come from studies of herbaceous models such as Arabidopsis and rice. However, transferring this information to woody plants often proves difficult, especially for genes expressed in woody stems. Here we report the use of induced somatic sector analysis (ISSA) for characterization of promoter expression patterns directly in the stems of Populus and Eucalyptus trees. As a case study, we used previously characterized primary and secondary cell wall-related cellulose synthase (CesA) promoters cloned from Eucalyptus grandis. We show that ISSA can be used to elucidate the phloem and xylem expression patterns of the CesA genes in Eucalyptus and Populus stems and also show that the staining patterns differ in Eucalyptus and Populus stems. These findings show that ISSA is an efficient approach to investigate promoter function in the developmental context of woody plant tissues and raise questions about the suitability of heterologous promoters for genetic manipulation in plant species.

Background: NAC domain transcription factors initiate secondary cell wall biosynthesis in Arabidopsis fibres and vessels by activating numerous transcriptional regulators and biosynthetic genes. NAC family member SND2 is an indirect target of a principal regulator of fibre secondary cell wall formation, SND1. A previous study showed that overexpression of SND2 produced a fibre cell-specific increase in secondary cell wall thickness in Arabidopsis stems, and that the protein was able to transactivate the cellulose synthase8 (CesA8) promoter. However, the full repertoire of genes regulated by SND2 is unknown, and the effect of its overexpression on cell wall chemistry remains unexplored., Results: We overexpressed SND2 in Arabidopsis and analyzed homozygous lines with regards to stem chemistry, biomass and fibre secondary cell wall thickness. A line showing upregulation of CesA8 was selected for transcriptome-wide gene expression profiling. We found evidence for upregulation of biosynthetic genes associated with cellulose, xylan, mannan and lignin polymerization in this line, in agreement with significant co-expression of these genes with native SND2 transcripts according to public microarray repositories. Only minor alterations in cell wall chemistry were detected. Transcription factor MYB103, in addition to SND1, was upregulated in SND2-overexpressing plants, and we detected upregulation of genes encoding components of a signal transduction machinery recently proposed to initiate secondary cell wall formation. Several homozygous T4 and hemizygous T1 transgenic lines with pronounced SND2 overexpression levels revealed a negative impact on fibre wall deposition, which may be indirectly attributable to excessive overexpression rather than co-suppression. Conversely, overexpression of SND2 in Eucalyptus stems led to increased fibre cross-sectional cell area., Conclusions: This study supports a function for SND2 in the regulation of cellulose and hemicellulose biosynthetic genes in addition of those involved in lignin polymerization and signalling. SND2 seems to occupy a subordinate but central tier in the secondary cell wall transcriptional network. Our results reveal phenotypic differences in the effect of SND2 overexpression between woody and herbaceous stems and emphasize the importance of expression thresholds in transcription factor studies.

This study examined the interspecific amplification of nuclear microsatellite loci developed mainly for eucalypts in the subgenus Symphyomyrtus across five species within the second most speciose subgenus, subgenus Eucalyptus. A set of eight to 10 loci, depending on taxon, have been identified that are highly variable and easily scored. The successful transfer of microsatellite loci to these eucalypt species sidesteps the expensive and time-consuming development of species-specific microsatellite libraries. This primer set will enable the examination and cross-species comparison of the genetic resources of commercially and ecologically important members of the subgenus Eucalyptus., (© 2008 The Authors. Journal compilation © 2008 Blackwell Publishing Ltd.)

Cellulose microfibrils are the major structural component of plant secondary cell walls. Their arrangement in plant primary cell walls, and its consequent influence on cell expansion and cellular morphology, is directed by cortical microtubules; cylindrical protein filaments composed of heterodimers of alpha- and beta-tubulin. In secondary cell walls of woody plant stems the orientation of cellulose microfibrils influences the strength and flexibility of wood, providing the physical support that has been instrumental in vascular plant colonization of the troposphere. Here we show that a Eucalyptus grandisbeta-tubulin gene (EgrTUB1) is involved in determining the orientation of cellulose microfibrils in plant secondary fibre cell walls. This finding is based on RNA expression studies in mature trees, where we identified and isolated EgrTUB1 as a candidate for association with wood-fibre formation, and on the analysis of somatically derived transgenic wood sectors in Eucalyptus. We show that cellulose microfibril angle (MFA) is correlated with EgrTUB1 expression, and that MFA was significantly altered as a consequence of stable transformation with EgrTUB1. Our findings present an important step towards the production of fibres with altered tensile strength, stiffness and elastic properties, and shed light on one of the molecular mechanisms that has enabled trees to dominate terrestrial ecosystems.

Large-scale functional analysis of genes and transgenes suspected to be involved in wood development in trees is hindered by long generation times, low transformation and regeneration efficiencies and difficulties with phenotypic assessment of traits, especially those that appear late in a tree's development. To avoid such obstacles many researchers have turned to model plants such as Arabidopsis thaliana (L.) Heynh., Zinnia elegans Jacq. and Nicotiana ssp., or have focused their attention on in vitro wood formation systems or in vivo approaches targeting primary meristems for transformation. Complementing such efforts, we report the use of Agrobacterium to introduce transgenes directly into cambial cells of glasshouse-grown trees in order to create transgenic somatic tissue sectors. These sectors are suitable for phenotypic evaluation and analysis of target gene function. In our experiments the wood formation zone containing the cambium of Eucalyptus, Populus and Pinus species of varying age was inoculated with Agrobacterium containing a CaMV 35S::GUS construct. Following an initial wound response, frequent and stable transformation was observed in the form of distinct GUS-staining patterns (sectors) in newly formed secondary tissues. Sector size and extent depended on the cell type transformed, the species and the length of time treated plants were allowed to grow (more than two years in some cases). Induced somatic sector analysis (ISSA) can now be efficiently used to study cell fate and gene function during secondary growth in stems of forest tree species.

In an attempt to devise a method for the rapid creation of somatic transgenic wood sectors of sufficient size that would allow us to detect and analyse altered wood characteristics within them, we have explored the manual wounding and subsequent infection with Agrobacterium of dormant lateral buds in poplar. Following treatment and transformation with a 35S-GUS construct, frequent stable transformation was found in the form of distinct and specific GUS staining patterns in the outer cortex, cambial region (including primary and secondary xylem and phloem) and pith. Sector frequency and size were consistent with anatomical features of dormant lateral buds at the time of manual wounding and Agrobacterium-infection. The suitability of somatic sector analysis for functional genomic studies as well as for studies investigating pattern formation and the developmental fate of various cell-types within poplar stems is discussed.

The genetic manipulation of perennial woody tree species presents a range of additional challenges compared to that of annual weedy crop species. These include long generation times and reproductive cycle, the heterogeneity of plants under investigation and, when investigating wood properties, a number of physical and biochemical limitations to microscopical and molecular experimentation. The use of in vitro wood formation systems for molecular studies and Agrobacterium-mediated introduction of transgenes overcomes many of these obstacles. Using a commercially relevant Eucalyptus species as model organism, we demonstrate here that in vitro wood formation systems can be readily employed to introduce transgenes into growing wood-producing tissue, initially leading to frequent transient gene expression in a range of cell types. Stable transformation events were observed as sectors of transformed tissue derived from primary transformation events in individual cells. The usefulness of such systems for the analysis of gene function during the process of wood formation and wood quality determination, as well as for constructing developmental fate maps of cambial derivatives, is discussed.

Two groups of mutants that affect the morphology of the lemma, a floral bract of barley, are described. The first comprises phenotypes associated with mutant alleles of calcaroides loci. On the lemma of these mutants, a well-organized neomorphic structure is formed, termed the sac. We provide a morphological description of wild-type (WT) and mutant lemmas, based on scanning electron microscopy (SEM), showing that both consist of similar tissues, but that the mutant is characterized by reversed growth polarity. The sac is a unique structure among grasses, and it is remarkable that recessive mutations at five different genetic loci lead to the same organ. The second group of mutants carry recessive alleles of two leafy lemma genes, both of which are necessary to cause the transformation of the lemma into a structure having all characteristics of a vegetative leaf, as shown by SEM analysis. The presence of sheath, blade, and ligule in the mutant lemma suggests that wild-type lemma development is interrupted at a leaf-like stage. The genes cal a, b, C, d, 23, lel1, and lel2 have now been mapped at precise positions on linkage groups 2, 7, 7, 3, 7, 5, and 7, respectively. The mutants considered in this article are unaffected in other floral organs. A model for lemma development is suggested.