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Summary: Forests make immense contributions to societies in the form of ecological services and sustainable industrial products. However, they face major challenges to their viability and economic use due to climate change and growing biotic and economic threats, for which recombinant DNA (rDNA) technology can sometimes provide solutions. But the application of rDNA technologies to forest trees faces major social and biological obstacles that make its societal acceptance a 'wicked' problem without straightforward solutions. We discuss the nature of these problems, and the social and biological innovations that we consider essential for progress. As case studies of biological challenges, we focus on studies of modifications in wood chemistry and transformation efficiency. We call for major innovations in regulations, and the dissolution of method‐based market barriers, that together could lead to greater research investments, enable wide use of field studies, and facilitate the integration of rDNA‐modified trees into conventional breeding programs. Without near‐term adoption of such innovations, rDNA‐based solutions will be largely unavailable to help forests adapt to the growing stresses from climate change and the proliferation of forest pests, nor will they be available to provide economic and environmental benefits from expanded use of wood and related bioproducts as part of an expanding bioeconomy. [ABSTRACT FROM AUTHOR]

Summary: Wide variation in amenability to transformation and regeneration (TR) among many plant species and genotypes presents a challenge to the use of genetic engineering in research and breeding. To help understand the causes of this variation, we performed association mapping and network analysis using a population of 1204 wild trees of Populus trichocarpa (black cottonwood).To enable precise and high‐throughput phenotyping of callus and shoot TR, we developed a computer vision system that cross‐referenced complementary red, green, and blue (RGB) and fluorescent‐hyperspectral images. We performed association mapping using single‐marker and combined variant methods, followed by statistical tests for epistasis and integration of published multi‐omic datasets to identify likely regulatory hubs.We report 409 candidate genes implicated by associations within 5 kb of coding sequences, and epistasis tests implicated 81 of these candidate genes as regulators of one another. Gene ontology terms related to protein–protein interactions and transcriptional regulation are overrepresented, among others.In addition to auxin and cytokinin pathways long established as critical to TR, our results highlight the importance of stress and wounding pathways. Potential regulatory hubs of signaling within and across these pathways include GROWTH REGULATORY FACTOR 1 (GRF1), PHOSPHATIDYLINOSITOL 4‐KINASE β1 (PI‐4Kβ1), and OBF‐BINDING PROTEIN 1 (OBP1). [ABSTRACT FROM AUTHOR]

Abstract Background Cytosine DNA methylation (5mC) is an epigenetic modification that is important to genome stability and regulation of gene expression. Perturbations of 5mC have been implicated as a cause of phenotypic variation among plants regenerated through in vitro culture systems. However, the pattern of change in 5mC and its functional role with respect to gene expression, are poorly understood at the genome scale. A fuller understanding of how 5mC changes during in vitro manipulation may aid the development of methods for reducing or amplifying the mutagenic and epigenetic effects of in vitro culture and plant transformation. Results We investigated the in vitro methylome of the model tree species Populus trichocarpa in a system that mimics routine methods for regeneration and plant transformation in the genus Populus (poplar). Using methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq), we compared the methylomes of internode stem segments from micropropagated explants, dedifferentiated calli, and internodes from regenerated plants. We found that more than half (56%) of the methylated portion of the genome appeared to be differentially methylated among the three tissue types. Surprisingly, gene promoter methylation varied little among tissues, however, the percentage of body-methylated genes increased from 9% to 14% between explants and callus tissue, then decreased to 8% in regenerated internodes. Forty-five percent of differentially-methylated genes underwent transient methylation, becoming methylated in calli, and demethylated in regenerants. These genes were more frequent in chromosomal regions with higher gene density. Comparisons with an expression microarray dataset showed that genes methylated at both promoters and gene bodies had lower expression than genes that were unmethylated or only promoter-methylated in all three tissues. Four types of abundant transposable elements showed their highest levels of 5mC in regenerated internodes. Conclusions DNA methylation varies in a highly gene- and chromosome-differential manner during in vitro differentiation and regeneration. 5mC in redifferentiated tissues was not reset to that in original explants during the study period. Hypermethylation of gene bodies in dedifferentiated cells did not interfere with transcription, and may serve a protective role against activation of abundant transposable elements.

Abstract Background DNA cytosine methylation is an epigenetic modification that has been implicated in many biological processes. However, large-scale epigenomic studies have been applied to very few plant species, and variability in methylation among specialized tissues and its relationship to gene expression is poorly understood. Results We surveyed DNA methylation from seven distinct tissue types (vegetative bud, male inflorescence [catkin], female catkin, leaf, root, xylem, phloem) in the reference tree species black cottonwood (Populus trichocarpa). Using 5-methyl-cytosine DNA immunoprecipitation followed by Illumina sequencing (MeDIP-seq), we mapped a total of 129,360,151 36- or 32-mer reads to the P. trichocarpa reference genome. We validated MeDIP-seq results by bisulfite sequencing, and compared methylation and gene expression using published microarray data. Qualitative DNA methylation differences among tissues were obvious on a chromosome scale. Methylated genes had lower expression than unmethylated genes, but genes with methylation in transcribed regions ("gene body methylation") had even lower expression than genes with promoter methylation. Promoter methylation was more frequent than gene body methylation in all tissues except male catkins. Male catkins differed in demethylation of particular transposable element categories, in level of gene body methylation, and in expression range of genes with methylated transcribed regions. Tissue-specific gene expression patterns were correlated with both gene body and promoter methylation. Conclusions We found striking differences among tissues in methylation, which were apparent at the chromosomal scale and when genes and transposable elements were examined. In contrast to other studies in plants, gene body methylation had a more repressive effect on transcription than promoter methylation.

Eucalyptus spp. are widely cultivated for the production of pulp, energy, essential oils, and as ornamentals. However, their dispersal from plantings, especially when grown as an exotic, can cause ecological disruptions. To provide new tools for prevention of sexual dispersal by pollen as well as to induce male‐sterility for hybrid breeding, we studied the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9‐mediated knockout of three floral genes in both FT‐expressing (early‐flowering) and non‐FT genotypes. We report male‐sterile phenotypes resulting from knockout of the homologs of all three genes, including one involved in meiosis and two regulating early stages of pollen development. The targeted genes were Eucalyptus homologs of REC8 (EREC8), TAPETAL DEVELOPMENT AND FUNCTION 1 (ETDF1), and HECATE3 (EHEC3‐like). The erec8 knockouts yielded abnormal pollen grains and a predominance of inviable pollen, whereas the etdf1 and ehec3‐like knockouts produced virtually no pollen. In addition to male‐sterility, both erec8 and ehec3‐like knockouts may provide complete sterility because the failure of erec8 to undergo meiosis is expected to be independent of sex, and ehec3‐like knockouts produce flowers with shortened styles and no visible stigmas. When comparing knockouts to controls in wild‐type (non‐early‐flowering) backgrounds, we did not find visible morphological or statistical differences in vegetative traits, including average single‐leaf mass, stem volume, density of oil glands, or chlorophyll in leaves. Loss‐of‐function mutations in any of these three genes show promise as a means of inducing male‐ or complete sterility without impacting vegetative development. [ABSTRACT FROM AUTHOR]

Summary: Ectomycorrhizas are an intrinsic component of tree nutrition and responses to environmental variations. How epigenetic mechanisms might regulate these mutualistic interactions is unknown.By manipulating the level of expression of the chromatin remodeler DECREASE IN DNA METHYLATION 1 (DDM1) and two demethylases DEMETER‐LIKE (DML) in Populus tremula × Populus alba lines, we examined how host DNA methylation modulates multiple parameters of the responses to root colonization with the mutualistic fungus Laccaria bicolor. We compared the ectomycorrhizas formed between transgenic and wild‐type (WT) trees and analyzed their methylomes and transcriptomes.The poplar lines displaying lower mycorrhiza formation rate corresponded to hypomethylated overexpressing DML or RNAi‐ddm1 lines. We found 86 genes and 288 transposable elements (TEs) differentially methylated between WT and hypomethylated lines (common to both OX‐dml and RNAi‐ddm1) and 120 genes/1441 TEs in the fungal genome suggesting a host‐induced remodeling of the fungal methylome. Hypomethylated poplar lines displayed 205 differentially expressed genes (cis and trans effects) in common with 17 being differentially methylated (cis).Our findings suggest a central role of host and fungal DNA methylation in the ability to form ectomycorrhizas including not only poplar genes involved in root initiation, ethylene and jasmonate‐mediated pathways, and immune response but also terpenoid metabolism. See also the Commentary on this article by Zimmermann & Gaillard, 238: 2259–2260. [ABSTRACT FROM AUTHOR]