Rare but diverse off-target and somatic mutations found in field and greenhouse grown trees expressing CRISPR/Cas9.

Introduction: CRISPR gene editing, while highly efficient in creating desired mutations, also has the potential to cause off-target mutations. This risk is especially high in clonally propagated plants, where editing reagents may remain in the genome for long periods of time or in perpetuity. We studied a diverse population of Populus and Eucalyptus trees that had CRISPR/Cas9-containing transgenes that targeted one or two types of floral development genes, homologs of LEAFY and AGAMOUS. Methods: Using a targeted sequence approach, we studied approximately 20,000 genomic sites with degenerate sequence homology of up to five base pairs relative to guide RNA (gRNA) target sites. We analyzed those sites in 96 individual tree samples that represented 37 independent insertion events containing one or multiples of six unique gRNAs. Results: We found low rates of off-target mutations, with rates of 1.2 × 10-9 in poplar and 3.1 × 10-10 in eucalypts, respectively, comparable to that expected due to sexual reproduction. The rates of mutation were highly idiosyncratic among sites and not predicted by sequence similarity to the target sites; a subset of two gRNAs showed off-target editing of four unique genomic sites with up to five mismatches relative to the true target sites, reaching fixation in some gene insertion events and clonal ramets. The location of off-target mutations relative to the PAM site were essentially identical to that seen with on-target CRISPR mutations. Discussion: The low rates observed support many other studies in plants that suggest that the rates of off-target mutagenesis from CRISPR/Cas9 transgenes are negligible; our study extends this conclusion to trees and other long-lived plants where CRISPR/Cas9 transgenes were present in the genome for approximately four years.

Social and biological innovations are essential to deliver transformative forest biotechnologies.

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.

Genome-wide association study and network analysis of in vitro transformation in Populus trichocarpa support key roles of diverse phytohormone pathways and cross talk.

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 ß1 (PI-4Kß1), and OBF-BINDING PROTEIN 1 (OBP1).

GWAS supported by computer vision identifies large numbers of candidate regulators of in planta regeneration in Populus trichocarpa.

Plant regeneration is an important dimension of plant propagation and a key step in the production of transgenic plants. However, regeneration capacity varies widely among genotypes and species, the molecular basis of which is largely unknown. Association mapping methods such as genome-wide association studies (GWAS) have long demonstrated abilities to help uncover the genetic basis of trait variation in plants; however, the performance of these methods depends on the accuracy and scale of phenotyping. To enable a large-scale GWAS of in planta callus and shoot regeneration in the model tree Populus, we developed a phenomics workflow involving semantic segmentation to quantify regenerating plant tissues over time. We found that the resulting statistics were of highly non-normal distributions, and thus employed transformations or permutations to avoid violating assumptions of linear models used in GWAS. We report over 200 statistically supported quantitative trait loci (QTLs), with genes encompassing or near to top QTLs including regulators of cell adhesion, stress signaling, and hormone signaling pathways, as well as other diverse functions. Our results encourage models of hormonal signaling during plant regeneration to consider keystone roles of stress-related signaling (e.g. involving jasmonates and salicylic acid), in addition to the auxin and cytokinin pathways commonly considered. The putative regulatory genes and biological processes we identified provide new insights into the biological complexity of plant regeneration, and may serve as new reagents for improving regeneration and transformation of recalcitrant genotypes and species.

GWAS identifies candidate genes controlling adventitious rooting in Populus trichocarpa.

Adventitious rooting (AR) is critical to the propagation, breeding, and genetic engineering of trees. The capacity for plants to undergo this process is highly heritable and of a polygenic nature; however, the basis of its genetic variation is largely uncharacterized. To identify genetic regulators of AR, we performed a genome-wide association study (GWAS) using 1148 genotypes of Populus trichocarpa. GWASs are often limited by the abilities of researchers to collect precise phenotype data on a high-throughput scale; to help overcome this limitation, we developed a computer vision system to measure an array of traits related to adventitious root development in poplar, including temporal measures of lateral and basal root length and area. GWAS was performed using multiple methods and significance thresholds to handle non-normal phenotype statistics and to gain statistical power. These analyses yielded a total of 277 unique associations, suggesting that genes that control rooting include regulators of hormone signaling, cell division and structure, reactive oxygen species signaling, and other processes with known roles in root development. Numerous genes with uncharacterized functions and/or cryptic roles were also identified. These candidates provide targets for functional analysis, including physiological and epistatic analyses, to better characterize the complex polygenic regulation of AR.

Variation in floral form of CRISPR knock-outs of the poplar homologs of LEAFY and AGAMOUS after FT heat-induced early flowering.

Plant migration and gene flow from genetically modified or exotic trees to nearby lands or by crossing with wild relatives is a major public and regulatory concern. Many genetic strategies exist to mitigate potential gene flow; however, the long delay in onset of flowering is a severe constraint to research progress. We used heat-induced FT overexpression to speed assessment of the expected floral phenotypes after CRISPR knockout of poplar homologs of the key floral genes, LEAFY and AGAMOUS. We selected events with previously characterized CRISPR-Cas9 induced biallelic changes then re-transformed them with the Arabidopsis thaliana FLOWERING LOCUS T (AtFT) gene under control of either a strong constitutive promoter or a heat-inducible promoter. We successfully obtained flowering in both a male and female clones of poplar, observing a wide range of inflorescence and floral forms among flowers, ramets, and insertion events. Overall, flowers obtained from the selected LFY and AG targeted events were consistent with what would be predicted for loss-of-function of these genes. LFY-targeted events showed small catkins with leaf-like organs, AG-targeted events had nested floral organs consistent with reduction in floral determinacy and absence of well-formed carpels or anthers. These findings demonstrate the great developmental plasticity of Populus flowers during genetically accelerated flowering, which may be of horticultural value. They also provide an informative early view of floral phenotypes and apparent sterility from knockouts of both these gene targets.

Knockout of floral and meiosis genes using CRISPR/Cas9 produces male-sterility in Eucalyptus without impacts on vegetative growth.

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.

DNA hypomethylation of the host tree impairs interaction with mutualistic ectomycorrhizal fungus.

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.

Robust High-Throughput Phenotyping with Deep Segmentation Enabled by a Web-Based Annotator.

The abilities of plant biologists and breeders to characterize the genetic basis of physiological traits are limited by their abilities to obtain quantitative data representing precise details of trait variation, and particularly to collect this data at a high-throughput scale with low cost. Although deep learning methods have demonstrated unprecedented potential to automate plant phenotyping, these methods commonly rely on large training sets that can be time-consuming to generate. Intelligent algorithms have therefore been proposed to enhance the productivity of these annotations and reduce human efforts. We propose a high-throughput phenotyping system which features a Graphical User Interface (GUI) and a novel interactive segmentation algorithm: Semantic-Guided Interactive Object Segmentation (SGIOS). By providing a user-friendly interface and intelligent assistance with annotation, this system offers potential to streamline and accelerate the generation of training sets, reducing the effort required by the user. Our evaluation shows that our proposed SGIOS model requires fewer user inputs compared to the state-of-art models for interactive segmentation. As a case study of the use of the GUI applied for genetic discovery in plants, we present an example of results from a preliminary genome-wide association study (GWAS) of in planta regeneration in Populus trichocarpa (poplar). We further demonstrate that the inclusion of a semantic prior map with SGIOS can accelerate the training process for future GWAS, using a sample of a dataset extracted from a poplar GWAS of in vitro regeneration. The capabilities of our phenotyping system surpass those of unassisted humans to rapidly and precisely phenotype our traits of interest. The scalability of this system enables large-scale phenomic screens that would otherwise be time-prohibitive, thereby providing increased power for GWAS, mutant screens, and other studies relying on large sample sizes to characterize the genetic basis of trait variation. Our user-friendly system can be used by researchers lacking a computational background, thus helping to democratize the use of deep segmentation as a tool for plant phenotyping.

High quality haplotype-resolved genome assemblies of Populus tomentosa Carr., a stabilized interspecific hybrid species widespread in Asia.

Populus has a wide ecogeographical range spanning the Northern Hemisphere, and interspecific hybrids are common. Populus tomentosa Carr. is widely distributed and cultivated in the eastern region of Asia, where it plays multiple important roles in forestry, agriculture, conservation, and urban horticulture. Reference genomes are available for several Populus species, however, our goals were to produce a very high quality de novo chromosome-level genome assembly in P. tomentosa genome that could serve as a reference for evolutionary and ecological studies of hybrid speciation throughout the genus. Here, combining long-read sequencing and Hi-C scaffolding, we present a high-quality, haplotype-resolved genome assembly. The genome size was 740.2 Mb, with a contig N50 size of 5.47 Mb and a scaffold N50 size of 46.68 Mb, consisting of 38 chromosomes, as expected with the known diploid chromosome number (2n = 2x = 38). A total of 59,124 protein-coding genes were identified. Phylogenomic analyses revealed that P. tomentosa is comprised of two distinct subgenomes, which we deomonstrate is likely to have resulted from hybridization between Populus adenopoda as the female parent and Populus alba var. pyramidalis as the male parent, with an origin of approximately 3.93 Ma. Although highly colinear, significant structural variation was found between the two subgenomes. Our study provides a valuable resource for ecological genetics and forest biotechnology.

RNAi Suppression of LEAFY Gives Stable Floral Sterility, and Reduced Growth Rate and Leaf Size, in Field-Grown Poplars.

The central floral development gene LEAFY (LFY), whose mutation leads to striking changes in flowering and often sterility, is commonly expressed in non-floral structures; however, its role in vegetative development is poorly understood. Sterility associated with suppression of LFY expression is an attractive means for mitigating gene flow by both seeds and pollen in vegetatively propagated forest trees, but the consequences of its suppression for tree form and wood production are unclear. To study the vegetative effects of RNAi suppression of LFY, we created a randomized, multiple-year field study with 30-40 trees (ramets) in each of two sterile gene insertion events, three transgenic control events, and a wild-type control population. We found that floral knock-down phenotypes were stable across years and propagation cycles, but that several leaf morphology and productivity traits were statistically and often substantially different in sterile vs. normal flowering RNAi-LFY trees. Though trees with suppressed LEAFY expression looked visibly normal, they appear to have reduced growth and altered leaf traits. LFY appears to have a significant role in vegetative meristem development, and evaluation of vegetative impacts from LFY suppression would be prudent prior to large-scale use for genetic containment.

Overexpression of SHORT VEGETATIVE PHASE-LIKE (SVL) in Populus delays onset and reduces abundance of flowering in field-grown trees.

The spread of transgenes and exotic germplasm from planted crops into wild or feral species is a difficult problem for public and regulatory acceptance of genetically engineered plants, particularly for wind-pollinated trees such as poplar. We report that overexpression of a poplar homolog of the floral repressor SHORT VEGETATIVE PHASE-LIKE (SVL), a homolog of the Arabidopsis MADS-box repressor SHORT VEGETATIVE PHASE (SVP), delayed the onset of flowering several years in three genotypes of field-grown transgenic poplars. Higher expression of SVL correlated with a delay in flowering onset and lower floral abundance, and did not cause morphologically obvious or statistically significant effects on leaf characteristics, tree form, or stem volume. Overexpression effects on reproductive and vegetative phenology in spring was modest and genotype-specific. Our results suggest that use of SVL and related floral repressors can be useful tools to enable a high level of containment for vegetatively propagated short-rotation woody energy or pulp crops.

RNAi suppression of DNA methylation affects the drought stress response and genome integrity in transgenic poplar.

Trees are long-lived organisms that continuously adapt to their environments, a process in which epigenetic mechanisms are likely to play a key role. Via downregulation of the chromatin remodeler DECREASED IN DNA METHYLATION 1 (DDM1) in poplar (Populus tremula × Populus alba) RNAi lines, we examined how DNA methylation coordinates genomic and physiological responses to moderate water deficit. We compared the growth and drought response of two RNAi-ddm1 lines to wild-type (WT) trees under well-watered and water deficit/rewatering conditions, and analyzed their methylomes, transcriptomes, mobilomes and phytohormone contents in the shoot apical meristem. The RNAi-ddm1 lines were more tolerant to drought-induced cavitation but did not differ in height or stem diameter growth. About 5000 differentially methylated regions were consistently detected in both RNAi-ddm1 lines, colocalizing with 910 genes and 89 active transposable elements. Under water deficit conditions, 136 differentially expressed genes were found, including many involved in phytohormone pathways; changes in phytohormone concentrations were also detected. Finally, the combination of hypomethylation and drought led to the mobility of two transposable elements. Our findings suggest major roles for DNA methylation in regulation of genes involved in hormone-related stress responses, and the maintenance of genome integrity through repression of transposable elements.

Genetic containment in vegetatively propagated forest trees: CRISPR disruption of LEAFY function in Eucalyptus gives sterile indeterminate inflorescences and normal juvenile development.

Eucalyptus is among the most widely planted taxa of forest trees worldwide. However, its spread as an exotic or genetically engineered form can create ecological and social problems. To mitigate gene flow via pollen and seeds, we mutated the Eucalyptus orthologue of LEAFY (LFY) by transforming a Eucalyptus grandis × urophylla wild-type hybrid and two Flowering Locus T (FT) overexpressing (and flowering) lines with CRISPR Cas9 targeting its LFY orthologue, ELFY. We achieved high rates of elfy biallelic knockouts, often approaching 100% of transgene insertion events. Frameshift mutations and deletions removing conserved amino acids caused strong floral alterations, including indeterminacy in floral development and an absence of male and female gametes. These mutants were otherwise visibly normal and did not differ statistically from transgenic controls in juvenile vegetative growth rate or leaf morphology in greenhouse trials. Genes upstream or near to ELFY in the floral development pathway were overexpressed, whereas floral organ identity genes downstream of ELFY were severely depressed. We conclude that disruption of ELFY function appears to be a useful tool for sexual containment, without causing statistically significant or large adverse effects on juvenile vegetative growth or leaf morphology.

Vegetative phase change in Populus tremula × alba.

Plants transition through juvenile and adult phases of vegetative development in a process known as vegetative phase change (VPC). In poplars (genus Populus) the differences between these stages are subtle, making it difficult to determine when this transition occurs. Previous studies of VPC in poplars have relied on plants propagated in vitro, leaving the natural progression of this process unknown. We examined developmental morphology of seed-grown and in vitro derived Populus tremula × alba (clone 717-1B4), and compared the phenotype of these to transgenics with manipulated miR156 expression, the master regulator of VPC. In seed-grown plants, most traits changed from node-to-node during the first 3 months of development but remained constant after node 25. Many traits remained unchanged in clones over-expressing miR156, or were enhanced when miR156 was lowered, demonstrating their natural progression is regulated by the miR156/SPL pathway. The characteristic leaf fluttering of Populus is one of these miR156-regulated traits. Vegetative development in plants grown from culture mirrored that of seed-grown plants, allowing direct comparison between plants often used in research and those found in nature. These results provide a foundation for further research on the role of VPC in the ecology and evolution of this economically important genus.

RNAi of AGAMOUS genes in sweetgum alters reproductive organ identity and decreases fruit persistence.

Sweetgums (Liquidambar), members of the family Altingiaceae (Altingiales), have inflorescences and floral organs that are distinctive in structure compared with other angiosperms in which the roles of floral homeotic genes have been studied. To begin to understand the role of AGAMOUS (AG)-a floral homeotic gene that has a major role in stamen and carpel development-in development of the monosexual flowers of sweetgum, we used RNAi to reduce the expression of two members of the AG subfamily. Because AG suppression should induce floral sterility, RNAi might also provide a tool to mitigate the risks of invasiveness-and to reduce the production of its nuisance fruits or allergenic pollen-when sweetgum is used as an exotic shade or forest tree. We tested 33 independent transgenic events and non-transgenic controls during 10 years in the field. The RNAi-AG sweetgum trees maintained normal growth, phenology, and vivid fall coloration during the 10 years of study, but 8 insertion events had highly modified inflorescence and floral morphology. The modified flowers had anthers and carpels that were converted to flat leaf-like structures lacking pollen grains and ovules, respectively. The female inflorescences developed into dry papery structures that failed to produce seeds. These infructescences were smaller than control infructescences, and lost a greater percentage of biomass in a controlled decay assay. RNAi against AG genes was highly effective at impairing fertility and modifying reproductive development without significant vegetative effects in sweetgum and gave phenotypes distinct from, but similar to, that of AG loss of function in other angiosperms.

Genotyping-by-sequencing and ecological niche modeling illuminate phylogeography, admixture, and Pleistocene range dynamics in quaking aspen (Populus tremuloides).

Populus tremuloides is the widest-ranging tree species in North America and an ecologically important component of mesic forest ecosystems displaced by the Pleistocene glaciations. Using phylogeographic analyses of genome-wide SNPs (34,796 SNPs, 183 individuals) and ecological niche modeling, we inferred population structure, ploidy levels, admixture, and Pleistocene range dynamics of P. tremuloides, and tested several historical biogeographical hypotheses. We found three genetic lineages located mainly in coastal-Cascades (cluster 1), east-slope Cascades-Sierra Nevadas-Northern Rockies (cluster 2), and U.S. Rocky Mountains through southern Canadian (cluster 3) regions of the P. tremuloides range, with tree graph relationships of the form ((cluster 1, cluster 2), cluster 3). Populations consisted mainly of diploids (86%) but also small numbers of triploids (12%) and tetraploids (1%), and ploidy did not adversely affect our genetic inferences. The main vector of admixture was from cluster 3 into cluster 2, with the admixture zone trending northwest through the Rocky Mountains along a recognized phenotypic cline (Utah to Idaho). Clusters 1 and 2 provided strong support for the "stable-edge hypothesis" that unglaciated southwestern populations persisted in situ since the last glaciation. By contrast, despite a lack of clinal genetic variation, cluster 3 exhibited "trailing-edge" dynamics from niche suitability predictions signifying complete northward postglacial expansion. Results were also consistent with the "inland dispersal hypothesis" predicting postglacial assembly of Pacific Northwestern forest ecosystems, but rejected the hypothesis that Pacific-coastal populations were colonized during outburst flooding from glacial Lake Missoula. Overall, congruent patterns between our phylogeographic and ecological niche modeling results and fossil pollen data demonstrate complex mixtures of stable-edge, refugial locations, and postglacial expansion within P. tremuloides. These findings confirm and refine previous genetic studies, while strongly supporting a distinct Pacific-coastal genetic lineage of quaking aspen.

Extensive transcriptome changes during seasonal leaf senescence in field-grown black cottonwood (Populus trichocarpa Nisqually-1).

To better understand the molecular control of leaf senescence, we examined transcriptome changes during seasonal leaf senescence in Populus trichocarpa Nisqually-1, the Populus reference genome, growing in its natural habitat. Using monthly (from May to October) transcriptomes for three years (2009, 2015, and 2016), we identified 17,974 differentially expressed genes (DEGs; false discovery rate <0.05; log-fold change cutoff = 0) from 36,007 expressed Populus gene models. A total of 14,415 DEGs were directly related to transitions between four major developmental phases - growth, senescence initiation, reorganization, and senescence termination. These DEGs were significantly (p < 0.05) enriched in 279 gene ontology (GO) terms, including those related to photosynthesis, metabolic process, catalytic activity, protein phosphorylation, kinase activity, pollination, and transport. Also, there were 881 differentially expressed transcription factor (TF) genes from 54 TF families, notably bHLH, MYB, ERF, MYB-related, NAC, and WRKY. We also examined 28 DEGs known as alternative splicing (AS) factors that regulate AS process, and found evidence for a reduced level of AS activity during leaf senescence. Furthermore, we were able to identify a number of promoter sequence motifs associated with leaf senescence. This work provides a comprehensive resource for identification of genes involved in seasonal leaf senescence in trees, and informs efforts to explore the conservation and divergence of molecular mechanisms underlying leaf senescence between annual and perennial species.

Trifluoromethylated Phenanthroline Ligands Reduce Excited-State Distortion in Homoleptic Copper(I) Complexes.

We report the synthesis and excited-state dynamics for a series of homoleptic copper(I) trifluoromethylated phenanthroline complexes with two, three, and four trifluoromethyl functional groups. Our analysis of the steady-state absorbance and emission, transient-absorption spectroscopy, and electronic-structure-theory calculations results enable in-depth analysis of the pseudo-Jahn-Teller distortion inhibition from increased steric hindrance of the trifluoromethyl functional group relative to the prototypical dimethyl phenanthroline complex. Surprisingly, our results demonstrate that the greatest degree of pseudo-Jahn-Teller distortion inhibition is achieved with trifluoromethylation of only the 2 and 9 positions by an unusual combination of steric hindrance and stabilization of a nondistorted 1MLCT manifold observed by transient kinetic lifetimes and optimized excited-state structures. The intersystem-crossing (ISC) lifetime for the 2,9-bis(trifluoromethyl)-1,10-phenanthroline Cu(I) complex is 69 ps, while the triplet excited-state lifetime and emission quantum yield are 106 ns and 4 × 10-3, respectively. Further trifluoromethylation of the phenanthroline yields a greater σ bond inductive withdrawing force on the phenanthroline nitrogens, ultimately resulting in weaker coordination to the copper. Last, the surprising success of the 2,9-bis(trifluoromethyl)-1,10-phenanthroline Cu(I) complex by adjusting both ligand sterics and electronic properties outlines a new strategy for developing long-lived Cu(I) charge-transfer complexes.

High productivity in hybrid-poplar plantations without isoprene emission to the atmosphere.

Hybrid-poplar tree plantations provide a source for biofuel and biomass, but they also increase forest isoprene emissions. The consequences of increased isoprene emissions include higher rates of tropospheric ozone production, increases in the lifetime of methane, and increases in atmospheric aerosol production, all of which affect the global energy budget and/or lead to the degradation of air quality. Using RNA interference (RNAi) to suppress isoprene emission, we show that this trait, which is thought to be required for the tolerance of abiotic stress, is not required for high rates of photosynthesis and woody biomass production in the agroforest plantation environment, even in areas with high levels of climatic stress. Biomass production over 4 y in plantations in Arizona and Oregon was similar among genetic lines that emitted or did not emit significant amounts of isoprene. Lines that had substantially reduced isoprene emission rates also showed decreases in flavonol pigments, which reduce oxidative damage during extremes of abiotic stress, a pattern that would be expected to amplify metabolic dysfunction in the absence of isoprene production in stress-prone climate regimes. However, compensatory increases in the expression of other proteomic components, especially those associated with the production of protective compounds, such as carotenoids and terpenoids, and the fact that most biomass is produced prior to the hottest and driest part of the growing season explain the observed pattern of high biomass production with low isoprene emission. Our results show that it is possible to reduce the deleterious influences of isoprene on the atmosphere, while sustaining woody biomass production in temperate agroforest plantations.