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Overgrowth of connective tissue and scar formation induced by the electrode array insertion increase the impedance and, thus, diminish the interactions between neural probes as like cochlear implants (CI) and the target tissue. Therefore, it is of great clinical interest to modify the carrier material of the electrodes to improve the electrode nerve interface for selective cell adhesion. On one side connective tissue growth needs to be reduced to avoid electrode array encapsulation, on the other side the carrier material should not compromise the interaction with neuronal cells. The present in vitro-study qualitatively and quantitatively characterises the interaction of fibroblasts, glial cells and spiral ganglion neurons (SGN) with ultrathin poly(N,N-dimethylacrylamide) (PDMAA), poly(2-ethyloxazoline) (PEtOx) and poly([2-methacryloyloxy)ethyl]trimethylammoniumchlorid) (PMTA) films immobilised onto glass surfaces using a photoreactive anchor layer. The layer thickness and hydrophilicity of the polymer films were characterised by ellipsometric and water contact angle measurement. Moreover the topography of the surfaces was investigated using atomic force microscopy (AFM). The neuronal and non-neuronal cells were dissociated from spiral ganglions of postnatal rats and cultivated for 48 h on top of the polymer coatings. Immunocytochemical staining of neuronal and intermediary filaments revealed that glial cells predominantly attached on PMTA films, but not on PDMAA and PEtOx monolayers. Hereby, strong survival rates and neurite outgrowth were only found on PMTA, whereas PDMAA and PEtOx coatings significantly reduced the SG neuron survival and neuritogenesis. As also shown by scanning electron microscopy (SEM) SGN strongly survived and retained their differentiated phenotype only on PMTA. In conclusion, survival and neuritogenesis of SGN may be associated with the extent of the glial cell growth. Since PMTA was the only of the polar polymers used in this study bearing a cationic charge, it can be assumed that this charge favours adhesion of both glial cells and SG neurons glial cells and SGN.

Forests face many threats. While traditional breeding may be too slow to deliver well-adapted trees, genomic selection (GS) can accelerate the process. We describe a comprehensive study of GS from proof of concept to operational application in western redcedar (WRC, Thuja plicata). Using genomic data, we developed models on a training population (TrP) of trees to predict breeding values (BVs) in a target seedling population (TaP) for growth, heartwood chemistry, and foliar chemistry traits. We used cross-validation to assess prediction accuracy (PACC) in the TrP; we also validated models for early-expressed foliar traits in the TaP. Prediction accuracy was high across generations, environments, and ages. PACC was not reduced to zero among unrelated individuals in TrP and was only slightly reduced in the TaP, confirming strong linkage disequilibrium and the ability of the model to generate accurate predictions across breeding generations. Genomic BV predictions were correlated with those from pedigree but displayed a wider range of within-family variation due to the ability of GS to capture the Mendelian sampling term. Using predicted TaP BVs in multi-trait selection, we functionally implemented and integrated GS into an operational tree-breeding program., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Nucleotide-binding domain and leucine-rich repeat (NLR) immune receptor genes form a major line of defense in plants, acting in both pathogen recognition and resistance machinery activation. NLRs are reported to form large gene clusters in limber pine (Pinus flexilis), but it is unknown how widespread this genomic architecture may be among the extant species of conifers (Pinophyta). We used comparative genomic analyses to assess patterns in the abundance, diversity, and genomic distribution of NLR genes. Chromosome-level whole genome assemblies and high-density linkage maps in the Pinaceae, Cupressaceae, Taxaceae, and other gymnosperms were scanned for NLR genes using existing and customized pipelines. The discovered genes were mapped across chromosomes and linkage groups and analyzed phylogenetically for evolutionary history. Conifer genomes are characterized by dense clusters of NLR genes, highly localized on one chromosome. These clusters are rich in TNL-encoding genes, which seem to have formed through multiple tandem duplication events. In contrast to angiosperms and nonconiferous gymnosperms, genomic clustering of NLR genes is ubiquitous in conifers. NLR-dense genomic regions are likely to influence a large part of the plant's resistance, informing our understanding of adaptation to biotic stress and the development of genetic resources through breeding., Competing Interests: Conflict of Interest The authors declare that they have no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The plant-specialized metabolite montbretin A (MbA) is being developed as a new treatment option for type-2 diabetes, which is among the ten leading causes of premature death and disability worldwide. MbA is a complex acylated flavonoid glycoside produced in small amounts in below-ground organs of the perennial plant Montbretia (Crocosmia × crocosmiiflora). The lack of a scalable production system limits the development and potential application of MbA as a pharmaceutical or nutraceutical. Previous efforts to reconstruct montbretin biosynthesis in Nicotiana benthamiana (Nb) resulted in low yields of MbA and higher levels of montbretin B (MbB) and montbretin C (MbC). MbA, MbB, and MbC are nearly identical metabolites differing only in their acyl moieties, derived from caffeoyl-CoA, coumaroyl-CoA, and feruloyl-CoA, respectively. In contrast to MbA, MbB and MbC are not pharmaceutically active. To utilize the montbretia caffeoyl-CoA biosynthesis for improved MbA engineering in Nb, we cloned and characterized enzymes of the shikimate shunt of the general phenylpropanoid pathway, specifically hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyl transferase (CcHCT), p-coumaroylshikimate 3'-hydroxylase (CcC3'H), and caffeoylshikimate esterase (CcCSE). Gene expression patterns suggest that CcCSE enables the predominant formation of MbA, relative to MbB and MbC, in montbretia. This observation is supported by results from in vitro characterization of CcCSE and reconstruction of the shikimate shunt in yeast. Using CcHCT together with montbretin biosynthetic genes in multigene constructs resulted in a 30-fold increase of MbA in Nb. This work advances our understanding of the phenylpropanoid pathway and features a critical step towards improved MbA production in bioengineered Nb., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Conifers are long-lived and slow-evolving, thus requiring effective defences against their fast-evolving insect natural enemies. The copy number variation (CNV) of two key acetophenone biosynthesis genes Ugt5/Ugt5b and βglu-1 may provide a plausible mechanism underlying the constitutively variable defence in white spruce (Picea glauca) against its primary defoliator, spruce budworm. This study develops a long-insert sequence capture probe set (Picea_hung_p1.0) for quantifying copy number of βglu-1-like, Ugt5-like genes and single-copy genes on 38 Norway spruce (Picea abies) and 40 P. glauca individuals from eight and nine provenances across Europe and North America respectively. We developed local assemblies (Piabi_c1.0 and Pigla_c.1.0), full-length transcriptomes (PIAB_v1 and PIGL_v1), and gene models to characterise the diversity of βglu-1 and Ugt5 genes. We observed very large copy numbers of βglu-1, with up to 381 copies in a single P. glauca individual. We observed among-provenance CNV of βglu-1 in P. glauca but not P. abies. Ugt5b was predominantly single-copy in both species. This study generates critical hypotheses for testing the emergence and mechanism of extreme CNV, the dosage effect on phenotype, and the varying copy number of genes with the same pathway. We demonstrate new approaches to overcome experimental challenges in genomic research in conifer defences., (© 2024. The Author(s).)

Black spruce (Picea mariana [Mill.] B.S.P.) is a dominant conifer species in the North American boreal forest that plays important ecological and economic roles. Here, we present the first genome assembly of P. mariana with a reconstructed genome size of 18.3 Gbp and NG50 scaffold length of 36.0 kbp. A total of 66,332 protein-coding sequences were predicted in silico and annotated based on sequence homology. We analyzed the evolutionary relationships between P. mariana and 5 other spruces for which complete nuclear and organelle genome sequences were available. The phylogenetic tree estimated from mitochondrial genome sequences agrees with biogeography; specifically, P. mariana was strongly supported as a sister lineage to P. glauca and 3 other taxa found in western North America, followed by the European Picea abies. We obtained mixed topologies with weaker statistical support in phylogenetic trees estimated from nuclear and chloroplast genome sequences, indicative of ancient reticulate evolution affecting these 2 genomes. Clustering of protein-coding sequences from the 6 Picea taxa and 2 Pinus species resulted in 34,776 orthogroups, 560 of which appeared to be specific to P. mariana. Analysis of these specific orthogroups and dN/dS analysis of positive selection signatures for 497 single-copy orthogroups identified gene functions mostly related to plant development and stress response. The P. mariana genome assembly and annotation provides a valuable resource for forest genetics research and applications in this broadly distributed species, especially in relation to climate adaptation., Competing Interests: Conflicts of interest statement The author(s) declare no conflicts of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

The biochemical profile of coffee beans translates directly into quality traits, nutraceutical and health promoting properties of the coffee beverage. Ent -kaurene is the ubiquitous precursor for gibberellin biosynthesis in plants, but it also serves as an intermediate in specialized (i.e., secondary) diterpenoid metabolism that leads to a diversity of more than 1,000 different metabolites. Nutraceutical effects on human health attributed to diterpenes include antioxidant, anticarcinogenic, and anti-inflammatory properties. Cafestol (CAF) and kahweol (KAH) are two diterpenes found exclusively in the Coffea genus. Our objective was to identify and functionally characterize genes involved in the central step of ent -kaurene production. We identified 17 putative terpene synthase genes in the transcriptome of Coffea arabica . Two ent -copalyl diphosphate synthase ( CaCPS ) and three kaurene synthase ( CaKS ) were selected and manually annotated. Transcript expression profiles of CaCPS1 and CaKS3 best matched the CAF and KAH metabolite profiles in different tissues. CaCPS1 and CaKS3 proteins were heterologously expressed and functionally characterized. CaCPS1 catalyzes the cyclization of geranylgeranyl diphosphate (GGPP) to ent -copalyl diphosphate ( ent -CPP), which is converted to ent -kaurene by CaKS3. Knowledge about the central steps of diterpene formation in coffee provides a foundation for future characterization of the subsequent enzymes involved in CAF and KAH biosynthesis.

Stone cells are a specialized, highly lignified cell type found in both angiosperms and gymnosperms. In conifers, abundance of stone cells in the cortex provides a robust constitutive physical defense against stem feeding insects. Stone cells are a major insect-resistance trait in Sitka spruce (Picea sitchensis), occurring in dense clusters in apical shoots of trees resistant (R) to spruce weevil (Pissodes strobi) but being rare in susceptible (S) trees. To learn more about molecular mechanisms of stone cell formation in conifers, we used laser microdissection and RNA sequencing to develop cell-type-specific transcriptomes of developing stone cells from R and S trees. Using light, immunohistochemical, and fluorescence microscopy, we also visualized the deposition of cellulose, xylan, and lignin associated with stone cell development. A total of 1293 genes were differentially expressed at higher levels in developing stone cells relative to cortical parenchyma. Genes with potential roles in stone cell secondary cell wall formation (SCW) were identified and their expression evaluated over a time course of stone cell formation in R and S trees. The expression of several transcriptional regulators was associated with stone cell formation, including a NAC family transcription factor and several genes annotated as MYB transcription factors with known roles in SCW formation., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

Background: The mountain pine beetle, Dendroctonus ponderosae, is an irruptive bark beetle that causes extensive mortality to many pine species within the forests of western North America. Driven by climate change and wildfire suppression, a recent mountain pine beetle (MPB) outbreak has spread across more than 18 million hectares, including areas to the east of the Rocky Mountains that comprise populations and species of pines not previously affected. Despite its impacts, there are few tactics available to control MPB populations. Beauveria bassiana is an entomopathogenic fungus used as a biological agent in agriculture and forestry and has potential as a management tactic for the mountain pine beetle population. This work investigates the phenotypic and genomic variation between B. bassiana strains to identify optimal strains against a specific insect., Results: Using comparative genome and transcriptome analyses of eight B. bassiana isolates, we have identified the genetic basis of virulence, which includes oosporein production. Genes unique to the more virulent strains included functions in biosynthesis of mycotoxins, membrane transporters, and transcription factors. Significant differential expression of genes related to virulence, transmembrane transport, and stress response was identified between the different strains, as well as up to nine-fold upregulation of genes involved in the biosynthesis of oosporein. Differential correlation analysis revealed transcription factors that may be involved in regulating oosporein production., Conclusion: This study provides a foundation for the selection and/or engineering of the most effective strain of B. bassiana for the biological control of mountain pine beetle and other insect pests populations., (© 2023. The Author(s).)

Vaccinium corymbosum (highbush blueberry) in British Columbia relies on honeybee pollination for a reliable fruit set. As floral volatiles may help explain pollinator preference for blueberry, we surveyed components of variation for volatiles using gas chromatography-mass spectrometry (GC/MS). Principal component analysis of GC chromatogram peaks revealed a grouping of cultivars by a biosynthetic pathway that also corresponds with their known pedigree. To identify genetic variance, we identified 34 chemicals with adequate sample sizes. We estimated "natural heritability" (using uncontrolled crosses in natural environments) in two ways: (1) as "clonal repeatability," which is equivalent to broad-sense heritability and is an upper bound for narrow-sense heritability, and (2) "marker-based heritability," which serves as a lower bound for narrow-sense heritability. Both methods indicate that heritability is relatively low, ca. 15%, and variable among traits. This is expected as the floral volatile release is changeable and dependent on environmental conditions. It might be possible to use highly heritable volatiles for breeding.

Diet-derived polysaccharides are an important carbon source for gut bacteria and shape the human gut microbiome. Acarbose, a compound used clinically to treat type 2 diabetes, is known to inhibit the growth of some bacteria on starches based on its activity as an inhibitor of α-glucosidases and α-amylases. In contrast to acarbose, montbretin A, a new drug candidate for the treatment of type 2 diabetes, has been reported to be more specific for the inhibition of α-amylase, notably human pancreatic α-amylase. However, the effects of both molecules on glycan metabolism across a larger diversity of human gut bacteria remain to be characterized. Here, we used ex vivo metabolic labeling of a human microbiota sample with fluorescent maltodextrin to identify gut bacteria affected by amylase inhibitors. Metabolic labeling was performed in the presence and absence of amylase inhibitors, and the fluorescently labeled bacteria were identified by fluorescence-activated cell sorting coupled with 16S rDNA amplicon sequencing. We validated the labeling results in cultured isolates and identified four gut bacteria species whose metabolism of maltodextrin is inhibited by acarbose. In contrast, montbretin A slowed the growth of only one species, supporting the fact that it is more selective. Metabolic labeling is a valuable tool to characterize glycan metabolism in microbiota samples and could help understand the untargeted impact of drugs on the human gut microbiota.

Western redcedar (WRC; Thuja plicata ) is a conifer of the Pacific Northwest of North America prized for its durable and rot-resistant wood. WRC has naturally low outcrossing rates and readily self-fertilizes in nature. Challenges faced in WRC breeding and propagation involve selecting trees for accelerated growth while also ensuring enhanced heartwood rot resistance and resistance to ungulate browsing, as well as mitigating potential effects of inbreeding depression. Terpenes, a large and diverse class of specialized metabolites, confer both rot and browse resistance in the wood and foliage of WRC, respectively. Using a Bayesian modelling approach, we isolated single nucleotide polymorphism (SNP) markers estimated to be associated with three different foliar terpene traits and four different heartwood terpene traits, as well as two growth traits. We found that all traits were complex, being associated with between 1700 and 3600 SNPs linked with putatively causal loci, with significant polygenic components. Growth traits tended to have a larger polygenic component while terpene traits had larger major gene components; SNPs with small or polygenic effect were spread across the genome, while larger-effect SNPs tended to be localized to specific linkage groups. To determine whether there was inbreeding depression for terpene chemistry or growth traits, we used mixed linear models for a genomic selection training population to estimate the effect of the inbreeding coefficient F on foliar terpenes, heartwood terpenes and several growth and dendrochronological traits. We did not find significant inbreeding depression for any assessed trait. We further assessed inbreeding depression across four generations of complete selfing and found that not only was inbreeding depression not significant but that selection for height growth was the only significant predictor for growth during selfing, suggesting that inbreeding depression due to selfing during operational breeding can be mitigated by increased selection intensity., Competing Interests: The authors declare no conflict of interest., (© 2023 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

We assembled the 9.8-Gbp genome of western redcedar (WRC; Thuja plicata ), an ecologically and economically important conifer species of the Cupressaceae. The genome assembly, derived from a uniquely inbred tree produced through five generations of self-fertilization (selfing), was determined to be 86% complete by BUSCO analysis, one of the most complete genome assemblies for a conifer. Population genomic analysis revealed WRC to be one of the most genetically depauperate wild plant species, with an effective population size of approximately 300 and no significant genetic differentiation across its geographic range. Nucleotide diversity, π, is low for a continuous tree species, with many loci showing zero diversity, and the ratio of π at zero- to fourfold degenerate sites is relatively high (approximately 0.33), suggestive of weak purifying selection. Using an array of genetic lines derived from up to five generations of selfing, we explored the relationship between genetic diversity and mating system. Although overall heterozygosity was found to decline faster than expected during selfing, heterozygosity persisted at many loci, and nearly 100 loci were found to deviate from expectations of genetic drift, suggestive of associative overdominance. Nonreference alleles at such loci often harbor deleterious mutations and are rare in natural populations, implying that balanced polymorphisms are maintained by linkage to dominant beneficial alleles. This may account for how WRC remains responsive to natural and artificial selection, despite low genetic diversity., (© 2022 Shalev et al.; Published by Cold Spring Harbor Laboratory Press.)

Spruces (Picea spp.) are coniferous trees widespread in boreal and mountainous forests of the northern hemisphere, with large economic significance and enormous contributions to global carbon sequestration. Spruces harbor very large genomes with high repetitiveness, hampering their comparative analysis. Here, we present and compare the genomes of four different North American spruces: the genome assemblies for Engelmann spruce (Picea engelmannii) and Sitka spruce (Picea sitchensis) together with improved and more contiguous genome assemblies for white spruce (Picea glauca) and for a naturally occurring introgress of these three species known as interior spruce (P. engelmannii × glauca × sitchensis). The genomes were structurally similar, and a large part of scaffolds could be anchored to a genetic map. The composition of the interior spruce genome indicated asymmetric contributions from the three ancestral genomes. Phylogenetic analysis of the nuclear and organelle genomes revealed a topology indicative of ancient reticulation. Different patterns of expansion of gene families among genomes were observed and related with presumed diversifying ecological adaptations. We identified rapidly evolving genes that harbored high rates of non-synonymous polymorphisms relative to synonymous ones, indicative of positive selection and its hitchhiking effects. These gene sets were mostly distinct between the genomes of ecologically contrasted species, and signatures of convergent balancing selection were detected. Stress and stimulus response was identified as the most frequent function assigned to expanding gene families and rapidly evolving genes. These two aspects of genomic evolution were complementary in their contribution to divergent evolution of presumed adaptive nature. These more contiguous spruce giga-genome sequences should strengthen our understanding of conifer genome structure and evolution, as their comparison offers clues into the genetic basis of adaptation and ecology of conifers at the genomic level. They will also provide tools to better monitor natural genetic diversity and improve the management of conifer forests. The genomes of four closely related North American spruces indicate that their high similarity at the morphological level is paralleled by the high conservation of their physical genome structure. Yet, the evidence of divergent evolution is apparent in their rapidly evolving genomes, supported by differential expansion of key gene families and large sets of genes under positive selection, largely in relation to stimulus and environmental stress response., (© 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Western redcedar (WRC) is an ecologically and economically important forest tree species characterized by low genetic diversity with high self-compatibility and high heartwood durability. Using sequence capture genotyping of target genic and non-genic regions, we genotyped 44 parent trees and 1520 offspring trees representing 26 polycross (PX) families collected from three progeny test sites using 45,378 SNPs. Trees were phenotyped for eight traits related to growth, heartwood and foliar chemistry associated with wood durability and deer browse resistance. We used the genomic realized relationship matrix for paternity assignment, maternal pedigree correction, and to estimate genetic parameters. We compared genomics-based (GBLUP) and two pedigree-based (ABLUP: polycross and reconstructed full-sib [FS] pedigrees) models. Models were extended to estimate dominance genetic effects. Pedigree reconstruction revealed significant unequal male contribution and separated the 26 PX families into 438 FS families. Traditional maternal PX pedigree analysis resulted in up to 51% overestimation in genetic gain and 44% in diversity. Genomic analysis resulted in up to 22% improvement in offspring breeding value (BV) theoretical accuracy, 35% increase in expected genetic gain for forward selection, and doubled selection intensity for backward selection. Overall, all traits showed low to moderate heritability (0.09-0.28), moderate genotype by environment interaction (type-B genetic correlation: 0.51-0.80), low to high expected genetic gain (6.01%-55%), and no significant negative genetic correlation reflecting no large trade-offs for multi-trait selection. Only three traits showed a significant dominance effect. GBLUP resulted in smaller but more accurate heritability estimates for five traits, but larger estimates for the wood traits. Comparison between all, genic-coding, genic-non-coding and intergenic SNPs showed little difference in genetic estimates. In summary, we show that GBLUP overcomes the PX limitations, successfully captures expected historical and hidden relatedness as well as linkage disequilibrium (LD), and results in increased breeding efficiency in WRC., Competing Interests: The authors declare that they have no conflict of interest, (© 2022 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

The mountain pine beetle epidemic has highlighted the complex interactions of bark beetles with conifer host defenses. In these interactions, oleoresin terpenoids and volatiles, produced and released by the host tree, can be both harmful and beneficial to the beetle's success in colonizing a tree and completing its life cycle. The insect spends almost its entire life, from egg to adult, within the bark and phloem of a pine host, exposed to large quantities of complex mixtures of oleoresin terpenoids. Conifer oleoresin comprises mostly monoterpenes and diterpene resin acids as well as many different sesquiterpenes. It functions as a major chemical and physical defense system. However, the insect has evolved host colonization behavior and enzymes for terpenoid metabolism and detoxification that allow it to overcome some of the terpenoid defenses and, importantly, to co-opt pine monoterpenes as cues for host search and as a precursor for its own pheromone system. The insect-associated microbiome also plays a role in the metabolism of conifer terpenoids.

The highly diverse insect family of true weevils, Curculionidae, includes many agricultural and forest pests. Pissodes strobi, commonly known as the spruce weevil or white pine weevil, is a major pest of spruce and pine forests in North America. Pissodes strobi larvae feed on the apical shoots of young trees, causing stunted growth and can destroy regenerating spruce or pine forests. Here, we describe the nuclear and mitochondrial Pissodes strobi genomes and their annotations, as well as the genome of an apparent Wolbachia endosymbiont. We report a substantial expansion of the weevil nuclear genome, relative to other Curculionidae species, possibly driven by an abundance of class II DNA transposons. The endosymbiont observed belongs to a group (supergroup A) of Wolbachia species that generally form parasitic relationships with their arthropod host., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Background: Montbretins are rare specialized metabolites found in montbretia (Crocosmia x crocosmiiflora) corms. Montbretin A (MbA) is of particular interest as a novel therapeutic for type-2 diabetes and obesity. There is no scalable production system for this complex acylated flavonol glycoside. MbA biosynthesis has been reconstructed in Nicotiana benthamiana using montbretia genes for the assembly of MbA from its various different building blocks. However, in addition to smaller amounts of MbA, the therapeutically inactive montbretin B (MbB) was the major product of this metabolic engineering effort. MbA and MbB differ in a single hydroxyl group of their acyl side chains, which are derived from caffeoyl-CoA and coumaroyl-CoA, respectively. Biosynthesis of both MbA and MbB also require coumaroyl-CoA for the formation of the myricetin core. Caffeoyl-CoA and coumaroyl-CoA are formed in the central phenylpropanoid pathway by acyl activating enzymes (AAEs) known as 4-coumaroyl-CoA ligases (4CLs). Here we investigated a small family of montbretia AAEs and 4CLs, and their possible contribution to montbretin biosynthesis., Results: Transcriptome analysis for gene expression patterns related to montbretin biosynthesis identified eight different montbretia AAEs belonging to four different clades. Enzyme characterization identified 4CL activity for two clade IV members, Cc4CL1 and Cc4CL2, converting different hydroxycinnamic acids into the corresponding CoA thioesters. Both enzymes preferred coumaric acid over caffeic acid as a substrate in vitro. While expression of montbretia AAEs did not enhance MbA biosynthesis in N. benthamiana, we demonstrated that both Cc4CLs can be used to activate coumaric and caffeic acid towards flavanone biosynthesis in yeast (Saccharomyces cerevisiae)., Conclusions: Montbretia expresses two functional 4CLs, but neither of them is specific for the formation of caffeoyl-CoA. Based on differential expression analysis and phylogeny Cc4CL1 is most likely involved in MbA biosynthesis, while Cc4CL2 may contribute to lignin biosynthesis. Both Cc4CLs can be used for flavanone production to support metabolic engineering of MbA in yeast., Competing Interests: The authors have declared that no competing interests exist.

Spruce weevil ( Pissodes strobi ) is a significant pest of regenerating spruce ( Picea ) and pine ( Pinus ) forests in North America. Weevil larvae feed in the bark, phloem, cambium, and outer xylem of apical shoots, causing stunted growth or mortality of young trees. We identified and characterized constitutive and weevil-induced patterns of Sitka spruce ( Picea sitchensis ) transcriptomes in weevil-resistant (R) and susceptible (S) trees using RNA sequencing (RNA-seq) and differential expression (DE) analyses. We developed a statistical model for the analysis of RNA-seq data from treatment experiments with a 2 × 3 factorial design to differentiate insect-induced responses from the effects of mechanical damage. Across the different comparisons, we identified two major transcriptome contrasts: A large set of genes that was constitutively DE between R and S trees, and another set of genes that was DE in weevil-induced S-trees. The constitutive transcriptome unique to R trees appeared to be attuned to defense, while the constitutive transcriptome unique to S trees was enriched for growth-related transcripts. Notably, a set of transcripts annotated as "fungal" was detected consistently in the transcriptomes. Fungal transcripts were identified as DE in the comparison of R and S trees and in the weevil-affected DE transcriptome of S trees, suggesting a potential microbiome role in this conifer-insect interaction., Competing Interests: The authors declare no conflict of interest., (© 2021 The Authors. Plant‐Environment Interactions published by John Wiley & Sons Ltd and New Phytologist Foundation.)