Dormancy by communication shutdown: Trees become dormant in winter, with encapsulated buds protected against harsh conditions. Tylewicz et al. found that, as the days get shorter, communication channels between cells in aspen trees shut down. The blocked plasmodesmata sequester the dormant meristems from growth signals. Growth-promoting signals can be turned on and off relatively rapidly, but the closed plasmodesmata are not so nimble. Thus, despite the occasional sunny day, the trees stay dormant until spring. Science , this issue p. 212

While several studies have investigated general properties of the genetic architecture of natural variation in gene expression, few of these have considered natural, outbreeding populations. In parallel, systems biology has established that a general feature of biological networks is that they are scale-free, rendering them buffered against random mutations. To date, few studies have attempted to examine the relationship between the selective processes acting to maintain natural variation of gene expression and the associated co-expression network structure. Here we utilised RNA-Sequencing to assay gene expression in winter buds undergoing bud flush in a natural population of Populus tremula, an outbreeding forest tree species. We performed expression Quantitative Trait Locus (eQTL) mapping and identified 164,290 significant eQTLs associating 6,241 unique genes (eGenes) with 147,419 unique SNPs (eSNPs). We found approximately four times as many local as distant eQTLs, with local eQTLs having significantly higher effect sizes. eQTLs were primarily located in regulatory regions of genes (UTRs or flanking regions), regardless of whether they were local or distant. We used the gene expression data to infer a co-expression network and investigated the relationship between network topology, the genetic architecture of gene expression and signatures of selection. Within the co-expression network, eGenes were underrepresented in network module cores (hubs) and overrepresented in the periphery of the network, with a negative correlation between eQTL effect size and network connectivity. We additionally found that module core genes have experienced stronger selective constraint on coding and non-coding sequence, with connectivity associated with signatures of selection. Our integrated genetics and genomics results suggest that purifying selection is the primary mechanism underlying the genetic architecture of natural variation in gene expression assayed in flushing leaf buds of P. tremula and that connectivity within the co-expression network is linked to the strength of purifying selection. Numerous studies have shown that many genomic polymorphisms contributing to phenotypic variation are located outside of protein coding regions, suggesting that they act by modulating gene expression. Furthermore, phenotypes are seldom explained by individual genes, but rather emerge from networks of interacting genes. The effect of regulatory variants and the interaction of genes can be described by co-expression networks, which are known to contain a small number of highly connected nodes and many more lowly connected nodes, making them robust to random mutation. While previous studies have examined the genetic architecture of gene expression variation, few were performed in natural populations with fewer still integrating the co-expression network. We undertook a study using a natural population of European aspen (Populus tremula), showing that highly connected genes within the co-expression network had lower levels of polymorphism, had polymorphisms segregating at lower frequencies and with lower than average effect sizes, suggesting purifying selection acts on central components of the network. Furthermore, the most highly connected genes within co-expression network hubs were underrepresented for identified expression quantitative trait loci, suggesting that purifying selection on individual SNPs is driven by stabilising selecting on gene expression. In contrast, genes in the periphery of the network displayed signatures of relaxed selective constraint. Highly connected genes are therefore buffered against large expression modulation, providing a mechanistic link between selective pressures and network topology, which act in cohort to maintain the robustness at the population level of the co-expression network derived from flushing buds in P. tremula.

Evolutionary theory suggests that males and females may evolve sexually dimorphic phenotypic and biochemical traits concordant with each sex having different optimal strategies of resource investment to maximise reproductive success and fitness. Such sexual dimorphism would result in sex biased gene expression patterns in non-floral organs for autosomal genes associated with the control and development of such phenotypic traits. We examined morphological, biochemical and herbivory traits to test for sexually dimorphic resource allocation strategies within collections of sexually mature and immature Populus tremula (European aspen) trees. In addition we profiled gene expression in mature leaves of sexually mature wild trees using whole-genome oligonucleotide microarrays and RNA-Sequencing. We found no evidence of sexual dimorphism or differential resource investment strategies between males and females in either sexually immature or mature trees. Similarly, single-gene differential expression and machine learning approaches revealed no evidence of large-scale sex biased gene expression. However, two significantly differentially expressed genes were identified from the RNA-Seq data, one of which is a robust diagnostic marker of sex in P. tremula.

Leaf shape is a defining feature of how we recognize and classify plant species. Although there is extensive variation in leaf shape within many species, few studies have disentangled the underlying genetic architecture. We characterized the genetic architecture of leaf shape variation in Eurasian aspen (Populus tremula L.) by performing genome‐wide association study (GWAS) for physiognomy traits. To ascertain the roles of identified GWAS candidate genes within the leaf development transcriptional program, we generated RNA‐Seq data that we used to perform gene co‐expression network analyses from a developmental series, which is publicly available within the PlantGenIE resource. We additionally used existing gene expression measurements across the population to analyze GWAS candidate genes in the context of a population‐wide co‐expression network and to identify genes that were differentially expressed between groups of individuals with contrasting leaf shapes. These data were integrated with expression GWAS (eQTL) results to define a set of candidate genes associated with leaf shape variation. Our results identified no clear adaptive link to leaf shape variation and indicate that leaf shape traits are genetically complex, likely determined by numerous small‐effect variations in gene expression. Genes associated with shape variation were peripheral within the population‐wide co‐expression network, were not highly connected within the leaf development co‐expression network, and exhibited signatures of relaxed selection. As such, our results are consistent with the omnigenic model.

Secretory Carrier-Associated Membrane Proteins (SCAMPs) are highly conserved 32–38 kDa proteins that are involved in membrane trafficking. A systems approach was taken to elucidate function of SCAMPs in wood formation of Populus trees. Phenotypic and multi-omics analyses were performed in woody tissues of transgenic Populus trees carrying an RNAi construct for Populus tremula x tremuloides SCAMP3 (PttSCAMP3; Potri.019G104000). The woody tissues of the transgenic trees displayed increased amounts of both polysaccharides and lignin oligomers, indicating increased deposition of both the carbohydrate and lignin components of the secondary cell walls. This coincided with a tendency towards increased wood density as well as significantly increased thickness of the suberized cork in the transgenic lines. Multivariate OnPLS (orthogonal projections to latent structures) modeling of five different omics datasets (the transcriptome, proteome, GC-MS metabolome, LC-MS metabolome and pyrolysis-GC/MS metabolome) collected from the secondary xylem tissues of the stem revealed systemic variation in the different variables in the transgenic lines, including changes that correlated with the changes in the secondary cell wall composition. The OnPLS model also identified a rather large number of proteins that were more abundant in the transgenic lines than in the wild type. Several of these were related to secretion and/or endocytosis as well as both primary and secondary cell wall biosynthesis. Populus SCAMP proteins were shown to influence accumulation of secondary cell wall components, including polysaccharides and phenolic compounds, in the woody tissues of Populus tree stems. Our multi-omics analyses combined with the OnPLS modelling suggest that this function is mediated by changes in membrane trafficking to fine-tune the abundance of cell wall precursors and/or proteins involved in cell wall biosynthesis and transport. The data provides a multi-level source of information for future studies on the function of the SCAMP proteins in plant stem tissues.