Loss of daylength sensitivity by splice site mutation in Cannabis pseudo-response regulator.

Photoperiod insensitivity (auto-flowering) in drug-type Cannabis sativa circumvents the need for short day (SD) flowering requirements making outdoor cultivation in high latitudes possible. However, the benefits of photoperiod insensitivity are counterbalanced by low cannabinoid content and poor flower quality in auto-flowering genotypes. Despite recent studies in cannabis flowering, a mechanistic understanding of photoperiod insensitivity is still lacking. We used a combination of genome-wide association study and genetic fine-mapping to identify the genetic cause of auto-flowering in cannabis. We then used gene expression analyses and transient transformation assays to characterize flowering time control. Herein, we identify a splice site mutation within circadian clock gene PSEUDO-RESPONSE REGULATOR 37 (CsPRR37) in auto-flowering cannabis. We show that CsPRR37 represses FT expression and its circadian oscillations transition to a less repressive state during SD as compared to long days (LD). We identify several key circadian clock genes whose expression is altered in auto-flowering cannabis, particularly under non-inductive LD. Research into the pervasiveness of this mutation and others affecting flowering time will help elucidate cannabis domestication history and advance cannabis breeding toward a more sustainable outdoor cultivation system.

Spruce giga-genomes: structurally similar yet distinctive with differentially expanding gene families and rapidly evolving genes.

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.

Genomic selection for resistance to spruce budworm in white spruce and relationships with growth and wood quality traits.

With climate change, the pressure on tree breeding to provide varieties with improved resilience to biotic and abiotic stress is increasing. As such, pest resistance is of high priority but has been neglected in most tree breeding programs, given the complexity of phenotyping for these traits and delays to assess mature trees. In addition, the existing genetic variation of resistance and its relationship with productivity should be better understood for their consideration in multitrait breeding. In this study, we evaluated the prospects for genetic improvement of the levels of acetophenone aglycones (AAs) in white spruce needles, which have been shown to be tightly linked to resistance to spruce budworm. Furthermore, we estimated the accuracy of genomic selection (GS) for these traits, allowing selection at a very early stage to accelerate breeding. A total of 1,516 progeny trees established on five sites and belonging to 136 full-sib families from a mature breeding population in New Brunswick were measured for height growth and genotyped for 4,148 high-quality SNPs belonging to as many genes along the white spruce genome. In addition, 598 trees were assessed for levels of AAs piceol and pungenol in needles, and 578 for wood stiffness. GS models were developed with the phenotyped trees and then applied to predict the trait values of unphenotyped trees. AAs were under moderate-to-high genetic control (h 2: 0.43-0.57) with null or marginally negative genetic correlations with other traits. The prediction accuracy of GS models (GBLUP) for AAs was high (PAAC: 0.63-0.67) and comparable or slightly higher than pedigree-based (ABLUP) or BayesCπ models. We show that AA traits can be improved and that GS speeds up the selection of improved trees for insect resistance and for growth and wood quality traits. Various selection strategies were tested to optimize multitrait gains.

Gymnosperm glandular trichomes: expanded dimensions of the conifer terpenoid defense system.

Glandular trichomes (GTs) are defensive structures that produce and accumulate specialized metabolites and protect plants against herbivores, pathogens, and abiotic stress. GTs have been extensively studied in angiosperms for their roles in defense and biosynthesis of high-value metabolites. In contrast, trichomes of gymnosperms have been described in fossilized samples, but have not been studied in living plants. Here, we describe the characterization of GTs on young stems of a hybrid white spruce. Metabolite and histological analysis of spruce GTs support a glandular function with accumulation of a diverse array of mono-, sesqui- and diterpenes including diterpene methylesters. Methylated diterpenes have previously been associated with insect resistance in white spruce. Headspeace analysis of spruce GTs showed a profile of volatiles dominated by monoterpenes and a highly diverse array of sesquiterpenes. Spruce GTs appear early during shoot growth, prior to the development of a lignified bark and prior to accumulation of terpenes in needles. Spruce GTs may provide an early, terpene-based chemical defense system at a developmental stage when young shoots are particularly vulnerable to foliage and shoot feeding insects, and before the resin duct system characteristic of conifers has fully developed.

Oleoresin defenses in conifers: chemical diversity, terpene synthases and limitations of oleoresin defense under climate change.

Conifers have evolved complex oleoresin terpene defenses against herbivores and pathogens. In co-evolved bark beetles, conifer terpenes also serve chemo-ecological functions as pheromone precursors, chemical barcodes for host identification, or nutrients for insect-associated microbiomes. We highlight the genomic, molecular and biochemical underpinnings of the large chemical space of conifer oleoresin terpenes and volatiles. Conifer terpenes are predominantly the products of the conifer terpene synthase (TPS) gene family. Terpene diversity is increased by cytochromes P450 of the CYP720B class. Many conifer TPS are multiproduct enzymes. Multisubstrate CYP720B enzymes catalyse multistep oxidations. We summarise known terpenoid gene functions in various different conifer species with reference to the annotated terpenoid gene space in a spruce genome. Overall, biosynthesis of terpene diversity in conifers is achieved through a system of biochemical radiation and metabolic grids. Expression of TPS and CYP720B genes can be specific to individual cell types of constitutive or traumatic resin duct systems. Induced terpenoid transcriptomes in resin duct cells lead to dynamic changes of terpene composition and quantity to fend off herbivores and pathogens. While terpenoid defenses have contributed much to the evolutionary success of conifers, under new conditions of climate change, these defences may become inconsequential against range-expanding forest pests.

An extended model of heartwood secondary metabolism informed by functional genomics.

The development of heartwood (HW) and the associated accumulation of secondary metabolites, which are also known as 'specialized metabolites' or 'extractives', is an important feature of tree biology. Heartwood development can affect tree health with broader implications for forest health. Heartwood development also defines a variety of wood quality traits that are important in the forest industry such as durability and colour of wood products. In the bioproducts industry, HW provides a source of high-value small molecules such as fragrances and antimicrobials. The HW properties of decay resistance in living trees, durability and colour of wood products, and small molecule bioproducts are largely defined by secondary metabolites, the biosynthesis of which appears to be activated during the onset of HW formation. Traditionally, it is thought that HW formation involves a spike in the activity of secondary metabolism in parenchyma cells in a transition zone between sapwood and HW, followed by programmed cell-death. The resulting HW tissue is thought to consist entirely of dead cells. Here, we discuss a variation of existing models of HW formation, based on the recent discovery of HW-specific transcriptome signatures of terpenoid biosynthesis in sandalwood (Santalum album L.) that invokes the activity of living cells in HW.

Cell-type- and tissue-specific transcriptomes of the white spruce (Picea glauca) bark unmask fine-scale spatial patterns of constitutive and induced conifer defense.

Plant defenses often involve specialized cells and tissues. In conifers, specialized cells of the bark are important for defense against insects and pathogens. Using laser microdissection, we characterized the transcriptomes of cortical resin duct cells, phenolic cells and phloem of white spruce (Picea glauca) bark under constitutive and methyl jasmonate (MeJa)-induced conditions, and we compared these transcriptomes with the transcriptome of the bark tissue complex. Overall, ~3700 bark transcripts were differentially expressed in response to MeJa. Approximately 25% of transcripts were expressed in only one cell type, revealing cell specialization at the transcriptome level. MeJa caused cell-type-specific transcriptome responses and changed the overall patterns of cell-type-specific transcript accumulation. Comparison of transcriptomes of the conifer bark tissue complex and specialized cells resolved a masking effect inherent to transcriptome analysis of complex tissues, and showed the actual cell-type-specific transcriptome signatures. Characterization of cell-type-specific transcriptomes is critical to reveal the dynamic patterns of spatial and temporal display of constitutive and induced defense systems in a complex plant tissue or organ. This was demonstrated with the improved resolution of spatially restricted expression of sets of genes of secondary metabolism in the specialized cell types.

Heartwood-specific transcriptome and metabolite signatures of tropical sandalwood (Santalum album) reveal the final step of (Z)-santalol fragrance biosynthesis.

Tropical sandalwood (Santalum album) produces one of the world's most highly prized fragrances, which is extracted from mature heartwood. However, in some places such as southern India, natural populations of this slow-growing tree are threatened by over-exploitation. Sandalwood oil contains four major and fragrance-defining sesquiterpenols: (Z)-α-santalol, (Z)-ß-santalol, (Z)-epi-ß-santalol and (Z)-α-exo-bergamotol. The first committed step in their biosynthesis is catalyzed by a multi-product santalene/bergamotene synthase. Sandalwood cytochromes P450 of the CYP76F sub-family were recently shown to hydroxylate santalenes and bergamotene; however, these enzymes produced mostly (E)-santalols and (E)-α-exo-bergamotol. We hypothesized that different santalene/bergamotene hydroxylases evolved in S. album to stereo-selectively produce (E)- or (Z)-sesquiterpenols, and that genes encoding (Z)-specific P450s contribute to sandalwood oil formation if co-expressed in the heartwood with upstream genes of sesquiterpene biosynthesis. This hypothesis was validated by the discovery of a heartwood-specific transcriptome signature for sesquiterpenoid biosynthesis, including highly expressed SaCYP736A167 transcripts. We characterized SaCYP736A167 as a multi-substrate P450, which stereo-selectively produces (Z)-α-santalol, (Z)-ß-santalol, (Z)-epi-ß-santalol and (Z)-α-exo-bergamotol, matching authentic sandalwood oil. This work completes the discovery of the biosynthetic enzymes of key components of sandalwood fragrance, and highlights the evolutionary diversification of stereo-selective P450s in sesquiterpenoid biosynthesis. Bioengineering of microbial systems using SaCYP736A167, combined with santalene/bergamotene synthase, has potential for development of alternative industrial production systems for sandalwood oil fragrances.

Intra-plastid protein trafficking: how plant cells adapted prokaryotic mechanisms to the eukaryotic condition.

Protein trafficking and localization in plastids involve a complex interplay between ancient (prokaryotic) and novel (eukaryotic) translocases and targeting machineries. During evolution, ancient systems acquired new functions and novel translocation machineries were developed to facilitate the correct localization of nuclear encoded proteins targeted to the chloroplast. Because of its post-translational nature, targeting and integration of membrane proteins posed the biggest challenge to the organelle to avoid aggregation in the aqueous compartments. Soluble proteins faced a different kind of problem since some had to be transported across three membranes to reach their destination. Early studies suggested that chloroplasts addressed these issues by adapting ancient-prokaryotic machineries and integrating them with novel-eukaryotic systems, a process called 'conservative sorting'. In the last decade, detailed biochemical, genetic, and structural studies have unraveled the mechanisms of protein targeting and localization in chloroplasts, suggesting a highly integrated scheme where ancient and novel systems collaborate at different stages of the process. In this review we focus on the differences and similarities between chloroplast ancestral translocases and their prokaryotic relatives to highlight known modifications that adapted them to the eukaryotic situation. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

Stoichiometry for binding and transport by the twin arginine translocation system.

Twin arginine translocation (Tat) systems transport large folded proteins across sealed membranes. Tat systems accomplish this feat with three membrane components organized in two complexes. In thylakoid membranes, cpTatC and Hcf106 comprise a large receptor complex containing an estimated eight cpTatC-Hcf106 pairs. Protein transport occurs when Tha4 joins the receptor complex as an oligomer of uncertain size that is thought to form the protein-conducting structure. Here, binding analyses with intact membranes or purified complexes indicate that each receptor complex could bind eight precursor proteins. Kinetic analysis of translocation showed that each precursor-bound site was independently functional for transport, and, with sufficient Tha4, all sites were concurrently active for transport. Tha4 titration determined that ∼26 Tha4 protomers were required for transport of each OE17 (oxygen-evolving complex subunit of 17 kD) precursor protein. Our results suggest that, when fully saturated with precursor proteins and Tha4, the Tat translocase is an ∼2.2-megadalton complex that can individually transport eight precursor proteins or cooperatively transport multimeric precursors.