The genomic and enzymatic basis for iridoid biosynthesis in cat thyme (Teucrium marum).

Iridoids are non-canonical monoterpenoids produced by both insects and plants. An example is the cat-attracting and insect-repelling volatile iridoid nepetalactone, produced by Nepeta sp. (catmint) and aphids. Recently, both nepetalactone biosynthetic pathways were elucidated, showing a remarkable convergent evolution. The iridoid, dolichodial, produced by Teucrium marum (cat thyme) and multiple insect species, has highly similar properties to nepetalactone but its biosynthetic origin remains unknown. We set out to determine the genomic, enzymatic, and evolutionary basis of iridoid biosynthesis in T. marum. First, we generated a de novo chromosome-scale genome assembly for T. marum using Oxford Nanopore Technologies long reads and proximity-by-ligation Hi-C reads. The 610.3 Mb assembly spans 15 pseudomolecules with a 32.9 Mb N50 scaffold size. This enabled identification of iridoid biosynthetic genes, whose roles were verified via activity assays. Phylogenomic analysis revealed that the evolutionary history of T. marum iridoid synthase, the iridoid scaffold-forming enzyme, is not orthologous to typical iridoid synthases but is derived from its conserved paralog. We discovered an enzymatic route from nepetalactol to diverse iridoids through the coupled activity of an iridoid oxidase cytochrome P450 and acetyltransferases, via an inferred acylated intermediate. This work provides a genomic resource for specialized metabolite research in mints and demonstration of the role of acetylation in T. marum iridoid diversity. This work will enable future biocatalytic or biosynthetic production of potent insect repellents, as well as comparative studies into iridoid biosynthesis in insects.

Advances in the Application of Single-Cell Transcriptomics in Plant Systems and Synthetic Biology.

Plants are complex systems hierarchically organized and composed of various cell types. To understand the molecular underpinnings of complex plant systems, single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool for revealing high resolution of gene expression patterns at the cellular level and investigating the cell-type heterogeneity. Furthermore, scRNA-seq analysis of plant biosystems has great potential for generating new knowledge to inform plant biosystems design and synthetic biology, which aims to modify plants genetically/epigenetically through genome editing, engineering, or re-writing based on rational design for increasing crop yield and quality, promoting the bioeconomy and enhancing environmental sustainability. In particular, data from scRNA-seq studies can be utilized to facilitate the development of high-precision Build-Design-Test-Learn capabilities for maximizing the targeted performance of engineered plant biosystems while minimizing unintended side effects. To date, scRNA-seq has been demonstrated in a limited number of plant species, including model plants (e.g., Arabidopsis thaliana), agricultural crops (e.g., Oryza sativa), and bioenergy crops (e.g., Populus spp.). It is expected that future technical advancements will reduce the cost of scRNA-seq and consequently accelerate the application of this emerging technology in plants. In this review, we summarize current technical advancements in plant scRNA-seq, including sample preparation, sequencing, and data analysis, to provide guidance on how to choose the appropriate scRNA-seq methods for different types of plant samples. We then highlight various applications of scRNA-seq in both plant systems biology and plant synthetic biology research. Finally, we discuss the challenges and opportunities for the application of scRNA-seq in plants.

Heat stress during seed development leads to impaired physiological function and plasticity in seed oil accumulation in Camelina sativa.

Camelina sativa, a member of the Brassicaceae, is a low-cost, renewable oilseed crop that produces seeds up to 40% oil by weight with high potential for use in food, feed, and biofuel applications. Camelina seeds contain high levels of the fatty acids α-linolenic acid (C18:3), linoleic acid (C18:2), oleic acid (C18:1), and gondoic acid (C20:1), which have high nutritional and industrial value. The impact of climate change, especially increased frequency and amplitude of heat waves, poses a serious threat to crop productivity. In this study, we evaluated the effect of elevated temperatures post-anthesis on the developing seeds of C. sativa and performed physiological, morphological, and chemical characterizations at 7, 14, 21, and 28 days post-anthesis (DPA), as well as at maturity. While the seed oil accumulation peaked at 21 DPA under control conditions, reaching 406mg/g dry weight, under heat stress it was only 186mg/g. Physiologically, transpiration rate (E) and internal CO2 concentration (Ci) increased between 2 to 9 days post-stress imposition and overall net photosynthesis was impaired. Seed yield, seed weight, and oil content reduced by 84.5%, 38.5% and 54.1% respectively. We demonstrate that post-anthesis heat stress causes severe yield losses and developmental plasticity in fatty acid accumulation in oilseeds.

Chromosome-scale genome assembly of the 'Munstead' cultivar of Lavandula angustifolia.

OBJECTIVES: Lavandula angustifolia (English lavender) is commercially important not only as an ornamental species but also as a major source of fragrances. To better understand the genomic basis of chemical diversity in lavender, we sequenced, assembled, and annotated the 'Munstead' cultivar of L. angustifolia. DATA DESCRIPTION: A total of 80 Gb of Oxford Nanopore Technologies reads was used to assemble the 'Munstead' genome using the Canu genome assembler software. Following multiple rounds of error correction and scaffolding using Hi-C data, the final chromosome-scale assembly represents 795,075,733 bp across 25 chromosomes with an N50 scaffold length of 31,371,815 bp. Benchmarking Universal Single Copy Orthologs analysis revealed 98.0% complete orthologs, indicative of a high-quality assembly representative of genic space. Annotation of protein-coding sequences revealed 58,702 high-confidence genes encoding 88,528 gene models. Access to the 'Munstead' genome will permit comparative analyses within and among lavender accessions and provides a pivotal species for comparative analyses within Lamiaceae.

Engineering custom morpho- and chemotypes of Populus for sustainable production of biofuels, bioproducts, and biomaterials.

Humans have been modifying plant traits for thousands of years, first through selection (i.e., domestication) then modern breeding, and in the last 30 years, through biotechnology. These modifications have resulted in increased yield, more efficient agronomic practices, and enhanced quality traits. Precision knowledge of gene regulation and function through high-resolution single-cell omics technologies, coupled with the ability to engineer plant genomes at the DNA sequence, chromatin accessibility, and gene expression levels, can enable engineering of complex and complementary traits at the biosystem level. Populus spp., the primary genetic model system for woody perennials, are among the fastest growing trees in temperate zones and are important for both carbon sequestration and global carbon cycling. Ample genomic and transcriptomic resources for poplar are available including emerging single-cell omics datasets. To expand use of poplar outside of valorization of woody biomass, chassis with novel morphotypes in which stem branching and tree height are modified can be fabricated thereby leading to trees with altered leaf to wood ratios. These morphotypes can then be engineered into customized chemotypes that produce high value biofuels, bioproducts, and biomaterials not only in specific organs but also in a cell-type-specific manner. For example, the recent discovery of triterpene production in poplar leaf trichomes can be exploited using cell-type specific regulatory sequences to synthesize high value terpenes such as the jet fuel precursor bisabolene specifically in the trichomes. By spatially and temporally controlling expression, not only can pools of abundant precursors be exploited but engineered molecules can be sequestered in discrete cell structures in the leaf. The structural diversity of the hemicellulose xylan is a barrier to fully utilizing lignocellulose in biomaterial production and by leveraging cell-type-specific omics data, cell wall composition can be modified in a tailored and targeted specific manner to generate poplar wood with novel chemical features that are amenable for processing or advanced manufacturing. Precision engineering poplar as a multi-purpose sustainable feedstock highlights how genome engineering can be used to re-imagine a crop species.

Cold stress induces differential gene expression of retained homeologs in Camelina sativa cv Suneson.

Camelina sativa (L.) Crantz, a member of the Brassicaceae, has potential as a biofuel feedstock which is attributable to the production of fatty acids in its seeds, its fast growth cycle, and low input requirements. While a genome assembly is available for camelina, it was generated from short sequence reads and is thus highly fragmented in nature. Using long read sequences, we generated a chromosome-scale, highly contiguous genome assembly (644,491,969 bp) for the spring biotype cultivar 'Suneson' with an N50 contig length of 12,031,512 bp and a scaffold N50 length of 32,184,682 bp. Annotation of protein-coding genes revealed 91,877 genes that encode 133,355 gene models. We identified a total of 4,467 genes that were significantly up-regulated under cold stress which were enriched in gene ontology terms associated with "response to cold" and "response to abiotic stress". Coexpression analyses revealed multiple coexpression modules that were enriched in genes differentially expressed following cold stress that had putative functions involved in stress adaptation, specifically within the plastid. With access to a highly contiguous genome assembly, comparative analyses with Arabidopsis thaliana revealed 23,625 A. thaliana genes syntenic with 45,453 Suneson genes. Of these, 24,960 Suneson genes were syntenic to 8,320 A. thaliana genes reflecting a 3 camelina homeolog to 1 Arabidopsis gene relationship and retention of all three homeologs. Some of the retained triplicated homeologs showed conserved gene expression patterns under control and cold-stressed conditions whereas other triplicated homeologs displayed diverged expression patterns revealing sub- and neo-functionalization of the homeologs at the transcription level. Access to the chromosome-scale assembly of Suneson will enable both basic and applied research efforts in the improvement of camelina as a sustainable biofuel feedstock.

Identification of genes associated with abiotic stress tolerance in sweetpotato using weighted gene co-expression network analysis.

Sweetpotato, Ipomoea batatas (L.), a key food security crop, is negatively impacted by heat, drought, and salinity stress. The orange-fleshed sweetpotato cultivar "Beauregard" was exposed to heat, salt, and drought treatments for 24 and 48 h to identify genes responding to each stress condition in leaves. Analysis revealed both common (35 up regulated, 259 down regulated genes in the three stress conditions) and unique sets of up regulated (1337 genes by drought, 516 genes by heat, and 97 genes by salt stress) and down regulated (2445 genes by drought, 678 genes by heat, and 204 genes by salt stress) differentially expressed genes (DEGs) suggesting common, yet stress-specific transcriptional responses to these three abiotic stressors. Gene Ontology analysis of down regulated DEGs common to both heat and salt stress revealed enrichment of terms associated with "cell population proliferation" suggestive of an impact on the cell cycle by the two stress conditions. To identify shared and unique gene co-expression networks under multiple abiotic stress conditions, weighted gene co-expression network analysis was performed using gene expression profiles from heat, salt, and drought stress treated 'Beauregard' leaves yielding 18 co-expression modules. One module was enriched for "response to water deprivation," "response to abscisic acid," and "nitrate transport" indicating synergetic crosstalk between nitrogen, water, and phytohormones with genes encoding osmotin, cell expansion, and cell wall modification proteins present as key hub genes in this drought-associated module. This research lays the groundwork for exploring to a further degree, mechanisms for abiotic stress tolerance in sweetpotato.

Transcriptome-based identification and functional characterization of iridoid synthase involved in monotropein biosynthesis in blueberry.

Blueberries (Vaccinium spp.) are well known for their nutritional quality, and recent work has shown that Vaccinium spp. also produce iridoids, which are specialized metabolites with potent health-promoting benefits. The iridoid glycoside monotropein, which has anti-inflammatory and antinociceptive activities, has been detected in several wild blueberry species but in only a few cultivated highbush blueberry cultivars. How monotropein is produced in blueberry and the genes involved in its biosynthesis remain to be elucidated. Using a monotropein-positive (M+) and monotropein-negative (M-) cultivar of blueberry, we employed transcriptomics and comparative genomics to identify candidate genes in the blueberry iridoid biosynthetic pathway. Orthology analysis was completed using de novo transcript assemblies for both the M+ and M- blueberry cultivars along with the known iridoid-producing plant species Catharanthus roseus to identify putative genes involved in key steps in the early iridoid biosynthetic pathway. From the identified orthologs, we functionally characterized iridoid synthase (ISY), a key enzyme involved in formation of the iridoid scaffold, from both the M+ and M- cultivars. Detection of nepetalactol suggests that ISY from both the M+ and M- cultivars produce functional enzymes that catalyze the formation of iridoids. Transcript accumulation of the putative ISY gene did not correlate with monotropein production, suggesting other genes in the monotropein biosynthetic pathway may be more directly responsible for differential accumulation of the metabolite in blueberry. Mutual rank analysis revealed that ISY is co-expressed with UDP-glucuronosyltransferase, which encodes an enzyme downstream of the ISY step. Results from this study contribute new knowledge in our understanding of iridoid biosynthesis in blueberry and could lead to development of new cultivars with increased human health benefits.

Genetic variation in a tepary bean (Phaseolus acutifolius A. Gray) diversity panel reveals loci associated with biotic stress resistance.

Tepary bean (Phaseolus acutifolius A. Gray), indigenous to the arid climates of northern Mexico and the Southwest United States, diverged from common bean (Phaseolus vulgaris L.), approximately 2 million years ago and exhibits a wide range of resistance to biotic stressors. The tepary genome is highly syntenic to the common bean genome providing a foundation for discovery and breeding of agronomic traits between these two crop species. Although a limited number of adaptive traits from tepary bean have been introgressed into common bean, hybridization barriers between these two species required the development of bridging lines to alleviate this barrier. Thus, to fully utilize the extant tepary bean germplasm as both a crop and as a donor of adaptive traits, we developed a diversity panel of 422 cultivated, weedy, and wild tepary bean accessions which were then genotyped and phenotyped to enable population genetic analyses and genome-wide association studies for their response to a range of biotic stressors. Population structure analyses of the panel revealed eight subpopulations and the differentiation of botanical varieties within P. acutifolius. Genome-wide association studies revealed loci and candidate genes underlying biotic stress resistance including quantitative trait loci for resistance to weevils, common bacterial blight, Fusarium wilt, and bean common mosaic necrosis virus that can be harnessed not only for tepary bean but also common bean improvement.

HT-B and S-RNase CRISPR-Cas9 double knockouts show enhanced self-fertility in diploid Solanum tuberosum.

The Gametophytic Self-Incompatibility (GSI) system in diploid potato (Solanum tuberosum L.) poses a substantial barrier in diploid potato breeding by hindering the generation of inbred lines. One solution is gene editing to generate self-compatible diploid potatoes which will allow for the generation of elite inbred lines with fixed favorable alleles and heterotic potential. The S-RNase and HT genes have been shown previously to contribute to GSI in the Solanaceae family and self-compatible S. tuberosum lines have been generated by knocking out S-RNase gene with CRISPR-Cas9 gene editing. This study employed CRISPR-Cas9 to knockout HT-B either individually or in concert with S-RNase in the diploid self-incompatible S. tuberosum clone DRH-195. Using mature seed formation from self-pollinated fruit as the defining characteristic of self-compatibility, HT-B-only knockouts produced little or no seed. In contrast, double knockout lines of HT-B and S-RNase displayed levels of seed production that were up to three times higher than observed in the S-RNase-only knockout, indicating a synergistic effect between HT-B and S-RNase in self-compatibility in diploid potato. This contrasts with compatible cross-pollinations, where S-RNase and HT-B did not have a significant effect on seed set. Contradictory to the traditional GSI model, self-incompatible lines displayed pollen tube growth reaching the ovary, yet ovules failed to develop into seeds indicating a potential late-acting self-incompatibility in DRH-195. Germplasm generated from this study will serve as a valuable resource for diploid potato breeding.

"Simple Tidy GeneCoEx": A gene co-expression analysis workflow powered by tidyverse and graph-based clustering in R.

Gene co-expression analysis is an effective method to detect groups (or modules) of co-expressed genes that display similar expression patterns, which may function in the same biological processes. Here, we present "Simple Tidy GeneCoEx", a gene co-expression analysis workflow written in the R programming language. The workflow is highly customizable across multiple stages of the pipeline including gene selection, edge selection, clustering resolution, and data visualization. Powered by the tidyverse package ecosystem and network analysis functions provided by the igraph package, the workflow detects gene co-expression modules whose members are highly interconnected. Step-by-step instructions with two use case examples as well as source code are available at https://github.com/cxli233/SimpleTidy_GeneCoEx.

Chromosome-scale assembly of the Verbenaceae species Queen's Wreath (Petrea volubilis L.).

OBJECTIVES: Petrea volubilis, a member of the Order Lamiales and the Verbenaceae family, is an important horticultural species that has been used in traditional folk medicine. To provide a genome sequence for comparative studies within the Order Lamiales that includes important families such as Lamiaceae (mints), we generated a long-read, chromosome-scale genome assembly of this species. DATA DESCRIPTION: Using a total of 45.5 Gb of Pacific Biosciences long read sequence, we generated a 480.2 Mb assembly of P. volubilis, of which, 93% is chromosome anchored. Representation of genic regions was robust with 96.6% of the Benchmarking of Universal Single Copy Orthologs present in the genome assembly. A total of 57.8% of the genome was annotated as a repetitive sequence. Using a gene annotation pipeline that included refinement of gene models using transcript evidence, 30,982 high confidence genes were annotated. Access to the P. volubilis genome will facilitate evolutionary studies in the Lamiales, a key order of Asterids that includes significant crop and medicinal plant species.

An image-based technique for automated root disease severity assessment using PlantCV.

Premise: Plant disease severity assessments are used to quantify plant-pathogen interactions and identify disease-resistant lines. One common method for disease assessment involves scoring tissue manually using a semi-quantitative scale. Automating assessments would provide fast, unbiased, and quantitative measurements of root disease severity, allowing for improved consistency within and across large data sets. However, using traditional Root System Markup Language (RSML) software in the study of root responses to pathogens presents additional challenges; these include the removal of necrotic tissue during the thresholding process, which results in inaccurate image analysis. Methods: Using PlantCV, we developed a Python-based pipeline, herein called RootDS, with two main objectives: (1) improving disease severity phenotyping and (2) generating binary images as inputs for RSML software. We tested the pipeline in common bean inoculated with Fusarium root rot. Results: Quantitative disease scores and root area generated by this pipeline had a strong correlation with manually curated values (R 2 = 0.92 and 0.90, respectively) and provided a broader capture of variation than manual disease scores. Compared to traditional manual thresholding, images generated using our pipeline did not affect RSML output. Discussion: Overall, the RootDS pipeline provides greater functionality in disease score data sets and provides an alternative method for generating image sets for use in available RSML software.

Genome Report: Genome sequence of 1S1, a transformable and highly regenerable diploid potato for use as a model for gene editing and genetic engineering.

Availability of readily transformable germplasm, as well as efficient pipelines for gene discovery are notable bottlenecks in the application of genome editing in potato. To study and introduce traits such as resistance against biotic and abiotic factors, tuber quality traits and self-fertility, model germplasm that is amenable to gene editing and regeneration is needed. Cultivated potato is a heterozygous autotetraploid and its genetic redundancy and complexity makes studying gene function challenging. Genome editing is simpler at the diploid level, with fewer allelic variants to consider. A readily transformable diploid potato would be further complemented by genomic resources that could aid in high throughput functional analysis. The heterozygous Solanum tuberosum Group Phureja clone 1S1 has a high regeneration rate, self-fertility, desirable tuber traits and is amenable to Agrobacterium-mediated transformation. We leveraged its amenability to Agrobacterium-mediated transformation to create a Cas9 constitutively expressing line for use in viral vector-based gene editing. To create a contiguous genome assembly, a homozygous doubled monoploid of 1S1 (DM1S1) was sequenced using 44 Gbp of long reads generated from Oxford Nanopore Technologies (ONT), yielding a 736 Mb assembly that encoded 31,145 protein-coding genes. The final assembly for DM1S1 represents a nearly complete genic space, shown by the presence of 99.6% of the genes in the Benchmarking Universal Single Copy Orthologs (BUSCO) set. Variant analysis with Illumina reads from 1S1 was used to deduce its alternate haplotype. These genetic and genomic resources provide a toolkit for applications of genome editing in both basic and applied research of potato.

Uncovering a miltiradiene biosynthetic gene cluster in the Lamiaceae reveals a dynamic evolutionary trajectory.

The spatial organization of genes within plant genomes can drive evolution of specialized metabolic pathways. Terpenoids are important specialized metabolites in plants with diverse adaptive functions that enable environmental interactions. Here, we report the genome assemblies of Prunella vulgaris, Plectranthus barbatus, and Leonotis leonurus. We investigate the origin and subsequent evolution of a diterpenoid biosynthetic gene cluster (BGC) together with other seven species within the Lamiaceae (mint) family. Based on core genes found in the BGCs of all species examined across the Lamiaceae, we predict a simplified version of this cluster evolved in an early Lamiaceae ancestor. The current composition of the extant BGCs highlights the dynamic nature of its evolution. We elucidate the terpene backbones generated by the Callicarpa americana BGC enzymes, including miltiradiene and the terpene (+)-kaurene, and show oxidization activities of BGC cytochrome P450s. Our work reveals the fluid nature of BGC assembly and the importance of genome structure in contributing to the origin of metabolites.

Leveraging prior biological knowledge improves prediction of tocochromanols in maize grain.

With an essential role in human health, tocochromanols are mostly obtained by consuming seed oils; however, the vitamin E content of the most abundant tocochromanols in maize (Zea mays L.) grain is low. Several large-effect genes with cis-acting variants affecting messenger RNA (mRNA) expression are mostly responsible for tocochromanol variation in maize grain, with other relevant associated quantitative trait loci (QTL) yet to be fully resolved. Leveraging existing genomic and transcriptomic information for maize inbreds could improve prediction when selecting for higher vitamin E content. Here, we first evaluated a multikernel genomic best linear unbiased prediction (MK-GBLUP) approach for modeling known QTL in the prediction of nine tocochromanol grain phenotypes (12-21 QTL per trait) within and between two panels of 1,462 and 242 maize inbred lines. On average, MK-GBLUP models improved predictive abilities by 7.0-13.6% when compared with GBLUP. In a second approach with a subset of 545 lines from the larger panel, the highest average improvement in predictive ability relative to GBLUP was achieved with a multi-trait GBLUP model (15.4%) that had a tocochromanol phenotype and transcript abundances in developing grain for a few large-effect candidate causal genes (1-3 genes per trait) as multiple response variables. Taken together, our study illustrates the enhancement of prediction models when informed by existing biological knowledge pertaining to QTL and candidate causal genes.

Toward product-based regulation of crops.

Current process-based approaches to regulation are no longer fit for purpose.