Carol Robin Buell

Poplar ( Populus spp.) is a model for tree biology and a platform for engineering woody biomass, biofuels, biomaterials, and bioproducts. Many relevant traits depend on tissue position, developmental stage, and cell type, yet these spatial relationships are difficult to recover from bulk or single-cell transcriptomes. Here, we generated a spatial transcriptome atlas of Populus tremula × P. alba INRA 717-1B4 across the shoot apex, axillary bud, stem, and petiole. After quality control, the atlas retained 29,687 spatial spots from 45 tissue sections and detected 58,748 genes. Histology-guided clustering and marker analysis resolved meristematic, epidermal, cortical, vascular, and organ-specific domains. Cross-organ comparisons assessed whether published markers retained tissue-associated expression across different anatomical contexts and developmental stages, while de novo analysis identified additional domain-enriched candidates. As case studies of the utility of the atlas, we examined the emergence of trichome-associated programs in the shoot apex and adaxial-abaxial expression differences in petioles. A trichome identity score based on poplar markers from the single-cell shoot atlas peaked along the inferred meristem-to-primordium trajectory, revealing spatially localized expression of trichome-associated programs during early leaf development. Petiole expression differences were concentrated in the epidermis and cortex and involved polarity-associated, auxin-responsive, and cell-wall-remodeling genes, with distinct expression profiles across leaf positions. Together, these data provide a spatial reference for investigating tissue differentiation and developmental patterning in a transformable poplar genotype.

Geophytes are plants that produce underground storage organs such as tubers, rhizomes, and bulbs, to facilitate asexual reproduction and withstand a myriad of environmental challenges. While the potato ( Solanum tuberosum L.) serves as the primary model for studying tuberization, the genetic mechanisms encoding this trait across diverse angiosperm lineages remains unclear. This study utilized a phylogenomic-transcriptomic approach to compare tuber development across nine tuberizing species with five nontuberizing sister taxa. We identified orthologs of key potato tuberization genes that exhibit similar expression in the stolons or tubers of these distant relatives. In nontuberizing species, these orthologs exhibit distinct expression profiles and are primarily expressed in the stem. This suggests that the independent evolution of tubers across angiosperms resulted from shifts in the expression of preexisting genes that led to their co-option. This process, also known as exaptation, occurs when existing genetic suites are recruited for entirely new biological functions. This mechanism stands in contrast to the repeated loss or gain of genes, which has been associated with the origin of other adaptive plant traits. Furthermore, the co-option of the same genes was observed in species with other stem-derived storage organs, such as rhizomes and runners. These findings reveal a conserved evolutionary model for the development of stem-derived geophyte organs that evolved independently across the flowering plants over the past 160 My.

Poplar (Populus spp.) is a model system for tree biology. Specifically, P. tremula x P. alba INRA 717-1B4 (hereafter poplar 717) has become an important platform for functional genomics and synthetic biology due to its rapid growth and ease of transgenesis. Here, we present a single-cell RNA-seq atlas of the poplar 717 shoot, including apical meristem, primary and secondary stems, and three stages of leaf development. Analysis of ca. 159,000 cells resolved 40 transcriptionally distinct clusters representing 7 major cell types, providing a high-resolution view of shoot development and tissue organization. We focused on the epidermis which constituted >15% of cells in the shoot atlas for in-depth characterization of epidermal heterogeneity. By integrating known marker genes with transcriptomic signatures consistent with established poplar leaf phytochemistry, we annotated epidermal cell subclusters corresponding to developmental stages, spatial location, and specialized cell types, including a distinct population of non-glandular trichomes. Coupling the single-cell RNA-seq atlas with bulk transcriptome data from glabrous mutants enabled the identification of novel trichome markers. Experimental validation of a representative trichome-specific promoter established a tool with potential to support cell type-targeted-metabolic engineering. We provide the poplar 717 atlas to the community through the BioPoplar Atlas Viewer (http://bio-poplar-atlas.com), providing a platform to explore the poplar transcriptome at single-cell resolution and a foundation for data-driven cell type-aware genetic engineering strategies in poplar.

Chromosome□scale genome engineering in plants typically relies on locus-specific recombination during meiosis to select desirable outcomes, limiting its application in species with long generation times. Here, we show that CRISPR targeting of abundant satellite DNA enables multiplexed chromosome restructuring in aspen, generating diverse large-scale structural variants in the first generation. These megabase rearrangements remain mitotically stable through clonal propagation and regeneration with no obvious growth defects.

SUMMARY: Plants produce specialized metabolites that function in plant defense and as attractants to pollinators and symbionts. One class of specialized metabolites are terpenoids that are synthesized from universal C ₅ building blocks via activities including terpene synthases, cytochromes P450, and glycosyl transferases. Some terpenes are highly valued for their use as insect repellants, fragrances, antimicrobial compounds, low calorie sweeteners, flavors, and medicines. Low abundance in target tissues, present in complex mixtures, as well as challenging extraction logistics are barriers to economic sustainable production of these compounds from their native species. While heterologous expression of terpenoid biosynthetic genes is feasible, the potential derivation of the products into conjugates via endogenous cytochromes P450 and glycosyl transferases limits this approach. In this project, we used multiplex gene editing technologies to overcome these challenges by creating novel tomato chassis with altered terpenoid biosynthetic capacity in fruit. Excluding central metabolic genes to minimalize impacts on growth and development, we selected 23 known and potential terpene-related genes expressed specifically in the fruit for gene editing. Fruit production and metabolic profiles of three chassis lines with alterations in the major classes of fruit specialized metabolites indicate loss of these genes is tolerated for fruit production. These combinatorial knockouts also showed modulation of native carbon reallocation toward endogenous sinks beneficial for a biosynthetic chassis. Establishing metabolite-modified fruit chassis demonstrates efficient combinatorial editing of entire branches of plant specialized metabolism, facilitating engineering of heterologous terpenes of industrial interest in tomato fruit.

Cinchona alkaloids, which have been studied for more than 250 years, are plant-derived natural products that have collectively had a substantial impact in medicine and basic science ^1–5 . Examples of cinchona alkaloids include quinine, a historically important antimalarial drug, and cinchonidine, a chiral catalyst widely used in process chemistry. However, it is still largely unknown how plants synthesize these well-known compounds. Here we report the discovery of genes responsible for the biosynthesis of the distinctive quinoline–quinuclidine scaffold of cinchona alkaloids. A combination of isotopic labelling, gene silencing, single-nucleus RNA sequencing and comparative transcriptomics revealed the involvement of several unexpected biosynthetic transformations. We also describe a previously unreported quaternary amine intermediate that is generated through an unusual enzymatic cyclization. We show that dihydroquini(di)none, dihydrocinchoni(di)none and cinchoni(di)none can be produced when these genes are heterologously expressed in Nicotiana benthamiana . Furthermore, we demonstrate that this N. benthamiana expression platform can convert non-native fluorinated and chlorinated tryptamine substrates into dihydrocinchoni(di)none analogues, which suggests that these biosynthetic enzymes can be leveraged to produce halogenated cinchona alkaloid derivatives. These discoveries uncover the long-standing mystery of how the cinchona alkaloid scaffold is biosynthesized and highlight prospects for access to these compounds through metabolic engineering approaches.

Fusarium oxysporum f. sp. batatas , the causal agent of Fusarium wilt disease, was once the most damaging pathogen of sweetpotato in the United States. Breeding for cultivar resistance has largely addressed this issue, however, little is known about the genetic basis for resistance. Historically, sweetpotato breeders have relied on the high heritability of Fusarium wilt resistance, so identification of a region controlling resistance would be a major first step in implementing marker‐assisted selection for this trait. We assayed a biparental mapping population, NCDM04‐0001 × ‘Covington’ (DC), consisting of a susceptible by resistant cross composed of 454 progenies, for resistance to Fusarium wilt disease using visual assessments and an ordinal disease severity rating scale. Parental and check lines performed as expected, and the DC population exhibited segregation for resistance across trials over 3 years and in a joint analysis. We next performed quantitative trait locus (QTL) analyses using a linkage map based on the Ipomoea trifida diploid reference genome. Across multiple trials, we repeatedly detected a major QTL on chromosome 10, herein named qIbFo‐10.1. This QTL had a heritability of 33.8%, suggesting that a single locus explains a large amount of variation for resistance to this critically important trait. A basic local alignment search tool revealed several candidate genes: itf10g19820 (transcriptional factor B3 family protein/auxin‐responsive factor AUX/IAA‐related), four LRR‐kinases (leucine‐rich repeat receptor kinase) (itf10g21910, itf10g19200, itf10g19260, and itf10g20000), and two toll‐interleukin‐resistance genes (itf10g20200 and itf10g20220). Future efforts should develop molecular tools for Fusarium wilt resistance breeding, resulting in shorter breeding cycles and faster variety releases.

Commercial potato ( Solanum tuberosum ) in North America is a clonal autotetraploid crop, which complicates breeding. Efforts are underway to convert potato to a diploid inbred‐hybrid crop, allowing breeders to more quickly meet market and environmental demands. With the goal of preserving haplotypes developed over 200 years of selection, diploid potato breeding in the United States started with the creation of diploids from tetraploid commercial varieties and advanced breeding lines through prickle pollination. This is an effective but slow method, which presents a barrier to entry for individual breeding programs. Therefore, we developed 97 publicly available dihaploids (diploids from prickle pollination of tetraploids) as a resource for diploid breeding in the United States. These clones contain the majority of alleles in the US breeding population for three market classes: chips, russets, and fresh market reds. To facilitate genomic informed breeding, all clones have been resequenced, and we have developed de novo assemblies for 20 individuals. As an illustration of how these data will be used in breeding, we explored the maturity locus ( StCDF1 ) and identified 15 different alleles. The majority of dihaploids were heterozygous for early and late alleles, resulting in intermediate maturity. Beyond informing breeding, these data facilitate investigations into potato genomics. The dihaploid population is both highly heterozygous and incredibly diverse on a population level. In particular, there is extensive structural diversity segregating within the population. This contrasts with a relatively low genome‐wide historical recombination rate ( ρ ), indicating that much of potato's high diversity is found within long linkage blocks.