The oleoresin defense system of loblolly pine (Pinus taeda) protects trees from insects and pathogens and is an important source of renewable biofuels and chemicals, but the genetic basis of oleoresin production is poorly understood. We characterized the genetic architecture of oleoresin flow, resin canal number, stem wood terpene content, and monoterpene composition in two clonal populations of P. taeda. We used quantitative genetic analyses, genome-wide association studies (GWASs), multiplex network learning, and gene expression profiling to elucidate shared gene networks underlying defense traits and to identify high-quality candidates for breeding and engineering loblolly pine. Genetic analyses revealed polygenic inheritance and trait-to-trait correlations provide strong evidence for shared genes regulating constitutive and induced oleoresin flow. We identified 236 single nucleotide polymorphisms associated with oleoresin flow, resin canal number, and terpene composition and highlight candidate genes likely involved in terpene biosynthesis, cambial meristem reprogramming, and pathogen perception and immune signaling. Fourteen GWAS candidates were methyl jasmonate-responsive in tissues where resin canals initiate and terpene production occurs. Integrating quantitative genetics, GWAS, gene expression, and multiplex network analyses enabled the prioritization of high-quality candidate genes. This work advances the development of more resilient loblolly pine optimized for ecological performance, renewable chemical, and biofuel production.

BACKGROUND:Drought significantly affects forests and woody crops by limiting their growth, increasing their susceptibility to diseases, and reducing productivity. Wood anatomical plasticity is a crucial adaptive mechanism that enables trees to cope with fluctuations in water availability. During severe drought, trees develop more and narrower vessels, enhancing hydraulic safety and reducing the risk of embolism. However, the molecular regulation of vessel formation is still not well understood. RESULTS:Using single-nucleus transcriptomics, we have generated a cell type-specific gene expression map of the mature poplar stem under well-watered and drought conditions. Our findings reveal extensive gene expression reprogramming in xylem-forming cells, with changes in auxin homeostasis identified as a key mechanism for anatomical adaptation. Specifically, we show that poplar WAT1-like genes control vessel spatial patterning. Additionally, the downregulation of WAT1-like gene expression in the dividing cells of the vascular cambium and the upregulation of MP-like gene expression in cells undergoing early vessel differentiation facilitate the formation of secondary xylem with narrower and more numerous vessels under drought. Furthermore, the wat2 mutant exhibits greater drought tolerance than wild-type trees, underscoring its potential for developing drought-resilient tree varieties. CONCLUSIONS:This study provides the first single-nucleus transcriptomic map of hybrid poplar stems under severe drought, uncovering auxin-driven hormonal networks that regulate xylem plasticity and enhance drought tolerance. These insights provide valuable targets for improving resilience in poplar and other woody species.

The extensive use of nitrogen fertilizers has detrimental environmental consequences, and it is essential for society to explore sustainable alternatives. One promising avenue is engineering root nodule symbiosis, a naturally occurring process in certain plant species within the nitrogen-fixing clade, into non-leguminous crops. Advancements in single-cell transcriptomics provide unprecedented opportunities to dissect the molecular mechanisms underlying root nodule symbiosis at the cellular level. This review summarizes key findings from single-cell studies in Medicago truncatula, Lotus japonicus, and Glycine max. We highlight how these studies address fundamental questions about the development of root nodule symbiosis, including the following findings: (i) single-cell transcriptomics has revealed a conserved transcriptional program in root hair and cortical cells during rhizobial infection, suggesting a common infection pathway across legume species; (ii) characterization of determinate and indeterminate nodules using single-cell technologies supports the compartmentalization of nitrogen fixation, assimilation, and transport into distinct cell populations; (iii) single-cell transcriptomics data have enabled the identification of novel root nodule symbiosis genes and provided new approaches for prioritizing candidate genes for functional characterization; and (iv) trajectory inference and RNA velocity analyses of single-cell transcriptomics data have allowed the reconstruction of cellular lineages and dynamic transcriptional states during root nodule symbiosis.

BACKGROUND:Single-cell genomics is revolutionizing plant developmental biology, enabling the transcriptome profiling of individual cells and their lineage relationships. However, plant cell walls polymers hamper the dissociation and analysis of intact cells. This rigid structure can conceal cell types embedded in complex, lignified, multi-cell layered tissues such as those undergoing secondary growth. Their absence leads to incomplete single-cell genomic atlases and lineage inferences. RESULTS:We isolate nuclei to capture transcripts representing the diversity of cells throughout the stem of the woody perennial Populus trichocarpa generating a high-resolution transcriptome atlas of cell types and lineage trajectories. RNA sequencing of 11,673 nuclei identifies 26 clusters representing cell types in the cambium, xylem, phloem, and periderm. Comparative analysis with protoplast-derived transcriptome data reveals significant biases, with nuclei-based sequencing providing a higher representation of cells in lignified inner xylem tissues. Among previously underrepresented types, we uncover vessel-associated cells (VAC), a largely uncharacterized parenchyma subtype and the terminus of a xylem cell lineage. Gene regulatory analysis identifies a VAC-specific network and the Populus MYB48 as its primary regulator. Functional validation of MYB48 knockout mutants show an increase in vessel number and size, pointing to a role of VACs in vessel development. CONCLUSIONS:Our study demonstrates the capture and transcriptome characterization of cell types embedded in plant secondary growth, identifying novel regulators of xylem development and stress adaptation. The discovery of MYB48 as a key regulator of VAC function highlights a previously uncharacterized mechanism influencing vessel development, with applications to improving wood formation and stress resilience.

Crassulacean acid metabolism (CAM) enhances drought tolerance by shifting carbon fixation to the night, improving water-use efficiency compared to C3 and C4 photosynthesis. However, the molecular regulators of CAM induction remain poorly understood. Here, we generate the first single-nucleus transcriptome atlas of a CAM species, Mesembryanthemum crystallinum , to resolve transcriptional dynamics at the cell-type level during the C3-to-CAM transition. Using snRNA-seq and a 24-hour time-course bulk RNA-seq dataset, we identify PPCK1 , a key CAM enzyme regulator, as part of a co-expression network enriched in circadian clock genes and salt-induced pathways. We demonstrate that the ice plant HY5 (McHY5) directly activates PPCK1 , a function absent in the C3 model species Arabidopsis thaliana . This discovery reveals a fundamental divergence in transcription factor activity between a CAM and a C3 species, suggesting that CAM evolution in M. crystallinum involved a rewiring of core regulatory elements underlying CAM. Identifying a transcription factor that directly controls a major CAM gene provides a key step toward decoding CAM regulatory architecture and opens new avenues for engineering drought-resilient crops. ### Competing Interest Statement The authors have declared no competing interest.

Objectives Escallonia (Escalloniaceae) belongs to the Escalloniales, a diverse clade of flowering plants with unclear placement in the tree of life. Escallonia species show impressive morphological and ecological diversity and are widely distributed across three hotspots of biodiversity in the Neotropics. To shed light on the genomic substrate of this radiation and the phylogenetic placement of Escalloniales as well as to generate useful data for comparative evolutionary genomics across flowering plants, we produced and annotated draft genomes for two species of Escallonia . Data description Genomic DNA from E. rubra and E. herrerae was sequenced with Oxford Nanopore sequencing chemistry, generating 3.4 and 12 million sequence reads with an average read length of 9.4 and 9.1 Kb (approximately 31 and 111 Gb of sequence data), respectively. In addition, we generated Illumina 100-bp paired-end short read data for E. rubra (approximately 75 Gb of sequence data). The Escallonia rubra genome was 566 Mb, with 3,233 contigs and an N50 of 285 Kb. The assembled genome for E. herrerae was 994 Mp, with 5,760 contigs and an N50 of 317 Kb. The genome sequences were annotated with 31,038 (E. rubra ) and 47,905 ( E. herrerea ) protein-coding gene models supported by transcriptome/protein evidence and/or Pfam domain content. BUSCO assessments indicated completeness levels of approximately 98% for the genome assemblies and 88% for the genome annotations.

ABSTRACTCrassulacean acid metabolism (CAM) is an evolutionary modification of the C3photosynthetic carbon dioxide fixation pathway used by approximately 7% of terrestrial plants to live in drought-prone environments. Facultative CAM species, such asMesembryanthemum crystallinum(common ice plant), possess the unique ability to switch from C3to CAM photosynthesis in response to high-salinity and water-deficit stress. Here we characterized the environmentally-triggered transition from C3to CAM in the ice plant using single nucleus RNA sequencing (snRNA-seq) to identify its putative regulators, supported by a novel high-quality assembled and annotated genome. Analysis of snRNA-seq datasets from ice plant leaves transitioning between C3and CAM collected at dawn and dusk revealed substantial transcriptional changes in mesophyll cells at the onset of CAM induction. Notably, our findings identify mesophyll sub-cell types engaged in either CAM or C3photosynthesis at dusk. Cell trajectory inference analysis reconstructed both 24-hour CAM and C3cycles, enabling a direct comparison of gene expression profiles in these pathways. This comparative study uncovered divergent expression patterns of key circadian clock genes in CAM and C3cell trajectories, pointing to a connection between circadian regulation and CAM induction.

Legumes establish a symbiotic relationship with nitrogen-fixing rhizobia by developing nodules. Nodules are modified lateral roots that undergo changes in their cellular development in response to bacteria, but the transcriptional reprogramming that occurs in these root cells remains largely uncharacterized. Here, we describe the cell-type-specific transcriptome response of Medicago truncatula roots to rhizobia during early nodule development in the wild-type genotype Jemalong A17, complemented with a hypernodulating mutant (sunn-4) to expand the cell population responding to infection and subsequent biological inferences. The analysis identifies epidermal root hair and stele sub-cell types associated with a symbiotic response to infection and regulation of nodule proliferation. Trajectory inference shows cortex-derived cell lineages differentiating to form the nodule primordia and, posteriorly, its meristem, while modulating the regulation of phytohormone-related genes. Gene regulatory analysis of the cell transcriptomes identifies new regulators of nodulation, including STYLISH 4, for which the function is validated.

ABSTRACTNitrogen is one of the most inaccessible plant nutrients, but certain species have overcome this limitation by establishing symbiotic interactions with nitrogen-fixing bacteria in the root nodule. This root nodule symbiosis (RNS) is restricted to species within a single clade of angiosperms, suggesting a critical evolutionary event at the base of this clade, which has not yet been determined. While genes implicated in the RNS are present in most plant species (nodulating or not), gene sequence conservation alone does not imply functional conservation – developmental or phenotypic differences can arise from variation in the regulation of transcription. To identify putative regulatory sequences implicated in the evolution of RNS, we aligned the genomes of 25 species capable of nodulation. We detected 3,091 conserved noncoding sequences (CNS) in the nitrogen-fixing clade that are absent from outgroup species. Functional analysis revealed that chromatin accessibility of 452 CNS significantly correlates with the differential regulation of genes responding to lipo-chitooligosaccharides inMedicago truncatula. These included 38 CNS in proximity to 19 known genes involved in RNS. Five such regions are upstream ofMtCRE1,Cytokinin Response Element 1,required to activate a suite of downstream transcription factors necessary for nodulation inM. truncatula. Genetic complementation of aMtcre1mutant showed a significant association between nodulation and the presence of these CNS, when they are driving the expression of a functional copy ofMtCRE1. Conserved noncoding sequences, therefore, may be required for the regulation of genes controlling the root nodule symbiosis inM. truncatula.

Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.

Background Symbiotic associations between bacteria and leguminous plants lead to the formation of root nodules that fix nitrogen needed for sustainable agricultural systems. Symbiosis triggers extensive genome and transcriptome remodeling in the plant, yet an integrated understanding of the extent of chromatin changes and transcriptional networks that functionally regulate gene expression associated with symbiosis remains poorly understood. In particular, analyses of early temporal events driving this symbiosis have only captured correlative relationships between regulators and targets at mRNA level. Here, we characterize changes in transcriptome and chromatin accessibility in the model legume Medicago truncatula , in response to rhizobial signals that trigger the formation of root nodules. Results We profiled the temporal chromatin accessibility (ATAC-seq) and transcriptome (RNA-seq) dynamics of M. truncatula roots treated with bacterial small molecules called lipo-chitooligosaccharides that trigger host symbiotic pathways of nodule development. Using a novel approach, dynamic regulatory module networks, we integrated ATAC-seq and RNA-seq time courses to predict cis -regulatory elements and transcription factors that most significantly contribute to transcriptomic changes associated with symbiosis. Regulators involved in auxin (IAA4-5, SHY2), ethylene (EIN3, ERF1), and abscisic acid (ABI5) hormone response, as well as histone and DNA methylation (IBM1), emerged among those most predictive of transcriptome dynamics. RNAi-based knockdown of EIN3 and ERF1 reduced nodule number in M. truncatula validating the role of these predicted regulators in symbiosis between legumes and rhizobia. Conclusions Our transcriptomic and chromatin accessibility datasets provide a valuable resource to understand the gene regulatory programs controlling the early stages of the dynamic process of symbiosis. The regulators identified provide potential targets for future experimental validation, and the engineering of nodulation in species is unable to establish that symbiosis naturally.

ABSTRACT Differentiation of stem cells in the plant apex gives rise to aerial tissues and organs. Presently, we lack a lineage map of the shoot apex cells in woody perennials – a crucial gap considering their role in determining primary and secondary growth. Here, we used single-nuclei RNA-sequencing to determine cell type-specific transcriptomes of the Populus vegetative shoot apex. We identified highly heterogeneous cell populations clustered into seven broad groups represented by 18 transcriptionally distinct cell clusters. Next, we established the developmental trajectories of the epidermis, leaf mesophyll and vascular tissue. Motivated by the high similarities between Populus and Arabidopsis cell population in the vegetative apex, we applied a pipeline for interspecific single-cell gene expression data integration. We contrasted the developmental trajectories of primary phloem and xylem formation in both species, establishing the first comparison of vascular development between a model annual herbaceous and a woody perennial plant species. Our results offer a valuable resource for investigating the principles underlying cell division and differentiation conserved between herbaceous and perennial species while also allowing us to examine species-specific differences at single-cell resolution.

Premise Commonly used molecular techniques such as next‐generation sequencing require reliable methods to extract DNA quickly and efficiently. Secondary compounds within plant tissues make this requirement all the more challenging, often forcing researchers to test different extraction methods tailored to their particular species of interest in order to obtain large amounts of high‐quality genomic DNA. The opportunities provided by high‐throughput, next‐generation sequencing only exacerbate these problems, especially when trying to extract DNA from multiple species at the same time. Several methods have attempted to resolve the challenges of obtaining suitable DNA from plants; however, a rapid, high‐yield, high‐quality, and highly scalable DNA extraction method is still needed. Methods and Results We present a rapid DNA extraction protocol that utilizes a buffer with relatively large amounts of cetyltrimethylammonium bromide (CTAB) and sodium chloride, combined with a silica maxi‐column cleanup of the extracted DNA. The new method is easy to implement using standard equipment and inexpensive reagents. The entire procedure (from grinding to the final elution) can be completed in less than two hours for a single sample and can be easily scaled to meet desired research goals. It works on diverse green plants with highly varied secondary chemistries (e.g., ferns, gymnosperms, and phylogenetically divergent angiosperms). Conclusions Application of the protocol to various plant species yielded DNA of high quality in less than two hours and can be adjusted to extract DNA at large (maxi‐preps) or small (96‐well minipreps) scales. We anticipate that our method will be of wide utility for rapidly isolating large quantities of quality genomic DNA from diverse plant species and will have broad applications in phylogenetic studies utilizing PCR and short‐read DNA sequencing.

Most legumes can establish a symbiotic association with soil rhizobia that triggers the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharide (LCO) signal in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) acts as an essential positive regulator of nodule organogenesis, and specific CK receptors are required for nodule formation. Temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In the present study, using a fluorescence-based CK sensor ( TCSn::nls:tGFP ), we performed a high-resolution tissue-specific temporal characterization of the CK response’s sequential activation during root infection and nodule development in M. truncatula after inoculation with S. meliloti . Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYL TRANSFERASE 3 ( IPT3 ) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula . Simultaneous live imaging of pIPT3::tdTOMATO and the CK sensor showed that IPT3 induction in the root stele at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula . One-sentence summary High-resolution spatiotemporal imaging of cytokinin signaling reveals IPT3 function during indeterminate nodule development in Medicago truncatula

Despite the enormous potential of novel approaches to explore gene expression at a single-cell level, we lack a high-resolution and cell type-specific gene expression map of the shoot apex in woody perennials. We use single-nuclei RNA sequencing to determine the cell type-specific transcriptome of the Populus vegetative shoot apex. We identified highly heterogeneous cell populations clustered into seven broad groups represented by 18 transcriptionally distinct cell clusters. Next, we established the developmental trajectories of epidermal cells, leaf mesophyll, and vascular tissue. Motivated by the high similarities between Populus and Arabidopsis cell population in the vegetative apex, we created and applied a pipeline for interspecific single-cell expression data integration. We contrasted the developmental trajectories of primary phloem and xylem formation in both species, establishing the first comparison of primary vascular development between a model annual herbaceous and a woody perennial plant species. Our results offer a valuable resource for investigating the basic principles underlying cell division and differentiation conserved between herbaceous and perennial species, which also allows the evaluation of the divergencies at single-cell resolution.

Prenylated benzoquinones are essential cofactors for the core bioenergetic processes in complex living organisms. Recently, we discovered that the gene product of rquA, a protein first discovered in Rhodospirillum rubrum, can convert ubiquinone to rhodoquinone, a rare prenylated benzoquinone. The presence of rhodoquinone in mitochondria facilitates anaerobic respiration by redirecting electrons in the respiratory chain toward the final electron acceptor fumarate. After reduction by complex I, rhodoquinol delivers electrons via fumarate reductase to fumarate. We predict that the production of rhodoquinone in plant mitochondria will confer the trait of anaerobic respiration providing a novel, synthetic biology approach to design flood tolerant agricultural plants. So far, I have discovered that a codon optimized, mitochondrially targeted RquA, under an inducible dexamethasone promoter, is able to convert ubiquinone-9 to rhodoquinone-9 in Arabidopsis. While this strategy has so far yielded an approximate 1% conversion rate of native ubiquinone to rhodoquinone, there have been no phenotypic impacts observed in plants where synthetic rhodoquinone production is occurring. One important conclusion from these results is that synthetic rhodoquinone production in plants appears to be nonlethal. These findings provide an impetus to continue to explore the synthetic production of rhodoquinone as a strategy for designing flood tolerant crops.

Single-cell transcriptome analysis has been extensively applied in humans and animal models to uncover gene expression heterogeneity between the different cell types of a tissue or an organ. It demonstrated its capability to discover key regulatory elements that determine cell fate during developmental programs. Single-cell analysis requires the isolation and labeling of the messenger RNA (mRNA) derived from each cell. These challenges were primarily addressed in mammals by developing microfluidic-based approaches. For plant species whose cells contain cell walls, these approaches have generally required the generation of isolated protoplasts. Many plant tissues’ secondary cell wall hinders enzymatic digestion required for individual protoplast isolation, resulting in an unequal representation of cell types in a protoplast population. This limitation is especially critical for cell types located in the inner layers of a tissue or the inner tissues of an organ. Consequently, single-cell RNA sequencing (scRNA-seq) studies using microfluidic approaches in plants have mainly been restricted to Arabidopsis roots, for which well-established procedures of protoplast isolation are available. Here we present a simple alternative approach to generating high-quality protoplasts from plant tissue by characterizing the mRNA extracted from individual nuclei instead of whole cells. We developed the protocol using two different plant materials with varying cellular complexity levels and cell wall structure, Populus shoot apices, and more lignified stems. Using the 10× Genomics Chromium technology, we show that this procedure results in intact mRNA isolation and limited leakage, with a broad representation of individual cell transcriptomes.

Running title: Sequence conservation in the nitrogen-fixing clade 4 5 Wendell J. Pereira, Sara Knaack, Daniel Conde, Sanhita Chakraborty, Ryan A. Folk, Paolo 6 M. Triozzi, Kelly M. Balmant, Christopher Dervinis, Henry W. Schmidt, Jean-Michel Ané, 7 Sushmita Roy, Matias Kirst. 8 9 1 School of Forest, Fisheries and Geomatics Sciences, University of Florida, Gainesville, FL 10 32611, USA. 11 2 Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison, 12 Madison, WI 53715, USA. 13 3 Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA. 14 4 Department of Biological Sciences, Mississippi State University, Starkville, MS 39762, USA. 15 5 Department of Agronomy, University of Wisconsin-Madison, Madison, WI 53706, USA. 16 6 Genetics Institute, University of Florida, Gainesville, FL 32611, USA. 17 18

Although the CRISPR/Cas9 system has been successfully used for crop breeding, its application remains limited in forest trees. Here, we describe an efficient gene editing strategy for hybrid poplar, (Populus tremula × alba INRA clone 717-1B4) based on the Golden Gate MoClo cloning. To test the system efficiency for generating single gene mutants, two single guide RNAs (sgRNAs) were designed and incorporated into the MoClo Tool Kit level 2 binary vector with the Cas9 expression cassette to mutate the SHORT ROOT (SHR) gene. Moreover, we also tested its efficiency for introducing mutations in two genes simultaneously by expressing one sgRNA targeting a single site of the YUC4 gene and the other sgRNA targeting the PLT1 gene. For a robust evaluation of the approach, we repeated the strategy to target the LBD12 and LBD4 genes simultaneously, using an independent construct. We generated hairy roots by Agrobacterium rhizogenes-mediated leaf transformation. Sequencing results confirmed the CRISPR/Cas9-mediated mutation in the targeted sites of PtaSHR. Biallelic and homozygous knockout mutations were detected. A deletion spanning both target sites and small insertions/deletions were the most common mutations. Out of the 22 SHR alleles sequenced, 21 were mutated. The phenotype's characterization showed that transgenic roots with biallelic mutations for the SHR gene lacked a defined endodermal single cell layer, suggesting a conserved gene function similar to its homolog in Arabidopsis Arabidopsis thaliana (L.) Heynh. Sequencing results also revealed the high efficiency of the system for generating double mutants. Biallelic mutations for both genes in the yuc4/plt1 and lbd12/lbd4 roots were detected in three (yuc4/plt1) and two (lbd12/lbd4) out of four transgenic roots evaluated. A small deletion or a single nucleotide insertion at the single target site was the most common mutations. This CRISPR/Cas9 strategy arises as a rapid, simple and standardized gene-editing tool to evaluate the gene role in essential developmental programs such as radial cell differentiation of poplar roots.