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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.

The study of plant biology has traditionally focused on investigations conducted at the tissue, organ, or whole plant level. However, single-cell transcriptomics has recently emerged as an important tool for plant biology, enabling researchers to uncover the expression profiles of individual cell types within a tissue. The application of this tool has revealed new insights into cell-to-cell gene expression heterogeneity and has opened new avenues for research in plant biology. A critical step in the successful application of single-cell and single-nuclei RNA-seq (scRNA-seq and snRNA-seq) is the isolation of individual cells or nuclei, respectively, from tissue to recover their transcriptional profile. A critical step during nuclei isolation for snRNA-seq studies is Fluorescent-Activated Cell Sorting (FACS). During this step, nuclei stained with DAPI (4′,6-diamidino-2-phenylindole) can be sorted and separated from cell debris and organelles. Leaf tissue presents a unique challenge due to its high content of chloroplasts, which can interfere with obtaining high-quality results. Because DAPI can also bind to the plastid genome, these organelles will be sorted as nuclei. Thus, in tissues with a high content of chloroplasts, we have a high contamination of these organelles and an overestimation of the number of nuclei. In this study, we introduce a straightforward alternative method for isolating nuclei from Zea mays leaves with reduced chloroplast contamination. By effectively removing chloroplasts during the FACS step of our protocol, using the autofluorescence from the chloroplasts, we achieved improved alignment of reads to the genome and transcriptome. Our enhanced protocol offers a valuable solution for applying snRNA-seq in tissues with a high content of chloroplasts.

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.

Breeding forest species can be a costly and slow process because of the extensive areas needed for field trials and the long periods (e.g., five years) that are required to measure economically and environmentally relevant phenotypes (e.g., adult plant biomass or plant height). Genomic selection (GS) and indirect selection using early phenotypes (e.g., phenotypes collected in greenhouse conditions) are two ways by which tree breeding can be accelerated. These approaches can both reduce the costs of field‐testing and the time required to make selection decisions. Moreover, these approaches can be highly synergistic. Therefore, in this study, we used a data set comprising DNA genotypes and longitudinal measurements of growth collected from a population of Populus deltoides W. Bartram ex Marshall (eastern cottonwood) in the greenhouse and the field, to evaluate the potential impact of integrating large‐scale greenhouse phenotyping with conventional GS. We found that the integration of greenhouse phenotyping and GS can deliver very early selection decisions that are moderately accurate. Therefore, we conclude that the adoption of these approaches, in conjunction with reproductive techniques that shorten the generation interval, can lead to an unprecedented acceleration of selection gains in P. deltoides and, potentially, other commercially planted tree species.

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.

Alternative splicing (AS) is a mechanism of regulation of the proteome via enabling the production of multiple mRNAs from a single gene. To date, the dynamics of AS and its effects on the protein sequences of individuals in a large and genetically unrelated population of trees have not been investigated. Here we describe the diversity of AS events within a previously genotyped population of 268 individuals of and their putative downstream functional effects. Using a robust bioinformatics pipeline, the AS events and resulting transcript isoforms were discovered and quantified for each individual in the population. Analysis of the AS revealed that, as expected, most AS isoforms are conserved. However, we also identified a substantial collection of new, unannotated splice junctions and transcript isoforms. Heritability estimates for the expression of transcript isoforms showed that approximately half of the isoforms are heritable. The genetic regulators of these AS isoforms and splice junction usage were then identified using a genome-wide association analysis. The expression of AS isoforms was predominately regulated while splice junction usage was generally regulated in . Additionally, we identified 696 genes encoding alternatively spliced isoforms that changed putative protein domains relative to the longest protein coding isoform of the gene, and 859 genes exhibiting this same phenomenon relative to the most highly expressed isoform. Finally, we found that 748 genes gained or lost micro-RNA binding sites relative to the longest protein coding isoform of a given gene, while 940 gained or lost micro-RNA binding sites relative to the most highly expressed isoform. These results indicate that a significant fraction of AS events are genetically regulated and that this isoform usage can result in protein domain architecture changes.

The radiation of angiosperms led to the emergence of the vast majority of today’s plant species and all our major food crops. Their extraordinary diversification occurred in conjunction with the evolution of a more efficient vascular system for the transport of water, composed of vessel elements. The physical dimensions of these water-conducting specialized cells have played a critical role in angiosperm evolution; they determine resistance to water flow, influence photosynthesis rate, and contribute to plant stature. However, the genetic factors that determine their dimensions are unclear. Here we show that a previously uncharacterized gene, ENLARGED VESSEL ELEMENT (EVE), contributes to the dimensions of vessel elements in Populus, impacting hydraulic conductivity. Our data suggest that EVE is localized in the plasma membrane and is involved in potassium uptake of differentiating xylem cells during vessel development. In plants, EVE first emerged in streptophyte algae, but expanded dramatically among vessel-containing angiosperms. The phylogeny, structure and composition of EVE indicates that it may have been involved in an ancient horizontal gene-transfer event.

Climate change as a result of increasing atmospheric CO affects plant growth and productivity. CO is not only a carbon donor for photosynthesis but also an environmental signal that can perturb cellular redox homeostasis and lead to modifications of redox-sensitive proteins. Although redox regulation of protein functions has emerged as an important mechanism in several biological processes, protein redox modifications and how they function in plant CO response remain unclear. Here a new iodoTMTRAQ proteomics technology was employed to analyze changes in protein redox modifications in suspension cells in response to bicarbonate (mimic of elevated CO ) in a time-course study. A total of 47 potential redox-regulated proteins were identified with functions in carbohydrate and energy metabolism, transport, ROS scavenging, cell structure modulation and protein turnover. This inventory of previously unknown redox responsive proteins in Arabidopsis bicarbonate responses lays a foundation for future research toward understanding the molecular mechanisms underlying plant CO responses.

Characterization of three DUF89 subfamilies representing all domains of life reveals that members function as metal-dependent phosphatases that help eliminate a wide range of damaged phosphometabolites. DUF89 family proteins occur widely in both prokaryotes and eukaryotes, but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), with subfamily II being split into stand-alone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metal-dependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S -sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast ( Saccharomyces cerevisiae ) subfamily III protein YMR027W revealed a novel phosphatase active site with fructose 6-phosphate and Mg 2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.