Best practices for the execution, analysis, and data storage of plant single-cell/nucleus transcriptomics.

Single-cell and single-nucleus RNA-sequencing technologies capture the expression of plant genes at an unprecedented resolution. Therefore, these technologies are gaining traction in plant molecular and developmental biology for elucidating the transcriptional changes across cell types in a specific tissue or organ, upon treatments, in response to biotic and abiotic stresses, or between genotypes. Despite the rapidly accelerating use of these technologies, collective and standardized experimental and analytical procedures to support the acquisition of high-quality data sets are still missing. In this commentary, we discuss common challenges associated with the use of single-cell transcriptomics in plants and propose general guidelines to improve reproducibility, quality, comparability, and interpretation and to make the data readily available to the community in this fast-developing field of research.

DIRT/µ - Automated extraction of root hair traits using combinatorial optimization.

Similar to any microscopic appendages, such as cilia or antennae, phenotyping of root hairs has been a challenge due to their complex intersecting arrangements in two-dimensional (2D) images and the technical limitations of automated measurements. Digital Imaging of Root Traits at Microscale (DIRT/µ) addresses this issue by computationally resolving intersections and extracting individual root hairs from 2D microscopy images. This solution enables automatic and precise trait measurements of individual root hairs. DIRT/µ rigorously defines a set of rules to resolve intersecting root hairs and minimizes a newly designed cost function to combinatorically identify each root hair in the microscopy image. As a result, DIRT/µ accurately measures traits such as root hair length (RHL) distribution and root hair density (RHD), which are impractical for manual assessment. We tested DIRT/µ on three datasets to validate its performance and showcase potential applications. By measuring root hair traits in a fraction of the time manual methods require, DIRT/µ eliminates subjective biases from manual measurements. Automating individual root hair extraction accelerates phenotyping and quantifies trait variability within and among plants, creating new possibilities to characterize root hair function and their underlying genetics.

A comparative genomic and transcriptomic analysis at the level of isolated root hair cells reveals new conserved root hair regulatory elements.

KEY MESSAGE: A comparative transcriptomic and genomic analysis between Arabidopsis thaliana and Glycine max root hair genes reveals the evolution of the expression of plant genes after speciation and whole genome duplication. Our understanding of the conservation and divergence of the expression patterns of genes between plant species is limited by the quality of the genomic and transcriptomic resources available. Specifically, the transcriptomes generated from plant organs are the reflection of the contribution of the different cell types composing the samples weighted by their relative abundances in the sample. These contributions can vary between plant species leading to the generation of datasets which are difficult to compare. To gain a deeper understanding of the evolution of gene transcription in and between plant species, we performed a comparative transcriptomic and genomic analysis at the level of one single plant cell type, the root hair cell, and between two model plants: Arabidopsis (Arabidopsis thaliana) and soybean (Glycine max). These two species, which diverged 90 million years ago, were selected as models based on the large amount of genomic and root hair transcriptomic information currently available. Our analysis revealed in detail the transcriptional divergence and conservation between soybean paralogs (i.e., the soybean genome is the product of two successive whole genome duplications) and between Arabidopsis and soybean orthologs in this single plant cell type. Taking advantage of this evolutionary study, we combined bioinformatics, molecular, cellular and microscopic tools to characterize plant promoter sequences and the discovery of two root hair regulatory elements (RHE1 and RHE2) consistently and specifically active in plant root hair cells.

The GmFWL1 (FW2-2-like) nodulation gene encodes a plasma membrane microdomain-associated protein.

The soybean gene GmFWL1 (FW2-2-like1) belongs to a plant-specific family that includes the tomato FW2-2 and the maize CNR1 genes, two regulators of plant development. In soybean, GmFWL1 is specifically expressed in root hair cells in response to rhizobia and in nodules. Silencing of GmFWL1 expression significantly reduced nodule numbers supporting its role during soybean nodulation. While the biological role of GmFWL1 has been described, its molecular function and, more generally, the molecular function of plant FW2-2-like proteins is unknown. In this study, we characterized the role of GmFWL1 as a membrane microdomain-associated protein. Specifically, using biochemical, molecular and cellular methods, our data show that GmFWL1 interacts with various proteins associated with membrane microdomains such as remorin, prohibitins and flotillins. Additionally, comparative genomics revealed that GmFWL1 interacts with GmFLOT2/4 (FLOTILLIN2/4), the soybean ortholog to Medicago truncatula FLOTILLIN4, a major regulator of the M. truncatula nodulation process. We also observed that, similarly to MtFLOT4 and GmFLOT2/4, GmFWL1 was localized at the tip of the soybean root hair cells in response to rhizobial inoculation supporting the early function of GmFWL1 in the rhizobium infection process.

Identification and functional characterization of soybean root hair microRNAs expressed in response to Bradyrhizobium japonicum infection.

Three soybean [Glycine max (L) Merr.] small RNA libraries were generated and sequenced using the Illumina platform to examine the role of miRNAs during soybean nodulation. The small RNA libraries were derived from root hairs inoculated with Bradyrhizobium japonicum (In_RH) or mock-inoculated with water (Un_RH), as well as from the comparable inoculated stripped root samples (i.e. inoculated roots with the root hairs removed). Sequencing of these libraries identified a total of 114 miRNAs, including 22 novel miRNAs. A comparison of miRNA abundance among the 114 miRNAs identified 66 miRNAs that were differentially expressed between root hairs and stripped roots, and 48 miRNAs that were differentially regulated in infected root hairs in response to B. japonicum when compared to uninfected root hairs (P ≤ 0.05). A parallel analysis of RNA ends (PARE) library was constructed and sequenced to reveal a total of 405 soybean miRNA targets, with most predicted to encode transcription factors or proteins involved in protein modification, protein degradation and hormone pathways. The roles of gma-miR4416 and gma-miR2606b during nodulation were further analysed. Ectopic expression of these two miRNAs in soybean roots resulted in significant changes in nodule numbers. miRNA target information suggested that gma-miR2606b regulates a Mannosyl-oligosaccharide 1, 2-alpha-mannosidase gene, while gma-miR4416 regulates the expression of a rhizobium-induced peroxidase 1 (RIP1)-like peroxidase gene, GmRIP1, during nodulation.

A Comparative Epigenomic Analysis of Polyploidy-Derived Genes in Soybean and Common Bean.

Soybean (Glycine max) and common bean (Phaseolus vulgaris) share a paleopolyploidy (whole-genome duplication [WGD]) event, approximately 56.5 million years ago, followed by a genus Glycine-specific polyploidy, approximately 10 million years ago. Cytosine methylation is an epigenetic mark that plays an important role in the regulation of genes and transposable elements (TEs); however, the role of DNA methylation in the fate/evolution of genes following polyploidy and speciation has not been fully explored. Whole-genome bisulfite sequencing was used to produce nucleotide resolution methylomes for soybean and common bean. We found that, in soybean, CG body-methylated genes were abundant in WGD genes, which were, on average, more highly expressed than single-copy genes and had slower evolutionary rates than unmethylated genes, suggesting that WGD genes evolve more slowly than single-copy genes. CG body-methylated genes were also enriched in shared single-copy genes (single copy in both species) that may be responsible for the broad and high expression patterns of this class of genes. In addition, diverged methylation patterns in non-CG contexts between paralogs were due mostly to TEs in or near genes, suggesting a role for TEs and non-CG methylation in regulating gene expression post polyploidy. Reference methylomes for both soybean and common bean were constructed, providing resources for investigating epigenetic variation in legume crops. Also, the analysis of methylation patterns of duplicated and single-copy genes has provided insights into the functional consequences of polyploidy and epigenetic regulation in plant genomes.

Genome sequence of the palaeopolyploid soybean.

Soybean (Glycine max) is one of the most important crop plants for seed protein and oil content, and for its capacity to fix atmospheric nitrogen through symbioses with soil-borne microorganisms. We sequenced the 1.1-gigabase genome by a whole-genome shotgun approach and integrated it with physical and high-density genetic maps to create a chromosome-scale draft sequence assembly. We predict 46,430 protein-coding genes, 70% more than Arabidopsis and similar to the poplar genome which, like soybean, is an ancient polyploid (palaeopolyploid). About 78% of the predicted genes occur in chromosome ends, which comprise less than one-half of the genome but account for nearly all of the genetic recombination. Genome duplications occurred at approximately 59 and 13 million years ago, resulting in a highly duplicated genome with nearly 75% of the genes present in multiple copies. The two duplication events were followed by gene diversification and loss, and numerous chromosome rearrangements. An accurate soybean genome sequence will facilitate the identification of the genetic basis of many soybean traits, and accelerate the creation of improved soybean varieties.

Xyloglucan, galactomannan, glucuronoxylan, and rhamnogalacturonan I do not have identical structures in soybean root and root hair cell walls.

MAIN CONCLUSION: Chemical analyses and glycome profiling demonstrate differences in the structures of the xyloglucan, galactomannan, glucuronoxylan, and rhamnogalacturonan I isolated from soybean ( Glycine max ) roots and root hair cell walls. The root hair is a plant cell that extends only at its tip. All other root cells have the ability to grow in different directions (diffuse growth). Although both growth modes require controlled expansion of the cell wall, the types and structures of polysaccharides in the walls of diffuse and tip-growing cells from the same plant have not been determined. Soybean (Glycine max) is one of the few plants whose root hairs can be isolated in amounts sufficient for cell wall chemical characterization. Here, we describe the structural features of rhamnogalacturonan I, rhamnogalacturonan II, xyloglucan, glucomannan, and 4-O-methyl glucuronoxylan present in the cell walls of soybean root hairs and roots stripped of root hairs. Irrespective of cell type, rhamnogalacturonan II exists as a dimer that is cross-linked by a borate ester. Root hair rhamnogalacturonan I contains more neutral oligosaccharide side chains than its root counterpart. At least 90% of the glucuronic acid is 4-O-methylated in root glucuronoxylan. Only 50% of this glycose is 4-O-methylated in the root hair counterpart. Mono O-acetylated fucose-containing subunits account for at least 60% of the neutral xyloglucan from root and root hair walls. By contrast, a galacturonic acid-containing xyloglucan was detected only in root hair cell walls. Soybean homologs of the Arabidopsis xyloglucan-specific galacturonosyltransferase are highly expressed only in root hairs. A mannose-rich polysaccharide was also detected only in root hair cell walls. Our data demonstrate that the walls of tip-growing root hairs cells have structural features that distinguish them from the walls of other roots cells.

Identification of microRNAs and their mRNA targets during soybean nodule development: functional analysis of the role of miR393j-3p in soybean nodulation.

Plant microRNAs (miRNAs) play important regulatory roles in a number of developmental processes. The present work investigated the roles of miRNAs during nodule development in the crop legume soybean (Glycine max). Fifteen soybean small RNA libraries were sequenced from different stages of nodule development, including young nodules, mature nodules and senescent nodules. In order to identify the regulatory targets of the miRNAs, five parallel analysis of RNA ends (PARE) libraries were also sequenced from the same stages of nodule development. Sequencing identified 284 miRNAs, including 178 novel soybean miRNAs. Analysis of miRNA abundance identified 139 miRNAs whose expression was significantly regulated during nodule development, including 12 miRNAs whose expression changed > 10-fold. Analysis of the PARE libraries identified 533 miRNA targets, including three nodulation-related genes and eight nodule-specific genes. miR393j-3p was selected for detailed analysis as its expression was significantly regulated during nodule formation, and it targeted a nodulin gene, Early Nodulin 93 (ENOD93). Strong, ectopic expression of miR393j-3p, as well as RNAi silencing of ENOD93 expression, significantly reduced nodule formation. The data indicate that miR393j-3p regulation of ENOD93 mRNA abundance is a key control point for soybean nodule formation.

The fate of duplicated genes in a polyploid plant genome.

Polyploidy is generally not tolerated in animals, but is widespread in plant genomes and may result in extensive genetic redundancy. The fate of duplicated genes is poorly understood, both functionally and evolutionarily. Soybean (Glycine max L.) has undergone two separate polyploidy events (13 and 59 million years ago) that have resulted in 75% of its genes being present in multiple copies. It therefore constitutes a good model to study the impact of whole-genome duplication on gene expression. Using RNA-seq, we tested the functional fate of a set of approximately 18 000 duplicated genes. Across seven tissues tested, approximately 50% of paralogs were differentially expressed and thus had undergone expression sub-functionalization. Based on gene ontology and expression data, our analysis also revealed that only a small proportion of the duplicated genes have been neo-functionalized or non-functionalized. In addition, duplicated genes were often found in collinear blocks, and several blocks of duplicated genes were co-regulated, suggesting some type of epigenetic or positional regulation. We also found that transcription factors and ribosomal protein genes were differentially expressed in many tissues, suggesting that the main consequence of polyploidy in soybean may be at the regulatory level.

Quantitative phosphoproteomic analysis of soybean root hairs inoculated with Bradyrhizobium japonicum.

Root hairs are single hair-forming cells on roots that function to increase root surface area, enhancing water and nutrient uptake. In leguminous plants, root hairs also play a critical role as the site of infection by symbiotic nitrogen fixing rhizobia, leading to the formation of a novel organ, the nodule. The initial steps in the rhizobia-root hair infection process are known to involve specific receptor kinases and subsequent kinase cascades. Here, we characterize the phosphoproteome of the root hairs and the corresponding stripped roots (i.e. roots from which root hairs were removed) during rhizobial colonization and infection to gain insight into the molecular mechanism of root hair cell biology. We chose soybean (Glycine max L.), one of the most important crop plants in the legume family, for this study because of its larger root size, which permits isolation of sufficient root hair material for phosphoproteomic analysis. Phosphopeptides derived from root hairs and stripped roots, mock inoculated or inoculated with the soybean-specific rhizobium Bradyrhizobium japonicum, were labeled with the isobaric tag eight-plex iTRAQ, enriched using Ni-NTA magnetic beads and subjected to nanoRPLC-MS/MS1 analysis using HCD and decision tree guided CID/ETD strategy. A total of 1625 unique phosphopeptides, spanning 1659 nonredundant phosphorylation sites, were detected from 1126 soybean phosphoproteins. Among them, 273 phosphopeptides corresponding to 240 phosphoproteins were found to be significantly regulated (>1.5-fold abundance change) in response to inoculation with B. japonicum. The data reveal unique features of the soybean root hair phosphoproteome, including root hair and stripped root-specific phosphorylation suggesting a complex network of kinase-substrate and phosphatase-substrate interactions in response to rhizobial inoculation.

Single-cell transcriptome atlases of soybean root and mature nodule reveal new regulatory programs that control the nodulation process.

The soybean root system is complex. In addition to being composed of various cell types, the soybean root system includes the primary root, the lateral roots, and the nodule, an organ in which mutualistic symbiosis with N-fixing rhizobia occurs. A mature soybean root nodule is characterized by a central infection zone where atmospheric nitrogen is fixed and assimilated by the symbiont, resulting from the close cooperation between the plant cell and the bacteria. To date, the transcriptome of individual cells isolated from developing soybean nodules has been established, but the transcriptomic signatures of cells from the mature soybean nodule have not yet been characterized. Using single-nucleus RNA-seq and Molecular Cartography technologies, we precisely characterized the transcriptomic signature of soybean root and mature nodule cell types and revealed the co-existence of different sub-populations of B. diazoefficiens-infected cells in the mature soybean nodule, including those actively involved in nitrogen fixation and those engaged in senescence. Mining of the single-cell-resolution nodule transcriptome atlas and the associated gene co-expression network confirmed the role of known nodulation-related genes and identified new genes that control the nodulation process. For instance, we functionally characterized the role of GmFWL3, a plasma membrane microdomain-associated protein that controls rhizobial infection. Our study reveals the unique cellular complexity of the mature soybean nodule and helps redefine the concept of cell types when considering the infection zone of the soybean nodule.

Identification and characterization of a temperature sensitive chlorotic soybean mutant.

Screening a transposon-mutagenized soybean population led to the discovery of a recessively inherited chlorotic phenotype. This "vir1" phenotype results in smaller stature, weaker stems, and a smaller root system with smaller nodules. Genome sequencing identified 15 candidate genes with mutations likely to result in a loss of function. Amplicon sequencing of a segregating population was then used to narrow the list to a single candidate mutation, a single-base change in Glyma.07G102300 that disrupts splicing of the second intron. Single cell transcriptomic profiling indicates that this gene is expressed primarily in mesophyll cells and RNA sequencing data indicates it is upregulated in germinating seedlings by cold stress. Previous studies have shown that mutations to Os05g34040, the rice homolog of Glyma.07G102300, produced a chlorotic phenotype that was more pronounced in cool temperatures. Growing soybean vir1 mutants at lower temperatures also resulted in a more severe phenotype. In addition, transgenic expression of wild type Glyma.07G102300 in the knockout mutant of the Arabidopsis homolog At4930720 rescues the chlorotic phenotype, further supporting the hypothesis that the mutation in Glyma.07G102300 is causal of the vir1 phenotype.

miR172 regulates soybean nodulation.

Micro-RNAs (miRNAs) play a pivotal role in the control of gene expression and regulate plant developmental processes. miRNA 172 (miR172) is a conserved miRNA in plants reported to control the expression of genes involved in developmental phase transition, floral organ identity, and flowering time. However, the specific role of miR172 in legume nodulation is undefined. Ectopic expression of soybean miR172 resulted in an increase in nodule numbers in transgenic roots and an increase in the expression of both symbiotic leghemoglobin and nonsymbiotic hemoglobin. These nodules showed higher levels of nitrogenase activity. Further analysis revealed a complex regulatory circuit in which miR156 regulates miR172 expression and controls the level of an AP2 transcription factor. The latter, either directly or indirectly, controls the expression of nonsymbiotic hemoglobin, which is essential for regulating the levels of nodulation.

14-3-3 proteins SGF14c and SGF14l play critical roles during soybean nodulation.

The soybean (Glycine max) genome contains 18 members of the 14-3-3 protein family, but little is known about their association with specific phenotypes. Here, we report that the Glyma0529080 Soybean G-box Factor 14-3-3c (SGF14c) and Glyma08g12220 (SGF14l) genes, encoding 14-3-3 proteins, appear to play essential roles in soybean nodulation. Quantitative reverse transcription-polymerase chain reaction and western-immunoblot analyses showed that SGF14c mRNA and protein levels were specifically increased in abundance in nodulated soybean roots 10, 12, 16, and 20 d after inoculation with Bradyrhizobium japonicum. To investigate the role of SGF14c during soybean nodulation, RNA interference was employed to silence SGF14c expression in soybean roots using Agrobacterium rhizogenes-mediated root transformation. Due to the paleopolyploid nature of soybean, designing a specific RNA interference sequence that exclusively targeted SGF14c was not possible. Therefore, two highly similar paralogs (SGF14c and SGF14l) that have been shown to function as dimers were silenced. Transcriptomic and proteomic analyses showed that mRNA and protein levels were significantly reduced in the SGF14c/SGF14l-silenced roots, and these roots exhibited reduced numbers of mature nodules. In addition, SGF14c/SGF14l-silenced roots contained large numbers of arrested nodule primordia following B. japonicum inoculation. Transmission electron microscopy further revealed that the host cytoplasm and membranes, except the symbiosome membrane, were severely degraded in the failed nodules. Altogether, transcriptomic, proteomic, and cytological data suggest a critical role of one or both of these 14-3-3 proteins in early development stages of soybean nodules.

Plant Single-Cell/Nucleus RNA-seq Workflow.

Single-cell transcriptomics technologies allow researchers to investigate how individual cells, in complex multicellular organisms, differentially use their common genomic DNA. In plant biology, these technologies were recently applied to reveal the transcriptomes of various plant cells isolated from different organs and different species and in response to environmental stresses. These first studies support the potential of single-cell transcriptomics technology to decipher the biological function of plant cells, their developmental programs, cell-type-specific gene networks, programs controlling plant cell response to environmental stresses, etc. In this chapter, we provide information regarding the critical steps and important information to consider when developing an experimental design in plant single-cell biology. We also describe the current status of bioinformatics tools used to analyze single-cell RNA-seq datasets and how additional emerging technologies such as spatial transcriptomics and long-read sequencing technologies will provide additional information on the differential use of the genome by plant cells.

The roles of a novel CDKB/KRP/FB3 cell cycle core complex in rice gametes and initiation of embryogenesis.

The cell cycle controls division and proliferation of all eukaryotic cells and is tightly regulated at multiple checkpoints by complexes of core cell cycle proteins. Due to the difficulty in accessing female gametes and zygotes of flowering plants, little is known about the molecular mechanisms underlying embryogenesis initiation despite the crucial importance of this process for seed crops. In this study, we reveal three levels of factors involved in rice zygotic cell cycle control and characterize their functions and regulation. Protein-protein interaction studies, including within zygote cells, and in vitro biochemical analyses delineate a model of the zygotic cell cycle core complex for rice. In this model, CDKB1, a major regulator of plant mitosis, is a cyclin (CYCD5)-dependent kinase; its activity is coordinately inhibited by two cell cycle inhibitors, KRP4 and KRP5; and both KRPs are regulated via F-box protein 3 (FB3)-mediated proteolysis. Supporting their critical roles in controlling the rice zygotic cell cycle, mutations in KRP4, KRP5 and FB3 result in the compromised function of sperm cells and abnormal organization of female germ units, embryo and endosperm, thus significantly reducing seed-set rate. This work helps reveal regulatory mechanisms controlling the zygotic cell cycle toward seed formation in angiosperms.

Identification of soybean proteins from a single cell type: the root hair.

Root hairs (RH) are a terminally differentiated single cell type, mainly involved in water and nutrient uptake from the soil. The soybean RH cell represents an excellent model for the study of single cell systems biology. In this study, we identified 5702 proteins, with at least two peptides, from soybean RH using an accurate mass and time tag approach, establishing a comprehensive proteome reference map of this single cell type. We also showed that trypsin is the most appropriate enzyme for soybean proteomic studies by performing an in silico digestion of the soybean proteome using different proteases. Although the majority of proteins identified in this study are involved in basal metabolism, the function of others are more related to RH formation/function and include proteins involved in nutrient uptake (transporters) or vesicular trafficking (cytoskeleton and ras-associated binding proteins). Interestingly, some of these proteins appear to be specifically detected in RH and constitute promising candidates for further studies to elucidate unique features of this single-cell model.

Soybean Knowledge Base (SoyKB): a web resource for soybean translational genomics.

BACKGROUND: Soybean Knowledge Base (SoyKB) is a comprehensive all-inclusive web resource for soybean translational genomics. SoyKB is designed to handle the management and integration of soybean genomics, transcriptomics, proteomics and metabolomics data along with annotation of gene function and biological pathway. It contains information on four entities, namely genes, microRNAs, metabolites and single nucleotide polymorphisms (SNPs). METHODS: SoyKB has many useful tools such as Affymetrix probe ID search, gene family search, multiple gene/metabolite search supporting co-expression analysis, and protein 3D structure viewer as well as download and upload capacity for experimental data and annotations. It has four tiers of registration, which control different levels of access to public and private data. It allows users of certain levels to share their expertise by adding comments to the data. It has a user-friendly web interface together with genome browser and pathway viewer, which display data in an intuitive manner to the soybean researchers, producers and consumers. CONCLUSIONS: SoyKB addresses the increasing need of the soybean research community to have a one-stop-shop functional and translational omics web resource for information retrieval and analysis in a user-friendly way. SoyKB can be publicly accessed at http://soykb.org/.

Enzymatic activity of the soybean ecto-apyrase GS52 is essential for stimulation of nodulation.

Nitrogen is an essential nutrient for plant growth. In the Rhizobium-legume symbiosis, root nodules are the sites of bacterial nitrogen fixation, in which atmospheric nitrogen is converted into a form that plants can utilize. While recent studies suggested an important role for the soybean (Glycine max) ecto-apyrase GS52 in rhizobial root hair infection and root nodule formation, precisely how this protein impacts the nodulation process remains undetermined. In this study, the biochemical characteristics of the GS52 enzyme were investigated. Computer modeling of the GS52 apyrase structure identified key amino acid residues important for catalytic activity, which were subsequently mutagenized. Although the GS52 enzyme exhibited broad substrate specificity, its activity on pyrimidine nucleotides and diphosphate nucleotides was significantly higher than on ATP. This result was corroborated by structural modeling of GS52, which predicted a low specificity for the adenine base within the substrate-binding pocket of the enzyme. The wild-type enzyme and its inactive mutant forms were expressed in soybean roots in order to evaluate the importance of GS52 enzymatic activity for nodulation. The results indicated a clear correlation between GS52 enzymatic activity and nodule number. Altogether, our study indicates that the catalytic activity of the GS52 apyrase, likely acting on extracellular nucleotides, is critical for rhizobial infection and nodulation.