Biology subjects

Meyers, B. C.

Publications and source records attributed to Meyers, B. C..

At least 19 recordsLinked to original sources

bioRxiv2026

Extracellular Vacuole-derived bodies (EVacs) mediate RNA secretion in plants

Extracellular RNAs are found in the plant extracellular space, but how they are exported from cells remains unclear. We found that the plant vacuole is a major source of extracellular RNA and identified a class of large extracellular vacuole-derived bodies, which we termed EVacs, that are key mediators of this transport. EVacs are marked by the vacuolar membrane (tonoplast) proteins {gamma}-TIP and V-ATPase and originate as intravacuolar structures formed by inward folding of the tonoplast, encapsulating intact cytoplasmic material, including both RNAs and proteins. These intravacuolar bodies then escape the vacuole and are subsequently released from the plasma membrane of mesophyll cells into the apoplast. These findings provide a novel mechanism for the unconventional secretion of macromolecules in plants.

plant biology↗

bioRxiv2026

Phasis: a software tool for register-resolved discovery of plant phased small RNA loci

Plant PHAS locus discovery remains challenging because phasiRNA-producing loci must be distinguished from other sRNA-producing regions with high abundance or apparent periodicity. This problem is especially acute for reproductive 24-PHAS loci, which occur within genomes that also produce abundant 24-nt siRNAs from non-PHAS regions. We present Phasis, an open-source Python software tool for plant PHAS-locus discovery from small RNA sequencing data. Phasis combines statistical evidence for phased accumulation with locus-level features and a Register-Resolved Locus Interpretation Layer that evaluates whether candidate loci show coherent phased architecture. Across diverse plant datasets, Phasis recovered validated or annotated 21- and 24-PHAS loci with a strong balance between call-level precision and reference-locus recall, and generally outperformed PhaseTank and ShortStack in matched benchmark analyses. The register-resolved interpretation layer reduced unsupported calls by separating coherent phased loci from ambiguous sRNA-producing regions. In maize dcl5 mutant libraries, Phasis showed strong depletion of 24-PHAS recovery, supporting DCL5-dependent recovery of reproductive 24-PHAS signal. Together, these results support Phasis as a biologically interpretable tool for large-scale discovery of plant DCL-dependent phasiRNA loci.

plant biology↗

bioRxiv2026

Stage-Specific RNA Turnover Drives Small RNA Dynamics in Arabidopsis-Colletotrichum Interactions

Small RNAs (sRNAs) are key regulators of plant defense and have been implicated in cross-kingdom interactions with pathogens. The hemibiotrophic fungus Colletotrichum higginsianum infects Arabidopsis thaliana through three stages: appressorial penetration, biotrophy, and necrotrophy. However, the dynamics of fungal and plant sRNA populations across these three stages have not been elucidated. Using high-throughput sequencing, we profiled sRNAs from A. thaliana and C. higginsianum during in planta appressorium (PA), biotrophic (BP), and necrotrophic (NP) phases, and compared them to fungal mycelia (MY) and in vitro appressoria (VA). Our analyses revealed stage-specific patterns in sRNA accumulation in both the plant and the pathogen. In C. higginsianum, sRNAs were dominated by 29 nt species in PA, BP, MY, and VA, but shifted to 18 nt in NP, consistent with RNA degradation during host cell death. In A. thaliana, sRNAs transitioned from 30-33-nt in PA/BP to a 21 nt dominant peak in NP. Also, TE-derived siRNAs and other regulatory sRNAs (miRNAs, ncRNA, snoRNAs and snRNAs) declined during NP. A total of 62 host miRNAs showed differential accumulation, including core plant developmental regulators active across infection stages, and stage-specific miRNAs such as miR396, miR170/171, miR472, and miR858b. tRFs displayed opposite trends in host and pathogen: fungal tRFs declined in NP, while host misc-tRFs, 5'-tRFs, and 3'-tRFs increased, suggesting contrasting regulatory roles. These results provide new insights into RNA-mediated plant-fungal interactions.

plant biology↗

bioRxiv2026

A multi-omics approach to maize (Zea mays) tassel development

The development of the male inflorescence in maize (Zea mays L.) is a complex and highly regulated process that is essential for reproductive success and yield. To gain a comprehensive understanding of the regulatory programs involved in tassel formation, including the initial specification of anther cell types, the transcriptomes, small RNAs (sRNAs), and proteomes were profiled at four developmental stages (0.5-2.0 cm). The RNAseq analysis indicates dynamic shifts in gene expression underlying the transition from indeterminate meristems to organ initiation and germinal cell determination by 2.0 cm. Complementing these data, sRNA sequencing uncovered 182 microRNAs (miRNAs) with distinct temporal patterns. A core set of 126 miRNAs was expressed throughout tassel development, while others displayed strong stage-specific enrichment. Notably, families of miRNAs that target auxin signaling were dynamically regulated, suggesting fine-tuning of hormone signaling in relation to meristem activity. Later stages were enriched for miR2275 and miR11969, which previously have both been associated with meiocytes, indicating the onset of reproductive sRNA pathways during the early stages of anther differentiation. Together, these datasets provide a broad overview of tassel development and form a backbone to enrich existing RNAseq and single cell RNAseq data sets of specific steps in tassel and initial anther development, while also adding new data on sRNA expression.

plant biology↗

bioRxiv2025

Elevated CO2 drives epigenetic reprogramming andchromatin dynamics in Arabidopsis thaliana

Atmospheric CO2 levels are rising rapidly, yet how plants respond at the genomic level remains poorly understood. The three-dimensional (3D) organization of chromatin in the nucleus is a key regulator of gene expression and environmental response, and changes in chromatin architecture can be transmitted across generations through epigenetic mechanisms. Here, we report that elevated CO2 is associated with broad 3D chromatin reorganization in Arabidopsis thaliana, spanning scales from chromosome-wide compartment identity to kilobase-resolution Local Chromatin Domains (LCDs) and LCD-Associated Loops (LALs). Using chromatin conformation capture and sequencing (Hi-C) across four mutant backgrounds, we show that these changes exhibit intergenerational persistence in the self-fertilized progeny of plants grown at elevated CO2, and that this persistence is sensitive to the Pol V branch of the RNA-directed DNA Methylation (RdDM) pathway rather than to upstream small RNA biogenesis. At kilobase resolution, we annotated 2,279 LCDs whose borders are enriched for transposable element loci occupied by the chromatin-silencing members Pol V and LHP1, and near a third of LCD borders are LALs. Elevated CO2 is associated with LAL attenuation and altered transcript abundance at LCD borders marked by H3K27me3, implicating coordinated RdDM and Polycomb group (PcG) activity in the chromatin response to elevated CO2. Integrated transcriptome and DNA methylation analysis across all five genetic backgrounds revealed widespread changes in gene and transposable element abundance. These findings support a model in which CO2-associated chromatin reorganization confers transcriptional plasticity through persistent chromatin states shaped by the RdDM and PcG pathways.

plant biology↗

bioRxiv2025

The Annotated Blueprint: Integrated Functional Genomic Resources for a Model Tetraploid Wheat Triticum turgidum cv. Kronos

Triticum turgidum cv. Kronos is a tetraploid wheat cultivar that underpins one of the richest community platforms for functional genomics. Over the past decade, about 3,000 exome- and promoter-capture datasets, linked to mutagenized seed stocks, and transcriptomic and phenotypic resources have accumulated, yet the absence of a reference genome has constrained their impact. Here, we present a chromosome-scale reference genome of Kronos with high-confidence annotations, including manual curation of over 1,000 disease resistance (NLR) genes. This reference revealed previously hidden NLR diversity and clarified their genomic organization at chromosomal ends. Re-analysis of exome- and promoter-capture datasets enabled high-resolution mutation discovery in genes and regulatory regions that were previously inaccessible, uncovering the full standing variation present in Kronos mutant lines. We further re-curated transcriptomic and small RNA datasets, generating improved, genome-wide maps of microRNAs and phasiRNAs important for wheat development. Collectively, these resources elevate Kronos to reference quality and establish it as a versatile platform for functional and translational wheat research.

plant biology↗

bioRxiv2025

Redesigning petal shape, size, and color in soybean reveals unexpected phenotypes for floral organ development

Soybean (Glycine max) has not yet benefited from large-scale hybrid breeding efforts due to its small, self-fertilizing flowers that are difficult to emasculate, and limited attractiveness to pollinators. This study explores targeted floral trait engineering to enhance pollinator attraction, aiming to overcome barriers to soybean hybridization. We generated a high-resolution floral organ expression atlas and H3K4 trimethylation ChIP-Seq dataset to identify candidate genes involved in petal development, nectar sugar content, and petal pigmentation. Using CRISPR-based activation and repression systems, we modified the expression of AINTEGUMENTA (GmANT), BIGPETAL (GmBPE), and SUCROSE TRANSPORTER2 (GmSUC2). Contrary to expectations based on Arabidopsis homologs, transcriptional activation of GmANT_B and repression of GmBPE led to reduced, rather than increased, petal size, highlighting divergent regulatory mechanisms in soybean. Complementation of the W1 gene that controls petal pigmentation successfully converted white petals to purple, with preliminary evidence indicating that this color conversion may increase pollinator visitation. These results underscore the complexity of floral development in soybean and provide foundational tools and resources for future efforts to engineer reproductive traits for hybrid seed production.

plant biology↗

bioRxiv2025

Transcriptional dynamics of nitrogen fixation and senescence in soybean nodules: A dual perspective on host and Bradyrhizobium regulation.

The Soybean-Bradyrhizobium symbiosis enables symbiotic nitrogen fixation (SNF) within root nodules, reducing reliance on synthetic N-fertilizers. However, nitrogen fixation is transient, peaking several weeks after Bradyrhizobium colonization and declining as nodules senesce in coordination with host development. To investigate the regulatory mechanisms governing SNF and senescence, we conducted a temporal transcriptomic analysis of soybean nodules inoculated with Bradyrhizobium diazoefficiens USDA110. Weekly nodule samples (2-10 weeks post-inoculation, wpi) were analyzed using RNA and small RNA sequencing, while acetylene reduction assays assessed nitrogenase activity from 4 to 7 wpi. We identified three major nodule developmental phases: early development (2-3 wpi), nitrogen fixation (3-8 wpi), and senescence (8-10 wpi). Soybean showed extensive transcriptional reprogramming during senescence, whereas Bradyrhizobium underwent major transcriptional shifts early in development before stabilizing during nitrogen fixation. We identified seven soybean genes and several microRNAs as candidate biomarkers of nitrogen fixation, including lipoxygenases (Lox), suggesting roles for oxylipin metabolism. Soy hemoglobin-2 (Hb2), previously classified as non-symbiotic, was upregulated during senescence, implicating oxidative stress responses within aging nodules. Upregulation of the Bradyrhizobium paa operon and rpoH during senescence suggested metabolic adaptation for survival beyond symbiosis. Additionally, Bradyrhizobium NIF gene expression showed stage-specific regulation, with nifK peaking at 2 wpi, nifD and nifA at 2 and 10 wpi, and nifH, nifW, and nifS at 10 wpi. These findings provide insights into SNF regulation and nodule aging, revealing temporal gene expression patterns that could inform breeding or genetic engineering strategies to enhance nitrogen fixation in soybeans and other legume crops.

plant biology↗

bioRxiv2025

Distinct classes of 21 and 24-nt phasiRNAs suggest diverse mechanisms of biogenesis and function in rice anther development

PhasiRNAs (phased small interfering RNAs) are a major class of plant small RNAs known to be key regulators in male reproductive development of maize (Zea mays) and rice (Oryza sativa), among other plants. Earlier research focused primarily on premeiotic 21-nt phasiRNAs and meiotic 24-nt phasiRNAs, while new studies uncovered a premeiotic class of 24-nt phasiRNAs. The biogenesis and function of these phasiRNAs remain unclear. We conducted an integrative analysis combining small RNA (sRNA) sequencing and transcriptomic profiling of sRNA-associated genes across ten developmental stages of anther in Kitaake rice to map associations between sRNA-related genes and phasiRNA classes. We identified previously undescribed classes of postmeiotic 21-nucleotide (nt) and 24-nt phasiRNA-producing loci and characterized their unique accumulation patterns. Additionally, our findings reveal distinct nucleotide composition and register accumulation among the phasiRNA classes, suggesting the presence of diverse mechanisms of biogenesis and function. Our results provide new insights into the regulatory complexity of phasiRNAs, establishing a foundation for further functional studies and advancing our understanding of their roles in anther development and their underlying mechanisms. Plain Language SummarySmall RNAs play important roles in controlling how plants develop, but their function in plant reproduction is still not well understood. In rice, small RNAs called phasiRNAs help regulate how pollen develops, but past research has mostly focused on just two types: one found before meiosis and one found during meiosis. In this study, we analyzed small RNA activity across ten stages of rice anther development and found new phasiRNA-producing genes that are active after meiosis. We also discovered that different types of phasiRNAs have distinct patterns of accumulation and composition, suggesting that they have specialized roles in reproductive development. These findings reveal new layers of complexity in how small RNAs function and set the stage for future research to understand their exact roles in pollen formation. This work may help improve our understanding of plant fertility and contribute to advancements in crop breeding. Core ideasO_LIStage-specific accumulation patterns suggest more specialized functions of phasiRNAs. C_LIO_LIMeiotic 24-nt phasiRNAs are compositionally distinct from other 24-nt phasiRNAs. C_LIO_LINewly described phasiRNA clusters may differ in mechanism of biogenesis and function. C_LI

plant biology↗

bioRxiv2024

miRScore: a rapid and precise microRNA validation tool

MicroRNAs (miRNAs) are small non-protein-coding RNAs that regulate gene expression in many eukaryotes. Next-generation sequencing of small RNAs (small RNA-seq) has accelerated discovery and annotation of novel miRNAs. Newly discovered miRNAs are typically submitted to databases such as the miRBase microRNA registry following the publication of a peer-reviewed study. However, genome-wide scans using small RNA-seq data often yield high rates of false-positive miRNA annotations, highlighting the need for more robust validation methods. miRScore was developed as an independent and efficient tool for evaluating miRNA annotations using sRNA-seq data. miRScore combines structural and expression-based analyses to provide rapid and reliable validation of miRNA annotations. By providing users with detailed metrics and visualization, miRScore enhances the ability to assess confidence in novel and existing miRNA annotations. miRScore has the potential to advance the overall quality of miRNA annotations by improving accuracy of new submissions to miRNA databases and serving as a resource for re-evaluating existing annotations.

bioinformatics↗

bioRxiv2024

Molecular evolution of a reproductive barrier in maize and related species

Three cross-incompatibility loci each control a distinct reproductive barrier in both domesticated maize (Zea mays ssp. mays) and its wild teosinte relatives. These three loci, Teosinte crossing barrier1 (Tcb1), Gametophytic factor1 (Ga1), and Ga2, each play a key role in preventing hybridization between incompatible populations and are proposed to maintain the barrier between domesticated and wild subspecies. Each locus encodes both a silk-active and a matching pollen-active pectin methylesterase (PMEs). To investigate the diversity and molecular evolution of these gametophytic factor loci, we identified existing and improved models of the responsible genes in a new genome assembly of maize line P8860 that contains active versions of all three loci. We then examined fifty-two assembled genomes from seventeen species to classify haplotype diversity and identify sites under diversifying selection during the evolution of these genes. We show that Ga2, the oldest of these three loci, was duplicated to form Ga1 at least 12 million years ago. Tcb1, the youngest locus, arose as a duplicate of Ga1 before or around the time of diversification of the Zea genus. We find evidence of positive selection during evolution of the functional genes at an active site in the pollen-expressed PME and predicted surface sites in both the silk- and pollen-expressed PMEs. The most common allele at the Ga1 locus is a conserved ga1 allele (ga1-Off), which is specific haplotype containing three full-length PME gene copies, all of which are non-coding due to conserved stop codons and are between 610 thousand and 1.5 million years old. We show that the ga1-Off allele is associated with and likely generates 24-nt siRNAs in developing pollen-producing tissue, and these siRNAs map to functional Ga1 alleles. In previously-published crosses, the ga1-Off allele was associated with reduced function of the typically dominant functional alleles for the Ga1 and Tcb1 barriers. Taken together, this seems to be an example of a type of epigenetic trans-homolog silencing known as paramutation functioning at a locus controlling a reproductive barrier.

genetics↗

bioRxiv2024

ExPOSE: A comprehensive toolkit to perform expansion microscopy in plant protoplast systems

Expansion microscopy (ExM) achieves nanoscale imaging by physical expansion of fixed biological tissues embedded in a swellable hydrogel, enhancing the resolution of any optical microscope several-fold. While ExM is commonly used in animal cells and tissues, there are few plant specific protocols. Protoplasts are a widely used cell system across plant species, especially in studying biomolecule localization. Here, we present an approach to achieve robust expansion of plant protoplasts, termed Expansion microscopy in plant PrOtoplast SystEms (ExPOSE). We demonstrate that coupling ExPOSE with other imaging techniques, immunofluorescence and in situ hybridization chain reaction to visualize proteins and mRNAs, respectively, greatly enhances the spatial resolution of endogenous biomolecules. Additionally, in this study, we tested the effectiveness and versatility of this technique to observe biomolecular condensates in Arabidopsis protoplasts and transcription factors in maize protoplasts at increased resolution. ExPOSE can be relatively inexpensive, fast, and simple to implement.

plant biology↗

bioRxiv2024

Premeiotic 24-nt phasiRNAs are present in the Zea genus and unique in biogenesis mechanism and molecular function

Reproductive phasiRNAs are broadly present in angiosperms and play crucial roles in sustaining male fertility. While the premeiotic 21-nt phasiRNAs and meiotic 24-nt phasiRNA pathways have been extensively studied in maize (Zea mays) and rice (Oryza sativa), a third putative category of reproductive phasiRNAs-named premeiotic 24-nt phasiRNAs-have recently been reported in barley (Hordeum vulgare) and wheat (Triticum aestivum). To determine whether premeiotic 24-nt phasiRNAs are also present in maize and related species and begin to characterize their biogenesis and function, we performed a comparative transcriptome and degradome analysis of premeiotic and meiotic anthers from five maize inbred lines and three teosinte species/subspecies. Our data indicate that a substantial subset of the 24-nt phasiRNA loci in maize and teosinte are already highly expressed at premeiotic phase. The premeiotic 24-nt phasiRNAs are similar to meiotic 24-nt phasiRNAs in genomic origin and dependence on DCL5 for biogenesis, however, premeiotic 24-nt phasiRNAs are unique in that they are likely (i) not triggered by microRNAs, (ii) not loaded by AGO18 proteins, and (iii) not capable of mediating cis-cleavage. In addition, we also observed a group of premeiotic 24-nt phasiRNAs in rice using previously published data. Together, our results indicate that the premeiotic 24-nt phasiRNAs constitute a unique class of reproductive phasiRNAs and are present more broadly in the grass family (Poaceae) than previously known. SIGNIFICANCEWe previously reported two classes of reproductive phasiRNAs in maize, the premeiotic 21-nt phasiRNAs and the meiotic 24-nt phasiRNAs. Here we report a third class of reproductive phasiRNAs - premeiotic 24-nt phasiRNAs - that are present in the Zea genus, including all five maize inbred lines and three teosinte species/subspecies that we examined, plus rice. We show that in the Zea genus the premeiotic 24-nt phasiRNAs are distinct from the meiotic 24-nt phasiRNAs in triggering mechanism, effector protein, and molecular function.

plant biology↗

bioRxiv2024

Fluorescence hybridization chain reaction enables localization of multiple molecular classes combined with plant cell ultrastructure

BackgroundRecent developments in hybridization chain reaction (HCR) have enabled robust simultaneous localization of multiple mRNA transcripts using fluorescence in situ hybridization (FISH). Once multiple split initiator oligonucleotide probes bind their target mRNA, HCR uses DNA base-pairing of fluorophore-labeled hairpin sets to self-assemble into large polymers, amplifying the fluorescence signal and reducing non-specific background. Few studies have applied HCR in plants, despite its demonstrated utility in whole mount animal tissues and cell culture. Our aim was to optimize this technique for sectioned plant tissues embedded with paraffin and methacrylate resins, and to test its utility in combination with immunolocalization and subsequent correlation with cell ultrastructure using scanning electron microscopy. ResultsApplication of HCR to 10 {micro}m paraffin sections of 17-day-old Setaria viridis (green millet) inflorescences using confocal microscopy revealed that the transcripts of the transcription factor KNOTTED 1 (KN1) were localized to developing floret meristem and vascular tissue while SHATTERING 1 (SH1) and MYB26 transcripts were co-localized to the breakpoint below the floral structures (the abscission zone). We also used methacrylate de-embedment with 1.5 {micro}m and 0.5 {micro}m sections of 3-day-old Arabidopsis thaliana seedlings to show tissue specific CHLOROPHYLL BINDING FACTOR a/b (CAB1) mRNA highly expressed in photosynthetic tissues and ELONGATION FACTOR 1 ALPHA (EF1) highly expressed in meristematic tissues of the shoot apex. The housekeeping gene ACTIN7 (ACT7) mRNA was more uniformly distributed with reduced signals using lattice structured-illumination microscopy. HCR using 1.5 {micro}m methacrylate sections was followed by backscattered imaging and scanning electron microscopy thus demonstrating the feasibility of correlating fluorescent localization with ultrastructure. ConclusionHCR was successfully adapted for use with both paraffin and methacrylate de-embedment on diverse plant tissues in two model organisms, allowing for concurrent cellular and subcellular localization of multiple mRNAs, antibodies and other affinity probe classes. The mild hybridization conditions used in HCR made it highly amenable to observe immunofluorescence in the same section. De-embedded semi-thin methacrylate sections with HCR were compatible with correlative electron microscopy approaches. Our protocol provides numerous practical tips for successful HCR and affinity probe labeling in electron microscopy-compatible, sectioned plant material.

plant biology↗

bioRxiv2023

The floral development of the allotetraploid Coffea arabica L. correlates with a small RNA dynamic reprogramming.

O_LINon-coding and coding RNAs are key regulators of plant growth, development, and stress responses. To investigate the types of transcripts accumulated during the vegetative to reproductive transition and floral development in the Coffea arabica L., we sequenced small RNA libraries from eight developmental stages, up to anthesis. C_LIO_LIWe combined this data with messenger RNA and PARE sequencing of two important development stages that marks the transition of an apparent latent to a rapid growth stage. In addition, we took advantage of multiple in silico tools to characterize genomic loci producing small RNAs such as phasiRNAs, miRNAs and tRFs. C_LIO_LIOur differential and co-expression analysis showed that some types of small RNAs such as tRNAs, snoRNAs, snRNAs and phasiRNAs preferentially accumulate in a stage- specific manner. C_LIO_LIMembers of the miR482/miR2118 superfamily and their 21-nucleotide phasiRNAs originating from resistance genes show a robust co-expression pattern that is maintained across all the evaluated developmental stages. Finally, the majority of miRNAs accumulate in a family-stage specific manner, related to modulated hormonal responses and transcription factors expression. C_LI Societal Impact StatementThis research holds potential to benefit millions of coffee-producing families in over 60 countries. We uncovered molecular regulatory mechanisms governing flower development, one of the causes for the Coffea arabicas uneven ripening. The absence of uniformity in coffee production, spanning from floral induction to branch senescence, has a detrimental impact on the final products quality. These insights will inform strategies for controlled coffee maturation, leading to improved, uniform harvests.

plant biology↗

bioRxiv2023

Coffee microsporogenesis and related small interfering RNAs biosynthesis have a unique pattern among eudicots suggesting a sensitivity to climate changes.

Recently, the siRNAs pathways, and especially reproductive phasiRNAs, have attracted attention in eudicots since their biological roles are still unknown and their biogenesis took different evolutionary pathways compared to monocots. In this work, we used Coffea arabica L., a recently allotetraploid formed from the hybridization of C. canephora and C. eugenioides unreduced gametes, to explore microsporogenesis and small RNAs related pathways in a eudicot crop. First, we identified the microsporogenesis stages during anther development revealing that pre-meiosis occurs in anthers of 1.5 mm inside floral buds (FBs), whereas meiosis between 1.5 and 4.2 mm FBs, and post-meiosis in FBs larger than 4.2mm. These stages coincide with the Brazilian winter, a period of FBs reduced growth which suggests temperature sensitivity. Next, we identified and quantified the expression of reproductive 21- and 24-nt phasiRNAs during coffee anther development together with their canonical and novel miRNA triggers, and characterized the DCL and AGO families. Our results showed that the pattern of reproductive phasiRNA abundance in C. arabica is unique among described eudicots and the canonical trigger car-miR2275 is involved in the processing of both 21 and 24 nt phasiRNAs. Fourteen DCL genes were identified, but DCL5, related to phasiRNA biosynthesis in monocots, was not according to its specificity for monocots. Thus, our work explored the knowledge gap about microsporogenesis and related siRNAs pathways in coffee, contributing to the control of reproductive development and to the improvement of fertility in eudicots.

plant biology↗

bioRxiv2023

Introduction of Barnase/Barstar in soybean produces a rescuable male sterility system for hybrid breeding

Hybrid breeding for increased vigor has been used for over a century to boost agricultural outputs without requiring higher inputs. While this approach has led to some of the most substantial gains in crop productivity, breeding barriers have fundamentally limited soybean (Glycine max) from reaping the benefits of hybrid vigor. Soybean makes inconspicuous flowers that self-fertilize before opening, and thus are not readily amenable to outcrossing. In this study, we demonstrate that the Barnase/Barstar male sterility/male rescue system can be used in soybean to produce hybrid seeds. By expressing the cytotoxic ribonuclease, Barnase, under a tapetum-specific promoter in soybean anthers, we are able to completely block pollen maturation, creating male-sterile plants. We show that fertility can be rescued in the F1 generation of these Barnase-expressing lines when they are crossed with pollen from plants that express the Barnase inhibitor, Barstar. Importantly, we found that the successful rescue of male fertility is dependent on the relative dosage of Barnase and Barstar. When Barnase and Barstar were expressed under the same tapetum-specific promoter, the F1 offspring remained male-sterile. When we expressed Barstar under a relatively stronger promoter than Barnase, we were able to achieve a successful rescue of male fertility in the F1 generation. This work demonstrates the successful implementation of a biotechnology approach to produce fertile hybrid offspring in soybean.

plant biology↗

bioRxiv2022

Plastid double-strand RNA transgenes trigger small RNA-based gene silencing of nuclear-encoded genes

Plastid transformation technology has been widely used to express traits of potential commercial importance, though the technology has been limited to traits that function while sequestered in the organelle. Prior research indicates that plastid contents can escape from the organelle, suggesting a possible mechanism for engineering plastid transgenes to function in other cellular locations. To test this hypothesis, we created tobacco plastid transformants that express a fragment of the nuclear-encoded Phytoene desaturase (PDS) gene capable of catalyzing post-transcriptional gene silencing if RNA escape to the cytoplasm occurs. We found multiple lines of direct evidence that plastid-encoded PDS transgenes affect nuclear PDS gene silencing: knockdown of the nuclear-encoded PDS mRNA and/or its apparent translational inhibition, biogenesis of 21-nucleotide (nt) phased small interfering RNAs (phasiRNAs), and pigment deficient plants. Furthermore, plastid-expressed double-stranded RNA (dsRNA) with no cognate nuclear-encoded pairing partner also produced abundant 21-nt phasiRNAs in the cytoplasm, demonstrating that a nuclear-encoded template is not required for siRNA biogenesis. Our results indicate that RNA escape from plastids to the cytoplasm occurs broadly, with functional consequences that include entry into the gene silencing pathway. Furthermore, we uncover a method to produce plastid-encoded traits with functions outside of the organelle and open new fields of study in plastid development, compartmentalization and small RNA biogenesis.

plant biology↗