Biology subjects

Walbot, V.

Publications and source records attributed to Walbot, V..

9 recordsLinked to original sources

bioRxiv2026

The Polyploid History of Cultivated Dahlia

The cultivated dahlia has a complex evolutionary history that is likely the driver for its diverse floral forms. Previous studies have proposed cultivated dahlia as an allotetraploid, autotetraploid, and in one study an auto-allooctoploid. Here, we used PacBio HiFi long-reads and Dovetail Omni-C reads to assemble and annotate a genome for a tetraploid Dahlia variabilis cultivar Edna C. We identified Edna C as a segmental tetraploid with signature of auto- and allo-polyploidy events based on subgenome-specific repeat partitioning and single-copy gene tree construction within homoeologous groups. Additionally, we identified a cultivated dahlia-specific centromeric monomer shared between Dahlia variabilis Edna C and the Dahlia pinnata Kelvin Floodlight. Based on finding three major chromosome clusters, we propose that Dahlia variabilis Edna C is a segmental tetraploid with evidence of auto- and allopolyploidy.

genomics↗

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

FX-Cell: a method for single-cell RNA sequencing on difficult-to-digest and cryopreserved plant samples

Single-cell RNA sequencing (scRNA-seq) in plants requires the isolation of high-quality protoplasts-- cells devoid of cell walls. However, many plant tissues and organs are resistant to enzymatic digestion, posing a significant barrier to advancing single-cell multi-omics research in plants. Furthermore, for many field-grown crops, the lack of immediate laboratory access presents another major challenge for protoplast preparation. To address these limitations, we developed the FX-Cell method and its derivatives, FXcryo-Cell and cryoFX-Cell, to enable scRNA-seq with both difficult-to-digest and cryopreserved plant samples. By optimizing the fixation buffer and minimizing RNA degradation, our approach ensures efficient cell wall digestion at high temperatures while maintaining high-quality single cells, even after long-term storage at -80{degrees}C, and circumvents use of nuclei, which are not representative of the pool of translatable mRNAs. Using these methods, we successfully constructed high-quality cell atlases for rice tiller nodes, rhizomes of wild rice, and maize crown roots grown in field conditions. Moreover, these methods enable the accurate reconstruction of plant acute wounding responses at single-cell resolution. Collectively, these advancements expand the applicability of plant single-cell genomics across a wider range of species and tissues, paving the way for comprehensive Plant Cell Atlases for plant species.

plant biology↗

bioRxiv2021

Quantitative cell release from plant tissues for single-cell genomics

Single-cell RNA-sequencing (scRNA-seq) can provide invaluable insight into cell development, cell type identification, and plant evolution. However, the resilience of the cell wall makes it difficult to dissociate plant tissues and release individual cells for single-cell analysis. Here, we show that plant organs can be rapidly and quantitatively dissociated into cells if fixed prior to enzymatic digestion. Fixation enables digestion at high temperatures at which enzymatic activity is optimal and stabilizes the plant cell cytoplasm, rendering cells resistant to mechanical shear force while maintaining high quality RNA. This protocol, FX-Cell, releases four to ten-fold more recoverable cells than optimized protoplasting methods applied to maize anthers or root tips with no cell type biases and can be readily applied to a variety of plant taxa and tissues with no optimization. FX-Cell and scRNA-seq analysis were applied to maize anthers for which 95% of the cells were dispersed and provided suitable scRNA-seq data for the identification of anther cell types with marker genes and well-understood biological functions, including rare meiocytes ([~]1% anther cells). In addition, the scRNA-seq data provided putative marker genes and gene ontology information for the identification of unknown cell types. FX-Cell also preserves the morphology of the isolated cells, permitting cell type identification without staining. Ultimately, FX-Cell can be applied to a range of plant species and tissues with minimal to no optimization paving the way for plant scRNA-seq analyses in non-model taxa and tissues.

plant biology↗

bioRxiv2021

24-nt phasiRNAs move from tapetal to meiotic cells in maize anthers

O_LIIn maize, 24-nt phased, secondary small interfering RNAs (phasiRNAs) are abundant in meiotic stage anthers, but their distribution and functions are not precisely known. C_LIO_LIUsing laser capture microdissection, we analyzed tapetal cells, meiocytes, and other somatic cells at several stages of anther development to establish the timing of 24-PHAS precursor transcripts and the 24-nt phasiRNA products. C_LIO_LIBy integrating RNA and small RNA profiling plus single-molecule and small RNA FISH (smFISH or sRNA-FISH) spatial detection, we demonstrate that the tapetum is the primary site of 24-PHAS precursor and Dcl5 transcripts and the resulting 24-nt phasiRNAs. Interestingly, 24-nt phasiRNAs accumulate in all cell types, with the highest levels in meiocytes, followed by tapetum. C_LIO_LIOur data support the conclusion that 24-nt phasiRNAs are mobile from tapetum to meiocytes and to other somatic cells. We discuss possible roles for 24-nt phasiRNAs in anther cell types. C_LI

plant biology↗

bioRxiv2021

Gametophyte genome activation occurs at pollen mitosis I in maize

Flowering plants alternate between multicellular haploid (gametophyte) and diploid (sporophyte) generations. One consequence of this life cycle is that plants face substantial selection during the haploid phase1-3. Pollen actively transcribes its haploid genome4, providing phenotypic diversity even among pollen grains from a single plant. Currently, the timing that pollen precursors first establish this independence is unclear. Starting with an endowment of transcripts from the diploid parent, when do haploid cells generated by meiosis begin to express genes? Here, we follow the shift to haploid expression in maize pollen using allele-specific RNA-sequencing (RNA-Seq) of single pollen precursors. We observe widespread biallelic expression for 11 days after meiosis, indicating that transcripts synthesized by the diploid sporophyte persist long into the haploid phase. Subsequently, there was a rapid and global conversion to monoallelic expression at pollen mitosis I (PMI), driven by active new transcription from the haploid genome. Genes expressed during the haploid phase showed reduced rates of nonsynonymous relative to synonymous substitutions (dn/ds) if they were expressed after PMI, but not before, consistent with purifying selection acting on the haploid gametophyte. This work establishes the timing with which haploid selection may act in pollen and provides a detailed time-course of gene expression during pollen development.

plant biology↗

bioRxiv2020

Environment-conditioned male fertility of HD-ZIP IV transcription factor mutant ocl4: impact on 21-nt phasiRNA accumulation in pre-meiotic maize anthers

Environment-conditioned genic male sterility is a key strategy used to produce hybrid seeds efficiently in many crops, with the exception of maize. The underlying molecular mechanisms of environment-conditioned sterility are poorly understood. Here, we report a derivative line of the male sterile outer cell layer 4 (ocl4) mutant of maize, in which fertility was restored and perpetuated over several generations, under warm growing conditions. Conditionally fertile ocl4 anthers exhibit the anatomical abnormality of a partially duplicated endothecial layer, just like their sterile counterparts. We profiled the dynamics of phased, small interfering RNAs (phasiRNAs) during pre-meiotic development in fully sterile and various grades of semi-fertile ocl4 anthers. We found that the biogenesis of 21-nt phasiRNAs is largely dependent on Ocl4 at three key steps: (1) production of PHAS precursor transcripts, (2) expression of miR2118 that modulates precursor processing, and (3) accumulation of 21-nt phasiRNAs. We propose that 21-nt reproductive phasiRNAs buffer development under unfavorable environmental conditions and are dispensable under favorable conditions.Competing Interest StatementThe authors have declared no competing interest.View Full Text

plant biology↗

bioRxiv2018

Dicer-like 5 deficiency confers temperature-sensitive male sterility in maize

Small RNAs play important roles during plant development by regulating transcript levels of target mRNAs, maintaining genome integrity, and reinforcing DNA methylation. Dicer-like 5 (Dcl5) is proposed to be responsible for precise slicing in many monocots to generate diverse 24-nt phased, secondary small interfering RNAs (phasiRNAs), which are exceptionally abundant in meiotic anthers of diverse flowering plants. The importance and functions of these phasiRNAs remain unclear. Here, we characterized several mutants of dcl5, including alleles generated by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas9 system and a transposon-disrupted allele. We report that dcl5 mutants have few or no 24-nt phasiRNAs, develop short anthers with defective tapetal cells, and exhibit temperature-sensitive male fertility. We propose that DCL5 and 24-nt phasiRNAs are critical for fertility under growth regimes for optimal yield.

plant biology↗

bioRxiv2018

Defining the developmental program leading to meiosis in maize

In multicellular organisms, the entry into meiosis is a complex process characterized by increasing levels of meiotic specialization. We used single-cell RNA-sequencing to reconstruct the developmental program into meiosis in maize. We observed a smooth continuum of expression stages leading up to meiosis, followed by a sharp reorganization of the transcriptome in early meiotic prophase. This latter transcriptional shift was dramatic, with 26.7% of expressed genes changing by 2 fold or more, and occurred just prior to a proposed cell cycle checkpoint. Changes in cell physiology accompanied the nuclear events of meiosis, including a decrease in protein translation capacity and increase in membrane-bound organelles. We further identified differences in gene expression between the mitotic and meiotic cell cycles. Our results uncover a multi-step pathway into meiosis and highlight the power of single cell RNA-seq to define developmental transitions.

plant biology↗