No abstract

The gynoecium is the most complex organ formed by the flowering plants. It encloses the ovules, provides a surface for pollen contact and self-incompatibility reactions, allows pollen tube growth, and, post fertilization, develops into the fruit. Consequently, the regulation of gynoecium morphogenesis is complex and appropriate timing of this process in part determines reproductive success. However, little is known about the global control of gynoecium development, even though many regulatory genes have been characterized. Here, we characterized dynamic gene expression changes using laser-microdissected gynoecium tissue from four developmental stages in Arabidopsis. We provide a high-resolution map of global expression dynamics during gynoecium morphogenesis and link these to the gynoecium interactome. We reveal groups of genes acting together early and others acting late in morphogenesis. Clustering of co-expressed genes enables comparisons between the leaf, shoot apex, and gynoecium transcriptomes, allowing the dissection of common and distinct regulators. Furthermore, our results lead to the discovery of genes with putative transcription factor activity (B3LF1, -2, DOFLF1), which, when mutated, lead to impaired gynoecium expansion, illustrating that global transcriptome analyses reveal yet unknown developmental regulators. Our data show that genes encoding highly interacting proteins, such as SEPALLATA3, AGAMOUS, and TOPLESS, are expressed evenly during development but switch interactors over time, whereas stage-specific proteins tend to have fewer interactors. Our analysis connects specific transcriptional regulator activities, protein interactions, and underlying metabolic processes, contributing toward a dynamic network model for gynoecium development.

BackgroundPlant development is controlled by the action of many, often connected gene regulatory networks. Differential gene expression controlled by internal and external cues is a major driver of growth and time specific differentiation in plants. Transcriptome analysis is the state-of-the-art method to detect spatio-temporal changes in gene expression during development. Monitoring changes in gene expression at early stages or in small plant organs and tissues requires an accurate technique of tissue isolation, which subsequently results in RNA of sufficient quality and quantity. Laser-microdissection enables such accurate dissection and collection of desired tissue from sectioned material at a microscopic level for RNA extraction and subsequent downstream analyses, such as transcriptome, proteome, genome or miRNA.ResultsA protocol for laser-microdissection, RNA extraction and RNA-seq was optimized and verified for three distant angiosperm species: Arabidopsis thaliana (Brassicaceae), Oryza sativa (Poaceae) and Eschscholzia californica (Papaveraceae). Previously published protocols were improved in processing speed by reducing the vacuum intensity and incubation time during tissue fixation and incubation time and cryoprotection and by applying adhesive tape. The sample preparation and sectioning of complex and heterogenous flowers produced adequate histological quality and subsequent RNA extraction from micro-dissected gynoecia reliably generated samples of sufficient quality and quantity on all species for RNA-seq. Expression analysis of growth stage specific A. thaliana and O. sativa transcriptomes showed distinct patterns of expression of chromatin remodelers on different time points of gynoecium morphogenesis from the initiation of development to post-meiotic stages.ConclusionHere we describe a protocol for plant tissue preparation, cryoprotection, cryo-sectioning, laser microdissection and RNA sample preparation for Illumina sequencing of complex plant organs from three phyletically distant plant species. We are confident that this approach is widely applicable to other plant species to enable transcriptome analysis with high spatial resolution in non-model plant species. The protocol is rapid, produces high quality sections of complex organs and results in RNA of adequate quality well suited for RNA-seq approaches. We provide detailed description of each stage of sample preparation with the quality and quantity measurements as well as an analysis of generated transcriptomes.

The gynoecium is the most complex organ formed by the flowering plants. It encloses the ovules, provides a surface for pollen contact and self-incompatibility reactions, allows pollen tube growth and, post fertilization, and develops into the fruit. Consequently, the regulation of gynoecium morphogenesis is complex and appropriate timing of this process in part determines reproductive success. However, little is known about the global control of gynoecium development, even though many regulatory genes have been characterized. Here, we characterized dynamic gene expression changes using laser-microdissected gynoecium tissue from four developmental stages in Arabidopsis. We provide a high-resolution map of global expression dynamics during gynoecium morphogenesis and link these to the gynoecium interactome. We reveal groups of genes acting together early and others acting late in morphogenesis. Clustering of co-expressed genes enables comparisons between the leaf, shoot apex, and gynoecium transcriptomes allowing the dissection of common and distinct regulators. Furthermore, our results lead to the discovery of the LESSER FERTILITY1-4 (LEF1-4) genes, which, when mutated, lead to impaired gynoecium expansion, illustrating that global transcriptome analyses reveal yet unknown developmental regulators. Our data show that highly interacting proteins, such as SEPALLATA3, AGAMOUS, and TOPLESS are expressed more evenly during development, but switch interactors in time, whereas stage-specific proteins have only few interactors. Our analysis connects specific transcriptional regulator activities, protein interactions, and underlying metabolic processes towards the development of a dynamic network model for gynoecium development.