laser-capture-microdissection-rna-sequencing-for-spatial-temporal

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

La Trobe University

The evening complex of ELF4-ELF3-LUX proteins is an integral component of a plant circadian clock. LUX ARRHYTHMO (LUX) is one of the key components of the evening complex, and that play a key role in circadian rhythms and flowering. Here, we report that diverged soybean LUX has the additional role in male reproductive development. We studied diurnal and circadian rhythms of soybean LUX (GmLUXa, GmLUXb, and GmLUXc) using qRT-PCR, and show its nuclear localisation by particle bombardment. Yeast-two hybrid (Y2H) studies indicate that both GmLUXb and GmLUXc form an evening complex with GmELF4b and GmELF3a, respectively. Ectopic expression of GmLUXb in Arabidopsis lux mutants can complement functions of AtLUX, whereas GmLUXc generates novel phenotypes of serrated leaves, stunted plants, shortened anther filament, and low seed set. Overall, our results suggest that the LUX gene has diverged in soybean where GmLUXb and GmLUXc share the role to control flowering time, but GmLUXc has evolved to regulate anther filament growth and seed set by regulating the Gibberellin hormone biosynthesis pathway.

A novel role of the soybean clock gene LUX ARRHYTHMO in male reproductive development.

Seed germination involves progression from complete metabolic dormancy to a highly active, growing seedling. Many factors regulate germination and these interact extensively, forming a complex network of inputs that control the seed-to-seedling transition. Our understanding of the direct regulation of gene expression and the dynamic changes in the epigenome and small RNAs during germination is limited. The interactions between genome, transcriptome and epigenome must be revealed in order to identify the regulatory mechanisms that control seed germination.

Extensive transcriptomic and epigenomic remodelling occurs during Arabidopsis thaliana germination.

The distinct functions of individual cell types require cells to express specific sets of genes. The germinating seed is an excellent model to study genome regulation between cell types since the majority of the transcriptome is differentially expressed in a short period, beginning from a uniform, metabolically inactive state. In this study, we applied laser-capture microdissection RNA-sequencing to small numbers of cells from the plumule, radicle tip and scutellum of germinating barley seeds every 8 h, over a 48 h time course. Tissue-specific gene expression was notably common; 25% (910) of differentially expressed transcripts in plumule, 34% (1876) in radicle tip and 41% (2562) in scutellum were exclusive to that organ. We also determined that tissue-specific storage of transcripts occurs during seed development and maturation. Co-expression of genes had strong spatiotemporal structure, with most co-expression occurring within one organ and at a subset of specific time points during germination. Overlapping and distinct enrichment of functional categories were observed in the tissue-specific profiles. We identified candidate transcription factors amongst these that may be regulators of spatiotemporal gene expression programs. Our findings contribute to the broader goal of generating an integrative model that describes the structure and function of individual cells within seeds during germination.

Lim Chee Liew

Department of Animal,
Plant and Soil Science,
AgriBio Building,
Australian Research Council Centre of Excellence in Plant Energy Biology,
Department of Animal, Plant and Soil Science, AgriBio Building

La Trobe University

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

Lim Chee Liew

Department of Animal,Plant and Soil Science,AgriBio Building,Australian Research Council Centre of Excellence in Plant Energy Biology,Department of Animal, Plant and Soil Science, AgriBio Building

La Trobe University

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

Department of Animal,
Plant and Soil Science,
AgriBio Building,
Australian Research Council Centre of Excellence in Plant Energy Biology,
Department of Animal, Plant and Soil Science, AgriBio Building