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Transcriptome Analysis of Intrusively Growing Flax Fibers Isolated by Laser Microdissection.

ABSTRACT: The intrusive growth, a type of plant cell elongation occurring in the depths of plant tissues, is characterized by the invasion of a growing cell between its neighbours due to a higher rate of elongation. In order to reveal the largely unknown molecular mechanisms of intrusive growth, we isolated primary flax phloem fibers specifically at the stage of intrusive growth by laser microdissection. The comparison of the RNA-Seq data from several flax stem parts enabled the characterization of those processes occurring specifically during the fiber intrusive elongation. The revealed molecular players are summarized as those involved in the supply of assimilates and support of turgor pressure, cell wall enlargement and modification, regulation by transcription factors and hormones, and responses to abiotic stress factors. The data obtained in this study provide a solid basis for developing approaches to manipulate fiber intrusive elongation, which is of importance both for plant biology and the yield of fiber crops.

SUBMITTER: Gorshkova T

PROVIDER: S-EPMC6167358 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Transcriptome Analysis of Intrusively Growing Flax Fibers Isolated by Laser Microdissection.

Gorshkova Tatyana T Chernova Tatyana T Mokshina Natalia N Gorshkov Vladimir V Kozlova Liudmila L Gorshkov Oleg O

Scientific reports 20181001 1

The intrusive growth, a type of plant cell elongation occurring in the depths of plant tissues, is characterized by the invasion of a growing cell between its neighbours due to a higher rate of elongation. In order to reveal the largely unknown molecular mechanisms of intrusive growth, we isolated primary flax phloem fibers specifically at the stage of intrusive growth by laser microdissection. The comparison of the RNA-Seq data from several flax stem parts enabled the characterization of those ...[more]

PMID: 30275452

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Project description:Different cell types and multiple cellular connections characterize the human brain. Gene expression analysis using a specific population of cells is more accurate than conducting analysis of the whole tissue homogenate, particularly in the context of neurodegenerative diseases, where a specific subset of cells is affected by the different pathology. Due to the difficulty of obtaining homogenous cell populations, gene expression in specific cell-types (neurons, astrocytes, etc.) has been understudied. To leverage the use of archive resources of frozen human brains in studies of neurodegenerative diseases, we developed and calibrated a method to quantify cell-type specific-neuronal, astrocytes-expression profiles of genes implicated in neurodegenerative diseases, including Parkinson's and Alzheimer's diseases. Archive human frozen brain tissues were used to prepare slides for rapid immunostaining using cell-specific antibodies. The immunoreactive-cells were isolated by Laser Capture Microdissection (LCM). The enrichment for a particular cell-type of interest was validated in post-analysis stage by the expression of cell-specific markers. We optimized the technique to preserve the RNA integrity, so that the RNA was suitable for downstream expression analyses. Following RNA extraction, the expression levels were determined digitally using nCounter Single Cell Gene Expression assay (NanoString Technologies®). The results demonstrated that using our optimized technique we successfully isolated single neurons and astrocytes from human frozen brain tissues and obtained RNA of a good quality that was suitable for mRNA expression analysis. We present here new advancements compared to previous reported methods, which improve the method's feasibility and its applicability for a variety of downstream molecular analyses. Our new developed method can be implemented in genetic and functional genomic research of neurodegenerative diseases and has the potential to significantly advance the field.

Project description: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.

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Transcriptome Analysis of Intrusively Growing Flax Fibers Isolated by Laser Microdissection.

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Transcriptome Analysis of Intrusively Growing Flax Fibers Isolated by Laser Microdissection.

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