Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of Arabidopsis phloem

ABSTRACT: This sudy focuses on the identification of transcripts in the shoot phloem of the model plant Arabidopsis thaliana. Transcripts expressed in the phloem tissue (parenchyma cell, companion cell, sieve element) were excised by laser microdissection pressure catapulting (LMPC). These were compared with transcripts isolated from leaf phloem exudates by EDTA-chelation technique. Optimization of sample harvest resulted in RNA of high quality from both sources. Modifications of the RNA amplification procedure obtained RNA of sufficient yield and quality for microarray experiments. Microarrays (Affymetrix, ATH1) hybridized with RNA derived from phloem tissue by LMPC or phloem sap allowed us to differentiate between phloem located and mobile transcript species. The datasets provide a search criterion for phloem-based signals and will facilitate reverse genetic studies and forward genetic screens for phloem and long distance RNA signaling mutants. Experiment Overall Design: Arabidopsis plants were cultivated under short day conditions (8 h light) until flowering. Phloem tissue was isolated by Lasermicrodissection and Pressure Catapulting (LMPC) and phloem exudate by EDTA-chelation technique. In both experiments poly-A-RNA was extracted and amplified before hybridization of microarrays (Affymetrix, ATH1). For both experiments three LMPC-derived phloem tissue (LMPC-derived phloem_1-3) and three phloem exudate samples (Phloem exudate_1-3) were analysed. Precise protocols of plant growth, sample harvest, RNA extraction and amplification are provided in Deeken et al., 2008.

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Rosalia Deeken

PROVIDER: E-GEOD-10247 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Identification of Arabidopsis thaliana phloem RNAs provides a search criterion for phloem-based transcripts hidden in complex datasets of microarray experiments.

Deeken Rosalia R Ache Peter P Kajahn Inga I Klinkenberg Joern J Bringmann Gerhard G Hedrich Rainer R

The Plant journal : for cell and molecular biology 20080514 5

<h4>Summary</h4>Phloem-mobile signals play a major role in plant nutrition, development and communication. In the latter context, phloem-mobile RNAs have been associated with signalling between plant tissues. In this study, we focused on the identification of transcripts in the shoot phloem of the model plant Arabidopsis thaliana. To isolate transcripts expressed in phloem parenchyma cells and in companion cell-sieve element complexes, we used laser microdissection coupled to laser pressure cata ...[more]

PMID: 18485061

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Project description:Phloem is the primary conduit through which photosynthates, hormones, and other biologically important molecules are distributed from aerial plant organs downward and throughout the plant. Increasing evidence suggests that phloem contents are diverse and play a critical role in biotic and abiotic stress adaptation. Drought causes the greatest decreases in agricultural crop productivity among all biotic and abiotic stresses, and the link between water deficiency and phloem protein contents is relatively unexplored. Here we employed the EDTA-facilitated phloem exudate collection method from Solanum lycopersicum leaves during a period of drought stress and recovery. Our analysis resulted in the confident identification and quantification of 2,558 proteins. Comparing our data with previous findings confirms that our exudate collection strategy enriched for known phloem proteins. Independent of drought, enrichment analysis of the total phloem exudate protein profiles from all samples suggest that the exchange of proteins into the phloem is more complex than previously thought, including additional protein chaperone systems, branched-chain amino acid synthesis proteins, trehalose metabolism, and RNA silencing proteins. During the experiment we observed 169 proteins whose abundance changed significantly within the phloem sap, the majority of which were impacted specifically in response to drought. Among these were proteins involved in lipid metabolism, chaperone-mediated protein folding, carboxylic acid metabolism, abscisic acid signaling, cytokinin biosynthesis, and amino acid metabolism are significantly upregulated during drought. Conversely, proteins involved in lipid signaling, sphingolipid metabolism, cell wall organization, carbohydrate metabolism, and a mitogen-activated protein kinase are downregulated in response to drought. Many of these observations are consistent with previous literature findings at the whole plant level but have not been localized to the vasculature in tomato, suggesting phloem plays a critical role in adaptation to drought stress in tomato .

Project description:The paraveinal mesophyll (PVM) of soybean leaves is a layer of laterally expanded cells sandwiched between the palisade and spongy mesophyll chlorenchyma. The vacuoles of PVM cells contain an abundance of a putative vegetative storage protein, VSP (α, β). VSP is is constitutively produced, but is up-regulated during sink limitation experiments involving flower, fruit, or vegetative bud removal. Soybean vegetative lipoxygenases (Vlx), consisting of 5 isozymes (Vlx, A-D), have been identified as potential storage proteins because they accumulate to high levels with experimental sink limitation and have been co-localized with VSP to the vacuoles of PVM cells. We re-investigated the sub-cellular locations of these enzymes with TEM immuno-cytochemistry. We employed laser micro-dissection to compared RNA expression of PVM cells with mesophyll chlorenchyma cells; and we performed a micro-array analysis of soybean leaf samples representing a time-course, sink-limitation, experiment. We found that none of the Vlx isozymes co-localize with putative storage proteins in PVM vacuoles, and that our sink limitation experiment (typical of those used in the past) induced a strong up-regulation of stress response genes, simultaneous with the up-regulation of the Vlx isozymes. Our findings do not support a storage function for soybean Vlx. The microarray results presented here represent our comparison of gene expression within PVM and palisade mesophyll paraenchyma (PP) cells from mature soybean leaves. For this part of our study both the PVM and palisade parenchyma (PP) cells were isolated from sections of soybean leaves by laser micro-dissection and pressure catapulting (LMPC). Total RNA was isolated from these cells and amplified for microarray analysis. Soybean leaves were fixed with a mixture of ethanol and acetic acid, embedded in paraffin and sectioned to provide samples for laser micro-dissection with pressure catapulting (LMPC). Slides were deparaffinized prior to LMPC. A total of six samples were collected: three biological replicates for PVM cells and three replicates for PP cells. After enough cells were collected for each samples, the cells were pooled for RNA extraction. RNA was amplified prior to microarray hybridization using the Whole-Transcriptome Ovation Pico RNA Amplification System from NuGEN Technologies, San Carlos, CA, USA.

Project description:The clubroot disease caused by the obligate biotrophic protist Plasmodiophora brassicae on host plants of the Brassicaceae family is characterized by enhanced cell division and cell expansion. Since a typical root section of an infected plant always includes different stages of the pathogen as well as uninfected cells, we were interested to investigate specific developmental stages of the pathogen and their effect on host transcriptional changes. We extended previous microarray studies on whole roots by using Laser Microdissection and Pressure Catapulting (LMPC) to isolate individual cells harboring defined developmental stages of the pathogen. In addition, we compared the central cylinder of infected to contol plants. We were especially interested to elucidate the stage-specific hormonal network. The upregulation of genes involved in auxin and cytokinin metabolism and signaling was confirmed. In addition, we found evidence that brassinosteroid (BR) synthesis and signal perception was in many cases upregulated in enlarged cells and the central cylinder. This was confirmed by qPCR and mutant analysis of the BR receptor mutant bri1-6, which exhibited less severe gall formation than the respective wild type. Our results identify novel hormone pathways involved in clubroot development. Using this method of single cell preparation combined with transcriptome analysis has been very useful to elucidate the regulation of gall growth by this obligate biotropic pathogen in a cell- and stage-specific manner. Transcription profiling was performed in isolated Arabidopsis thaliana root cells harboring different developmental stages of Plasmodiophora brassicae at two time points after inoculation (dai) (14 and 21 dai), as well as in infected central cylinder tissue from roots at 14 dai (days after inoculation). Control samples were taken from uninfected roots. Host cells were dissected from paraffin embedded roots using Laser Microdissection and Pressure Catapulting (LMPC). 8 samples have been analyzed.

Dataset Information

Transcription profiling of Arabidopsis phloem

Publications

Identification of Arabidopsis thaliana phloem RNAs provides a search criterion for phloem-based transcripts hidden in complex datasets of microarray experiments.

Similar Datasets

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