RNA-seq of FAC sorted leaf palisade cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Arabidopsis thaliana
PROVIDER: GSE182414 | GEO | 2022/08/11
REPOSITORIES: GEO
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Project description:RNA-seq of FAC sorted leaf palisade cells
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Project description:Microarray experiments were performed using FAC-sorted young photoreceptors to analyze their transcriptome in comparison to remaining retinal cells at same developmental stage and retinal progenitors. For each replicate, retinae of 6 to 8 postnatal day 0 pNestin-GFP or postnatal day 4 rhoEGFP mice were dissected and FAC-sorted based on GFP expression. RNA of fractions was isolated and subsequently analyzed with microarray experiment.
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2019-04-13 | MSV000083683 | MassIVE
Project description:A number of taxa utilize C4 photosynthesis, to limit the impact of photorespiration upon photosynthetic performance. In order to achieve a local elevation of CO2 concentration, maize plants possess two photosynthetic cell types. Rubisco accumulation is restricted to bundle sheath (BS) cells that surround the leaf veins. Carbon fixation occurs initially in adjacent mesophyll (ME) cells. C4 compounds are transported into the BS cells where they are subsequently decarboxylated, releasing CO2. Although the major components of the C4 pathway have been well characterized, less is known about further metabolic partitioning in the maize leaf. Microarray hybridizations have been performed in order to further investigate metabolic differences between BS and ME cell types. BS strands and ME protoplasts were isolated from the leaves of 10 day old maize seedlings by mechanical disruption and enzymatic digestion respectively. To control for differences arising from these different protocols, total leaf (TO) and total leaf stress (ST) samples were also isolated. The ST sample was subjected to the same treatments as the ME sample, with the omission of cell-wall degrading enzymes. Leaves for the TO sample were harvested as for the BS strand sample. An interwoven loop design was used to compare the four treatment groups. A biological group consisted of a growth of plants from which pooled individuals were taken for the four treatments. Six biological replicates (groups) were used. Labeling was performed using the Genisphere Array 900-MPX kit according to the manufacturer's protocol. Post hybridization washes were performed according to the recommendations of the Maize Oligo Array Project. Scan settings were used for detection of moderate to high expression signals (gain ~ 60%. power 90%). Following hybridization with TO cDNA, ~1/3 of features provided signal above twice background and below saturation. A number of taxa utilize C4 photosynthesis, to limit the impact of photorespiration upon photosynthetic performance. In order to achieve a local elevation of CO2 concentration, maize plants possess two photosynthetic cell types. Rubisco accumulation is restricted to bundle sheath (BS) cells that surround the leaf veins. Carbon fixation occurs initially in adjacent mesophyll (ME) cells. C4 compounds are transported into the BS cells where they are subsequently decarboxylated, releasing CO2. Although the major components of the C4 pathway have been well characterized, less is known about further metabolic partitioning in the maize leaf. Microarray hybridizations have been performed in order to further investigate metabolic differences between BS and ME cell types. BS strands and ME protoplasts were isolated from the leaves of 10 day old maize seedlings by mechanical disruption and enzymatic digestion respectively. To control for differences arising from these different protocols, total leaf (TO) and total leaf stress (ST) samples were also isolated. The ST sample was subjected to the same treatments as the ME sample, with the omission of cell-wall degrading enzymes. Leaves for the TO sample were harvested as for the BS strand sample. An interwoven loop design was used to compare the four treatment groups. A biological group consisted of a growth of plants from which pooled individuals were taken for the four treatments. Six biological replicates (groups) were used. Labeling was performed using the Genisphere Array 900-MPX kit according to the manufacturer's protocol. Post hybridization washes were performed according to the recommendations of the Maize Oligo Array Project. Scan settings were used for detection of moderate to high expression signals (gain ~ 60%. power 90%). Following hybridization with TO cDNA, ~1/3 of features provided signal above twice background and below saturation. Keywords: Gene expression profiling of bundle sheath and mesophyll cell types
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Project description:Hakea prostrata (Proteaceae) maintains a high photosynthetic rate at low leaf phosphorus and exhibits delayed leaf greening, a convergent adaptation found in many plant families where investment in photosynthetic machinery is delayed until after the leaf structure is built, saving on nutrient investment. To identify the dynamics of gene expression underlying delayed greening, we made a de novo transcriptome assembly for H. prostrata using roots and leaves from hydroponically grown plants, and leaves from five morphologically distinct stages of leaf development from wild plants. Abundance of unique transcripts was compared among the developing leaf stages to identify differentially expressed genes, which were separated into groups based on co-expression patterns. In general, transcripts encoding functions associated with leaf structure decreased in abundance across leaf development, with this concomitant with the decline in foliar P and N concentrations. An enrichment of differentially expressed genes associated with photosynthetic function increased in abundance at or after leaf expansion and much later in development than in species that do not have delayed greening. The abundance of transcripts encoding cytoplasmic and mitochondrial ribosomal subunits generally declined across leaf development, whilst transcripts encoding chloroplast ribosomal subunits increased. Taken together, these results indicate a clear shift in protein synthesis apparatus, moving from a leaf developmental stage and into the photosynthetically-active stage.
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Project description:Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of leaf color at different development stages. The goals of this study are to compare chlorophyll metabolism and chloroplast organization transcriptome profiling (RNA-seq) to microarray and quantitative reverse transcription polymerase chain reaction (qRT–PCR) methods and to evaluate protocols for optimal high-throughput data analysis Methods: leaf mRNA profiles of 12 RNA sequencing libraries (S1, S2, S3_S, and S3_C) were generated by deep sequencing, in triplicate, using an Illumina HiSeq 4000 system. After removing reads of low quality, those that remained were mapped to the reference genome (ftp://ftp.ensemblgenomes.org/pub/release-38/plants/genbank/brassica_oleracea/) using the HISAT package, allowing for a maximum of two mismatches and multiple alignments per read (up to 20 by default). qRT–PCR validation was performed using SYBR Green assays Results: Using an optimized data analysis workflow, we mapped about 571.74 million sequence reads per sample to the the reference genome (ftp://ftp.ensemblgenomes.org/pub/release-38/plants/genbank/brassica_oleracea/) and identified 1028, 4323, 428, and 1033 DEGs were detected in pairwise comparison (S2 vs. S1, S3_S vs. S2, S3_S vs. S2, and S3_S vs. S3_C, respectively). The DEGs were associated with ‘photosynthesis’, ‘carbon fixation in photosynthetic organisms’, ‘porphyrin and chlorophyll metabolism’ and other pathways in the Kyoto Encyclopedia of Genes and Genomes database; DEGs related to chloroplast organization were identified in the Gene Ontology analysis. The DEGs identified by RNA sequencing were confirmed by qRT-PCR analysis, indicating that the data were reliable. These findings provide information that can be useful for investigating the molecular basis for leaf variegation in ornamental kale and other plants. Conclusions: The results presented here reveal changes in the transcriptome profile of a variegated leaf kale. DEGs related to chlorophyll metabolism and chloroplast organization were detected. These results demonstrate that leaf color at different stages of development is influenced by chloroplast and pigment metabolism, providing a foundation for investigating the molecular basis for leaf variegation in ornamental kale and other plants.
2021-03-27 | GSE112493 | GEO
Project description:In this report, we describe a successful protocol for isolating and expression-profiling live fluorescent- protein-labelled neurons from zebrafish embryos. As a proof-of-principle for this method, we FAC-sorted and RNA-profiled GFP-labelled spinal CiA interneurons and compared the expression profile of these cells to those of post-mitotic spinal neurons in general and to all trunk cells. We show that RNA of sufficient quality and quantity to uncover both expected and novel transcription profiles via Affymetrix microarray analysis can be extracted from 5,700 to 20,000 FAC-sorted cells. As part of this study, we also further confirm the genetic homology of mammalian and zebrafish V1 interneurons, by demonstrating that zebrafish V1 cells (CiAs) express genes that encode for the transcription factors Lhx1a and Lhx5. This protocol for dissociating, sorting and RNA-profiling neurons from organogenesis-stage zebrafish embryos should also be applicable to other developing organs and tissues and potentially other model organisms.
2020-02-26 | GSE145916 | GEO
Project description:Early leaf growth is sustained by cell proliferation and subsequent cell expansion that initiates at the leaf tip and proceeds in a basipetal direction. Using detailed kinematic and gene expression studies to map these stages during early development of the third leaf of Arabidopsis thaliana we showed that the cell cycle arrest front did not progress gradually down the leaf, but rather was established and abolished abruptly. Interestingly, leaf greening and stomatal patterning followed a similar basipetal pattern, but proliferative pavement cell and formative meristemoid divisions were uncoordinated in respect to onset and persistence. Genes differentially expressed during the transition from cell proliferation to expansion were enriched in genes involved in cell cycle, photosynthesis, and chloroplast retrograde signaling. Proliferating primordia treated with norflurazon, a chemical inhibitor of retrograde signaling, showed inhibited onset of cell expansion. Hence, differentiation of the photosynthetic machinery is important for regulating the exit from proliferation.
2012-01-17 | GSE33936 | GEO
Project description:Maize and rice are the two most economically important grass crops and utilize distinct forms of photosynthesis to fix carbon: C4 and C3 respectively. Relative to C3 photosynthesis, C4 photosynthesis reduces photorespiration and affords higher water and nitrogen use efficiencies under hot arid conditions. To define key innovations in C4 photosynthesis, we profiled metabolites and gene expression along a developing leaf gradient. A novel statistical method was implemented to compare transcriptomes from these two species along a unified leaf developmental gradient and define candidate cis-regulatory elements and transcription factors driving photosynthetic gene expression. We also present comparative primary and secondary metabolic profiles along the gradients that provide new insight into nitrogen and carbon metabolism in C3 and C4 grasses. These resources, including community viewers to access and mine these datasets, will enable the elucidation and engineering of C4 photosynthetic networks to improve the photosynthetic capacity of C3 and C4 grasses.
2014-10-12 | GSE54274 | GEO