Dataset Information

Cell Type-Specific Imaging of Calcium Signaling in Arabidopsis thaliana Seedling Roots Using GCaMP3.

ABSTRACT: Cytoplasmic calcium ([Ca2+]cyt) is a well-characterized second messenger in eukaryotic cells. An elevation in [Ca2+]cyt levels is one of the earliest responses in plant cells after exposure to a range of environmental stimuli. Advances in understanding the role of [Ca2+]cyt in plant development has been facilitated by the use of genetically-encoded reporters such as GCaMP. Most of these studies have relied on promoters such as Cauliflower Mosaic Virus (35S) and Ubiquitin10 (UBQ10) to drive expression of GCaMP in all cell/tissue types. Plant organs such as roots consist of various cell types that likely exhibit unique [Ca2+]cyt responses to exogenous and endogenous signals. However, few studies have addressed this question. Here, we introduce a set of Arabidopsis thaliana lines expressing GCaMP3 in five root cell types including the columella, endodermis, cortex, epidermis, and trichoblasts. We found similarities and differences in the [Ca2+]cyt signature among these root cell types when exposed to adenosine tri-phosphate (ATP), glutamate, aluminum, and salt, which are known to trigger [Ca2+]cyt increases in root cells. These cell type-targeted GCaMP3 lines provide a new resource that should enable more in depth studies that address how a particular environmental stimulus is linked to specific root developmental pathways via [Ca2+]cyt.

SUBMITTER: Krogman W

PROVIDER: S-EPMC7503278 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Publications

Cell Type-Specific Imaging of Calcium Signaling in Arabidopsis thaliana Seedling Roots Using GCaMP3.

Krogman William W Sparks J Alan JA Blancaflor Elison B EB

International journal of molecular sciences 20200902 17

Cytoplasmic calcium ([Ca2+]cyt) is a well-characterized second messenger in eukaryotic cells. An elevation in [Ca2+]cyt levels is one of the earliest responses in plant cells after exposure to a range of environmental stimuli. Advances in understanding the role of [Ca2+]cyt in plant development has been facilitated by the use of genetically-encoded reporters such as GCaMP. Most of these studies have relied on promoters such as ...[more]

PMID: 32887481

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Project description:The purpose of the McrBC methylation microarray* assay is to determine which regions of a genome are methylated versus those that are unmethylated in a single Arabidopsis thanliana genotype. McrBC is a methylation-sensitive enzyme that restricts DNA only at purine-Cmethyl half sites when separated between* 50bp and 3kb. A designated amount of DNA from a particular genotype is sheared to a size range of 1kb-10kb using nebulization. We restrict half of the nebulized DNA with McrBC, and the methylated fraction is then removed from the unmethylated fraction through gel purification of DNA fragments greater than 1kb.* The remaining nebulized DNA is subjected to the same gel purification scheme, but with no McrBC treatment. In a single hybridization, the untreated sample is labeled with Cy5 and the McrBC-treated sample with Cy3.* Thus, after labeling and microarray hybridization, the ratio of normalized Cy5 to normalized Cy3 represents the relative methylation at the sequence represented by the spot on the microarray.* Dye swap analysis is carried out to take account of experimental variation by repeating the hybridization with identical samples labeled with Cy3 and Cy5, respectively. The two samples in this series are complementary hybridizations in a dye-swap analysis. wild-type seedlings, 9 days old. Below is the normalized result of the paired dye swap samples VC134 and VC136. The ANOVA model of Kerr, Martin and Churchill (2000) was used to analyze the data from the dye-swap experiments, with terms included to account for gene, dye-by-gene, treatment-by-gene, and random error terms. The style of hypothesis test proposed by Black and Doerge (2002) was applied to each of the features represented on each array, with rejection of the null hypothesis indicating a significant change in fluorescence intensity. To account for the number of hypothesis tests being made, and thus provide some level of error rate control, significance was assessed using both family-wise error rate (FWER) and false discovery rate (FDR) controlling methods. The step-down multiple comparisons procedure of Holm (1979) was used to control the FWER below alpha = 0.01, while the step-up procedure of Benjamini and Hochberg (1995) was used to control the FDR below alpha = 0.01. For the purposes of this experiment, the hypotheses were assumed to be independent. Features found to be significantly enriched for DNA methylation after hypothesis-testing with a controlled error rate are flagged in the last two columns. Keywords: other

Dataset Information

Cell Type-Specific Imaging of Calcium Signaling in Arabidopsis thaliana Seedling Roots Using GCaMP3.

Publications

Cell Type-Specific Imaging of Calcium Signaling in <i>Arabidopsis thaliana</i> Seedling Roots Using GCaMP3.

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