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Updated: Jun 25, 2025
Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants
Published on: August 5, 2020
MicroRNAs potentially targeting DDR-related genes are differentially expressed upon exposure to γ-rays during seed
Sri Amarnadh Gupta Tondepu1, Vasilissa Manova2, Dhanalakshmi Vadivel3
1Department of Biology and Biotechnology "L. Spallanzani", University of Pavia, Via Adolfo Ferrata 9, 27100, Pavia, Italy.
Abstract:
DNA damage response (DDR), a complex network of cellular pathways that cooperate to sense and repair DNA lesions, is regulated by several mechanisms, including microRNAs. As small, single-stranded RNA molecules, miRNAs post-transcriptionally regulate their target genes by mRNA cleavage or translation inhibition. Knowledge regarding miRNAs influence on DDR-associated genes is still scanty in plants. In this work, an in silico analysis was performed to identify putative miRNAs that could target DDR sensors, signal transducers and effector genes in wheat. Selected putative miRNA-gene pairs were tested in an experimental system where seeds from two wheat mutant lines were irradiated with 50 Gy and 300 Gy gamma(γ)-rays. To evaluate the effect of the treatments on wheat germination, phenotypic and molecular (DNA damage, ROS accumulation, gene/miRNA expression profile) analyses have been carried out. The results showed that in dry seeds ROS accumulated immediately after irradiation and decayed soon after while the negative impact on seedling growth was supported by enhanced accumulation of DNA damage. When a qRT-PCR analysis was performed, the selected miRNAs and DDR-related genes were differentially modulated by the γ-rays treatments in a dose-, time- and genotype-dependent manner. A significant negative correlation was observed between the expression of tae-miR5086 and the RAD50 gene, involved in double-strand break sensing and homologous recombination repair, one of the main processes that repairs DNA breaks induced by γ-rays. The results hereby reported can be relevant for wheat breeding programs and screening of the radiation response and tolerance of novel wheat varieties.
Insights
This study identifies microRNAs regulating the DNA damage response (DDR) in wheat. Gamma radiation affects miRNA and DDR gene expression, with tae-miR5086 negatively correlating with RAD50, impacting DNA repair.
Area of Science:
- Plant molecular biology
- Genetics
- Biochemistry
Background:
- MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression post-transcriptionally.
- The DNA damage response (DDR) network in plants, including its regulation by miRNAs, is not well understood.
- Investigating miRNA roles in plant DDR is crucial for understanding stress responses.
Purpose of the Study:
- To identify wheat miRNAs targeting DDR-associated genes.
- To experimentally validate the effect of gamma radiation on miRNA and DDR gene expression in wheat.
- To explore the relationship between specific miRNAs and DDR genes in response to radiation stress.
Main Methods:
- In silico analysis to predict miRNA-gene interactions in wheat DDR pathways.
- Gamma irradiation of wheat seeds (50 Gy and 300 Gy).
- Phenotypic analysis, DNA damage assessment, reactive oxygen species (ROS) measurement, and quantitative real-time PCR (qRT-PCR) for gene and miRNA expression profiling.
Main Results:
- Gamma irradiation induced ROS accumulation and DNA damage in wheat seeds, negatively impacting seedling growth.
- Selected miRNAs and DDR genes showed differential expression in response to gamma rays in a dose-, time-, and genotype-dependent manner.
- A significant negative correlation was found between tae-miR5086 and the RAD50 gene, crucial for DNA double-strand break repair.
Conclusions:
- This study provides insights into miRNA-mediated regulation of DDR in wheat.
- The findings highlight the complex interplay between miRNAs, DDR genes, and radiation response in wheat.
- Results are relevant for wheat breeding programs aiming to improve radiation response and tolerance.
