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Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana
Published on: May 21, 2019
Plant-specific environmental and developmental signals regulate the mismatch repair protein MSH6 in Arabidopsis
Valentina Gonzalez1, Nicolás R Figueroa1, Claudia P Spampinato1
1Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531, Rosario 2000, Argentina.
Abstract:
The DNA mismatch repair (MMR) is a postreplicative system that guarantees genomic stability by correcting mispaired and unpaired nucleotides. In eukaryotic nuclei, MMR is initiated by the binding of heterodimeric MutS homologue (MSH) complexes to the DNA error or lesion. Among these proteins, MSH2-MSH6 is the most abundant heterodimer. Even though the MMR mechanism and proteins are highly conserved throughout evolution, physiological differences between species can lead to different regulatory features. Here, we investigated how light, sugar, and/or hormones modulate Arabidopsis thaliana MSH6 expression pattern. We first characterized the promoter region of MSH6. Phylogenetic shadowing revealed three highly conserved regions. These regions were analyzed by the generation of deletion constructs of the MSH6 full-length promoter fused to the β-glucuronidase (GUS) gene. Combined, our in silico and genetic analyses revealed that a 121-bp promoter fragment was necessary for MSH6 expression and contained potential cis-acting elements involved in light- and hormone-responsive gene expression. Accordingly, light exposure or sugar treatment of four-day old A. thaliana seedlings triggered an upregulation of MSH6 in shoot and root apical meristems. Appropriately, MSH6 was also induced by the stem cell inducer WUSCHEL. Further, the stimulatory effect of light was dependent on the presence of phyA. In addition, treatment of seedlings with auxin or cytokinin also caused an upregulation of MSH6 under darkness. Consistent with auxin signals, MSH6 expression was suppressed in the GATA23 RNAi line compared with the wild type. Our results provide evidence that endogenous factors and environmental signals controlling plant growth and development regulate the MSH6 protein in A. thaliana.
Insights
Environmental signals like light and sugar, along with hormones, regulate the MSH6 gene in Arabidopsis thaliana, crucial for DNA mismatch repair (MMR) and genomic stability.
Area of Science:
- Molecular Biology
- Plant Science
- Genetics
Background:
- The DNA mismatch repair (MMR) system ensures genomic stability by correcting DNA replication errors.
- MutS homologue (MSH) complexes initiate MMR in eukaryotes, with MSH2-MSH6 being the most abundant.
Purpose of the Study:
- To investigate how light, sugar, and hormones modulate the expression pattern of the MSH6 gene in Arabidopsis thaliana.
- To identify regulatory elements within the MSH6 promoter responsive to environmental and hormonal cues.
Main Methods:
- Phylogenetic shadowing to identify conserved regions in the MSH6 promoter.
- Generation of MSH6 promoter deletion constructs fused to the GUS reporter gene.
- Analysis of MSH6 expression in response to light, sugar, hormones (auxin, cytokinin), and specific genetic lines (WUSCHEL, GATA23 RNAi).
Main Results:
- A 121-bp MSH6 promoter fragment is essential for gene expression and contains cis-acting elements for light and hormone response.
- Light exposure, sugar treatment, and WUSCHEL induced MSH6 expression in meristems.
- Light-induced MSH6 expression is dependent on phyA; auxin and cytokinin also upregulated MSH6 under darkness.
- Auxin signaling influences MSH6 expression, as shown by suppression in the GATA23 RNAi line.
Conclusions:
- Environmental signals (light, sugar) and plant hormones (auxin, cytokinin) play significant roles in regulating MSH6 expression in Arabidopsis thaliana.
- The identified 121-bp promoter region is critical for mediating these responses, linking growth and development signals to DNA repair.
- These findings highlight the intricate regulation of DNA repair mechanisms by endogenous and exogenous factors in plants.
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