Abstract
Ataxia Telangiectasia and Rad-3-related protein (ATR) is a DNA damage signaling kinase required for the monitoring of DNA integrity. Together with ATM and SOG1, it is a key player in the transcriptional regulation of DNA damage response (DDR) genes in plants. In this study, we describe the role of ATR in the DDR pathway in barley and the function of the HvATR gene in response to DNA damages induced by aluminum toxicity. Aluminum is the third most abundant element in the Earth’s crust. It becomes highly phytotoxic in acidic soils, which comprise more than 50% of arable lands worldwide. At low pH, Al is known to be a genotoxic agent causing DNA damage and cell cycle arrest. We present barley mutants, hvatr.g and hvatr.i, developed by TILLING strategy. The hvatr.g mutant carries a G6054A missense mutation in the ATR gene, leading to the substitution of a highly conserved amino acid in the protein (G1015S). The hvatr.g mutant showed the impaired DDR pathway. It accumulated DNA damages in the nuclei of root meristem cells when grown in control conditions. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis revealed that 60% of mutant nuclei possessed DNA nicks and breaks, whereas in the wild type only 2% of the nuclei were TUNEL-positive. The high frequency of DNA damages did not lead to the inhibition of the cell cycle progression, but the mutant showed an increased number of cells in the G2/M phase. In response to treatments with different Al doses, hvatr.g showed a high level of tolerance. The retention of root growth, which is the most evident symptom of Al toxicity, was not observed in the mutant, as it was in its parent variety. Furthermore, Al treatment increased the level of DNA damages, but did not affect the mitotic activity and the cell cycle profile in the hvatr.g mutant. A similar phenotype was observed for the hvatr.i mutant, carrying another missense mutation leading to G903E substitution in the HvATR protein. Our results demonstrate that the impaired mechanism of DNA damage response may lead to aluminum tolerance. They shed a new light on the role of the ATR-dependent DDR pathway in an agronomically important species.
Highlights
Aluminum (Al) is the most abundant metal and the third most abundant chemical element in the Earth’s crust that comprises approximately 8% of its mass (Bojórquez-Quintal et al, 2017)
We have identified barley TILLING mutants, hvatr.g and hvatr.i, carrying different missense mutations in the HvATR gene that showed an impaired repair of DNA lesions, but the Al-tolerant phenotype
The high frequency of DNA damages observed in the mutants already in the control conditions did not lead to the inhibition of cell cycle progression
Summary
Aluminum (Al) is the most abundant metal and the third most abundant chemical element (after oxygen and silicon) in the Earth’s crust that comprises approximately 8% of its mass (Bojórquez-Quintal et al, 2017). The toxicity of Al3+ in acidic soils is an important agricultural problem that has been identified more than 100 years ago (Miyake, 1916). In tropical and subtropical regions, soil acidity is one of the most important constrains that hinder the increase of food production, and in the temperate zones of eastern North America and throughout Europe (where acidic soils reach up to 80% of the total area), Al stress may significantly affect crop yields (von Uexküll and Mutert, 1995; Aggarwal et al, 2015). Aluminum toxicity is considered as the main growth-limiting factor in acidic soils and the second, after drought, most serious abiotic stress to crop production worldwide (Kochian et al, 2015)
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