A multi-level response to DNA damage induced by aluminium

Abstract

Aluminium (Al) ions are one of the primary growth-limiting factors for plants on acid soils, globally restricting agriculture. Despite its impact, little is known about Al action in planta. Earlier work has indicated that, among other effects, Al induces DNA damage. However, the loss of major DNA damage response regulators, such SOG1, partially suppressed the growth reduction in plants seen on Al-containing media. This raised the question whether Al actually causes DNA damage and, if so, how. Here, we provide cytological and genetic data corroborating that exposure to Al leads to DNA double-strand breaks. We find that the Al-induced damage specifically involves homology-dependent (HR) recombination repair. Using an Al toxicity assay that delivers higher Al concentrations than used in previous tests, we find that sog1 mutants become highly sensitive to Al. This indicates a multi-level response to Al-induced DNA damage in plants.

Keywords: ATM; ATR; Arabidopsis thaliana; CDKB1; DNA damage; RAD51; SOG1; aluminium; growth; homologous recombination repair.

Figure 1

Detection of Al‐induced DNA damage in long‐term growth assays. (a) Immunofluorescence analysis of γH2AX accumulation (green) in DAPI‐stained nuclei (blue) of wild‐type (WT) Arabidopsis root tips grown for 10 days on Al‐containing medium. (b) Quantification of γH2AX foci in WT plants after Al treatment. One‐hundred nuclei per line per experiment were grouped into six classes according to their number of γH2AX foci: nuclei containing no γH2AX foci, 1–2, 3–5, 6–10 and 11–20 γH2AX foci. (c) Seedling growth of WT, cdkb1, ku70 and rad51 mutants. Seeds were germinated on soaked gel medium containing 0, 1 and 1.5 mm Al (pH 4.2), and grown for 10 days. Scale bar: 1 cm. (d) Root growth measurements of WT, cdkb1, ku70 and rad51 mutants grown on soaked gel medium containing 0, 1 and 1.5 mm Al (pH 4.2) for 10 days. Data are presented as mean ± SD in three independent experiments. Significant differences from WT were determined by independent samples t‐test: *P < 0.05.

Figure 2

Detection of Al‐induced DNA damage in short‐term growth assays. (a) Six‐day‐old seedlings of wild‐type (WT), cdkb1, ku70 and rad51 mutants were transferred to 0 or 1.5 mm Al‐containing hydroponics (pH 4.2), and treated for 12 h. Treated seedlings were planted on agar plates without Al and grown for 5 subsequent days. Scale bar: 1 cm. (b) Root growth measurements of WT, cdkb1, ku70 and rad51. Al‐treated seedlings were transferred to agar plates without Al, and root lengths were measured for 5 days. Data are presented as mean ± SD in three independent experiments. Significant differences from WT were determined by independent samples t‐test: *P < 0.05. (c) Immunofluorescence analysis of γH2AX accumulation (green) in nuclei, stained with DAPI (DNA, blue), of root tips of WT, cdkb1, ku70 and rad51 plants after 3 days of growth on 1.5 mm Al‐containing soaked gel medium (pH 4.2), or 12 h in 1.5 mm Al‐containing hydroponic solution (pH 4.2). (d) Quantification of γH2AX foci in WT and cdkb1 plants after Al treatment. One‐hundred nuclei per line per experiment were grouped into six classes according to their number of γH2AX foci: nuclei containing no γH2AX foci, 1–2, 3–5, 6–10 and 11–20 γH2AX foci.

Figure 3

Short‐term Al treatment triggers CDKB1‐dependent homology‐dependent repair (HR). (a) Wild‐type (WT) plants containing the recombination reporter IC9C show blue spots on leaves after 24 h of incubation in 1.5 mm Al‐containing hydroponics. Arrows indicate representative blue sectors. (b) Number of blue sectors per plant grown without or with Al treatment. Data are presented as mean ± SD in three independent experiments. The significance of the difference was determined by independent samples t‐test: *P < 0.05. (c) WT IC9C and cdkb1 mutant plants containing the recombination reporter IC9C show blue spots on the leaves after 24 h of incubation in 1.5 mm Al‐containing hydroponics (pH 4.2). Arrows indicate representative blue sectors. (d) Numbers of blue sectors per plant grown without or with Al treatment. Data are presented as mean ± SD in three independent experiments. Significant differences from WT IC9C were determined by independent samples t‐test: *P < 0.05.

Figure 4

Reduction of root meristem size after Al treatment. (a) Five‐day‐old seedlings were transferred to 0 or 1.5 mm Al‐containing hydroponics (pH 4.2) and treated for 12 h. (b) Cortex cell number between the quiescent centre and the first elongated cell was counted after 12 h. Data are presented as mean ± SD in three independent experiments. Significant differences from wild‐type (WT) were determined by independent samples t‐test: *P < 0.05. (c) Root growth measurements of WT, cdkb1 and als3; 12 h Al‐treated seedlings were transferred to agar plates without Al and root lengths were measured for 5 days. Data are presented as mean ± SD in three independent experiments. Significant differences from WT were determined by independent samples t‐test: *P < 0.05.

Figure 5

Mutants in B1‐type cyclins are sensitive to Al in short‐term growth assays. (a) Six‐day‐old seedlings of wild‐type (WT), cdkb1, cycb1;1, cycb1;2, cycb1;3 and cycb1;4 mutants were transferred to 1.5 mm Al‐containing hydroponics (pH 4.2) and treated for 12 h. Treated seedlings were planted on agar plates without Al and grown for 5 days. Scale bar: 1 cm. (b) Root growth measurements of WT, cdkb1, cycb1;1, cycb1;2, cycb1;3 and cycb1;4. Al‐treated seedlings were transferred to agar plates without Al and root length was monitored for 5 days. Data are presented as mean ± SD in three independent experiments. Different letters indicate significant differences by independent samples t‐test: *P < 0.05.

Figure 6

Mutants in ATM and SOG1 are sensitive to Al in short‐term growth assays. (a) Six‐day‐old seedlings of wild‐type (WT), cdkb1, atr, atm and sog1 mutants were transferred to 1.5 mm Al‐containing hydroponics (pH 4.2) and treated for 12 h. Treated seedlings were subsequently planted on agar plates without Al and grown for 5 days. Scale bar: 1 cm. (b) Root growth measurements of WT, cdkb1, atr, atm and sog1. Al‐treated seedlings were transferred to agar plates without Al and root length was monitored for 5 days. Data are presented as mean ± SD in three independent experiments. Different letters indicate significant differences by independent samples t‐test: *P < 0.05. (c) Immunofluorescence analysis of γH2AX accumulation (green) in nuclei stained with DAPI (DNA, blue) of root tips of WT, atr, atm, and sog1 mutants after 12 h of incubation in 1.5 mm Al‐containing hydroponics (pH 4.2). (d) Quantification of γH2AX foci in root tips after Al treatment. One‐hundred nuclei per line per experiment were grouped into six classes according to their counted number of γH2AX foci: nuclei containing no γH2AX foci, 1–2, 3–5, 6–10 and 11–20 γH2AX foci.

Figure 7

Expression analysis of DNA damage response (DDR) genes after short‐term and long‐term Al treatments. (a) RNA was prepared from 10‐day‐old wild‐type (WT), atm, atr and sog1 seedlings, either untreated or treated with 1.5 mm Al‐containing hydroponics (pH 4.2) for 3 h. Relative expression levels of the indicated genes are shown as mean values from three biological repeats and with the untreated WT value set as 1. Error bars indicate SD. Significant differences from untreated value were determined by one‐way anova analysis, P < 0.05. (b) RNA was prepared from seeds germinated and grown on soaked gel medium containing 0 and 1.5 mm Al (pH 4.2) for 3 days. Relative expression levels of the indicated genes are shown as mean values from three biological repeats and with the untreated WT value set as 1. Error bars indicate SD. Significant differences from untreated plants were determined by one‐way anova analysis, P < 0.05. (c) RNA was prepared from seeds germinated and grown on soaked gel medium containing 0 and 1.5 mm Al (pH 4.2) for 10 days. Relative expression levels of the indicated genes are shown as mean values from three biological repeats and with the untreated WT value set as 1. Error bars indicate SD. Significant differences from untreated value were determined by one‐way anova analysis, P < 0.05.

Figure 8

Model of multi‐level response to DNA damage induced by Al. Four different scenarios are compared that result in different plant phenotypes upon DNA damage. (a and b) DNA damage response (DDR) in wild‐type (WT) plants at low and high levels of DNA damage, respectively. (a) Already low levels of DNA damage presumably trigger a signalling cascade via ATR and SOG1 resulting in a full‐blown DDR that involves the induction of DNA repair genes as well as other responses, in particular the downregulation of cell proliferation activity. As a result, plant growth is reduced (green plant). Grey shaded plant indicates growth of a plant under conditions that do not damage DNA. (b) High levels of DNA damage give rise to even further reduced plant growth (green plant). Grey shaded plant indicates growth of a plant under conditions that do not damage DNA. (c and d) DDR in mutant plants at low and high levels of DNA damage. (c) Elimination of SOG1 and ATR can result in increased growth (green plant) in comparison to the WT (grey plant) under mildly DNA‐damaging conditions as long as DNA repair mechanisms [i.e. homology‐dependent repair (HR) pathways] remain functional. (d) During high levels of DNA damage, DNA repair alone is not sufficient anymore to sustain survival and growth, and the full repertoire of DDR is needed, for example, cell proliferation activities have to be adjusted. Grey shaded plant indicates the growth of a WT plant under these conditions. X indicates a yet to be identified upstream regulator that is postulated to act at a similar level as SOG1. Moreover, ATR and ATM appear to have different response thresholds for DNA damage, at least for the damage induced by Al. For details, see Discussion.