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. 2019 Jun 27;9(1):9369.
doi: 10.1038/s41598-019-44789-w.

Abnormal leaf development of rpt5a mutant under zinc deficiency reveals important role of DNA damage alleviation for normal leaf development

Naoyuki Sotta  1 , Takuya Sakamoto  2 , Sachihiro Matsunaga  2 , Toru Fujiwara  3
Affiliations

Abnormal leaf development of rpt5a mutant under zinc deficiency reveals important role of DNA damage alleviation for normal leaf development

Naoyuki Sotta et al. Sci Rep. .

Abstract

Leaf development in plants, including dorsoventral (adaxial-abaxial) patterning, is tightly regulated. The involvement of several subunits of the 26S proteasome in adaxial-abaxial polarity establishment has been reported. In the present study, we revealed that in Arabidopsis thaliana, a mutation in RPT5A, a subunit of 26S proteasome, causes abnormally narrow true leaves under zinc deficiency. mRNA accumulations of DNA damage marker genes in leaves were elevated by zinc deficiency. PARP2, a single-strand break (SSB) inducible gene, was more strongly induced by zinc deficiency in rpt5a mutants compared with the wild type. A comet assay indicated that SSB is enhanced in mutants grown under the zinc deficiency condition. These results suggest that SSB accumulation is accompanied by abnormal leaf development. To test if DNA damage is a sole cause of abnormal leaf development, we treated the wild type grown under normal zinc conditions with zeocin, a DNA damage-inducing reagent, and found that narrow leaves developed, suggesting that DNA damage is sufficient to induce the development of abnormally narrow leaves. Taken together with the observation of the abnormal leaf morphology of our mutant plant under zinc deficiency, we demonstrated that the alleviation of DNA damage is important for normal leaf development.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Leaf shape of rpt5a-4 under zinc deficiency. (A) Fourteen-day-old seedlings of wild type, rpt5a-4, and rpt5a-4 complementation line grown under control and zinc deficiency conditions. Bar; 5 mm. (B–E) Leaf shapes were categorized into four groups, round tip (B), edged tip (C), narrow (D), and filamentous (E), as illustrated in (B) from left to right. Bar; 5 mm. (F) Frequency was calculated from observation of at least 20 individual plants for each condition. The numbers with “#” are identifiers for independent transformant lines in the complementation experiment. There is no significant difference in the ratio of (“round tip” + “edged tip”): (“narrow” + “filamentous”) at p < 0.05 among groups sharing the same letter by Fisher’s exact test with Holm correction.
Figure 2
Figure 2
Aberrant leaf development in rpt5a-4 leaves under zinc deficiency. (A) Filamentous true leaf of rpt5a-4 under zinc deficiency. Note that no trichome has been developed. (B) rpt5a-4 leaf under control (1 µM zinc) condition (C) SAM of rpt5a-4 under zinc deficiency. Arrowhead indicates abnormal horn-like structure developed from emerging leaf. (D,E) Cross-sections of leaves of wild type under control (D) and zinc deficiency (E) conditions. (F,G) Cross-sections of leaves of rpt5a-4 under control (F) and zinc deficiency (G) conditions. (H) Cross-section of most severely filamentous leaf of rpt5a-4 under zinc deficiency. Blue stain is marker ink put on adaxial leaf surface before slicing. (I,J) Vascular bundle of wild type under control (I) and zinc deficiency (J) conditions. (K–M) Vascular bundle of rpt5a-4 under control conditions (K) and filamentous leaves under zinc deficiency (L,M). Bars, (A,B) 1 mm; (C) 500 µm; (D–H) 100 µm; (I–M) 50 µm.
Figure 3
Figure 3
mRNA accumulation in rpt5a-4 mutant under zinc deficiency. Seedlings were grown for 14 days and total RNA was extracted from the first pair of true leaves with the representative shapes; for the control condition, the wild type and rpt5a-4 were represented by “round tip” and “edged tip”, respectively, and for the zinc deficiency condition, the wild type was represented by “round tip” and “edged tip,” whereas rpt5a-4 was represented by “narrow” or “filamentous.” mRNA accumulations were normalized by the geometric mean of Actin8, PEX4, and SAND. Values represent mean ± standard deviation of four biological replicates. Groups sharing the same letter were not significantly different from each other at p < 0.05 by Tukey’s multiple test.
Figure 4
Figure 4
DSB and SSB accumulations in shoot of rpt5a-4 under zinc deficiency. Nuclei were extracted from whole shoots of 16-day-old seedlings and were subjected to a comet assay. (A) N/N comet assay to detect DSB. (B) A/N comet assay to detect DSB and SSB. At least 100 nuclei were observed for each treatment. Median and first and third quartiles are shown in cross bars and vertical bars represent range of data points obtained. There is no significant difference at p < 0.05 between groups sharing the same letter by Steel–Dwass test. (C–J) Representative nuclei images from comet assay with N/N method (C–F) and A/N method (G–J). Nuclei with median Tail DNA [%] are shown with their values. (C,G) Wild type, control, (D,H) wild type, zinc deficiency, (E,I) rpt5a-4, control, (F,J) rpt5a-4, zinc deficiency. Bars, 50 µm. Contrast and brightness were adjusted for visualization purposes. A pseudo-color scale is shown at the bottom.
Figure 5
Figure 5
DNA damage-inducing treatment caused filamentous leaves in a dose-dependent manner. Seedlings were grown under treatment with various concentrations of zeocin, a DNA damage-inducing reagent. Zeocin was added to the normal medium (MGRL medium with normal zinc). (A) Frequency of aberrant leaves observed under treatment with various concentrations of zeocin. For each condition, 52 seedlings were observed. There is no significant difference in the ratio of (“round tip” + “edged tip”): (“narrow” + “filamentous”) at p < 0.05 among groups sharing the same letter by Fisher’s exact test with Holm correction. (B) Seventeen-day-old wild type under 6.6 µM zeocin treatment. (C) rpt5a-4 under 3.3 µM zeocin treatment. Bars, 5 mm. (D,E) DNA damage accumulations in shoot of rpt5a-4 grown under 3.3 µM zeocin treatment: (D) the N/N comet assay to detect double strand break, (E) the A/N comet assay to detect double and single strand break. Nuclei were extracted from whole shoots of 14-day-old seedlings and were subjected to comet assay. At least 100 nuclei were observed for each treatment. There is no significant difference at p < 0.05 between groups sharing the same alphabets by Steel-Dwass’ test. (F–M) Representative nuclei images from the comet assay with N/N method (F–I) or A/N method (J–M). Nuclei which give median Tail DNA [%] are shown with its value. (F,J) Wild type, control, (G,K) wild type, 3.3 µM zeocin, (H,L) rpt5a-4, control, (I,M) rpt5a-4, 3.3 µM zeocin. Bar, 50 µm. Contrast and brightness were adjusted for visualization purpose. Pseudo-color scale is shown in the bottom.
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