III-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 54 (2021)

RIKEN Accel. Prog. Rep. 54 (2021)

Argon-ion-induced mutant of Arabidopsis thaliana exhibiting
accelerated leaf chlorosis†
A. Sanjaya,∗1 Y. Kazama,∗2,∗3 K. Ishii,∗2 S. Ohbu,∗2 T. Abe,∗2 and M. Fujiwara∗1,∗2
Chloroplast development in leaf tissues is crucial forplant growth and productivity. Plant leaves are composed of the epidermis, which forms the outermost layer,
and the mesophyll and vasculature inside. In the model
plant Arabidopsis thaliana, chloroplasts are distributedin leaf epidermal pavement and guard cells, as well as
in mesophyll cells. Despite vast research on the structure and function of leaf chloroplasts, there is limited
knowledge about their regulations in the plant life cycle.
To explore possible novel gene functions for leaf chloroplast development, we attempted a forward genetic approach. We created mutants of A. thaliana by exposing dry seeds of ecotype Col-0 to accelerated argon ions(290 keV/µm, 50 Gy) at RIBF.1) Following two rounds ofcultivation and selfing, as well as a macroscopic screening of the M3 generation of plants, we isolated a newmutant, designated as Ar50-33-pg1.
During seedling development, Ar50-33-pg1 produced
slightly pale cotyledons and leaves under standard plant
growth conditions (Fig. 1A, B). As the mutant leaves expanded and matured, their chlorosis became prominent(Fig. 1C). This observation of accelerated leaf chlorosis was supported by the chlorophyll measurement of
detached leaves through spectrophotometry (data notshown). Furthermore, Ar50-33-pg1 exhibited abnormalities at the reproductive stage, including phenotypes oflate flowering, flower longevity, and low seed production(data not shown).
Microscopic examination of expanding leaves of wild-(A)(C)(B)

Fig. 1. Growth and morphology of seedlings of Arabidopsis. (A) 2-week-old wild-type seedling. (B) 2-week-old
Ar50-33-pg1 seedling. In (A) and (B), images of chlorophyll autoﬂuorescence were taken at the same excitation condition by stereoﬂuorescence microscopy. Scalebar = 5 mm. (C) 4-week-old wild-type and Ar50-33-pg1
seedlings. Scale bar = 5 cm.
†∗1∗2∗3Condensed from the article in Plants 10, 848 (2021)
Department of Biology, Sophia University
RIKEN Nishina Center
Department of Biosciences and Biotechnology, Fukui Prefectural University

DIC

Guard cell
Chlorophyll

Pavement cellDIC
ChlorophyllAB

Fig. 2. Fluorescence microscopy of leaf epidermal guard cellsand pavement cells of Arabidopsis. (A) Wild type. (B)Ar50-33-pg1. Diﬀerential interference contrast (DIC) andchlorophyll autoﬂuorescence (colored in red) images ofcells from epidermal peels of growing rosette leaves. Scalebar = 10 µm.

type and Ar50-33-pg1 plants under normal observation
conditions revealed that a certain population of mutant epidermal chloroplasts lacked chlorophyll autofluorescence signal (Fig. 2). Meanwhile, mutant mesophyll
chloroplasts retained chlorophyll autofluorescence until
later stages of leaf senescence. Therefore, the chlorosis leaf phenotype of Ar50-33-pg1 might involve tissuedependent chloroplast abnormalities between the epidermis and mesophyll.
Backcrossing and segregation analyses indicated thatthe chlorotic phenotype of Ar50-33-pg1 is associatedwith impaired epidermal chloroplasts and is caused bya single nuclear recessive allele. Through whole-genome
resequencing, Ar50-33-pg1 was revealed to contain a∼0.9 Mb deletion from position 12621534 to 13561533
in chromosome V, which spans approximately 40 putative or well-characterized protein-coding genes. One ofthem encoded a known chloroplast membrane-localized,
ATP-independent metalloprotease, Ethylene-dependent
Gravitropism-deficient and Yellow-green 1 (EGY1).2)
Ar50-33-pg1 was then crossed with an egy1 mutant3)
in both directions. All F1 seedlings were similar to bothparents in that they commonly exhibited accelerated leaf
chlorosis with epidermal chloroplast defects. F1 plant
phenotypes at the reproductive stage were all wild typelike. Therefore, EGY1 was deemed to be the main causal
gene for the impaired chloroplast phenotypes in Ar50-33pg1.
References

1) Y. Kazama et al., The Plant J. 92, 1020 (2017).
2) G. Chen et al., Plant J. 41, 364 (2005).
3) T. Hirano et al., Mutat. Res. 735, 19 (2012).

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