I. HIGHLIGHTS OF THE YEARRIKEN Accel. Prog. Rep. 55 (2022)

RIKEN Accel. Prog. Rep. 55 (2022)

Argon-ion-induced mutations in Arabidopsis EGY1 gene aﬀectchloroplast development in leaf guard cells†
A. Sanjaya,∗1 R. Muramatsu,∗1 S. Sato,∗1 M. Suzuki,∗1 S. Sasaki,∗1 H. Ishikawa,∗1 Y. Fujii,∗1 M. Asano,∗1R. Itoh,∗2 K. Kanamaru,∗3 S. Ohbu,∗4 T. Abe,∗4 Y. Kazama,∗4,∗5 and M. Fujiwara∗1,∗4Leaf tissues of higher plants such as Arabidopsis
thaliana contain chloroplasts in the mesophyll and epidermis. Until now, it has been indicated that thereexists a tissue- or cell-type-dependent control of chloroplast division in leaves. This raises a fundamental question about the control of chloroplast development in theleaf epidermis, or, more speciﬁcally, whether chloroplastbiogenesis-related factors in leaf mesophyll cells play anequivalent role in the leaf epidermis.
To address this issue, we examined the phenotypeof leaf epidermal chloroplasts using two argon ion
irradiation-derived, pale green mutants of A. thaliana,Ar50-33-pg1 and Ar-28-pg1 (egy1-4 ).1–3) Both represent loss-of-function mutants of the Ethylene-dependent
Gravitropism-deﬁcient and Yellow-green 1 (EGY1 )gene, which encodes a thylakoid membrane-localized
protease for chloroplast development in the mesophyll.4) Moreover, the defects in the thylakoid structure
and pigmentation in the mutant mesophyll chloroplasts
are known to be pronounced during the later stages ofleaf (seedling) development.3,4)Fluorescence microscopy observations indicated severe chlorophyll deﬁciency in the chloroplasts of leaf
guard cells and guard mother cells of egy1 (Fig. 1).
This ﬁnding was in contrast to that observed in mesophyll cells, which retained a considerable amount ofchlorophyll. Labeling of plastids with stroma-targeted
ﬂuorescent proteins revealed that egy1 guard cells contained a normal number of plastids; however, their size
was moderately reduced compared to those of the wildtype guard cells (Fig. 1). Transmission electron microscopy further revealed that, in the egy1 chloroplasts,thylakoid development was impaired not only in mature
guard cells but also in the guard mother cell (Fig. 2).
Thus, the disorganization of the thylakoid structure andthe reduction in the chlorophyll contents showed a positive correlation.
Collectively, these observations demonstrate that
EGY1 is critical for chloroplast diﬀerentiation in guard
cells: egy1 guard cell chloroplasts apparently exhibit
permanent defects, starting from their development.
This prompts a revision to the current understanding ofchloroplast development, which has been based mainly
from on studies using mesophyll cells.
†∗1
∗2
∗3
∗4
∗5Condensed from the article in Plants 10, 1254 (2021)
Faculty of Science and Technology, Sophia University
Faculty of Science, University of the Ryukyus
Faculty of Agriculture, Kobe University
RIKEN Nishina Center
Faculty of Bioscience and Biotechnology, Fukui PrefecturalUniversity

Fig. 1. Fluorescence microscopy images of chloroplasts during guard cell development in Ar50-33-pg1 expressing
a stroma-targeted GFP. Bright ﬁeld (DIC), chlorophyll
autoﬂuorescence (chl), and GFP images of leaf epidermis
from 18-day-old seedlings are shown. Bar = 10 μm.
Wild-typeAr50-33-pg1

Fig. 2. Transmission electron microscopy analysis of chloroplasts in dividing guard mother cells of expanding leaves.
Primary leaves of 10-day-old wild-type and Ar50-33-pg1
plants were analyzed. Guard mother cells (A) and thechloroplasts therein (B). cp, chloroplast; n, nucleus; s,starch; v, vacuole. Bar = 2 μm (A) and 0.5 μm (B).

References
1) Y. Kazama et al., RIKEN Accel. Prog. Rep. 43, 283(2010).
2) T. Hirano et al., Mutat. Res. 735, 19 (2012).
3) A. Sanjaya et al., Plants 10, 848 (2021).
4) G. Chen et al., Plant J. 41, 364 (2005).

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