RIKEN Accel. Prog. Rep. 50 (2017)

Ⅲ-4. Radiation Chemistry & Biology

A nalysis of a temperature sensitive virescent mutantof rice induced by heavy-ion beam †R. Morita,1 M. Nakagawa, 2 H. Takehisa, 3 Y. Hayashi, 1 H. Ichida, 1 S. Usuda, 1 K. Ichinose, 1
H. Abe, 1 Y. Shirakawa, 1 T. Sato, 1,4 M. Fujiwara, 1,5 R. Itoh 6 and T. Abe 1
In plants, several leaf-color mutants such as albino,xantha, pale green, stripe, and virescence have been found.
Among them, virescent mutants are intriguing because theyexhibit white chlorotic leaves during the early growth stages,
but produce normal green leaves as they grow. To date, anincreasing number of genes responsible for virescentphenotypes have been identified from a number of species.
For example, eight genes were recognized as causative
genes for the virescent phenotype in rice. Among them,
only three genes were reported until 2010, and theremaining five genes were identified recently (since 2011).
However, little is known about the mechanism underlyingthis phenotype. To gain insight into the molecularmechanism of the virescent phenotype, we isolated andcharacterized a novel rice mutant named 22-4Y.
22-4Y was obtained from a mutant population generated
by heavy-ion irradiation (12C6+ ions, 20 Gy, LET: 22.5 keVμm-1).1) This mutant exhibited chlorotic leaves only during
an early growth period, especially the second, third, andfourth leaves at 25°C. After the fifth-leaf stage, the mutants
produced normal green leaves. In addition, the mutant
shows temperature sensitivity. At 30°C, 22-4Y produced
third and fourth leaves with slight chlorosis. However, at20°C, almost all leaves of the mutant were white.
Interestingly, the mutant transferred from 30°C to 20°C
conditions at the fifth leaf stage produced chlorotic leaves
as the sixth and subsequent leaves, indicating that thismutant responded to low temperature in the early growth
and tillering stages. We performed transmission electron
microscopy to compare the chloroplast ultrastructure of themutant grown at 30°C and 20°C. At 30°C, mutant plants
possessed normally developed chloroplasts. However, thechloroplasts in the mutant at 20°C displayed undeveloped
membrane structures. These observations demonstrate thatthe chlorotic phenotype of the mutant is attributable toaberrant chloroplast development during leaf formation.
Genetic analysis revealed that there are two genes alteredby translocation (deletion for LOC_Os05g34040 andinsertionfor
LOC_Os12g31810)
(Fig.
1).
A
complementation test revealed that the causative gene forthe virescent phenotype is LOC_Os05g34040.2) Since
LOC_Os05g34040 is a newly identified causative gene for†

Condensed from the article in Biosci. Biotech. Biochem. 81, 271 (2017)
RIKEN Nishina Center
2
Faculty of Science and Technology, Ishinomaki Senshu University
1

3

Institute of Crop Science, National Agriculture and Food Research Organization

4

Graduate School of Agricultural Science, Tohoku University
Faculty of Science and Technology, Sophia University

5
6

the virescent phenotype, we named this gene Cold Sensitive
Virescent1 (CSV1). The protein domain analysis of CSV1
showed that this protein belongs to the large family ofFAD-dependent pyridine nucleotide reductases (FADPNR).
Since the FADPNR family includes a broad range ofoxidoreductase enzymes, it is difficult to infer the molecularfunction of CSV1 simply from its amino acid sequencehomology. ChloroP program predicted that the N-terminal
region of the CSV1 protein might function as a chloroplasttransit peptide. To verify its ability, we constructed a fusion
gene expressing the N-terminal region of CSV1 fused to theN-terminus of the cyan fluorescent protein (CFP), under thecontrol of a strong promoter (named 35S::TPCSV1-CFP). We
co-introduced 35S::TPCSV1-CFP with 35S::TPAtFtsZ1-YFP,
positive control encoding the plastid-targeted yellow
fluorescent protein (YFP), into an onion epidermal cell. The
fluorescent signals of CFP were clearly co-localized withthose of YFP (Fig. 2). In contrast, the fluorescent signals
were observed in the cytoplasm and nucleus when we
introduced the plasmid DNA for a non-fused CFP or YFP
as a control. These observations demonstrated that CSV1 isa plastid-localizing protein. Our findings indicate that CSV1
is an important gene for chloroplast development under cold
stress, both in the early growth stage and in the tilleringstage.

Fig. 1. Schematic representation of the translocationinduced in 22-4Y. The boxes indicate exons. ATG andSTOP indicate the start and stop codon, respectively.

Fig. 2. Intracellular localization of CSV1. 35S::TPCSV1-CFP,
CFP with the putative transit peptide of CSV1(pseudo-coloured in green); 35S::TPAtFtsZ1-YFP, YFP withthe transit peptide of plastid-targeted AtFtsZ1 (magenta);
Merged, merged images of the CFP and YFP images; DIC,differential interference contrast. Scale bars = 50 μm.
References1) M. Nakagawa et al.: RIKEN Accel. Prog. Rep. 43, 281 (2010).
2) R. Morita et al.: RIKEN Accel. Prog. Rep. 46, 259 (2013).

Faculty of Science, University of the Ryukyus- 272 -