Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 48 (2015)

Functional analysis of a new virescent mutant in rice induced byheavy-ion beam irradiation
H. Abe,＊1 R. Morita,＊1 S. Kogure,＊1 K. Ichinose,＊1 Y. Hayashi,＊1 and T. Abe＊1
It is evident that heavy-ion beam irradiation effectively
produced many phenotypic mutants in rice (Oryza sativa L.
cv. Nipponbare)1, 2); thus, the method has been applied torice breeding as a new technology. Although many ricemutants have been isolated to date, the functions of anumber of their causative genes have not been completelyelucidated. One such mutant, 22-4Y in rice, has beenobtained as a virescent mutant by irradiating seeds with acarbon-ion beam (20 Gy, LET: 22.5 keV/µm)3). virescentis a chlorotic mutant of higher plants that reduces thechlorophyll content in young leaves, but the chlorophyll
content of the leaves recover as the plants grow4). Certainclasses of the virescent mutants are low-temperature
conditional4); the 22-4Y mutant also shows chlorotic leaves
phenotype when grown at 20 ○C but not at 30 ○C during theearly growth stages3). As the 22-4Y functions are relatedwith chlorophyll synthesis and/or chloroplast development,
both of which are important for crop production, wedecided to investigate the 22-4Y gene and to analyze itsfunctions.

Fig. 1. Schematic representation of the 22-4Y protein andits fragments expressed in E. coli.
The predicted 22-4Y protein (the upper line) has 720
amino acids (M1-Y720) with a calculated molecular massof 78.2 kDa.
Each fragment of 22-4Y(C58-Y720),(C58-R417), (D418-Y720), (C58-P239) and (R240-R417)
is shown. Positions of the FAD/NAD(P)-binding motif(GXGXXG) are shown in red bars. A region withhomology to pyridine nucleotide-disulfide oxidoreductase
family enzymes is shown in green.
The predictedchloroplast transit peptide is shown in dark blue.
＊1

Genetic analysis of the 22-4Y mutant revealed that gene
LOC_Os05g34040 on the long arm of chromosome 5
showed a deletion of 13095 bp 3). Morita et al.3) confirmed
by using a genetic complementation test that the causative
gene of 22-4Y is LOC_05g34040.
In order to know the gene functions, in many cases, it ismost important and necessary to reveal the functions of theprotein encoding to the corresponding gene. BLAST
searches of the complete Oryza sativa sequence revealed
that only one copy of the 22-4Y gene is present in thenuclear genome, which encodes a putative polypeptide of
720 amino acids with a calculated molecular mass of 78.2
kDa (Fig.1, the upper line). Pfam search predicted that the22-4Y
protein
hadaputativeconserved
FAD/NAD(P)-binding motif (GXGXXG) (Fig.1, red bars)and a homology to pyridine nucleotide-disulfideoxidoreductase family enzymes (Fig1, green region). Thismeans that 22-4Y is a new class of VIRECENT protein
among so far identified4) (V1, chloroplast-localized RNAbinding protein; V2, guanylate kinase; V3, large subunit ofribonucleotide reductase). TargetP program predicted that
the N-terminal 57 amino acids sequence of the 22-4Y
protein was a chloroplast transit peptide. Actually, when
the N-terminal 76 amino acids sequence of the 22-4Y
protein was fused to cyan fluorescent protein (CFP) andtransiently expressed in an onion epidermal cell, the fusion
protein was localized to chloroplast. Thus, the N-terminal
sequence of the 22-4Y protein was functional as achloroplast targeting peptide.
Electron microscopic
observation revealed that chloroplast development wasarrested when 22-4Y mutant was grown at 20 ○C but not at
30 ○C during the early growth stages. These results
suggest that the 22-4Y protein was functional in chloroplastand had some important regulatory functions in chloroplastdevelopment.
To further reveal and analyze the protein function of22-4Y, we decided to purify some recombinant proteins of22-4Y by using an E. coli expression system (Fig. 1). It is
important to demonstrate the enzymatic activity of the22-4Y protein, as well as to identify the target (substrate)
protein(s) to reveal the regulatory functions of the 22-4Y
protein during chloroplast development.
References

1) Y. Hayashi et al.: RIKEN Accel. Prog. Rep. 43, 282 (2010).
2) Y. Hayashi et al.: RIKEN Accel. Prog. Rep. 46, xxiv (2013).
3) R. Morita et al.: RIKEN Accel. Prog. Rep. 46, 259 (2013).
4) K. Kusumi and K. Iba: Front Plant Sci. 5, 386 (2014).

RIKEN Nishina Center- 313 -

完全版2014_本文.indd 313

15/10/16 18:11