Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 53

A novel domain of LONG GRAIN 1 in rice possesses transcriptionalactivation activity
H. Abe,∗1 R. Morita,∗1 Y. Shirakawa,∗1 Y. Hayashi,∗1 H. Ichida,∗1 and T. Abe∗1
We previously identiﬁed the gene LONG GRAIN 1(LIN1 ), which regulates seed development, and a mutation of the gene (lin1 ), which causes long grain size.1)
The lin1 mutant was obtained from rice (Oryza sativa
L. ssp. japonica cv. Nipponbare) M2 population derived
from imbibed seeds irradiated with argon-ion beams(5 Gy, 95 MeV/nucleon, LET: 286 keV m−1 ) in theRIKEN RI-beam factory.1) LIN1 is predicted to encode
the 412 amino acid protein, LIN1, with calculated MW
42,579 and pI 10.8. InterPro search of LIN1 revealed
that LIN1 has no known functional domain and/or motif.
To gain more insight into LIN1 functions for seeddevelopment, we conducted yeast two-hybrid screening
for isolation of LIN1 interaction proteins (LIPs).2) We
obtained several LIPs, and one LIP was MADS1, thetranscription factor for ﬂoral organ development. During the course of the yeast two-hybrid assay for LIN1
and MADS1 interaction, we noticed that mutant lin1
was more actively bound to MADS1 than full length
LIN1 (Figs. 2.3 and 4). As sequence alignments of
LIN1 homologs in rice and Arabidopsis revealed that
LIN1 has 5 short stretch conserved domains (I∼V)(Fig. 1.1), we divided LIN1 into several N- and Cterminal fragments, each containing several conserved
domains, and performed yeast two-hybrid assays. Unexpectedly, yeast cells transformed with BD-fusion construct of N-terminal fragment of LIN1-M120 (Fig. 1.3)
containing only conserved domain I were grown on aselection plate of SD/-Leu/-Trp/-His/-Ade not only inthe presence of AD-MADS1 but also in an AD-empty
construct (Figs. 2.6 and 7). On the contrary, the BD1
234Fig. 1. Schematic diagram of LIN1, lin1 and LIN1 fragmentprotein sequence. Dark blue color with Roman numerals(I∼V) indicates positions of conserved domains. 1. Fulllength LIN1 protein (M1-G412). 2. Mutant lin1 proteincaused by 1 bp deletion of LIN1 gene. The frame shift
mutation result for an extended sequence (orange color)and a premature stop codon. 3. N-terminal fragmentof LIN1-M120 containing the domain I. 4. C-terminal
fragment of P121-LIN1 without the N-terminal portion(3) of LIN1.
∗1

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Fig. 2. Yeast two-hybrid assays. Combination of BD-fusion
constructs and AD-fusion constructs are indicated (1-8).
Both constructs were transformed into Y2H-Gold yeast
competent cells, and growth assays on SD/-Leu/-Trp andSD/-Leu/-Trp/-His/-Ade plates were performed.

fusion construct of the C-terminal fragment of P121LIN1 (Fig. 1.4) had no such activity (Fig. 2.8). These
results indicate that the N-terminal fragment of LIN1M120 had transcriptional activation activity for the reporter gene expression in yeast cells.
Considering the results, we hypothesize that LIN1might be a novel transcription factor family protein with
N-terminal transcriptional activation activity for regulating seed development. Elucidation of LIN1-regulated
target genes should be provided for important information not only for understanding seed development but
also for application of plant breeding.
References

1) R. Morita et al., Mol. Breeding 39, 135 (2019).
2) H. Abe et al., unpublished.

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