Ⅲ-4. Radiation Chemistry & Biology

RIKEN Accel. Prog. Rep. 51 (2018)

Isolation of C4 Flaveria bidentis mutants with reduced quenching ofchlorophyll fluorescence from heavy-ion-beam-mutagenized M2
population
T. Ogawa,∗1 M. Yamane,∗1 Y. Taniguchi,∗1 T. Furumoto,∗2 T. Abe,∗3 and Y. N. Munekage∗1Plants using C4 photosynthesis exhibit higher CO2 assimilation rates than plants using C3 photosynthesis under low CO2 conditions. This C4 photosynthesis (exceptfor single-cell C4 photosynthesis) is usually achieved bythe operation of the C4 metabolic cycle between mesophyll (M) and bundle-sheath (BS) cells, which concentrates CO2 at the site of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) in BS cells.1) With an
aim to analyze the regulation of light-energy conversion
and metabolic cycle between two cells, we screened mutants with a reduced quenching of chlorophyll ﬂuorescence from a heavy-ion-beam-mutagenized M2 population of C4 Flaveria bidentis (Asteraceae) which carries
NADP-malic enzyme-type C4 photosynthesis.
We ﬁrst investigated the survival ratio of mutagenized
seedlings to evaluate the optimum condition for mutagenesis by the irradiation of a carbon-ion beam. F. bidentis seeds were irradiated at diﬀerent dose levels ranging
from 25 to 300 Gy with a linear energy transfer (LET)
value of 30 keV/ µm (Table 1). The Number of seedlings
forming true leaves/total number of seedlings (true leaf
formation ratio), average true leaf length in 13-days-old
seedlings, and survival ratio 1 month after germination
decreased with the increase of dose level. Although thetrue leaf formation ratio was only slightly aﬀected at
50 Gy, the survival ratio was lower at 50 Gy than at
25 Gy. Therefore, we determined 25 Gy as an optical condition for mutagenesis. A large number of seeds
were mutagenized with a 25 Gy carbon-ion beam (M1 )and grown in a green house, and the next population ofseeds (M2 ) was corrected for screening.
Table 1. Survival ratio in F. bidentis M1 population at different dose levels. Average ±SD is shown for the true leaflength. ∗P < 0.01.
Dose level,GyNonirradiation
2550
100
150
200300

True leaf
formation ratio, %(n=50-70)
100

True leaf
length, mm(n=25)
3.4 ± 2.2

Survivalratio, %(n=50-70)1009797596258
3.9

2.4 ± 1.8 *3.4 ± 1.8
1.4 ± 3.1 *1.3 ± 2.0 *1.4 ± 2.2 *0.3 ± 0.8 *

948310400We used a chlorophyll ﬂuorescence imaging system,
Maxi-Imaging-PAM (Walz, Germany), for screening mutants. Chlorophyll ﬂuorescence emitted from photosystem II reﬂects the photosynthetic electron transport∗1∗2∗3

School of Science and Technology, Kwansei Gakuin University
Faculty of Agriculture, Ryukoku University
RIKEN Nishina Center

Fig. 1. Chlorophyll fluorescence imaging after exposure toblue light of 500 µmol photons m−2 s−1 for 1 min. Orange
shows lower NPQ levels than green.

Fig. 2. Time courses of NPQ induction in white-light illumination at 270 µmol photons m−2 s−1 and relaxation ofNPQ in 3 min in dark.

state and light energy dissipation induced by the luminal
acidiﬁcation of the thylakoid membrane, which is monitored as nonphotochemical quenching (NPQ) of chlorophyll ﬂuorescence.2) In the imaging system, the majority of detected chlorophyll ﬂuorescence was assumed tobe derived from chloroplasts in palisade M cells and aminority was assumed to be derived from BS chloroplast because BS cells surround the vasculature immediately beneath the palisade cells within leaves and contain chloroplasts with reduced grana and PSII activity.
Six mutants named q1 ∼q6 with reduced NPQ wereisolated from a population of 860 M2 plants, which wereobtained from 180 M1 parents (Figs. 1 and 2). q1 ∼q3 orq4 and q5 can possess identical mutation because thosewere derived from the same parental batch, but at least
3 independent mutants were isolated from this screening
system. q1 and q4 showed lower CO2 assimilation rates
than the wild type (data not shown). Since coordinatedmetabolism in M and BS cells is required for C4 photosynthesis, mutants isolated with this screening method
are expected to include defects in not only genes directlyrelated to NPQ induction but also genes related to theregulation of C4 photosynthesis.
References
1) M. D. Hatch, Biochim. Biophys. Acta 895, 81 (1987).
2) T. Shikanai et al., Plant Cell Physiol. 40, 1134 (1999).

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