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Plant Cell Physiol. 45(11): 1557–1565 (2004)
JSPP © 2004
Radial Expansion of Root Cells and Elongation of Root Hairs of Arabidopsis
thaliana Induced by Massive Doses of Gamma Irradiation
Toshifumi Nagata 1, 3, Setsuko Todoriki 2 and Shoshi Kikuchi 1
1
2
Department of Molecular Genetics, National Institute of Agrobiological Sciences, Kannondai 2-1-2, Tsukuba, Ibaraki, 305-8602 Japan
National Food Research Institute, Kannondai 2-1-12, Tsukuba, Ibaraki, 305-0856 Japan
;
Radial expansion of root cells and elongation of root
hairs were induced within 3 d of a massive dose (3 kGy) of
gamma irradiation to Arabidopsis thaliana. Because treatment with the antioxidant n-propyl gallate before irradiation suppressed these changes, gamma irradiation partially
rescued the rhd2 mutant (defective in NADPH oxidase); the
superoxide-generating reagent paraquat induced similar
root morphogenesis. These responses appeared to be
induced by the active oxygen species (AOS) generated by
water radiolysis. Ethylene production was induced immediately after gamma irradiation and reached a steady level
after about 2 h. Addition of the ethylene precursor 1aminocyclopropane-1-carboxylic acid partly induced a similar expansion of root cells and elongation of root hairs.
Addition of an inhibitor of ethylene biosynthesis, aminoethoxyvinylglycine, before gamma irradiation completely
suppressed the formation of abnormal structures. These
results suggest that the AOS is involved in the root morphological changes through the ethylene biosynthesis induced
by gamma irradiation in Arabidopsis.
Keywords: Active oxygen species — Ethylene — Gamma
irradiation — Root hair.
Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid;
AOS, active oxygen species; AVG, aminoethoxyvinylglycine; MAPKKK, mitogen-activated protein kinase kinase kinase; n-PG, n-propyl
gallate.
root morphogenesis. Cell expansion and root hair differentiation are controlled by active oxygen species (AOS) generated
by RHD2 NADPH oxidase (Foreman et al. 2003). The AOS are
synthesized by NADPH oxidase at the elongation site of differentiating root cells. After many steps, they causes calcium ions
to flood into the cells by activating the calcium channel on the
plasma membrane, and induce cell elongation and root hair differentiation. Therefore, the generation of a large amount of
AOS may induce many physiological changes in root tissues.
We have been studying the regulation and location of AOS in
living plants in order to gain an understanding of the signal
transduction process.
The phytohormone ethylene plays a key role in the radial
swelling of roots; root swelling has been used as a sensitive
bioassay to test for contamination of soils with household gas,
thereby indicating a gas leak (Harvey and Rose 1915). Ethylene
regulates root elongation, as well as root hair initiation and
expansion (Cao et al. 1999, Cho and Cosgrove 2002, Ma et al.
2003). Ethylene production can be induced by gamma irradiation of plants (Young 1965, Abdel-Kader et al. 1968, Lee et al.
1968, Akamine and Goo 1971, Romani 1984, Larrigaudiere et
al. 1990). Identification of the ethylene biosynthetic pathway
therefore provides a basis for investigating the action of gamma
irradiation. However, little is known about the molecular mechanism of ethylene induction by gamma irradiation. In the
present report, through biochemical and genetic analyses, we
show that root morphological changes induced by gamma irradiation are mediated by ethylene production and that ethylene
production is mediated by AOS generated by water radiolysis.
Introduction
Results
Massive doses (1–3 kGy) of gamma irradiation produce
severe abiotic stress in Arabidopsis thaliana. The plants
respond by accumulating anthocyanin and ascorbic acid and
forming new trichomes (Nagata et al. 1999, Nagata et al. 2003).
In the present study, we focused on the morphological changes,
namely, radial expansion of root cells and elongation of root
hairs, observed in the tip regions of roots. Arabidopsis roots
consist of single layers of epidermis, cortex, endodermis and
pericycle surrounding a vascular bundle (Dolan et al. 1993,
Benfey and Scheres 2000). Roots of abnormal shape or size
have been genetically screened to identify genes that regulate
Gamma irradiation induces radial expansion of root cells and
elongation of root hairs in Arabidopsis
Photomicrographs of root morphological changes after
gamma irradiation (3 kGy) are shown in Fig. 1. These changes
appear to consist of termination of root extension, swelling of
the root and change of the outgrowth direction of root hairs.
These changes appeared 12 h after irradiation (Fig. 1A-2, B-2)
and were complete by about 72 h after irradiation. Even
500 Gy could induce these alterations (Fig. 1A-4, B-4). These
changes were induced more easily in the tip region of young
and short lateral roots than in long and mature roots. Scanning
3
Corresponding author: E-mail, [email protected]; Fax, +81-298-38-7007.
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Enlargement of Arabidopsis root hairs by irradiation
The root of Arabidopsis is made up of organized cell layers. To see which cells or cell layers were changed after gamma
irradiation, we made transverse and longitudinal sections of
roots (Fig. 3). The cells in the epidermal and cortical cell layers expanded radially. The pericycle cell layer also expanded
slightly, and the meristematic region was reduced (Fig. 3A-2,
B-2). Furthermore, the diameter of the irradiated plant’s root
(240±23 µm) was twice that of the control plant’s root
(110±8 µm). Root hairs also were initiated and elongated in the
swollen root region.
Fig. 1 Gamma-radiation-induced morphological changes in individual root tips of A. thaliana (ecotype Columbia). (A) Tip region of the
main root. (B) Tip region of a short lateral root: (1) before irradiation;
(2) 1 d after irradiation; (3) 2 d after irradiation; (4) 3 d after irradiation. Bar = 0.4 mm.
Ethylene production attributed to the gamma-radiation-induced
morphological changes in roots
Because similar root morphological changes appear to be
mediated by the phytohormone ethylene, we treated Arabidopsis plants with the ethylene precursor 1-aminocyclopropane-1carboxylic acid (ACC) and the ethylene biosynthesis inhibitor
aminoethoxyvinylglycine (AVG), then observed the root structure by means of scanning electron microscopy. ACC solution
(100 µl, 5 mM) was dropped onto the base of plants. The plants
were grown for 2 d, then the root structure was observed (Fig.
4). Moderate morphological changes were observed (Fig. 4B):
a slight increase in root diameter, slight elongation of root hairs
and conspicuous ectopic root hair outgrowth in the distal region
of the root (Fig. 4C). In comparison, treatment with 1 mM
AVG for 3 d before gamma irradiation completely suppressed
the root morphological changes (Fig. 4D). These data suggest
that ethylene may play an important role in root morphological
changes.
We also examined the contribution of AOS to the root
morphological changes. Two experiments were performed, one
to test the inhibitory effect of the antioxidant n-propyl gallate (n-PG), and the other to test the additive effect of the
radical-generating reagents paraquat and menadione (Fig. 5).
Treatment with the antioxidant completely inhibited the radiation-induced root morphological changes (Fig. 5E), and treatment with paraquat or menadione slightly induced the
radiation-associated radial expansion of root epidermal cells
and outgrowth of root-hair-like structures (Fig. 5C, D). In these
structures, the cell file rule appears to be maintained. These
observations suggest that root morphological changes are mediated by AOS.
electron micrographs of the changes are shown in Fig. 2.
Radial expansion of root epidermal cells in the elongation zone
of the root (Schiefelbein and Benfy 1991) appeared first (Fig.
2B–F). Root extension was stopped, probably because of the
destruction of DNA in the root meristem by the gamma irradiation. Radial expansion of epidermal cells looks like the initiation of outgrowth of root hairs. In the root-hair-like outgrowths
that appeared, the cell file rule appeared to be maintained. Root
hair elongation was also detected in the swollen root region.
The columella root cap region was not changed, even after
completion of the expansion of epidermal cells.
Gamma irradiation of Arabidopsis induces ethylene production
Many external agents—including wounding, anaerobiosis, viral infection, auxin treatment, chilling, injury,
drought, and Cd2+ and Li+—induce ethylene production (Yang
and Hoffman 1984, Abeles et al. 1992). Gamma irradiation
also induces ethylene production in tomato (Abdel-Kader et al.
1968) and avocado (Young 1965). Ethylene production was
measured by means of gas chromatography. Every hour after
irradiation, 1 ml of air in the growth chamber (about 9 cm ×
9 cm × 0.5 cm) was taken by syringe, then injected into the gas
chromatograph. The kinetics of ethylene production after
Enlargement of Arabidopsis root hairs by irradiation
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Fig. 2 Scanning electron micrographs
of root structural changes after gamma
irradiation. (A–I) Temporal pattern of
root morphological changes. (A) Unirradiated root. (B) 12 h after irradiation
(3 kGy). (C) 24 h after irradiation. (D)
36 h after irradiation. (E) 48 h after irradiation. (F) 72 h after irradiation. (G–I)
Close-up view of the root surface structures. (G) Normal root tip region. (H)
24 h after gamma irradiation. (I) 72 h
after gamma irradiation. Tissues were
fixed and dried as described in Materials
and Methods. (A, B, E, F) Bar =
100 µm. (C, D) Bar = 150 µm. (G–I)
Bar = 50 µm.
gamma irradiation were measured first. Unirradiated plants did
not produce ethylene, whereas ethylene production was
induced within 1 h after gamma irradiation and continued for at
least 3 h (Fig. 6).
Are AOS involved in gamma-radiation-induced ethylene
production? To answer this question, we treated plants with an
antioxidant (n-PG) and paraquat. We used 5-week-old plants
that had already bolted and flowered. About 1.5 kGy of gamma
rays was applied. Three hours after irradiation, ethylene production was measured. Then we treated plants with n-PG and
the ethylene biosynthesis inhibitor AVG for 3 d before irradiation, as described in Materials and Methods. Gamma irradiation increased ethylene production to 4–5 times that of the
control (Table 1). Addition of n-PG in the absence of irradiation gave no change in the basal level of ethylene biosynthesis.
However, treatment with n-PG before gamma irradiation suppressed ethylene production. AVG suppressed basal ethylene
production and gamma-radiation-induced ethylene overproduction. The addition of paraquat (10 µM) caused ethylene production to double. These data suggest that gamma-radiationinduced ethylene production is suppressed by pretreatment
with antioxidant and that ethylene production is inducible by
the addition of paraquat. Therefore, gamma-radiation-induced
ethylene production may be mediated by AOS generated by
water radiolysis.
Gamma-irradiation-induced morphological changes in mutant
backgrounds
To identify which genes contribute to gamma-radiationinduced changes in root morphology, we examined root structural alteration in several mutants. RHD1, RHD2, RHD3,
RHD4 and RHD6 all play roles in root hair initiation and elongation (Schiefelbein and Somerville 1990, Masucci and Schiefelbein 1994, Wang H et al. 2002). We tested the mutant strains
rhd1-1, rhd2-1, rhd3-1 and rhd4-1. The rhd mutants showed
immature outgrowth of root hairs (Fig. 7A, C, E, G). However,
gamma irradiation induced a slight extension of the root hairs
(Fig. 7B, D, F, H), especially in rhd2-1 and rhd4-1. Most plants
showed radial expansion of root cells. The rhd2-1 phenotype
was partially rescued by gamma irradiation: that is, gamma
irradiation induced new trichome formation on the adaxial surface of the glabra rosette leaves of rhd2-1, as we reported previously (Nagata et al. 1999).
We isolated 15 ethylene-responsive mutants by tripleresponse screening and characterized them. The results
revealed that ETR1 and EIN4 act before CTR1 as negative regulators. ETR1 encodes an ethylene receptor, and EIN4 might be
a ‘second component’ (Chang et al. 1993, Schaller and Bleeker
1995, Bleecker et al. 1998). CTR1 is a central component in the
ethylene signal-transduction pathway; it acts as a negative regulator of EIN2, EIN3, EIN5, EIN6, EIN7, EIR1 and HLS1. In
addition, CTR1 shows amino acid similarity to the Raf family
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Enlargement of Arabidopsis root hairs by irradiation
Fig. 3 Transverse and longitudinal sections of roots. Transverse (A) and longitudinal (B) sections of normal (1) and
irradiated (2) roots at the same magnification. Staining, solidification and sectioning procedures are described in
Materials and Methods. Bar = 50 µm.
of protein kinases; its structure is similar to that of mitogenactivated protein kinase kinase kinase (MAPKKK; Kieber et al.
1993). The ctr1 mutant exhibits multiple changes in the root
epidermis, including a modest increase in the proportion of root
hair cells and a significant reduction in epidermal cell length
(Masucci and Schiefelbein 1996). The effects of gamma irradiation on the roots of these mutants are shown in Fig. 8. The
etr1 and ein4 mutants showed a modest increase in root diameter after gamma irradiation. Ectopic root hair initiation and
elongation of root hairs were suppressed (Fig. 8D, F). The ctr1
mutant showed an excessive increase in root diameter and a
slight extension of root hairs (Fig. 8H). The ein2, ein3 and ein5
mutants showed a slight increase in root diameter, but elongation of root hairs was suppressed (Fig. 8J, L, N).
Auxin enhances ethylene production in roots (Abeles et al.
1992). AXR2 represents an important component of the
hormone response pathway that affects root hair initiation
(Masucci and Schiefelbein 1996). The axr1 mutant is auxin
insensitive (Estelle and Somerville 1987, Lincoln et al. 1990)
and slightly ethylene insensitive (Timpte et al. 1995); AXR1 is
related to ubiquitin-conjugating enzymes (Leyser et al. 1993).
Mutant aux1 is auxin and ethylene insensitive in the roots
(Pickett et al. 1990). The axr4 mutant is auxin resistant
(Hobbie and Estelle 1995). We tested mutants aux1-7, axr2 and
Fig. 4 Effects of an ethylene precursor (ACC) or an ethylene biosynthesis inhibitor (AVG) on root morphological changes. (A) Unirradiated root. (B) Gamma-irradiated (72 h) root. (C) Root treated with
ACC. (D) Root treated with AVG and irradiated. The procedures for
treatment with ACC and AVG are described in Materials and Methods. Root tissues were fixed at 72 h after irradiation or treatment. Bar
= 150 µm.
Enlargement of Arabidopsis root hairs by irradiation
Fig. 5 Effects of treatment with paraquat, menadione or an antioxidant (n-PG) on root morphological changes. (A) Normal root. (B)
Gamma-irradiated (24 h) root. (C) Paraquat-treated root. (D) Menadione-treated root. (E) Root treated with n-PG and gamma irradiated.
The procedures for treatment with paraquat, menadione and n-PG are
described in Materials and Methods. All root tissues except (A) and
(B) were fixed at 72 h after treatment or irradiation. Bar = 150 µm.
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Fig. 7 Gamma-radiation-induced root morphological changes in rhd
mutants. Left-hand images: unirradiated plants. Right-hand images:
irradiated plants. (A, B) rhd1-1. (C, D) rhd2-1. (E, F) rhd3-1. (G, H)
rhd4-1. Tissues were fixed 72 h after irradiation (3 kGy). (A) Bar =
250 µm. (B–D) Bar = 200 µm. (E, F) Bar = 50 µm. (G, H) Bar =
150 µm.
These results indicate that the ETR1 ethylene-responsive
cascade (Ecker 1995) plays an important role in gammaradiation-induced root morphological changes in Arabidopsis.
Two other ethylene-responsive cascades have been reported:
the AUX1 cascade and the AXR1 cascade (Timpte et al. 1995,
Masucci and Schiefelbein 1996). The slight increase in root
diameter in the etr1 and ein4 mutants may be due to the action
Fig. 6 Kinetics of ethylene production after gamma irradiation.
Twenty plates containing 36 plants grown on MS-agar medium for
34 d were irradiated. Every hour after irradiation, ethylene production
during that hour was measured. One milliliter of air was sampled, and
the amount of ethylene was measured by means of gas chromatography.
axr4-2. As shown in Fig. 9, aux1-7 and axr4-2 showed radial
expansion of root cells like that of the wild type, but no elongation of root hairs (Fig. 9B, F). In comparison, axr2 showed little radial expansion of root cells but noticeable elongation of
root hairs (Fig. 9D).
Table 1 Ethylene production after gamma irradiation
Treatment
Control
Gamma irradiation (1.5 kGy)
n-PG
n-PG + gamma irradiation
AVG
AVG + gamma irradiation
Paraquat
a
Concentration (nl ml–1) a
0.3890 ± 0.0197
1.9141 ± 0.1068
0.4408 ± 0.0182
0.4998 ± 0.0421
0.0614 ± 0.0023
0.3821 ± 0.0123
1.0338 ± 0.0436
Mean ± 1 standard deviation of three independent experiments.
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Enlargement of Arabidopsis root hairs by irradiation
Fig. 8 Gamma-radiation-induced root morphological changes in ethylene-responsive mutants. (A) Unirradiated root of wild type. (B) Gammairradiated root of wild type. (C) Unirradiated root of etr1-1. (D) Irradiated root of etr1-1. (E) Unirradiated root of ein4. (F) Irradiated root of ein4.
(G) Unirradiated root of ctr1-1. (H) Irradiated root of ctr1-1. (I) Unirradiated root of ein2-1. (J) Irradiated root of ein2-1. (K) Unirradiated root of
ein3-1. (L) Irradiated root of ein3-1. (M) Unirradiated root of ein5-1. (N) Irradiated root of ein5-1. All irradiated root tissues were fixed at 72 h
after gamma irradiation (3 kGy). (A, B) Bar = 100 µm. (C–N) Bar = 150 µm.
of these cascades. Not only ethylene but also auxin appears to
play an important role in the root morphological changes
induced by gamma irradiation of Arabidopsis.
Discussion
Gamma irradiation induces physiological and morphological
changes in developing Arabidopsis roots
The responses of Arabidopsis roots after gamma irradiation represent growth regulation by AOS. Therefore, the formation of root hairs and expansion of epidermal cells are
controlled by AOS signal-transduction systems. Root hair differentiation and expansion are thought to be controlled by AOS
generated by NADPH oxidase (RHD2) under normal conditions (Foreman et al. 2003). Our results support this hypothesis
and suggest that AOS regulate the expansion of epidermal
cells. Gamma-irradiation-associated control of differentiation
of epidermal cells also occurred in leaves (Nagata et al. 1999).
Trichomes on the adaxial surface of the leaves increased in
number a few days after gamma irradiation. Therefore, there
seem to exist common systems that control both root and leaf
epidermal cells (Schellmann et al. 2002). The differentiation of
root hairs and trichomes is regulated by genes common to both
root and leaf epidermal cells [TTG1 (WD40 protein), GL2
(homeodomain protein), GL3 (bHLH), CPC (MYB), TRY
(MYB)] and by those that are similar between the two types of
cell [WER (MYB) → root hair differentiation, GL1 (MYB) →
trichome differentiation]. TTG and GL2 negatively regulate the
differentiation of root hair cells (Walker et al. 1999, Ohashi et
al. 2003). We gamma-irradiated the mutants ttg1-1 and gl2 and
Enlargement of Arabidopsis root hairs by irradiation
Fig. 9 Gamma-radiation-induced root morphological changes in
auxin-responsive mutants. (A) Unirradiated root of aux1-7. (B) Irradiated root of aux1-7. (C) Unirradiated root of axr2. (D) Irradiated root
of axr2. (E) Unirradiated root of axr4-2. (F) Irradiated root of axr4-2.
All irradiated root tissues were fixed at 72 h after gamma irradiation
(3 kGy). Bar = 150 µm.
observed that the root hair pattern of these mutants was altered.
However, the radial expansion of root cells and extension of
root hairs were similar to those of the wild type (data not
shown). Although the root and trichome differ at the histological level, both tissues seem to have a common cell differentiation system. Epidermal cells of both are induced to differentiate
exserted tissues; therefore, the same regulation systems may be
conserved between root and leaf. In addition, TTG1 regulates
root hair formation, trichome formation and anthocyanin synthesis. Therefore, transcription systems regulated by AOS may
use the common hierarchical regulatory system of WD40 →
MYB → homeodomain protein.
Contribution of ethylene production to gamma-radiationinduced morphological changes
Massive doses (1–3 kGy) of gamma irradiation induce
marked responses in A. thaliana. We previously characterized
these responses genetically and analyzed them physiologically.
We also analyzed anthocyanin and ascorbic acid biosynthesis
and trichome formation and reported the contribution of AOS
to the signal-transduction pathways, the biological significance
of these responses and the genetic components that contribute
to these responses (Nagata et al. 1999, Nagata et al. 2003). In
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the present study, we have shown that ethylene production
influences these responses by means of four independent
experiments: ethylene production after gamma irradiation,
treatment with an ethylene precursor (ACC), treatment with an
inhibitor of ethylene biosynthesis (AVG), and analysis with
ethylene- and auxin-responsive mutants.
The results of these experiments support the hypothesis
that ethylene production participates in gamma-radiationinduced root morphological changes and is mediated by AOS.
Ethylene is a gaseous phytohormone that influences many
aspects of plant growth and development (Abeles et al. 1992).
It promotes germination of seeds and senescence and abscission of flowers and leaves. The contribution of ethylene to root
cellular organization and especially to root hair formation has
been reported recently (Masucci and Schiefelbein 1996, Zhang
et al. 2003). Ethylene has also been implicated in the response
to a wide variety of stresses, including pathogen attack, wounding, anaerobiosis, heat shock, cold treatment, UV irradiation
and ionizing radiation (Wang K.L. et al. 2002). Stress-induced
ethylene has two effects. One is the induction of senescence of
damaged tissue, and the other is the induction of the expression of self-protective genes. The ethylene biosynthetic pathway has been elucidated (Yang and Hoffman 1984). The two
key enzymes are ACC synthase and ACC oxidase. ACC synthase is the rate-limiting step in ethylene biosynthesis. This
enzyme’s activity is highly regulated and closely parallels the
level of ethylene biosynthesis. In Arabidopsis, there are at least
five ACC synthase genes that show distinct expression patterns
in different tissues and that respond unequally to various inducers. This pattern suggests that the different ACC enzymes are
functionally distinct. Yang and Hoffman (1984) were unable to
elucidate which gene products are involved in the two types of
ethylene production (developmental and stress-induced) because
of the relatively low abundance of the gene products. Although
the results of our antioxidant treatment before gamma irradiation and of our treatment with paraquat and menadione imply
the possible contribution of AOS to some gamma-radiationinduced responses, we have shown that gamma-radiationinduced ethylene production is mediated by AOS.
Microarray analysis has revealed that the expression of
ethylene response genes, development control genes and redox
genes was dramatically changed after gamma irradiation
(Nagata et al. 2003). Therefore, the responses to AOS-induced
stress have been shown at the molecular level. As a next step
we will analyze those genes to reveal the AOS signal-transduction systems.
Materials and Methods
Arabidopsis strains and growth conditions
The following genetic stocks (with associated stock numbers)
were obtained from the Arabidopsis Biological Resource Center (Ohio
State University, Columbus, OH, U.S.A.): ttg1-1 (CS89), gl2-1 (CS65),
rhd1-1 (CS2257), rhd2-1 (CS2259), rhd3-1 (CS2260), rhd4-1
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Enlargement of Arabidopsis root hairs by irradiation
(CS2261), etr1-1 (CS237), ein4 (CS8053), ctr1-1 (CS8057), ein2-1
(CS3071), ein3-1(CS8052), ein5-1 (CS8054), ein6 (CS8055), ein7
(CS8056), eir1-1 (CS8058), hls1-1 (CS3073), aux1-7 (CS3074), axr2
(CS3077) and axr4-2 (CS8019). Arabidopsis thaliana (ecotype
Columbia) seeds were sown on agar-solidified Murashige and Skoog
medium (Nagata et al. 1999). After germination, plants were cultured
for 2–3 weeks under a 16-h light (4,000 lux)/8-h dark cycle at 22°C.
For the observation of root structure, 12 seedlings were grown in a
9 cm × 9 cm Petri dish. For the assay of ethylene production, 36 seedlings were grown in the same type of dish.
Gamma irradiation
Three-week-old plants on agar medium were irradiated with
gamma rays from cobalt-60 in a gamma cell (3.0 × 103 Gy h–1, 1.3 ×
102 TBq; Nordion Intl., Ontario, Canada). After irradiation, plants
were grown under the same conditions as already described.
Treatment with reagents
n-PG (Sigma-Aldrich, Tokyo, Japan) was used as the antioxidant. For 3 d before gamma irradiation, 100 µL of a 1-mM aqueous
solution was added to the surface of the solidified medium once per
day. About 16 h after the last treatment with antioxidant, gamma irradiation was started. Paraquat (2 µM), menadione (1 mM), ACC
(1 mM) and AVG (1 mM) (all Sigma products) were added similarly.
Microscopy
Root tissues were stained with 0.05% toluidine blue, washed
with distilled water and solidified in 1% agarose (Funakoshi, Tokyo,
Japan). Sections (30–50 µm) were made with a Microslicer DTK-1000
microtome (Do-han EM, Kyoto, Japan) fitted with a disposable steel
blade. Sections were mounted on Microphoto-FXA (Nikon, Tokyo,
Japan) and photographed with an FX35DX camera (Nikon) using Fujichrome Sensia-100 film.
Scanning electron microscopy
Roots were fixed for 4 h with 3% paraformaldehyde and 0.25%
glutaraldehyde in 50 mM sodium phosphate buffer (pH 7.2). After two
washes in distilled water, tissues were dehydrated in ethanol and
embedded in 3-methylbutyl acetate. The tissues were dried with a critical-point dryer (HCP-2; Hitachi, Tokyo, Japan) and ion-coated with an
ion sputter source (E-1010; Hitachi). Tissues were observed with a
scanning electron microscope (S-2040N; Hitachi).
Measurement of ethylene production
After gamma irradiation or treatment with reagents, 1 ml of the
air in the culture vessel was collected into a syringe through a hole
made previously. The air was transferred to a gas chromatograph (GC7AG, C-R5A; Shimadzu, Kyoto, Japan), and the peak area corresponding to ethylene was measured.
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(Received December 15, 2003; Accepted August 12, 2004)