Rice Science, 2004, 11(3): 125–128
http://www.ricescience.info
125
Effect of Indoleacetic acid (IAA) on the Negative Phototropism
of Rice Root
MO Yi-wei, WANG Zhong, QIAN Shan-qin, GU Yun-jie
(Agronomy Department, Agricultural College, Yangzhou University, Yangzhou 225009, China)
Abstract: To explore the effects of IAA on negative phototropism of rice （Oryza sativa L.） root，agar block containing IAA
was unilaterally applied on root tip to examine the phototropic response of root to exogenous IAA, and microstructure of the
bending part was observed with an optical microscope. The growth of seminal roots could be regulated by exogenous IAA as
well as light, as a result the root bent towards the site treated, causing asymmetric growth of the root cells at the elongation
zone and consequently bending growth. IAA concentration in the shaded side of adventitious root increased much greater at
1.5 h after the start of irradiation. The unequal lateral IAA distribution can be concluded to be the main cause for negative
phototropism of rice root.
Key words: rice; root tip; negative phototropism; indoleacetic acid
Light, as an environmental signal, has a profound
effect on the growth and development of plant [1]. Many
investigations on the positive phototropism of plant stem
have been made previously [2], but there were very few
reports on the negative phototropism of plant roots
before Okada’s report on Arabidopsis root in 1992 [3].
Wang et al. discovered that the seminal roots and
adventitious roots of the rice, including the branch roots,
had the characteristics of negative phototropism, and the
negative phototropic bending was caused by the
asymmetric growth of the cells on the irradiated and
shaded side of the root tip. IAA played a significant role
in the negative phototropism of rice root, and the
phototropic curvature was concluded to be resulted from
the light-induced unequal lateral distribution of IAA [4,5],
but the exact mechanism was still unknown and needed
to be investigated. In this paper, we proved that the
negative phototropism of rice root resulted from the
unequal lateral IAA distribution in the root tip.
long) were filled up with water, close end of which was
inserted into the foamed mass, and the germinating seed
was inserted into the open end in such a way that only
the germinated embryo was exposed to the air. Foamed
plastic mass then was hung up in a glass tank with water
inside and the tank was covered with a plastic film to
maintain the humidity (Fig. 1). Six centimeters long
roots were unilaterally applied with agar blocks
containing different concentrations of IAA on tip, then
were exposed to irradiation or kept in dark for 24 h, and
photographed at 24 h after the treatment.
Growth of adventitious root and measurement of
endogenous IAA in root tip
Rice seeds were split-sown in the paddy field.
Different leaf age seedlings were cultured in the glass
tank for producing adventitious roots according to the
experiment plan. Seedlings at tillering stage were
inserted in the foamed plastic mass in the glass tank after
the removal of tillers and roots, and then water or culture
MATERIALS AND METHODS
Germinating seed in plastic tube
Plant materials
Indica rice (Oryza sativa L.) variety Yangdao 6 was
used as plant materials in the investigation undertaken in
this study.
Foamed plastic mass
Agar block containing IAA
Plastic film
Growth of the seminal roots
Glass tank
Rice seeds were pre-germinated on damp gauze, the
orderly germinating ones were chosen to culture seminal
roots by aeroponic method. Hard plastic tubes (2-cm
Received: 15 May 2003; Accepted: 25 August 2003
Lamp source
water
Bending towards the side of application
Vertical growing root
Corresponding author: WANG Zhong([email protected])
Fig. 1. Device for the aeroponic culture of seminal roots.
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Rice Science, Vol. 11, No. 3, 2004
solution was added to the tank for ensuring the bottom of
stem being 2 cm beneath the water surface. The roots
were subjected to continuous unilateral irradiation when
they attained a growth of 10 mm in 2 d, and the vertical
root showed distinct negative phototropic bending at 1.5
hours after irradiation. The negative bending part was
evenly cut in the longitudinal direction under low
temperature and weak light conditions, and 0.5 g of each
side was taken to measure the IAA concentration on the
irradiated and shaded side by immunoassay of ELISA
(Enzyme linked Immunosorbent Assay) [6,7].
Microstructure of the bending part
The bending part of seminal root, which was
induced by the unilateral IAA application, was cut into
fractions approximately 2 mm in length. The fractions
were pre-fixed for 3 h with 2.0% glutaradehyde, 1.0%
paraformaldehyde, 0.05 mol/L sodium cacodyate (pH
7.2). After the pre-fixation, these were post-fixed with
1.0% OsO4 for several hours. Specimens were
dehydrated with a graded ethanolic series followed by
replacement with epihydrin and embedding in Spurr’s
resin. Semi-thin sections of 1 µm were sliced, stained
with 1% tobuidine blue (TBO), and observed with an
optical microscope.
RESULTS
Effects of exogenous IAA on root growth
Table 1 indicated that the growth of the root tip was
regulated by exogenous IAA as well as light. The root
tip bent toward the side of application, suggesting that
the increase of IAA concentration would result in the
increase of curvature (Fig. 2). Table 1 also showed that
exogenous IAA had an important effect on the growth of
seminal root, regardless of whether it was under light or
dark conditions, i.e. low concentration of IAA promoted
and high one suppressed the growth of root, and a rise in
IAA concentration led to the enhancement of
suppression.
The length of 30-cell from the treated side with IAA
agar block and another one from the untreated side were
Fig. 2. Effects of IAA on the growth of rice seminal roots.
1. Effects of different concentrations of IAA on the curvature of
seminal roots. The seminal root bent toward the site treated with agar
block containing IAA, the curvature of the root under 5 mg/L IAA is
larger than that under 1 mg/L IAA, the photographs were taken at 24
hous after IAA agar block treatment (×3);
2. Microstructure of the bending part in seminal root induced by
exogenous IAA, the side treated with IAA agar block is on the left
(×100).
randomly measured. The average length of 30-cell from
the untreated side was 58.5±11.4 µm, while from the
treated side was 43.6±16.4 µm. This also highlighted
that it was the exogenous IAA leading to the bending
growth of root tip.
The redistribution of endogenous IAA in the
light-exposed root tip
The IAA concentration in the adventitious root of
rice was measured at 1 h after unilateral irradiation. The
results evidenced that with the increase in light intensity,
Table 1. Effects of exogenous IAA on the negative phototropism of rice seminal roots.
Treatment
Curvature (°)
2
Unilateral irradiation (Light intensity: 60 µmol/m · s)
2
Irradiate the side with IAA treated (Light intensity: 60 µmol/m · s)
Dark
Root growth status
39.6±4.8 (45)
Negative phototropic bending
43.7±5.6 (44)
Increase in negative phototropic curvature
0 (20)
Vertical growth
Dark (unilateral treated with 5 mg/L IAA)
41.3±5.9 (43)
Grow toward the side of application
Dark (unilateral treated with 1 mg/L IAA)
37.5±9.8 (52)
Grow toward the side of application
Values in this table are means±SE with 5 replicates and the numbers in the parentheses represent the number of roots measured.
MO Yi-wei, et al. Effect of Indoleacetic Acid(IAA) on Negative Phototropism of Rice Root
127
Table 2. Changes of IAA concentration on irradiated side and shaded side of rice root after being exposed to light.
Treatment
Irradiated side (ng/g FW)
2
Light (40 µmol/m · s)
2
210.47±109.66 (4)
Shaded side (ng/g FW)
t value
421.73±211.60 (4)**
5.15
4.28
Light (100 µmol/m · s)
184.79±165.88 (5)
498.59±408.29 (5)**
Dark
418.64±27.90 (4)
418.64±27.90 (4)
Data in the table are means±SE and the number in the parentheses represents the replicates. The rice root tip is so small that it can not be cut
very evenly, which results in the large difference in the data. And ** shows there is significant difference between irradiated side and shaded side at
1% level.
the IAA concentration dropped remarkably on the
irradiated side, and this might be resulted from either the
unilateral IAA transport to the shaded side, or the
photooxidation of IAA on the irradiated side.
The microstructure of the bending part of root
It is known that under normal conditions the root
cells grow evenly on both sides of the elongation zone.
Asymmetric growth occurs in the elongation zone of the
seminal root when unilaterally applied with agar block
containing IAA, the cells of treated side were much
smaller in size than those on the other side (Fig. 2–2).
This supported the view that the suppression of high
IAA concentration on cell growth caused asymmetric
growth and ultimately resulted in bending toward the
treated side.
DISCUSSION
It is often believed that phototropic and gravitropic
stimuli, somehow, result in an unequal lateral IAA
distribution and the tropic response of plant. For
example, unilateral irradiation will cause an unequal
lateral IAA distribution within Avena coleoptile, and
result in higher IAA concentration on the shaded side,
lead to uneven differentiation and phototropic curvature.
Horizontal positioning of roots results in supra-optimal
IAA concentration on the lower side, inhibits growth and
leads to gravitropic curvature. Young reported that IAA
played a significant role in gravitropism, which was first
carried to root cap from the growth zone. If the young
roots of maize (Zea mays L.) were placed horizontally,
gravitropic stimulus first led to the asymmetric
distribution of IAA in root cap with higher IAA
concentration on the lower side. The unequal lateral
distributed IAA concentration was carried to the growth
zone, and caused an asymmetric growth and gravitropic
curvature [8,9]. IAA is the only known plant hormone
with polar transport activity, the unequal lateral IAA
distribution results from the polar efflux of IAA
carrier[10]，the asymmetric cellular localization of IAA
carrier is consistent with a role in controlling the polar
efflux of IAA from cells [11]. The polarity of IAA
transport is believed to be controlled by the localization
of IAA carrier, with both putative efflux carriers and
influx carriers (PIN1 and PIN2) having asymmetric
distribution acting at plasma membrane [12]. A recent
report by Friml presented evidence that both asymmetric
distribution and dynamic redistribution of PIN3 protein
were involved in the lateral redistribution of IAA in
response to tropic stimuli [13]. Gaba and Black found that
exogenous hormone can substitute for light to mediate
plant growth [14] and on the other hand endogenous
hormone can serve as the second messenger in the
transduction of light signal. For example, IAA works as
an intermediate in the light-regulated elongation of plant
stem [15], and also in the response of phototropism and
gravitropism. It is suggested that IAA have two putative
acting models, one is called fast model, rapid IAA
effects on H+-pumping across plasma membrane precede
IAA-induced relaxation and elongation of the cell
wall[16]. The other is named as slow response, which
means the asymmetric growth that results from
regulation of IAA in nuclear gene expression and the
consequent composition of new substance. Kufman
pointed out that blue light can change the endogenous
hormone level to control plant growth [17]. IAA was also
considered as one signal following the activation of
phytochrome in the transduction process. IAA can cause
alteration in rates of cell division and elongation, alter
H+-fluxes across membrane, cue changes in patterning
and differentiation, and affect the expression of hundreds
of genes [18,19].
Yu reported that the activity of IAA oxidase was
enhanced in blue light-treated seedlings, resulting in a
decrease of endogenous free IAA, and inhibiting the
seedling growth [20]. Wang found that rice root would
show no more negative phototropism when the root cap
was excised, suggesting that root cap was light
perception site for the polar IAA transport when being
exposed to irradiation [4]. It can be concluded from our
results that, the light-induced unequal lateral distribution
of IAA is the main reason for the negative phototropism
of rice root.
Bruinsma assumed that some light-induced
inhibitors could lead to the plant phototropism, such as
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raphansimide, raphansine and MBOA (6-methoxy-2benzoxazolinone) etc., light stimulus could cause higher
inhibitor concentration on the irradiated side, suppress
the cell growth and result in the positive phototropism of
plant stem [21, 22]. Being unilaterally applied MBOA and
other inhibitors, the plant bent towards the side of factor
applied [23].
We conducted aeroponics to culture the seminal root
of rice in the dark for 24 h with agar blocks containing
IAA unilaterally applied on root tip, and the curvature
was found to be correlated with the concentration of
applied IAA. Thus, we concluded that the bending
growth was not caused by the light-induced inhibitors
rather by the unequal lateral distribution of IAA.
However, as far as whether there are any lightstimulated inhibitors in root tip still remains unknown.
It could be concluded that the mechanism of
negative phototropism, i.e. the photoreceptors acting at
the cell membrane activated the signal transduction
downstream after accepting light signals, caused polar
transport through efflux of IAA transport protein
resulting in unequal lateral IAA distribution and
negative phototropism of rice root. As for how the
photoreceptors at the membrane activated the polar
efflux of IAA transport after light signal perception, and
how the regulations continued after the unequal lateral
distribution of IAA? More investigations will be
required in this regard.
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ACKNOWLEDGEMENTS
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This work was supported by the National Natural Science
Foundation of China (30270795) and the open foundation of
the State Key Laboratory of Plant Physiology and
Biochemistry (2002001).
REFERENCES
1
2
3
4
5
Briggs W R, Olney M A. Photoreceptors in plant photomorphogenesis to date, five phytochromes, two
cryptochromes, one phototropin, and one super chrome. Plant
Physiol, 2001, 125: 85－88.
Vitha S, Zhao L, Sack F D. Interaction of root gravitropism
and phototropism in Arabidopsis wild-type and starchless
mutants. Plant Physiol, 2000, 122: 453－461.
Okada K, Shimura Y.
Mutational analysis of root
gravitropism and phototropism of Arabidopsis thaliana
seedlings. Aust J Plant Physiol, 1992, 19: 439－448.
Wang Z, Gu Y J , Chen L, Chen G, Xiong F, Liang G B.
Negative phototropism of rice root. Chinese Rice Res Newsl,
2001, 9 (3): 9－11.
Wang Z, Mo Y W, Qian S Q, Gu Y J. Negative
phototropism of rice root and its influencing factors. Sci in
China (Series C ) , 2002, 45: 485－496.
17
18
19
20
21
22
23
He Z P. Guidance to Experiment on Chemical Control in
Crop Plant. Beijing: Beijing Agricultural University Press,
1993. 60－68.
Liu W Q, Ding Y L, Zhang L H. Study on the relationship
between hormonal leveling root and metabolism of
substance in tobacco before and after topping. Plant Physiol
Comm, 2002, 38: 330－332. (in Chinese)
Young L M, Evans M L, Hertel R. Correlations between
gravitropic curvature and auxin movement across
gravistimulated roots of Zea mays. Plant Physiol, 1990, 92:
792－796.
Rashotte A M, Brady S R, Reed R C. Basipetal auxin
transports is required for gravitropisms in roots of
Arabidopsis. Plant Physiol, 2000, 122: 481－490.
Ni W M, Chen X Y, Xu Z H, Xue H W. Advance in study
of polar auxin transport. Acta Bot Sin, 2000, 42: 221－228.
(in Chinese with English abstract)
Estelle M. Transporters on the move. Nature, 2001, 413: 372
－375.
Geldner N, Friml J, Stierhof Y D, Jurgens G, Palme K. Auxin
transport inhibitors block PIN1 cycling and vescicle
trafficking. Nature, 2001, 413: 425－428.
Friml J, Wisniewska J, Benkava E, Mendgen K, Palme K.
Lateral relocation of auxin efflux regulator PIN3 mediates
tropism in Arabidopsis. Nature, 2002, 415: 806－809.
Gaba V, Black M. The control of cell growth by light. In: Jr.
Shropshire W, Mohr H. Encyclopedia of Plant Physiology,
New series, Vol. 16A. Berlin: Springer-Verlag, 1983. 338－
342.
Behriger F J, Davies P J. Indole-3-acid level after
phytochrome-mediated changes in the stem elongation rate of
dark and light-grown Pisum seedlings. Planta, 1992, 188: 85
－89.
Coener C, Bierfreund N, Lüthen H, Neuhaus G.
Developmental regulation of H+-ATPaes-dependent auxin
responses in the diageotropica mutant of tomato
(Lycopersicm esculeutum). Physiol Plant, 2002, 114: 461－
471.
Kaufmam L S. Transduction of blue light signals. Plant
Physiol, 1993, 102: 33－38.
Bao F, Li J Y. Evidence that the auin signaling pathway
interact with plant stress response. Acta Bot Sin, 2002, 44:
537－540.
Kepinski S, Leyser O. Ubiquitination and auxin signaling:
A degrading story. Plant Cell, 2002, 14(suppl): S81－S95.
Yu R C, Pan R C. Effects of blue light on the growth and
levels of endogenous phytohormones. Acta Phytophysiol Sin,
1997, 23: 175－180.
Bruinsma J, Hasegawa K. A new theory of theory of
phototropism－its regulation by a light-induced gradient of
auxin－inhibiting substrance. Physiol Plant, 1990, 79: 700－
704.
Chen R M. Unequal distribution of 6-methoxy-2benzoazolinone (MBOA) is the main reason for phototropism
in maize coleoptiles. Acta Bot Sin, 1999, 41: 296－300.
Chen R M. Physiological effects of 6-methoxy-2-benzoxazolinone(MBOA) from maize coleoptiles. J Trop & Subtrop Bot,
1999, 7: 70－76.