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Plant Physiol. (1999) 121: 53-60
Root Formation in Ethylene-Insensitive Plants1
David G. Clark*,
Erika K. Gubrium,
James E. Barrett,
Terril A. Nell, and
Harry J. Klee
Environmental Horticulture Department, P.O. Box 110670 (D.G.C.,
E.K.G., J.E.B., T.A.N.), and Horticultural Sciences Department, P.O.
Box 110690 (H.J.K.), University of Florida, Gainesville, Florida
32611-0670
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ABSTRACT |
Experiments
with ethylene-insensitive tomato (Lycopersicon
esculentum) and petunia (Petunia × hybrida) plants were conducted to determine if normal or
adventitious root formation is affected by ethylene insensitivity.
Ethylene-insensitive Never ripe (NR) tomato plants
produced more belowground root mass but fewer aboveground adventitious roots than wild-type Pearson plants. Applied auxin (indole-3-butyric acid) increased adventitious root formation on
vegetative stem cuttings of wild-type plants but had little or no
effect on rooting of NR plants. Reduced adventitious root formation was
also observed in ethylene-insensitive transgenic petunia plants.
Applied 1-aminocyclopropane-1-carboxylic acid increased adventitious
root formation on vegetative stem cuttings from NR and wild-type
plants, but NR cuttings produced fewer adventitious roots than
wild-type cuttings. These data suggest that the promotive effect of
auxin on adventitious rooting is influenced by ethylene responsiveness.
Seedling root growth of tomato in response to mechanical impedance was
also influenced by ethylene sensitivity. Ninety-six percent of
wild-type seedlings germinated and grown on sand for 7 d grew
normal roots into the medium, whereas 47% of NR seedlings displayed
elongated taproots, shortened hypocotyls, and did not penetrate the
medium. These data indicate that ethylene has a critical role in
various responses of roots to environmental stimuli.
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INTRODUCTION |
The plant hormone ethylene is known to play a critical role in
many diverse physiological processes, such as leaf and flower senescence, abscission of organs, flower initiation, fruit ripening, and seed germination (for review, see Abeles et al., 1992 ). Since the
initial discovery that ethylene has stimulatory effects on adventitious
root formation in many plant species (Zimmerman and Hitchcock, 1933), a
wide array of experiments have been conducted to determine its role in
root initiation and development. Results of these studies have been
highly variable. For example, in experiments using applied ethylene or
ethylene-generating compounds to examine adventitious rooting in mung
bean, researchers showed that ethylene stimulates rooting (Robbins et
al., 1983), inhibits rooting (Geneve and Heuser, 1983), or has no
effect (Mudge and Swanson, 1978). Similar experiments in tomato
(Lycopersicon esculentum) have shown stimulatory effects
(Hitchcock and Zimmerman, 1940; Orion and Minz, 1969; Phatak et al.,
1981) and inhibitory effects (Roy et al., 1972; Coleman et al., 1980).
Since the initial report that auxin induces ethylene synthesis in many
plant species and tissues (Zimmerman and Wilcoxin, 1935), there have
been numerous attempts to determine if interactions exist between auxin
and ethylene during adventitious root formation and development. In
experiments designed to correlate the magnitude of rooting stimulation
with the magnitude of auxin-induced ethylene synthesis, there has been
no clear correlation between the two (Mullins, 1972; Batten and
Mullins, 1978; Coleman et al., 1980; Geneve and Heuser, 1982). Mudge
(1988) pointed out that the lack of correlation between auxin-induced
ethylene synthesis could be misleading, and hypothesized that if a low
saturating concentration for ethylene-stimulated rooting exists, then
auxin-stimulated ethylene production above this level would have no
additional effect on rooting.
Reports of the variable rooting response of many plant systems to
ethylene compared with ubiquitous reports of auxin-stimulated rooting
have suggested that ethylene is less often a limiting factor or is less
directly involved in the rooting process than auxin (Mudge, 1988). With
the development of chemicals designed to block ethylene synthesis and
perception, ideas for the role of ethylene sensitivity in adventitious
root formation have become clearer. Inhibitors of ethylene
biosynthesis, such as aminoethoxyvinylglycine, have been shown to
reduce the number of adventitious roots of mung bean cuttings (Robbins
et al., 1983; Jusaitis, 1986). Inhibitors of ethylene perception such
as silver thiosulfate and 2,5-norbornadiene have been shown to reduce
root number on mung bean cuttings (Robbins et al., 1985) and to reduce
the responsiveness of sunflower hypocotyls to endogenous and exogenous
auxin (Liu and Reid, 1992). Support for this type of interaction
between auxin and ethylene comes from experiments with waterlogged
Rumex palustris plants (Visser et al., 1996), in which
higher tissue ethylene concentrations increased the sensitivity of
root-forming tissues to endogenous IAA.
Different sensitivity of plants to ethylene at different stages of
development could account for much of the reported variability in the
rooting response to ethylene. Maximal rooting of bean cuttings in
response to auxin-induced ethylene synthesis occurred during the first
44 h after excision (Linkins et al., 1973). Conversely, maximal
rooting response due to the apparent accumulation of ethylene sensitivity over time of mung bean (Robbins et al., 1985) and sunflower
(Fabijan et al., 1981) has been observed. These contradictory results
suggest that ethylene has a role in the adventitious rooting response
of many different plants. However, it is likely that regulation of the
rooting response may differ greatly from species to species depending
on the developmental stage of the experimental tissue. Attempts to
clarify the roles of auxin and ethylene in adventitious rooting will
likely depend on newly characterized mutants that are insensitive to
these hormones.
Experiments conducted with Arabidopsis wild-type and mutant seedlings
to determine the roles of ethylene and auxin have offered clues to the
complex regulation of these two hormones in root hair development.
Treatment of wild-type seedlings with increased concentrations of ACC
resulted in increased root hair formation (Tanimoto et al., 1995;
Masucci and Schiefelbein, 1996). Seedlings treated with inhibitors of
ethylene synthesis (aminoethoxyvinylglycine) or perception (silver
thiosulfate) exhibited reduced root hair formation with increased
inhibitor concentration (Tanimoto et al., 1995).
In a series of experiments conducted with auxin and ethylene response
mutants, Masucci and Schiefelbein (1996) showed that neither the
ethylene response mutations etr1 and ein2 nor the auxin response mutations aux1 and axr1 affected
root hair initiation. Interestingly, the aux1/etr1 double
mutant was shown to produce fewer root hairs than wild-type seedlings
or either single mutant. The reduced root hair phenotype in
aux1/etr1 could be reversed by treatment with IAA but not by
treatment with ACC. These results led Masucci and Schiefelbein (1996)
to propose that both AUX1 and ETR1 may normally act in separate
redundant pathways to promote root hair formation. Furthermore, they
proposed that ethylene could be involved in at least two, and possibly
three, signal transduction pathways leading to root hair formation. To
further expand the role of ethylene in root hair development, Pitts et al. (1999) reported that root hair elongation in Arabidopsis was positively regulated by both auxin and ethylene.
In recent years, a number of physiological and molecular
experiments have been conducted with ethylene-insensitive Arabidopsis and tomato mutants to elucidate the mechanism of ethylene perception and action in plants (for review, see Kieber, 1997). The main physiological response systems used for much of this work have been the
seedling triple response and fruit ripening: Ethylene-insensitive (etr1-1) Arabidopsis and the tomato
mutant Never ripe (NR) produce seeds that do not display an
ethylene-mediated triple response (Bleecker et al., 1988; Lanahan et
al., 1994), and NR tomato plants produce fruit that never completely
ripen even when treated with exogenous ethylene (Giovannoni, 1993).
Isolation and characterization of the genes responsible for ethylene
insensitivity in these plants led to the discovery that the
etr1-1 gene from Arabidopsis and the NR gene from
tomato encode mutant ethylene receptor proteins (Schaller and Bleecker,
1995; Wilkinson et al., 1995). Evidence of the conserved role for the
control of ethylene sensitivity was demonstrated using genetic
transformation of the etr1-1 mutant gene into the
heterologous species tomato, petunia (Petunia × hybrida), and tobacco. Using the constitutive CaMV 35S
promoter to drive expression of the dominant mutant
etr1-1 gene, Wilkinson et al. (1997) were able to
transform all of these different plant species and obtain various
ethylene-insensitive phenotypes: wild-type tomato plants transformed
with etr1-1 displayed delayed flower senescence
and a NR fruit phenotype, and etr1-1 petunias
produced flowers with delayed senescence after ethylene treatment and
pollination (Wilkinson et al., 1997).
The purpose of this study was to examine the effects of genetically
manipulated ethylene insensitivity on the adventitious rooting
responses of tomato and petunia in response to applied auxin and
ethylene. Using NR tomato and petunia plants engineered for
constitutive ethylene insensitivity (CaMV
35S/etr1-1) we show that endogenous ethylene
sensitivity is necessary for the formation of adventitious roots on
vegetative stem cuttings. We also demonstrate that ethylene sensitivity
is required for normal seedling root growth in response to mechanical
impedance, but is not required for normal root growth of plants under
greenhouse conditions.
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MATERIALS AND METHODS |
Plant Materials and Culture Methods
Inbred cv Mitchell Diploid (wild-type) and transgenic (line 44568;
Wilkinson et al., 1997) petunia (Petunia × hybrida) plants were grown under greenhouse conditions with
a day/night temperature regime of 25°C/18°C in commercial potting
medium (Fafard 2B, Conrad Fafard, Agawam, MA) in 15-cm, 1.5-L
pots, and were fertilized at each irrigation with 150 mg
L 1 N from 15:7:14.1 soluble fertilizer
(Peter's Fertilizer Products, Fogelsville, PA). Experiments using
inbred cv Pearson (wild type) and mutant Never ripe (NR)
tomato (Lycopersicon esculentum) plants were grown with
cultural and environmental conditions similar to those described for
petunia. The only notable exception was that tomato plants used in
experiments to determine the effects of indole-3-butyric acid (IBA) and
ACC on adventitious root formation of vegetative stem cuttings received
day/night temperature regimes of 28°C/21°C.
Growth Data of Tomato Plants
To determine if differences in growth exist between wild-type and
NR tomato plants, roots from 5-week-old plants were excised at the soil
level and washed in tap water. Data for root fresh weights and stem
diameters of 50 35-d-old plants of each genotype were recorded, and the
means and SE values were calculated. To quantify
differences in above ground adventitious roots on plant stems between
wild-type and NR tomato plants, 25-cm sections of stems from 20 stems
of 77-d-old plants were excised at the soil level. Data for the number
of root initials for each genotype were recorded, and the means and
SE values were calculated.
Response of Tomato Seedlings to Mechanical Impedance
To determine the influence of mechanical impedance on growth of
wild-type and NR seedlings, 96 seeds of each genotype were arranged in
a completely randomized design on either silica sand or commercial
potting mix and covered with 1 cm of vermiculite. During 7 d of
growth, seedlings received intermittent mistings with tap water during
daylight hours for 5 s every 30 min. Mist house temperatures were
approximately 25°C during the day and 22°C at night. After 7 d, taproot and seedling hypocotyl lengths were measured for all
seedlings, and means and SE values were calculated. To
observe gross differences in root hair growth of tomato seedlings,
wild-type and NR seeds were plated on 2% agar plates and germinated in
the dark for 5 d. Typical seedlings treated in this manner were
documented graphically on a dissecting microscope (data not shown).
Adventitious Root Formation
Tomato
For all experiments using stem cuttings from tomato, vegetative
cuttings were taken from 35-d-old wild-type and NR plants at the
developmental stage of six to seven true leaves. To determine the
effect of auxin on adventitious root formation, the basal ends of
cuttings that were approximately 10 cm in length were dipped in talcum
powder containing 0, 500, 1,000, or 1,500 µg g1 IBA (Rhizopan AA#2, Hortus USA, New York).
Ten cuttings were taken from plants of each genotype per treatment, and
cuttings were propagated in four-pack cells (each cell = 100 mL)
containing perlite and arranged in a completely randomized design in a
mist house. Misting schedules and mist house temperatures were the same
as described previously. After 3 weeks, the total number of root
initials and root lengths for each cutting were measured.
To determine the effect of ethylene on adventitious root formation, the
basal ends of cuttings that were approximately 10 cm in length were
dipped in talcum powder containing 0, 1, 3, 10, 30, 100, 300, or 1,000 µg g1 ACC (Sigma). Eight cuttings were taken
from plants of each genotype per treatment, and cuttings were
propagated as described for previous IBA treatments. After 3 weeks, the
total number of root initials and root lengths for each cutting were
measured, and the means and SE values were calculated.
Petunia
Vegetative stem cuttings were taken from approximately 8-week-old
wild-type and T1 generation petunia plants from
44568 (Wilkinson et al., 1997). The basal ends of cuttings that were
approximately 5 cm in length and had two fully expanded leaves were
dipped in talcum powder (0 µg g1; control) or
1,000 µg g1 IBA. Ten cuttings per genotype
were propagated in six-pack cells (each cell = 70 mL) containing
perlite, and were arranged randomly in a mist house. During 3 weeks of
propagation, cuttings received intermittent mist with tap water during
daylight hours for 5 s every 15 min for the first week, then for
5 s every 30 min for 2 subsequent weeks. Mist house temperatures
for all rooting experiments were approximately 25°C during the day
and 22°C at night. After 3 weeks, the total number of root initials
and root lengths for each cutting were measured, and the means and
SE values were calculated. There were no observed
adventitious roots on intact stems of either wild-type or transgenic
44568 plants at any point in development from seed germination to
seed production.
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RESULTS |
Growth of Tomato Plants
In an effort to characterize gross differences in root morphology
and plant development between wild-type and NR tomato plants, root
fresh weights of 35-d-old seedling plants were measured. Observations
of belowground root growth indicated no obvious morphological differences between genotypes (Fig. 1A).
However, upon closer investigation we observed that NR plants produced
approximately 13% more root mass than wild-type plants (data not
shown). We also noticed that there were significant differences between
NR and wild-type plants for the number of adventitious roots produced on the lower portion of plant stems (Fig. 1B). To quantify this observation, we counted the number of adventitious roots on the basal-most 25 cm of stems from 77-d-old NR and wild-type plants, and
found that wild-type plants produced almost 20 times more adventitious
roots on stems than NR plants (data not shown).
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| Figure 1.
A, Thirty-five-day-old mutant NR and wild-type
tomato plants. Whole-plant root morphologies are not visually
distinguishable. B, Seventy-seven-day-old mutant NR tomato stems with
reduced adventitious root formation compared with wild-type stems. C,
Response of 7-d-old mutant NR and wild-type seedlings germinated and
grown on sand. Approximately one-half of NR seedlings grew horizontally
and had longer taproots and shorter hypocotyls than wild-type seedlings
(denoted by arrows). D, Reduced adventitious root formation in mutant
NR vegetative cuttings compared with wild-type cuttings. Stem cuttings
were propagated for 21 d. E, Reduced adventitious root formation
in transgenic 44568 petunia vegetative cuttings compared with wild-type
cuttings. Stem cuttings were propagated for 21 d.
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Response of Tomato Seedlings to Physical Impedance
When germinating and growing tomato seeds on sand, we observed a
tendency for many of the NR seedlings to grow abnormally compared with
wild-type seedlings. All of the wild-type seedlings and most of the NR
seedlings grown on commercial potting medium had normal vertical stems
and roots that penetrated the medium. On sand, most of the wild-type
seedlings grew normally, but many of the NR seedlings grew horizontally
and their roots did not penetrate the sand (Fig. 1C). To quantify this
response, NR and wild-type seeds were germinated and grown for 7 d
in a mist house on either sand or commercial potting medium covered
with vermiculite to maintain consistent moisture. After 7 d, most
of the NR seedlings (96%) and all of the wild-type seedlings
germinated and grown on commercial potting medium produced a normal
phenotype (Table I). Taproot and shoot
lengths of NR and wild-type seedlings grown on potting soil and showing
a normal phenotype did not differ greatly due to genotype and were
visually indistinguishable. When germinated and grown on sand, 96% of
wild-type seedlings grew normally, while only 53% of NR seedlings
displayed a normal erect phenotype and roots penetrating the medium
(Table I). The remaining 47% of the NR seedlings showed the abnormal
phenotype, with significantly longer taproots and shorter hypocotyls
than those with normal phenotypes (Table I; Fig. 1C).
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Table I.
Seedling root formation of wild-type and NR tomato
plants germinated and grown for 7 d on sand (S) or potting media
(PM)
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The Effects of IBA, ACC, and Ethylene on Adventitious Root
Formation in Tomato
To investigate the effects of ethylene insensitivity on
adventitious rooting of tomato, a series of propagation experiments was
conducted on vegetative cuttings taken from 35-d-old wild-type and NR
plants at the six to seven true leaf stage. In the first experiment, NR
and wild-type cuttings were treated with a range of IBA concentrations
to determine if applied auxin affected adventitious rooting. Ethylene
insensitivity affected how cuttings responded to IBA concentration for
the number of adventitious roots (Table II). With increased applied IBA
concentration, wild-type cuttings were more prolific in rooting,
producing approximately 50% more adventitious roots with 1,000 or
1,500 µg g1 IBA treatments than controls. NR
cuttings did not produce more adventitious roots with increased applied
IBA concentration, and never produced as many adventitious roots as
wild-type cuttings rooted at any IBA concentration (Fig. 1D). Lengths
of adventitious roots produced by wild-type cuttings were reduced with
higher IBA concentrations, while lengths of NR roots were unchanged
(Table II). Although significant, the magnitude of the difference in length of both wild-type and NR cuttings in response to IBA was slight,
with observed growth differences of less than 1 cm due to any
particular treatment.
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Table II.
Number and length of adventitious roots of
wild-type and NR vegetative tomato cuttings treated with various
concentrations of IBA
Data were taken 3 weeks after the onset of propagation.
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In a second experiment, NR and wild-type cuttings were treated with a
range of ACC concentrations to determine if applied ethylene affected
adventitious rooting. ACC had a promotive effect on adventitious
rooting for both genotypes (Table III).
Wild-type cuttings treated with 30 µg g1 ACC
produced approximately 22% more adventitious roots than control wild-type cuttings. NR cuttings treated with 10 µg
g1 ACC produced approximately 41% more
adventitious roots than control NR cuttings. NR cuttings never produced
as many adventitious roots as wild-type cuttings at any applied ACC
concentration (Table III).
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Table III.
Number of adventitious roots of wild-type and NR
vegetative tomato cuttings treated with various concentrations of ACC
Data were taken 3 weeks after the onset of propagation.
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Adventitious Root Formation of Petunia after Treatment with IBA
and Ethylene
In an effort to investigate the conserved nature of endogenous
ethylene insensitivity on adventitious rooting, we conducted an
experiment to determine the effects of applied auxin on adventitious rooting of wild-type and 44568 petunia (Wilkinson et al., 1997) cuttings. Based on preliminary adventitious rooting experiments, we
chose to treat cuttings with 0 or 1,000 µg g1
IBA. As was observed in the previous experiment on tomato, 44568 petunia cuttings had consistently fewer adventitious roots than wild-type cuttings after both treatments (Fig. 1E). At 1,000 µg g1 IBA, wild-type cuttings were more prolific
in rooting, producing almost twice as many adventitious roots than
controls (Table IV); 44568 cuttings also
produced slightly more adventitious roots with increased applied IBA
concentration, but never produced as many adventitious roots as control
wild-type cuttings. Lengths of adventitious roots produced by both
wild-type and 44568 cuttings were unaffected by IBA (Table IV).
Although the length of adventitious roots produced by 44568 cuttings
was reduced compared with the wild type, the low number of roots on
44568 cuttings may not allow for a valid comparison of root length
between the two genotypes.
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Table IV.
Adventitious root formation of wild-type and
transgenic T1 generation 44568 petunia cuttings treated
with 0 or 1,000 µg/g1 IBA
Data were taken 3 weeks after the onset of propagation.
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DISCUSSION |
Using ethylene-insensitive plants, we have shown that ethylene
must be perceived by tomato plants to induce normal seedling root
growth and normal levels of adventitious rooting on plant stems and
vegetative cuttings. We have also shown that petunia plants genetically
engineered with constitutive ethylene insensitivity show reduced
adventitious root formation similar to that of NR tomato.
In terms of normal plant growth, 35-d-old NR plants had more
belowground root mass than wild-type tomato plants. Until now, this
phenomenon has been unreported to our knowledge, but given that NR
seedlings are insensitive to the ethylene-mediated seedling triple
response (Lanahan et al., 1994), it is not surprising that plants could
show differences in gross morphology during subsequent development as
well. It is important that these plants were started and grown in
potting soil under optimal greenhouse conditions that promote maximal
growth. Future experiments designed to characterize the growth of
NR and wild-type plants under less than optimal soil and environmental
conditions may lead to a better understanding of the role of ethylene
in belowground root development.
Adventitious rooting of vegetative cuttings from NR tomato and 44568 petunia plants was greatly inhibited. We also observed that 77-d-old
wild-type tomato plants had 20 times more aboveground adventitious
roots than NR plants. When combined with the observations of normal
belowground root growth in NR, these data suggest a fundamentally
different regulation of control for normal root formation versus
adventitious root formation.
Under less than optimal soil medium conditions (i.e. sand versus
commercial potting media), 7-d-old NR seedlings performed significantly
different than wild-type tomato seedlings and with less consistency.
Normal growth was observed on 100% of wild-type and 96% of NR
seedlings germinated and grown on commercial potting medium. When
germinated and grown on sand, 96% of wild-type seedlings grew
normally, while 47% of NR seedlings grew elongated roots and shorter
hypocotyls, and never penetrated the medium. The commercial potting
medium used in this experiment had a bulk density of approximately 100 lb/yd3, whereas the sand had a bulk density of
approximately 1,600 lb/yd3, meaning that there
was much greater physical resistance to downward growth.
A similar phenomenon has been reported previously with tomato seedlings
grown on agar plates in the presence of inhibitors of ethylene action.
Zacarias and Reid (1992) showed that seedlings germinated on 2%
agar plates containing silver thiosulfate or in the presence of
2,5-norbornadiene failed to insert their radicles into the medium,
whereas seedlings germinated on 0.5% agar with similar treatments
penetrated the medium. Seedlings that did not grow into the medium had
a corkscrew morphology, with increased root length and decreased
hypocotyl elongation, and these characters were all observed in our
experiments as well. We do not know why 53% of NR seedlings grown on
sand in the present study displayed a normal phenotype. Since sand is a
less homogenous medium than agar, it is likely that some roots were
able to grow downward between the sand particles. This idea would
support the results of Zacarias and Reid (1992), who hypothesized that
inhibition of ethylene action may reduce developmental movement of the
root cap, thus decreasing the ability of the root to penetrate through compacted media.
When we germinated and grew wild-type and NR tomato seedlings in the
dark on 2% agar, we observed a reduction in root hair length on NR
roots compared with wild-type roots (data not shown). This observation
supports the results of Pitts et al. (1999), who reported that ethylene
is a positive regulator of root hair elongation in Arabidopsis. It is
possible that decreased root hair formation reduces the ability of NR
seedlings to anchor in soil during emergence. In real terms, these
results suggest that ethylene-insensitive plants may be hindered in
normal root growth under adverse growing conditions. Further
experiments need to be conducted to determine the full extent of this
response, because it could be a major limitation in determining the
utility of genetically manipulated ethylene insensitivity in
horticultural crops.
Over a range of applied IBA concentrations that induced maximal numbers
of adventitious roots in wild-type tomato cuttings, NR cuttings did not
show corresponding increases in rooting. These results were further
supported in studies with 44568 petunia plants treated with auxin,
which showed no increase in adventitious root formation at IBA
concentrations that doubled the number of roots in control wild-type
cuttings. The fact that cuttings of ethylene-insensitive plants never
produced comparable numbers of adventitious roots compared with
controls under any auxin treatment is clear evidence of a central role
for ethylene in adventitious root formation of tomato and petunia
cuttings.
We also observed that increased auxin concentration decreased root
length of wild-type tomato adventitious roots, but not of NR roots. It
is possible that this observation was due to a higher demand for carbon
allocation because of more adventitious roots to support in wild-type
cuttings treated with higher IBA concentrations. In experiments with
vegetative tomato cuttings, we observed that rooting was not totally
inhibited in NR cuttings, and application of ACC to both wild-type and
NR cuttings increased the number of adventitious roots formed. This
observation was likely due to the fact that the NR tomato mutation is
semidominant or "leaky" (Yen et al., 1995), and has a small amount
of endogenous ethylene sensitivity. When constitutive ethylene
insensitivity was engineered into petunia by use of the CaMV
35S-etr1-1 construct (Wilkinson et al., 1997),
rooting was almost completely inhibited. It is possible that
CaMV35S-driven ethylene insensitivity is more effective than an
endogenous mutation, or observed differences may reflect the inherent
differences in adventitious root formation between petunia and tomato.
These observations led us to conclude that the commercial utility of
plants that have been genetically manipulated for ethylene
insensitivity is chiefly dependent on the ability to drive
tissue-specific expression in plants if they need to be vegetatively
propagated.
It is apparent from these experiments that ethylene may have a
different role(s) in normal root formation than in adventitious root
formation. Ethylene sensitivity appears to be temporally regulated
during normal root formation of plants grown from seed. Morphological
differences were observed between NR and wild-type seedlings for root
hair growth and for their response to mechanical impedance, suggesting
a critical role for ethylene during early seedling root development. We
predict that with further investigation of NR seedlings, many of the
characteristic root hair responses seen with
etr1-1 (Masucci and Schiefelbein, 1996) will be
observed. Although indistinguishable by visual appearance,
greenhouse-grown 35-d-old NR plants actually had more total root mass
than wild-type tomato plants. This result suggests that ethylene does
not play a major role in root formation of plants established in soil
and growing under optimal growth conditions. Future experiments to determine how ethylene-insensitive plants respond to adverse soil conditions leading to abiotic stress should help further characterize the role of ethylene in normal root formation and growth at the whole-plant level.
The role of ethylene during adventitious root formation appears to be
more centralized. On tomato stems, adventitious root formation was
greatly reduced by ethylene insensitivity. A similar reduction in
adventitious rooting was observed with vegetative cuttings, and could
not be overcome with application of auxin at levels that increased root
formation in wild-type plants. Results from these experiments led to
the hypothesis that ethylene has a significant role in adventitious
root formation of tomato and petunia. It is likely that similar
experiments with auxin-insensitive mutants will be needed to help
clarify the interaction between ethylene and auxin during adventitious
root formation.
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FOOTNOTES |
1
This work was supported in part by the Fred C. Gloeckner Foundation. This paper is University of Florida Journal
Series no. R-06707.
*
Corresponding author; e-mail dgc{at}gnv.ifas.ufl.edu; fax
352-392-3870.
Received January 11, 1999;
accepted May 19, 1999.
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Abstract
Introduction