Directory UMM :Data Elmu:jurnal:T:Tree Physiology:Vol15.1995:
Tree Physiology 15, 27--34
© 1994 Heron Publishing----Victoria, Canada
Interaction between indole-3-acetic acid and ethylene in the control of
tracheid production in detached shoots of Abies balsamea
LEIF EKLUND1 and C. H. ANTHONY LITTLE2
1
Department of Engineering and Natural Sciences, Växjö University, S-351 95 Växjö, Sweden
2
Natural Resources Canada, Canadian Forest Service, P.O. Box 4000, Fredericton, New Brunswick E3B 5P7, Canada
Received December 16, 1993
Summary We investigated the interaction between indole-3acetic acid (IAA) and ethylene in the regulation of the seasonal
periodicity of tracheid production in 1-year-old balsam fir
(Abies balsamea (L.) Mill.) cuttings collected at different times
during the dormant period. The cuttings were left with their
buds intact or were debudded and treated either apically with
IAA or 2-chloroethylphosphonic acid (Ethrel) in lanolin, laterally with IAA or Ethrel in lanolin, or basally with Ethrel, Co2+
or Ag+ in deionized water. The treated cuttings were then
cultured for up to 5 weeks under controlled environment conditions favorable for cambial growth. No change in ethylene
evolution was detected during the rest--quiescence transition,
when IAA-induced tracheid production increased. The induction of cambial reactivation by IAA was associated with a rise
in ethylene evolution, but there was no consistent relationship
among IAA concentration, tracheid number and ethylene emission. Neither Ethrel, Co2+ nor Ag+ affected tracheid production
when applied basally, except for 10 and 100 µM Ethrel and 100
µM Co2+, which were inhibitory. In contrast, ethylene evolution
was promoted by Ethrel and inhibited by Co2+, whereas Ag+
had no effect. Similarly, applying Ethrel apically or laterally
increased ethylene evolution, but did not promote tracheid
production except in the treatment in which 1 mg Ethrel g −1
lanolin was applied laterally to cuttings treated apically with
0.1 mg IAA g −1 lanolin, and in the treatment in which 10 mg
Ethrel g −1 lanolin was applied laterally to budded cuttings. We
conclude that (1) ethylene evolution is not specifically associated with IAA-induced tracheid production, (2) ethylene does
not mimic the promoting effect of IAA on tracheid production,
and (3) ethylene can promote tracheid production, but only
when its application results in a localized unphysiologically
high concentration in the cambial region, which, in turn, induces an accumulation of IAA.
Keywords: cambium, 2-chloroethylphosphonic acid, cobalt,
dormancy, Ethrel, IAA, silver.
Introduction
Considerable evidence indicates that indole-3-acetic acid
(IAA) is involved in the mechanism regulating xylem production in tree stems. For example, it has been shown that in
1-year-old shoots of Abies balsamea and Pinus sylvestris,
debudding concomitantly decreases the production of tracheids and the concentration of IAA, whereas both are increased by applying IAA either apically to debudded shoots or
laterally to budded shoots (Little and Savidge 1987, Sundberg
and Little 1990, Little and Sundberg 1991). Other research has
demonstrated that IAA transport is basipetally polar in the
cambial region, and that stem girdling both increases tracheid
production and the IAA concentration above the girdle while
reducing both below (Little and Savidge 1987). However, the
rate and duration of tracheid production are not clearly related
to the concentration of IAA in the cambial region (Sundberg et
al. 1991, 1993). Moreover, the cambium’s ability to produce
tracheids in response to IAA decreases with increasing age
(Little and Savidge 1987, Little and Sundberg 1991) and varies
with the time of IAA application. Studies with Abies balsamea
and Pinus sylvestris have shown that exogenous IAA promotes
tracheid production under environmental conditions favorable
for growth if applied during the winter when the cambium is
in the quiescence stage of dormancy imposed externally by
low temperature, but not if applied during the autumn when the
cambium is in the resting stage of dormancy maintained by
internal agents or conditions (Little and Savidge 1987, Sundberg et al. 1987, Little et al. 1990, Mellerowicz et al. 1992).
The reduced response to IAA by the resting cambium cannot
be attributed to either the accumulation of abscisic acid (Little
and Wareing 1981) or the inhibition of IAA transport (Little
1981), nor is it altered by exogenous gibberellic acid or kinetin
(Little and Bonga 1974). It is not known whether ethylene
affects the seasonal variation in cambial responsiveness to
IAA, or if it acts as an intermediate in IAA-induced tracheid
production.
There is evidence that ethylene plays a role in the regulation
of xylem production in tree stems. First, the application of the
ethylene-releasing compound 2-chloroethylphosphonic acid
(Ethrel) increases tracheid production in Pinus species.
(Barker 1979, Telewski and Jaffe 1986, Yamamoto and Kozlowski 1987a, 1987b), as well as xylem increment in several
woody angiosperms (Robitaille and Leopold 1974, Phelps et
al. 1980, Yamamoto and Kozlowski 1987c, Yamamoto et al.
1987). Ethrel also alters the cell wall composition of Picea
abies hypocotyls, increasing the content of cellulose and de-
28
EKLUND AND LITTLE
creasing that of noncellulosic polysaccharides (Ingemarsson et
al. 1991a, Eklund 1991a), and promotes xylem differentiation
in Lactuca pith explants (Miller et al. 1984, 1985). ReindersGouwentak (1965) reported that cambial rest in Fraxinus
shoots was overcome by ethylene chlorohydrin treatment. In
addition, Co2+, which decreases ethylene production (Yu and
Yang 1979, Ingemarsson et al. 1991a), inhibited cell wall
deposition, whereas applying Ag+, an inhibitor of ethylene
action (Veen and Van de Geijn 1978), increased tracheid size
but did not alter cell wall composition in Picea abies (Ingemarsson et al. 1991a, Eklund 1991a). Second, in several tree
species, ethylene evolution from the stem is higher during the
period of xylem production than when the cambium is dormant
(Yamanaka 1985, Eklund 1990, 1991b, 1993b, Ingemarsson et
al. 1991b, Eklund et al. 1992). Moreover, mechanical perturbation increases both ethylene evolution and tracheid production in Pinus taeda (Telewski 1990), and there is evidence that
cambial region cells of Abies balsamea can convert an ethylene
precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), to
ethylene (Savidge 1988).
This study was undertaken to determine if ethylene interacts
with IAA in the regulation of the annual cycle of cambial
activity and dormancy in balsam fir stems, as measured by
seasonal changes in tracheid production.
Materials and methods
around the shoot circumference at a point located 8--9 cm
(budded cuttings) or 7--8 cm (debudded cuttings) below the
apex, the application of about 0.5 g of lanolin mixture spread
evenly over the surface of the application site, and the covering
of this site with aluminum foil. The cuttings were placed
upright in beakers with their bases immersed in deionized
water (containing Ethrel, Co2+ or Ag+ when these were applied
basally) and cultured for up to 5 weeks in a controlled environment chamber providing a photon flux density of 90 µmol m −2
s −1 from combined fluorescent and incandescent lamps, a 16-h
photoperiod and a day/night temperature of 23/13 °C. At
weekly intervals, basal solutions and IAA-lanolin mixtures
were replaced after removing a 1-mm slice from the basal or
apical cut surface.
Measurement of tracheid production
Tracheid production was determined in transverse, hand-cut
sections obtained from the midpoint of the cutting’s length,
unless indicated otherwise. The sections were stained in an
aqueous solution of phloroglucinol in 20% hydrochloric acid,
which reacts with lignin, and mounted in glycerol. Tracheid
production was measured as the number of new cells per radial
file on the xylem side of the cambium, recorded at eight
equidistant points around the circumference and starting from
the last-formed tracheids in the previous year’s latewood. The
measurement included only those cells reacting with the stain,
i.e., in which at least some lignification had occurred.
Plant material, application of growth regulators and culture
conditions
Measurement of ethylene evolution
Current-year shoots were collected from the upper crown of
balsam fir (Abies balsamea (L.) Mill.) trees approximately 25
years old and 4 to 7 m tall, located in a spaced stand at the
University of New Brunswick forest in Fredericton, New
Brunswick, Canada. In one experiment, cuttings were collected from the same trees on September 9, October 21 and
December 5, 1992, corresponding approximately to the cambial dormancy stages of rest, partial quiescence and full quiescence, respectively (Little and Bonga 1974, Sundberg et al.
1987). In all other experiments, the cuttings were collected in
January to March 1993, during quiescence. On each collection
date, enough cuttings were obtained to ensure that every tree
was represented in all treatments per experiment. The cuttings
were trimmed under tap water at the proximal end to a length
of 13 cm below the apical whorl of buds, and subsequently
were either left with their buds intact or debudded (final length
of debudded cuttings = 11 cm). They then were treated (1)
apically, either with 0, 0.1 or 1 mg IAA g −1 lanolin, or with 1
or 10 mg Ethrel g −1 lanolin, (2) laterally, either with 0, 1 or
10 mg Ethrel g −1 lanolin (neutralized Ethrel solution mixed
with lanolin), or with 1 mg IAA g −1 lanolin, or (3) basally,
either with 0, 0.1, 1, 10, 100, 1000 or 10,000 µM Ethrel, or with
1, 10 or 100 µM cobalt chloride, or with 1, 10 or 50 µM silver
thiosulfate in deionized water. Apical treatment involved complete debudding, the removal of the needles borne on the
uppermost 1 cm of the shoot, and the application of about 0.8 g
of lanolin mixture to the apical cut surface. Lateral treatment
involved the careful removal of the needles and periderm from
After wiping away any lanolin mixture or basal solution,
ethylene evolution was determined using either the entire cutting or two 4-cm-long segments. In the latter case, the length
of the first segment was measured starting at the basal end of
the cutting (denoted the basal segment, or the treated segment
if a lateral application site was present). The second segment
was positioned immediately above the first, and excluded the
apical application site or the terminal whorl of buds, whichever
was present (denoted the apical segment, or the control segment if the subjacent segment had a lateral application site). In
one experiment involving debudded cuttings, all cut surfaces
were covered with a silicone grease that we demonstrated did
not produce ethylene. Each cutting or segment was placed in a
50- or 25-ml glass vial, respectively, which was sealed with a
non-ethylene-releasing rubber stopper. The concentration of
ethylene was measured after incubation for 30 min at room
temperature. This short incubation period was chosen to avoid
the production of stress ethylene induced by wounding and
water deficit (Yamanaka 1985, 1986, Morgan et al. 1990). A
1-ml air sample was drawn from the headspace with a gas-tight
syringe and injected into a Hewlett Packard gas chromatograph (HP 5890) equipped with a 3.0 m glass column packed
with 80/100 mesh Porapak N (Supelco), a flame ionization
detector and an integrator. Nitrogen was used as the carrier gas
at 45 ml min −1, and the temperatures of the injector, column
and detector were 200, 65 and 250 °C, respectively. The retention time of ethylene was 1.5 min. The concentration of the
sample on a fresh weight basis was calculated based on a
IAA AND ETHYLENE CONTROL OF TRACHEID PRODUCTION
1 ppm ethylene standard (Valley Oxygen, Fredericton, Canada) after measuring and subtracting the volume of the needles
and stem from the vial volume, and determining the fresh
weight of the needles and stem.
Statistical analysis
Analysis of variance was performed on each data set to determine the significance of the effects of concentration, tree and
time or position, depending on the experiment. These effects
were fixed in the model, except for tree, which was treated as
random replication. Where appropriate, the significance (P ≤
0.05) of the difference in effect induced by individual concentrations at a particular time or position was determined by
Fisher’s Least Significant Difference test or a paired t-test.
Results
Ethylene evolution during IAA-induced cambial reactivation
in quiescent cuttings
29
higher in the budded cuttings.
The evolution of ethylene from the budded control cuttings
increased gradually through Week 3, then declined (Figure 1B). In contrast, in all debudded cuttings regardless of IAA
concentration, ethylene evolution increased during the first
week and subsequently decreased. The only between-treatment difference was evident after one week, when debudded
cuttings evolved more ethylene than budded cuttings.
Effect of basal application of Ethrel, Ag2+ and Co2+ on
IAA-induced tracheid production and ethylene evolution
during the rest--quiescence transition
The production of tracheids induced by 1 mg IAA g −1 lanolin
in cuttings treated basally with only water increased during the
autumn, most noticeably between September 9 and October 21
(Figure 2), reflecting the transition between the cambial dormancy states of rest and quiescence. Tracheid production was
not affected by Ethrel, Co2+ or Ag+ on September 9 and October
Bud burst and cambial reactivation occurred in the budded
control cuttings, and tracheids were present by Week 2 (Figure 1A). As expected, tracheid production was inhibited by
debudding and stimulated in debudded cuttings by applying
IAA apically, and the extent of the stimulation was positively
related to the exogenous IAA concentration. During the first
three weeks, the rate of tracheid production in the budded
cuttings was similar to that in the debudded cuttings treated
with 0.1 mg IAA g −1 lanolin, but subsequently the rate was
Figure 1. Tracheid number (A) in, and ethylene evolution (B) from,
cuttings that were either left intact (budded control, n) or debudded
and treated apically with 0 (s), 0.1 (h) or 1 (j) mg IAA g −1 lanolin.
Mean ± SE, n = 9; within each week, means accompanied by the same
letter are not significantly different.
Figure 2. Tracheid number in cuttings that were collected on September 9, October 21 and December 5, then debudded and treated apically
with 1 mg IAA g − 1 lanolin and basally with an aqueous solution of
Ethrel (A), silver thiosulfate (B) or cobalt chloride (C) for 4 weeks.
Ethrel at 100 mM was applied only on December 5. Mean ± SE, n =
6; within each collection date and chemical treatment, means accompanied by the same letter are not significantly different.
30
EKLUND AND LITTLE
21, but 10--100 µM Ethrel and 100 µM Co2+ were inhibitory
on December 5.
Ethylene evolution at the time of collection (Week 0) was
undetectable on every collection date (Figure 3). However,
after one week of culture, the evolution of ethylene from the
cuttings treated basally with only water was about 25 pmol
gFW−1 h −1 for the material collected on September 9, and about
twice that for the material collected on October 21 and December 5. Subsequently, ethylene evolution from these cuttings
declined, as observed also in the previous experiment (Figure 1). Applying Ethrel basally increased ethylene evolution to
about 1000 pmol gFW−1 h −1, and the extent of the increase was
positively related to the Ethrel concentration (Figures 3A--C).
Ethylene evolution from all Ethrel-treated cuttings collected
on September 9 and October 21 attained a maximum on Weeks
2 and 1, respectively, and subsequently declined. In contrast,
ethylene evolution from the Ethrel-treated cuttings collected
on December 5 remained high throughout. Applying Ag+
basally did not affect ethylene evolution from the cuttings
collected on any date (Figures 3D--F). In contrast, basal application of Co2+ inhibited ethylene evolution on all collection
dates, but the degree of inhibition did not vary with the Co2+
concentration (Figures 3G--I).
We investigated if the decrease in tracheid production and
stimulation of ethylene evolution induced by the 10 and 100
µM Ethrel (Figures 2A and 3A--C) were attributable to the high
concentrations of Ethrel decreasing the pH of the basal solution (Figure 4A). Adjustment to pH 7 reduced ethylene evolution, but only at the very high concentrations of 1 and 10 mM
Ethrel, which also caused massive defoliation and inhibited
tracheid production (Figures 4B--D). However, neutralization
did not alter the effect that Ethrel at any concentration had on
tracheid production or defoliation.
We also determined if ethylene emanated from the wounds
caused by excision and debudding, and if ethylene evolution
was greater from the stem or the needles. At the normal low
rate of ethylene evolution, as well as at an Ethrel-induced
elevated rate, the evolution of ethylene was not affected by
sealing all cut surfaces with a non-ethylene-releasing silicone
grease, and the stem evolved about five times more ethylene
than the needles (Table I).
Effect of applying IAA and Ethrel laterally or apically on
tracheid production and ethylene evolution in quiescent
cuttings
In the absence of Ethrel, tracheid production increased with
increasing concentration of apically applied IAA (Figure 5A),
whereas ethylene evolution did not (Figure 5D). Lateral appli-
Figure 3. Ethylene evolution from
cuttings that were collected on
September 9 (A, D, G), October
21 (B, E, H) and December 5 (C,
F, I), then debudded and treated
apically with 1 mg IAA g − 1 lanolin and basally with an aqueous
solution of Ethrel (A--C), silver
thiosulfate (D--F) or cobalt chloride (G--I) for 4 weeks. For each
collection date, chemical treatment and concentration, the same
cuttings were measured each
week. Mean ± SE, n = 6; within
each collection date, chemical
treatment and week, means accompanied by the same letter are
not significantly different.
IAA AND ETHYLENE CONTROL OF TRACHEID PRODUCTION
31
moted by lateral application of 0.1 mg and 1 mg IAA g −1
lanolin at, and by 1 mg IAA g −1 lanolin below, the application
site (Figure 6A). Lateral application of Ethrel at 10 mg g −1
lanolin inhibited tracheid production above and below the
application site. Ethylene evolution was not altered by 0.1 mg
IAA g −1 lanolin, whereas 1 mg IAA g −1 lanolin promoted
ethylene evolution in the treated segment by about an order of
magnitude (Figure 6B). Treatment with 1 mg Ethrel g −1 lanolin
had the same effect, whereas 10 mg Ethrel g −1 lanolin stimulated the evolution of ethylene from the treated segment by a
second order of magnitude and also promoted ethylene evolution from the control segment.
We also investigated the effect on tracheid production and
ethylene evolution of separately applying IAA and Ethrel in
lanolin to the apex of debudded cuttings. Treatment with 1 mg
IAA g −1 lanolin promoted tracheid production along the cutting length, but only increased ethylene evolution from the
basal segment (Figure 7). Neither 1 mg nor 10 mg Ethrel g −1
lanolin induced tracheid production, although the higher concentration stimulated the evolution of ethylene from the basal
segment to the same extent as did 1 mg IAA g −1 lanolin and,
in addition, increased ethylene evolution from the apical segment.
Discussion
Figure 4. Ethrel solution pH (A), ethylene evolution (B), tracheid
number (C) and percentage defoliation (D) in cuttings that were
debudded and treated apically with 1 mg IAA g −1 lanolin and basally
with a nonneutralized (s) or neutralized (d) aqueous solution of
Ethrel for 4 weeks. Mean ± SE, n = 6; within each Ethrel concentration, means that are significantly different are indicated with an asterisk.
cation of 1 mg Ethrel g −1 lanolin increased the number of
tracheids induced by 0.1 mg IAA g −1 lanolin, but only at the
Ethrel application site (Figures 5A and 5B). Ethrel at 10 mg
g −1 lanolin inhibited IAA-induced tracheid production
throughout the cutting (Figures 5A and 5C). Regardless of
IAA concentration, 1 and 10 mg Ethrel g −1 lanolin increased
ethylene evolution by about 1 and 2 orders of magnitude,
respectively (Figures 5D--F). The Ethrel-induced increases
were greater in the treated segment than in the control segment.
In stems of cuttings whose buds, the major source of endogenous IAA, were not removed, tracheid production was pro-
About 20% of the ethylene evolved from debudded cuttings
emanated from the needles, and undetectable amounts were
produced by, and transmitted through, the wounds created by
excising the buds and the cutting itself (Table 1). Thus, the bulk
of the ethylene evolved from the defoliated stem, but the
relative amounts emanated from the needle scars and the unwounded stem parts is not known. The major source of ethylene in the stem is the combined phloem and cambial tissue
(Yamanaka 1985, Telewski 1990). Hence, we assume that the
bulk of the ethylene evolved from a cutting in the absence of
Ethrel reflects production by the cambial region. We also
assume that the evolution of ethylene induced by applying
Ethrel, IAA, Co2+ and Ag+ reflects the level of ethylene in the
cambial region.
Our results provide evidence for and against the hypothesis
that ethylene interacts with IAA in regulating tracheid production. The hypothesis is supported by the finding that lateral
application of 1 mg Ethrel g −1 lanolin to debudded cuttings
treated apically with 0.1 mg IAA g −1 lanolin (Figure 5) and
lateral application of 10 mg Ethrel g −1 lanolin to budded
cuttings (Figure 6) increased ethylene evolution and tracheid
production at the application site. Lateral Ethrel application
has also been observed to promote tracheid production locally
in other conifers (Barker 1979, Telewski and Jaffe 1986,
Yamamoto and Kozlowski 1987a, 1987b). However, four other
observations indicate that IAA-induced tracheid production
does not involve ethylene. First, there was no consistent relationship between ethylene evolution and exogenous IAA concentration or tracheid production along the length of either
debudded cuttings treated apically with IAA (Figures 1, 5 and
7) or budded cuttings treated laterally with IAA (Figure 6).
32
EKLUND AND LITTLE
Table 1. Ethylene evolution from cuttings that were debudded and treated apically with 1 mg IAA g − 1 lanolin and basally with water containing 0
or 10 µM Ethrel for 4 weeks. Ethylene evolution was measured before and after covering all cut surfaces with silicone grease (Experiment 1), and
before and after separating the cutting into stem and needles (Experiment 2). Mean ± SE, n = 6; within each row, means accompanied by the same
letter are not significantly different.
Experiment
Ethrel (µM)
Ethylene (pmol gFW− 1 h −1)
Cut surfaces
Uncovered
Covered
Stem
Needles
1
0
10
79 ± 11a
512 ± 98a
80 ± 11a
487 ± 103a
---
---
2
0
10
160 ± 14a
426 ± 47a
---
124 ± 11b
369 ± 48b
59 ± 10c
62 ± 14c
Figure 5. Tracheid number (A--C) in, and
ethylene evolution (D--F) from, cuttings
that were debudded and treated apically
with 0 (s, white), 0.1 (h, brick) or 1 (j,
black) mg IAA g − 1 lanolin and laterally
with 0, 1 or 10 mg Ethrel g − 1 lanolin for
4 weeks. Mean ± SE, n = 5. The thick
horizontal bar indicates where the Ethrel
was applied. The thin horizontal bars indicate the position of the two segments
used to measure ethylene evolution.
Within each sampling position, means accompanied by the same letter are not significantly different. The asterisk indicates
where 1 mg Ethrel g −1 lanolin increased
tracheid production induced by 0.1 mg
IAA g − 1 lanolin (P < 0.05).
Second, varying ethylene evolution by basal application of
Ethrel and Co2+ to cuttings in which the cambium was in
different stages along the rest--quiescence transition had no
corresponding effect on IAA-induced tracheid production.
Ethrel always increased ethylene evolution, as expected, but
never tracheid production (Figures 2A, 3A--C and 4). Application of Co2+ caused the expected decrease in ethylene evolution, but tracheid production was not inhibited except by 100
µM Co2+ in cuttings collected in December, which we speculate was a toxic concentration at that time (Figures 2C and
3G--I). Third, Ag+, which blocks ethylene action, never inhibited ethylene evolution or IAA-induced tracheid production
(Figures 2B and 3D--F). Finally, applying Ethrel apically to
debudded cuttings stimulated ethylene evolution, but not tracheid production (Figure 7). It follows from the above observations that Ethrel promoted tracheid production only when
applied both laterally and below an IAA source, and that this
promotion was restricted to the Ethrel application point.
The different effects of basally and laterally applied Ethrel
on tracheid production may reflect the way in which the two
IAA AND ETHYLENE CONTROL OF TRACHEID PRODUCTION
Figure 6. Tracheid number (A) in, and ethylene evolution (B) from,
budded cuttings treated laterally either with 0 (s, white), 0.1 (h,
brick) or 1 (j, black) mg IAA g −1 lanolin, or with 1 (n, checkered)
or 10 (m, hearts) mg Ethrel g − 1 lanolin for 4 weeks. Mean ± SE, n =
5. The thick horizontal bar indicates where the lanolin mixtures were
applied. The thin horizontal bars indicate the position of the two
segments used to measure ethylene evolution. Within each sampling
position, means accompanied by the same letter are not significantly
different.
methods of application supplied ethylene to the cambial region. All solutions applied basally (Figure 4) and used to make
the lanolin mixtures (data not shown) had a pH > 4, thus Ethrel
decomposed to ethylene nonbiologically (Biddle et al. 1976)
before and after being taken up by the cutting. After uptake,
Ethrel and ethylene in the solution applied basally moved
upwards in the transpiration stream (Jackson and Campbell
1975, Foster et al. 1992, Eklund 1993a) and passed into the
cambial region along the entire transpiration pathway. In contrast, laterally applied Ethrel and derived ethylene reached the
cambial region after localized passage through the cortex and
phloem. We speculate that applying Ethrel laterally, but not
basally, raised the ethylene concentration in the cambial region
to an unphysiologically high level sufficient to induce a local
accumulation of IAA, which, in turn, caused the increase in
tracheid production (Sundberg and Little 1990, Little and
Sundberg 1991). The cause of the presumptive increase in IAA
concentration is not known, because the IAA concentration in
a particular tissue is determined by the relative rates of biosynthesis, conjugation, catabolism and transport, and ethylene is
known to affect each of these processes (Abeles et al. 1992).
33
Figure 7. Tracheid number (A) in, and ethylene evolution (B) from,
cuttings that were debudded and treated apically either with 0 (s,
white), 1 (n, checkered) or 10 (m, hearts) mg Ethrel g −1 lanolin, or
with 1 (j, brick) mg IAA g −1 lanolin for 5 weeks. Mean ± SE, n = 3.
The thin horizontal bars indicate the position of the two segments used
to measure ethylene production. Within each sampling position,
means accompanied by the same letter are not significantly different.
However, the possibility that Ethrel affects tracheid production
in a way other than, or in addition to, an ethylene--IAA interaction, e.g., directly or through a metabolite other than ethylene, cannot be excluded.
The poor relationship between ethylene evolution and tracheid production (Figures 1, 5--7) suggests that the higher rate
of ethylene evolution observed when the cambium is active
than when it is dormant (Figures 1--3; Yamanaka 1985, Eklund
1990, 1991b, 1993b, Ingemarsson et al. 1991b, Eklund et al.
1992) is not associated specifically with tracheid production.
This is supported by the finding that ethylene evolution from
the control cuttings collected in September, in which no tracheids were produced, also increased during the experimental
period (Figures 2 and 3). Furthermore, the magnitude of the
increase in ethylene evolution from IAA-treated cuttings,
which formed many tracheids, was similar to that from cuttings treated with lanolin only, in which tracheid production
did not occur (Figure 1).
Acknowledgment
Supported by a grant from the Swedish Council for Forestry and
Agriculture Research to L.E. We thank Dr. R.A. Savidge for the use of
a gas chromatograph, Ms. S.A. Martin for statistical advice, and
Rhône-Poulenc Canada Ltd. for a gift of Ethrel.
34
EKLUND AND LITTLE
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© 1994 Heron Publishing----Victoria, Canada
Interaction between indole-3-acetic acid and ethylene in the control of
tracheid production in detached shoots of Abies balsamea
LEIF EKLUND1 and C. H. ANTHONY LITTLE2
1
Department of Engineering and Natural Sciences, Växjö University, S-351 95 Växjö, Sweden
2
Natural Resources Canada, Canadian Forest Service, P.O. Box 4000, Fredericton, New Brunswick E3B 5P7, Canada
Received December 16, 1993
Summary We investigated the interaction between indole-3acetic acid (IAA) and ethylene in the regulation of the seasonal
periodicity of tracheid production in 1-year-old balsam fir
(Abies balsamea (L.) Mill.) cuttings collected at different times
during the dormant period. The cuttings were left with their
buds intact or were debudded and treated either apically with
IAA or 2-chloroethylphosphonic acid (Ethrel) in lanolin, laterally with IAA or Ethrel in lanolin, or basally with Ethrel, Co2+
or Ag+ in deionized water. The treated cuttings were then
cultured for up to 5 weeks under controlled environment conditions favorable for cambial growth. No change in ethylene
evolution was detected during the rest--quiescence transition,
when IAA-induced tracheid production increased. The induction of cambial reactivation by IAA was associated with a rise
in ethylene evolution, but there was no consistent relationship
among IAA concentration, tracheid number and ethylene emission. Neither Ethrel, Co2+ nor Ag+ affected tracheid production
when applied basally, except for 10 and 100 µM Ethrel and 100
µM Co2+, which were inhibitory. In contrast, ethylene evolution
was promoted by Ethrel and inhibited by Co2+, whereas Ag+
had no effect. Similarly, applying Ethrel apically or laterally
increased ethylene evolution, but did not promote tracheid
production except in the treatment in which 1 mg Ethrel g −1
lanolin was applied laterally to cuttings treated apically with
0.1 mg IAA g −1 lanolin, and in the treatment in which 10 mg
Ethrel g −1 lanolin was applied laterally to budded cuttings. We
conclude that (1) ethylene evolution is not specifically associated with IAA-induced tracheid production, (2) ethylene does
not mimic the promoting effect of IAA on tracheid production,
and (3) ethylene can promote tracheid production, but only
when its application results in a localized unphysiologically
high concentration in the cambial region, which, in turn, induces an accumulation of IAA.
Keywords: cambium, 2-chloroethylphosphonic acid, cobalt,
dormancy, Ethrel, IAA, silver.
Introduction
Considerable evidence indicates that indole-3-acetic acid
(IAA) is involved in the mechanism regulating xylem production in tree stems. For example, it has been shown that in
1-year-old shoots of Abies balsamea and Pinus sylvestris,
debudding concomitantly decreases the production of tracheids and the concentration of IAA, whereas both are increased by applying IAA either apically to debudded shoots or
laterally to budded shoots (Little and Savidge 1987, Sundberg
and Little 1990, Little and Sundberg 1991). Other research has
demonstrated that IAA transport is basipetally polar in the
cambial region, and that stem girdling both increases tracheid
production and the IAA concentration above the girdle while
reducing both below (Little and Savidge 1987). However, the
rate and duration of tracheid production are not clearly related
to the concentration of IAA in the cambial region (Sundberg et
al. 1991, 1993). Moreover, the cambium’s ability to produce
tracheids in response to IAA decreases with increasing age
(Little and Savidge 1987, Little and Sundberg 1991) and varies
with the time of IAA application. Studies with Abies balsamea
and Pinus sylvestris have shown that exogenous IAA promotes
tracheid production under environmental conditions favorable
for growth if applied during the winter when the cambium is
in the quiescence stage of dormancy imposed externally by
low temperature, but not if applied during the autumn when the
cambium is in the resting stage of dormancy maintained by
internal agents or conditions (Little and Savidge 1987, Sundberg et al. 1987, Little et al. 1990, Mellerowicz et al. 1992).
The reduced response to IAA by the resting cambium cannot
be attributed to either the accumulation of abscisic acid (Little
and Wareing 1981) or the inhibition of IAA transport (Little
1981), nor is it altered by exogenous gibberellic acid or kinetin
(Little and Bonga 1974). It is not known whether ethylene
affects the seasonal variation in cambial responsiveness to
IAA, or if it acts as an intermediate in IAA-induced tracheid
production.
There is evidence that ethylene plays a role in the regulation
of xylem production in tree stems. First, the application of the
ethylene-releasing compound 2-chloroethylphosphonic acid
(Ethrel) increases tracheid production in Pinus species.
(Barker 1979, Telewski and Jaffe 1986, Yamamoto and Kozlowski 1987a, 1987b), as well as xylem increment in several
woody angiosperms (Robitaille and Leopold 1974, Phelps et
al. 1980, Yamamoto and Kozlowski 1987c, Yamamoto et al.
1987). Ethrel also alters the cell wall composition of Picea
abies hypocotyls, increasing the content of cellulose and de-
28
EKLUND AND LITTLE
creasing that of noncellulosic polysaccharides (Ingemarsson et
al. 1991a, Eklund 1991a), and promotes xylem differentiation
in Lactuca pith explants (Miller et al. 1984, 1985). ReindersGouwentak (1965) reported that cambial rest in Fraxinus
shoots was overcome by ethylene chlorohydrin treatment. In
addition, Co2+, which decreases ethylene production (Yu and
Yang 1979, Ingemarsson et al. 1991a), inhibited cell wall
deposition, whereas applying Ag+, an inhibitor of ethylene
action (Veen and Van de Geijn 1978), increased tracheid size
but did not alter cell wall composition in Picea abies (Ingemarsson et al. 1991a, Eklund 1991a). Second, in several tree
species, ethylene evolution from the stem is higher during the
period of xylem production than when the cambium is dormant
(Yamanaka 1985, Eklund 1990, 1991b, 1993b, Ingemarsson et
al. 1991b, Eklund et al. 1992). Moreover, mechanical perturbation increases both ethylene evolution and tracheid production in Pinus taeda (Telewski 1990), and there is evidence that
cambial region cells of Abies balsamea can convert an ethylene
precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), to
ethylene (Savidge 1988).
This study was undertaken to determine if ethylene interacts
with IAA in the regulation of the annual cycle of cambial
activity and dormancy in balsam fir stems, as measured by
seasonal changes in tracheid production.
Materials and methods
around the shoot circumference at a point located 8--9 cm
(budded cuttings) or 7--8 cm (debudded cuttings) below the
apex, the application of about 0.5 g of lanolin mixture spread
evenly over the surface of the application site, and the covering
of this site with aluminum foil. The cuttings were placed
upright in beakers with their bases immersed in deionized
water (containing Ethrel, Co2+ or Ag+ when these were applied
basally) and cultured for up to 5 weeks in a controlled environment chamber providing a photon flux density of 90 µmol m −2
s −1 from combined fluorescent and incandescent lamps, a 16-h
photoperiod and a day/night temperature of 23/13 °C. At
weekly intervals, basal solutions and IAA-lanolin mixtures
were replaced after removing a 1-mm slice from the basal or
apical cut surface.
Measurement of tracheid production
Tracheid production was determined in transverse, hand-cut
sections obtained from the midpoint of the cutting’s length,
unless indicated otherwise. The sections were stained in an
aqueous solution of phloroglucinol in 20% hydrochloric acid,
which reacts with lignin, and mounted in glycerol. Tracheid
production was measured as the number of new cells per radial
file on the xylem side of the cambium, recorded at eight
equidistant points around the circumference and starting from
the last-formed tracheids in the previous year’s latewood. The
measurement included only those cells reacting with the stain,
i.e., in which at least some lignification had occurred.
Plant material, application of growth regulators and culture
conditions
Measurement of ethylene evolution
Current-year shoots were collected from the upper crown of
balsam fir (Abies balsamea (L.) Mill.) trees approximately 25
years old and 4 to 7 m tall, located in a spaced stand at the
University of New Brunswick forest in Fredericton, New
Brunswick, Canada. In one experiment, cuttings were collected from the same trees on September 9, October 21 and
December 5, 1992, corresponding approximately to the cambial dormancy stages of rest, partial quiescence and full quiescence, respectively (Little and Bonga 1974, Sundberg et al.
1987). In all other experiments, the cuttings were collected in
January to March 1993, during quiescence. On each collection
date, enough cuttings were obtained to ensure that every tree
was represented in all treatments per experiment. The cuttings
were trimmed under tap water at the proximal end to a length
of 13 cm below the apical whorl of buds, and subsequently
were either left with their buds intact or debudded (final length
of debudded cuttings = 11 cm). They then were treated (1)
apically, either with 0, 0.1 or 1 mg IAA g −1 lanolin, or with 1
or 10 mg Ethrel g −1 lanolin, (2) laterally, either with 0, 1 or
10 mg Ethrel g −1 lanolin (neutralized Ethrel solution mixed
with lanolin), or with 1 mg IAA g −1 lanolin, or (3) basally,
either with 0, 0.1, 1, 10, 100, 1000 or 10,000 µM Ethrel, or with
1, 10 or 100 µM cobalt chloride, or with 1, 10 or 50 µM silver
thiosulfate in deionized water. Apical treatment involved complete debudding, the removal of the needles borne on the
uppermost 1 cm of the shoot, and the application of about 0.8 g
of lanolin mixture to the apical cut surface. Lateral treatment
involved the careful removal of the needles and periderm from
After wiping away any lanolin mixture or basal solution,
ethylene evolution was determined using either the entire cutting or two 4-cm-long segments. In the latter case, the length
of the first segment was measured starting at the basal end of
the cutting (denoted the basal segment, or the treated segment
if a lateral application site was present). The second segment
was positioned immediately above the first, and excluded the
apical application site or the terminal whorl of buds, whichever
was present (denoted the apical segment, or the control segment if the subjacent segment had a lateral application site). In
one experiment involving debudded cuttings, all cut surfaces
were covered with a silicone grease that we demonstrated did
not produce ethylene. Each cutting or segment was placed in a
50- or 25-ml glass vial, respectively, which was sealed with a
non-ethylene-releasing rubber stopper. The concentration of
ethylene was measured after incubation for 30 min at room
temperature. This short incubation period was chosen to avoid
the production of stress ethylene induced by wounding and
water deficit (Yamanaka 1985, 1986, Morgan et al. 1990). A
1-ml air sample was drawn from the headspace with a gas-tight
syringe and injected into a Hewlett Packard gas chromatograph (HP 5890) equipped with a 3.0 m glass column packed
with 80/100 mesh Porapak N (Supelco), a flame ionization
detector and an integrator. Nitrogen was used as the carrier gas
at 45 ml min −1, and the temperatures of the injector, column
and detector were 200, 65 and 250 °C, respectively. The retention time of ethylene was 1.5 min. The concentration of the
sample on a fresh weight basis was calculated based on a
IAA AND ETHYLENE CONTROL OF TRACHEID PRODUCTION
1 ppm ethylene standard (Valley Oxygen, Fredericton, Canada) after measuring and subtracting the volume of the needles
and stem from the vial volume, and determining the fresh
weight of the needles and stem.
Statistical analysis
Analysis of variance was performed on each data set to determine the significance of the effects of concentration, tree and
time or position, depending on the experiment. These effects
were fixed in the model, except for tree, which was treated as
random replication. Where appropriate, the significance (P ≤
0.05) of the difference in effect induced by individual concentrations at a particular time or position was determined by
Fisher’s Least Significant Difference test or a paired t-test.
Results
Ethylene evolution during IAA-induced cambial reactivation
in quiescent cuttings
29
higher in the budded cuttings.
The evolution of ethylene from the budded control cuttings
increased gradually through Week 3, then declined (Figure 1B). In contrast, in all debudded cuttings regardless of IAA
concentration, ethylene evolution increased during the first
week and subsequently decreased. The only between-treatment difference was evident after one week, when debudded
cuttings evolved more ethylene than budded cuttings.
Effect of basal application of Ethrel, Ag2+ and Co2+ on
IAA-induced tracheid production and ethylene evolution
during the rest--quiescence transition
The production of tracheids induced by 1 mg IAA g −1 lanolin
in cuttings treated basally with only water increased during the
autumn, most noticeably between September 9 and October 21
(Figure 2), reflecting the transition between the cambial dormancy states of rest and quiescence. Tracheid production was
not affected by Ethrel, Co2+ or Ag+ on September 9 and October
Bud burst and cambial reactivation occurred in the budded
control cuttings, and tracheids were present by Week 2 (Figure 1A). As expected, tracheid production was inhibited by
debudding and stimulated in debudded cuttings by applying
IAA apically, and the extent of the stimulation was positively
related to the exogenous IAA concentration. During the first
three weeks, the rate of tracheid production in the budded
cuttings was similar to that in the debudded cuttings treated
with 0.1 mg IAA g −1 lanolin, but subsequently the rate was
Figure 1. Tracheid number (A) in, and ethylene evolution (B) from,
cuttings that were either left intact (budded control, n) or debudded
and treated apically with 0 (s), 0.1 (h) or 1 (j) mg IAA g −1 lanolin.
Mean ± SE, n = 9; within each week, means accompanied by the same
letter are not significantly different.
Figure 2. Tracheid number in cuttings that were collected on September 9, October 21 and December 5, then debudded and treated apically
with 1 mg IAA g − 1 lanolin and basally with an aqueous solution of
Ethrel (A), silver thiosulfate (B) or cobalt chloride (C) for 4 weeks.
Ethrel at 100 mM was applied only on December 5. Mean ± SE, n =
6; within each collection date and chemical treatment, means accompanied by the same letter are not significantly different.
30
EKLUND AND LITTLE
21, but 10--100 µM Ethrel and 100 µM Co2+ were inhibitory
on December 5.
Ethylene evolution at the time of collection (Week 0) was
undetectable on every collection date (Figure 3). However,
after one week of culture, the evolution of ethylene from the
cuttings treated basally with only water was about 25 pmol
gFW−1 h −1 for the material collected on September 9, and about
twice that for the material collected on October 21 and December 5. Subsequently, ethylene evolution from these cuttings
declined, as observed also in the previous experiment (Figure 1). Applying Ethrel basally increased ethylene evolution to
about 1000 pmol gFW−1 h −1, and the extent of the increase was
positively related to the Ethrel concentration (Figures 3A--C).
Ethylene evolution from all Ethrel-treated cuttings collected
on September 9 and October 21 attained a maximum on Weeks
2 and 1, respectively, and subsequently declined. In contrast,
ethylene evolution from the Ethrel-treated cuttings collected
on December 5 remained high throughout. Applying Ag+
basally did not affect ethylene evolution from the cuttings
collected on any date (Figures 3D--F). In contrast, basal application of Co2+ inhibited ethylene evolution on all collection
dates, but the degree of inhibition did not vary with the Co2+
concentration (Figures 3G--I).
We investigated if the decrease in tracheid production and
stimulation of ethylene evolution induced by the 10 and 100
µM Ethrel (Figures 2A and 3A--C) were attributable to the high
concentrations of Ethrel decreasing the pH of the basal solution (Figure 4A). Adjustment to pH 7 reduced ethylene evolution, but only at the very high concentrations of 1 and 10 mM
Ethrel, which also caused massive defoliation and inhibited
tracheid production (Figures 4B--D). However, neutralization
did not alter the effect that Ethrel at any concentration had on
tracheid production or defoliation.
We also determined if ethylene emanated from the wounds
caused by excision and debudding, and if ethylene evolution
was greater from the stem or the needles. At the normal low
rate of ethylene evolution, as well as at an Ethrel-induced
elevated rate, the evolution of ethylene was not affected by
sealing all cut surfaces with a non-ethylene-releasing silicone
grease, and the stem evolved about five times more ethylene
than the needles (Table I).
Effect of applying IAA and Ethrel laterally or apically on
tracheid production and ethylene evolution in quiescent
cuttings
In the absence of Ethrel, tracheid production increased with
increasing concentration of apically applied IAA (Figure 5A),
whereas ethylene evolution did not (Figure 5D). Lateral appli-
Figure 3. Ethylene evolution from
cuttings that were collected on
September 9 (A, D, G), October
21 (B, E, H) and December 5 (C,
F, I), then debudded and treated
apically with 1 mg IAA g − 1 lanolin and basally with an aqueous
solution of Ethrel (A--C), silver
thiosulfate (D--F) or cobalt chloride (G--I) for 4 weeks. For each
collection date, chemical treatment and concentration, the same
cuttings were measured each
week. Mean ± SE, n = 6; within
each collection date, chemical
treatment and week, means accompanied by the same letter are
not significantly different.
IAA AND ETHYLENE CONTROL OF TRACHEID PRODUCTION
31
moted by lateral application of 0.1 mg and 1 mg IAA g −1
lanolin at, and by 1 mg IAA g −1 lanolin below, the application
site (Figure 6A). Lateral application of Ethrel at 10 mg g −1
lanolin inhibited tracheid production above and below the
application site. Ethylene evolution was not altered by 0.1 mg
IAA g −1 lanolin, whereas 1 mg IAA g −1 lanolin promoted
ethylene evolution in the treated segment by about an order of
magnitude (Figure 6B). Treatment with 1 mg Ethrel g −1 lanolin
had the same effect, whereas 10 mg Ethrel g −1 lanolin stimulated the evolution of ethylene from the treated segment by a
second order of magnitude and also promoted ethylene evolution from the control segment.
We also investigated the effect on tracheid production and
ethylene evolution of separately applying IAA and Ethrel in
lanolin to the apex of debudded cuttings. Treatment with 1 mg
IAA g −1 lanolin promoted tracheid production along the cutting length, but only increased ethylene evolution from the
basal segment (Figure 7). Neither 1 mg nor 10 mg Ethrel g −1
lanolin induced tracheid production, although the higher concentration stimulated the evolution of ethylene from the basal
segment to the same extent as did 1 mg IAA g −1 lanolin and,
in addition, increased ethylene evolution from the apical segment.
Discussion
Figure 4. Ethrel solution pH (A), ethylene evolution (B), tracheid
number (C) and percentage defoliation (D) in cuttings that were
debudded and treated apically with 1 mg IAA g −1 lanolin and basally
with a nonneutralized (s) or neutralized (d) aqueous solution of
Ethrel for 4 weeks. Mean ± SE, n = 6; within each Ethrel concentration, means that are significantly different are indicated with an asterisk.
cation of 1 mg Ethrel g −1 lanolin increased the number of
tracheids induced by 0.1 mg IAA g −1 lanolin, but only at the
Ethrel application site (Figures 5A and 5B). Ethrel at 10 mg
g −1 lanolin inhibited IAA-induced tracheid production
throughout the cutting (Figures 5A and 5C). Regardless of
IAA concentration, 1 and 10 mg Ethrel g −1 lanolin increased
ethylene evolution by about 1 and 2 orders of magnitude,
respectively (Figures 5D--F). The Ethrel-induced increases
were greater in the treated segment than in the control segment.
In stems of cuttings whose buds, the major source of endogenous IAA, were not removed, tracheid production was pro-
About 20% of the ethylene evolved from debudded cuttings
emanated from the needles, and undetectable amounts were
produced by, and transmitted through, the wounds created by
excising the buds and the cutting itself (Table 1). Thus, the bulk
of the ethylene evolved from the defoliated stem, but the
relative amounts emanated from the needle scars and the unwounded stem parts is not known. The major source of ethylene in the stem is the combined phloem and cambial tissue
(Yamanaka 1985, Telewski 1990). Hence, we assume that the
bulk of the ethylene evolved from a cutting in the absence of
Ethrel reflects production by the cambial region. We also
assume that the evolution of ethylene induced by applying
Ethrel, IAA, Co2+ and Ag+ reflects the level of ethylene in the
cambial region.
Our results provide evidence for and against the hypothesis
that ethylene interacts with IAA in regulating tracheid production. The hypothesis is supported by the finding that lateral
application of 1 mg Ethrel g −1 lanolin to debudded cuttings
treated apically with 0.1 mg IAA g −1 lanolin (Figure 5) and
lateral application of 10 mg Ethrel g −1 lanolin to budded
cuttings (Figure 6) increased ethylene evolution and tracheid
production at the application site. Lateral Ethrel application
has also been observed to promote tracheid production locally
in other conifers (Barker 1979, Telewski and Jaffe 1986,
Yamamoto and Kozlowski 1987a, 1987b). However, four other
observations indicate that IAA-induced tracheid production
does not involve ethylene. First, there was no consistent relationship between ethylene evolution and exogenous IAA concentration or tracheid production along the length of either
debudded cuttings treated apically with IAA (Figures 1, 5 and
7) or budded cuttings treated laterally with IAA (Figure 6).
32
EKLUND AND LITTLE
Table 1. Ethylene evolution from cuttings that were debudded and treated apically with 1 mg IAA g − 1 lanolin and basally with water containing 0
or 10 µM Ethrel for 4 weeks. Ethylene evolution was measured before and after covering all cut surfaces with silicone grease (Experiment 1), and
before and after separating the cutting into stem and needles (Experiment 2). Mean ± SE, n = 6; within each row, means accompanied by the same
letter are not significantly different.
Experiment
Ethrel (µM)
Ethylene (pmol gFW− 1 h −1)
Cut surfaces
Uncovered
Covered
Stem
Needles
1
0
10
79 ± 11a
512 ± 98a
80 ± 11a
487 ± 103a
---
---
2
0
10
160 ± 14a
426 ± 47a
---
124 ± 11b
369 ± 48b
59 ± 10c
62 ± 14c
Figure 5. Tracheid number (A--C) in, and
ethylene evolution (D--F) from, cuttings
that were debudded and treated apically
with 0 (s, white), 0.1 (h, brick) or 1 (j,
black) mg IAA g − 1 lanolin and laterally
with 0, 1 or 10 mg Ethrel g − 1 lanolin for
4 weeks. Mean ± SE, n = 5. The thick
horizontal bar indicates where the Ethrel
was applied. The thin horizontal bars indicate the position of the two segments
used to measure ethylene evolution.
Within each sampling position, means accompanied by the same letter are not significantly different. The asterisk indicates
where 1 mg Ethrel g −1 lanolin increased
tracheid production induced by 0.1 mg
IAA g − 1 lanolin (P < 0.05).
Second, varying ethylene evolution by basal application of
Ethrel and Co2+ to cuttings in which the cambium was in
different stages along the rest--quiescence transition had no
corresponding effect on IAA-induced tracheid production.
Ethrel always increased ethylene evolution, as expected, but
never tracheid production (Figures 2A, 3A--C and 4). Application of Co2+ caused the expected decrease in ethylene evolution, but tracheid production was not inhibited except by 100
µM Co2+ in cuttings collected in December, which we speculate was a toxic concentration at that time (Figures 2C and
3G--I). Third, Ag+, which blocks ethylene action, never inhibited ethylene evolution or IAA-induced tracheid production
(Figures 2B and 3D--F). Finally, applying Ethrel apically to
debudded cuttings stimulated ethylene evolution, but not tracheid production (Figure 7). It follows from the above observations that Ethrel promoted tracheid production only when
applied both laterally and below an IAA source, and that this
promotion was restricted to the Ethrel application point.
The different effects of basally and laterally applied Ethrel
on tracheid production may reflect the way in which the two
IAA AND ETHYLENE CONTROL OF TRACHEID PRODUCTION
Figure 6. Tracheid number (A) in, and ethylene evolution (B) from,
budded cuttings treated laterally either with 0 (s, white), 0.1 (h,
brick) or 1 (j, black) mg IAA g −1 lanolin, or with 1 (n, checkered)
or 10 (m, hearts) mg Ethrel g − 1 lanolin for 4 weeks. Mean ± SE, n =
5. The thick horizontal bar indicates where the lanolin mixtures were
applied. The thin horizontal bars indicate the position of the two
segments used to measure ethylene evolution. Within each sampling
position, means accompanied by the same letter are not significantly
different.
methods of application supplied ethylene to the cambial region. All solutions applied basally (Figure 4) and used to make
the lanolin mixtures (data not shown) had a pH > 4, thus Ethrel
decomposed to ethylene nonbiologically (Biddle et al. 1976)
before and after being taken up by the cutting. After uptake,
Ethrel and ethylene in the solution applied basally moved
upwards in the transpiration stream (Jackson and Campbell
1975, Foster et al. 1992, Eklund 1993a) and passed into the
cambial region along the entire transpiration pathway. In contrast, laterally applied Ethrel and derived ethylene reached the
cambial region after localized passage through the cortex and
phloem. We speculate that applying Ethrel laterally, but not
basally, raised the ethylene concentration in the cambial region
to an unphysiologically high level sufficient to induce a local
accumulation of IAA, which, in turn, caused the increase in
tracheid production (Sundberg and Little 1990, Little and
Sundberg 1991). The cause of the presumptive increase in IAA
concentration is not known, because the IAA concentration in
a particular tissue is determined by the relative rates of biosynthesis, conjugation, catabolism and transport, and ethylene is
known to affect each of these processes (Abeles et al. 1992).
33
Figure 7. Tracheid number (A) in, and ethylene evolution (B) from,
cuttings that were debudded and treated apically either with 0 (s,
white), 1 (n, checkered) or 10 (m, hearts) mg Ethrel g −1 lanolin, or
with 1 (j, brick) mg IAA g −1 lanolin for 5 weeks. Mean ± SE, n = 3.
The thin horizontal bars indicate the position of the two segments used
to measure ethylene production. Within each sampling position,
means accompanied by the same letter are not significantly different.
However, the possibility that Ethrel affects tracheid production
in a way other than, or in addition to, an ethylene--IAA interaction, e.g., directly or through a metabolite other than ethylene, cannot be excluded.
The poor relationship between ethylene evolution and tracheid production (Figures 1, 5--7) suggests that the higher rate
of ethylene evolution observed when the cambium is active
than when it is dormant (Figures 1--3; Yamanaka 1985, Eklund
1990, 1991b, 1993b, Ingemarsson et al. 1991b, Eklund et al.
1992) is not associated specifically with tracheid production.
This is supported by the finding that ethylene evolution from
the control cuttings collected in September, in which no tracheids were produced, also increased during the experimental
period (Figures 2 and 3). Furthermore, the magnitude of the
increase in ethylene evolution from IAA-treated cuttings,
which formed many tracheids, was similar to that from cuttings treated with lanolin only, in which tracheid production
did not occur (Figure 1).
Acknowledgment
Supported by a grant from the Swedish Council for Forestry and
Agriculture Research to L.E. We thank Dr. R.A. Savidge for the use of
a gas chromatograph, Ms. S.A. Martin for statistical advice, and
Rhône-Poulenc Canada Ltd. for a gift of Ethrel.
34
EKLUND AND LITTLE
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