Tree Physiology 23, 73–83
© 2003 Heron Publishing—Victoria, Canada
Effects of exogenous gibberellin and auxin on shoot elongation and
vegetative bud development in seedlings of Pinus sylvestris and Picea
glauca
C. H. A. LITTLE1,2 and JOANNE E. MACDONALD1
1
Natural Resources Canada, Canadian Forest Service–Atlantic Forestry Centre, P.O. Box 4000, Fredericton, NB E3B 5P7, Canada
2
Author to whom correspondence should be addressed ([email protected])
Received November 26, 2001; accepted July 20, 2002; published online January 2, 2003
Summary The hormonal control of stem unit (foliar appendage and axillary structure, if present, plus subtending internode) number and length was investigated in shoots of Scots
pine (Pinus sylvestris L.) and white spruce (Picea glauca
(Moench) Voss). Seedlings were treated with six gibberellins
(GA1, GA3, GA 4, GA 5, GA 9 and GA20) and two auxins (indole-3-acetic acid (IAA) and naphthaleneacetic acid (NAA))
when either neoformed growth was occurring or the terminal
vegetative bud was developing. Hormones were applied by
drenching the shoot tip, injecting the stem or spraying the
foliage. Combined results for all three application methods
indicated that shoot elongation in first-year seedlings (i.e.,
neoformed growth) was promoted in both species by GA1,
GA3, GA 4 and, less obviously, by GA9. This promotion was attributable to an increase in length, rather than number, of stem
units. However, the number of stem units formed during terminal bud development, as reflected in the number of needles
(white spruce) or cataphylls (Scots pine) present on the shoot
resulting from the terminal bud, was stimulated by GA1, GA3
and GA 4 in both species and by GA9 in Scots pine. The GA-induced increase in the number of preformed stem units was associated with increased bud width in white spruce and increased bud length and resulting shoot length in Scots pine. In
contrast, application of IAA or NAA either did not affect or inhibited both neoformed growth and terminal bud stem unit
number, depending on the application method and concentration. We conclude that, in the Pinaceae, (1) GA stimulates the
activity of both the subapical meristem during neoformed
growth and the apical meristem during vegetative bud development, and (2) the early non-hydroxylation pathway, via GA9, is
the major route of GA biosynthesis. The role of auxin in the
control of stem unit number and length remains to be resolved.
Keywords: indole-3-acetic acid, naphthaleneacetic acid,
neoformed (free) growth, preformed (fixed) growth, stem unit.
Introduction
In Pinaceae species, annual shoot length depends primarily on
the number of stem units, rather than their length (Cannell et
al. 1976, Lanner 1976, Bongarten 1986, Smith et al. 1993,
Lascoux et al. 1994, Norgren et al. 1996). Stem unit number
reflects the activity of the apical meristem, whereas the extent
of cell division and accompanying cell elongation in the subapical meristem determine stem unit length (Cannell et al.
1976, Owens and Simpson 1988, Brown and Sommer 1992).
In first-year seedlings, shoot elongation typically proceeds as
neoformed (free) growth (Hallé et al. 1978), where the initiation and elongation of stem units occur simultaneously. Eventually, stem unit elongation ceases, typically in response to
declining photoperiod, and stem units are initiated within developing terminal buds. After bud development ends, the buds
overwinter in a dormant state, and during the following growing season, the stem units elongate. The elongation of these
stem units is denoted preformed (fixed) growth (Hallé et al.
1978). Under favorable environmental conditions, however, a
terminal bud can elongate during the year of its development,
resulting in a lammas shoot (Rudolph 1964, Cannell et al.
1976).
Accumulating evidence from investigations involving species of Pinus and Picea implicates gibberellin (GA) in the control of shoot elongation in the Pinaceae. First, twelve endogenous GAs (GA1, GA3, GA 4, GA7, GA8, GA9, GA12, GA15, GA20,
GA29, GA 34 and GA51) have been unequivocally identified in
elongating shoots (Little and Pharis 1995, Moritz 1995, Wang
et al. 1996). Second, experiments involving the application of
stable isotope-labeled GAs to elongating shoots showed that
GAs are readily translocated and metabolized, and that the
early non-hydroxylation pathway, where 13-hydroxylation
occurs subsequent to formation of GA 9, is likely the only route
of GA biosynthesis (Moritz et al. 1989, Moritz and Odén
1990, Odén et al. 1995, Wang et al. 1996). Third, high concentrations of endogenous GA1, GA3, GA 4 and GA9 have been
observed during rapid shoot elongation (Moritz et al. 1990,
Pharis et al. 1992, Moritz 1995, Wang et al. 1995b, 1996).
Fourth, both neoformed and preformed shoot elongation can
be promoted by exogenous GA1, GA3, GA 4 or GA 4/7 (Little
and Pharis 1995, Moritz 1995, Wang et al. 1997) and decreased by applying inhibitors of GA biosynthesis (Ross et
al. 1983, Graham et al. 1994, Wang et al. 1995b). However,
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LITTLE AND MACDONALD
whether treatment with exogenous GA or GA-biosynthesis inhibitors acts by altering the number or the length of stem units
has not been reported.
Although there is abundant evidence that GAs play a causal
role in reproductive bud development in conifers (Pharis 1991,
Smith 1998), scant attention has been paid to the possibility
that GAs are also involved in the control of stem unit initiation
in vegetative terminal buds developing at the tip of currentyear shoots. Owens et al. (1985) found that applying GA 4/7
during the early period of leaf initiation in Pseudotsuga menziesii (Mirb.) Franco did not affect either the number of leaves
initiated or shoot elongation the following year. However,
GA 4/7 application increased bud width and the length of the
resulting shoot in Picea sitchensis (Bong.) Carr. (Philipson
1983), as well as bud length and resulting shoot length and
stem unit number in Pinus contorta Dougl. (Longman 1982).
The evidence implicating auxin in the control of shoot elongation in the Pinaceae is sparse, compared with GA, and contradictory. The endogenous auxin, indole-3-acetic acid (IAA),
has been identified in many species, but high concentrations
have been correlated with both rapid and decreased shoot
elongation (Little and Pharis 1995). Defoliation of elongating
shoots concomitantly lowered the IAA concentration and inhibited longitudinal growth in Abies balsamea (L.) Mill.
(Sundberg and Little 1987) and Pinus sylvestris L. (Wang et al.
1997). In contrast, decapitation of elongating Pinus sylvestris
shoots also decreased IAA concentration, but did not affect
longitudinal growth (Wang et al. 1997). Finally, exogenous
IAA increased, decreased or did not affect shoot elongation in
several species (Ross et al. 1983, Wang et al. 1997), whereas it
consistently promoted elongation in sections excised from hypocotyls of Pinus spp. (Zakrzewski 1975, Carpita and Tarmann 1982, Terry et al. 1982) and in intact nonwoody species
(Yang et al. 1993, Haga and Iino 1997).
The purpose of this work was to determine if exogenous GA
or auxin affected the number or length of stem units produced
during neoformed growth or vegetative bud development in
Pinus sylvestris and Picea glauca (Moench) Voss, species
known to differ in shoot and bud morphology. Six GAs (GA1,
GA3, GA 4, GA5, GA 9 and GA20) were tested for activity. The
gibberellins GA1, GA3 and GA 4 are known effectors of shoot
elongation (Little and Pharis 1995, Moritz 1995, Wang et al.
1995b, 1997). Metabolic experiments with stable-isotope labeled GAs have shown that GA9 is converted to GA 4 and
GA20, and that GA20 and GA5 are potential precursors of GA1
and GA3, respectively (Moritz et al. 1989, Moritz and Odén
1990, Odén et al. 1995, Wang et al. 1995b, 1996). Two auxins
were applied: naturally occurring IAA and a synthetic auxin,
naphthaleneacetic acid (NAA), which is known to be more stable than IAA when applied exogenously. Three application
methods, namely shoot-tip drench, foliar spray and stem injection, were compared for efficacy.
Materials and methods
Scots pine (Pinus sylvestris) and white spruce (Picea glauca)
seedlings were used in two series of experiments, one per-
formed in 1999–2000 and the other in 2000–2001. The same
seed lots (Scots pine of unknown origin, obtained from the
New Brunswick Department of Natural Resources and Energy; white spruce from a British Columbia Ministry of Forests open-pollinated seed orchard) and culturing procedures
were used for each series. In late February, seeds were sown on
a mixture of peat and vermiculite (2:1, v/v) in individual Ray
Leach containers (115 ml) to enable the resulting seedlings to
be sorted without disturbing the root systems. Seedlings were
reared in a greenhouse compartment with a photon flux density of 600 µmol m –2 s –1 on sunny days, a photoperiod extended to 20 h with 400-W high-pressure sodium lamps
(Sylvania) yielding a photon flux density of 30 µmol m –2 s –1, a
day/night temperature set at 25/18 °C, and an overhead travelling boom system for watering. The water delivered “starter”
fertilizer (11:41:8, N:P:K, with N at 50 ppm) during the establishment stage (about 5 weeks) and “grower” fertilizer
(20:8:20, with N at 100 ppm) during the subsequent rearing
period.
In mid-May, seedlings whose epicotyl was 5 ± 1 cm long
and elongating rapidly were selected. These seedlings were
grouped such that the average epicotyl length was the same for
each group per treatment in a particular experiment. The seedlings in these groups were treated with exogenous GAs and
auxins, as described below, to investigate their effect on neoformed shoot growth. Immediately before the first treatment,
the seedlings were transferred to another greenhouse compartment with the same environmental conditions as the compartment used to rear them, except that the water plus “grower”
fertilizer solution was supplied directly to the roots from below the container by means of an “ebb-and-flow” system.
Consequently, the hormones applied to the shoot were not subsequently washed away. At the end of the treatment period, the
seedlings were harvested to measure stem unit number and
length.
Seedlings left in the rearing compartment in May were allowed to grow until the end of June, when they were used to investigate the effect of exogenous GAs and auxins on the development of vegetative terminal buds. To induce the cessation of
neoformed growth and the start of bud development, seedlings
were transferred to a third greenhouse compartment where
they were subjected to “finisher” fertilizer (8:20:30, with N at
35 ppm) and a short photoperiod (8 h) for up to 25 days. Seedlings were then returned to the compartment with the ebband-flow watering system and exposed to the “finisher” fertilizer under a 14-h photoperiod to maximize bud development
while minimizing lammas growth. Seedlings were sorted into
matched groups such that the average epicotyl length was the
same for each group per treatment in a particular experiment.
Exogenous GAs and auxins were applied as described below.
At the end of the hormone treatments, the seedlings were subjected to the “finisher” fertilizer under ambient (external) photoperiod and temperature. After bud size appeared to be maximal, the size of the terminal bud was measured and some buds
were harvested for stereomicroscopic examination. In December, the dormant seedlings were transferred to storage at 5 °C
to ensure that they received sufficient chilling to enable nor-
TREE PHYSIOLOGY VOLUME 23, 2003
HORMONAL CONTROL OF SHOOT AND BUD DEVELOPMENT
mal bud flushing. In March, the seedlings were returned to the
initial rearing compartment to induce growth. Stem unit number and length were measured after shoot elongation ceased.
Seedlings undergoing neoformed growth or terminal bud
development were treated with GAs and auxins in one of three
ways. (1) With a micropipette, each shoot tip was drenched
with 10 µl of 70% ethanol in water containing 0, 0.1, 1, 10 or
100 µg of GA1, GA3, GA 4, GA5, GA 9 or GA20, or with 0,
0.001, 0.01, 0.1 or 1 µg of IAA or NAA, applied weekly for up
to 10 weeks. (2) Foliage was sprayed to runoff with 0 or 0.1%
GA3, or with 0, 0.0005, 0.005 or 0.05% NAA, in water containing 0.05% Tween 20, applied twice weekly for up to
7 weeks. (3) In Scots pine seedlings only, the stem was injected 1 to 2 cm below the shoot tip with 2 µl of 70% ethanol in
water containing 0 or 20 µg of GA1, GA3, GA 4, GA5, GA9 or
GA20, or with 0, 0.2, 2 or 20 µg of IAA or NAA, applied
weekly for up to 10 weeks. In a preliminary experiment, it was
determined for both white spruce and Scots pine that drenching the shoot tip with 10 µl was the optimum volume for completely covering the neoformed primordia immediately surrounding the shoot apical meristem or the developing terminal
bud, while minimizing runoff.
Shoot elongation of seedlings in neoformed growth was
monitored by measuring epicotyl growth at weekly intervals
during the hormone application period, and for one additional
week thereafter. Bud size was determined by measuring terminal bud length in Scots pine and terminal bud width in white
spruce. The culture conditions for Scots pine, more often than
not, induced a modified type of lammas growth, resulting in
the formation of a long bud (Rudolph 1964). In long buds,
elongation occurs in the portion of the bud bearing sterile cataphylls, whereas the fertile cataphyll portion of the bud remains
closed. Accordingly, terminal bud length in Scots pine was determined by measuring the elongated (if present) and closed
portions separately. The number of stem units in the epicotyl,
and in the shoot resulting from the overwintered terminal bud,
was determined in white spruce by counting needle number,
and in Scots pine by counting the number of primary needles
and cataphylls having axillary secondary needle fascicles (fertile cataphylls) or lacking such axillary fascicles (sterile cataphylls). Buds were sectioned longitudinally by hand and
examined under a stereomicroscope to evaluate their morphology and anatomy. The terminology of MacDonald and Owens
(1993) was used to describe white spruce buds.
One-way analysis of variance was applied to each data set
collected on the final measurement date and, where appropriate (P < 0.05, unless noted otherwise), the significance of the
difference between means was determined by the Duncan
New Multiple Range Test as performed by the SuperANOVA
statistics software package (1989; Abacus Concepts, Berkeley, CA). To facilitate presentation, with some exceptions,
only means ± standard error (SE) pertaining to treatments that
had a stimulatory effect are presented in the figures or tables;
inhibitory or ineffective treatments are summarized in the text.
75
Results
Neoformed growth
Drenching the shoot tip with GA1, GA3 or GA 4 stimulated
shoot elongation in both Scots pine and white spruce, and in
white spruce the extent of stimulation increased with increasing GA concentration (Figures 1 and 2). The GA-induced promotion of shoot length was associated with an increase in stem
unit length, but not in stem unit number (Table 1). Shoot elongation ceased in Scots pine during the treatment period and
drenching the shoot tip with GA1, GA3 or GA 4 did not prevent
this cessation (Figure 1). In contrast, shoot elongation in white
spruce continued long after the application of GA stopped;
nevertheless, the stimulatory effect of GA application was still
evident at the end of the growing season (Figure 3). Drenching
the shoot tip with GA 9 stimulated shoot elongation in white
spruce but not in Scots pine, whereas neither GA5 nor GA20 affected longitudinal growth in either species (Figures 1 and 2).
Drenching the shoot tip with IAA or NAA also had no effect
on shoot elongation in either Scots pine or white spruce (data
not shown), except for the highest concentration (1 µg), which
was inhibitory (P < 0.02).
Spraying the foliage with GA3 promoted shoot elongation in
both Scots pine and white spruce, and the increased shoot
length in Scots pine was a result of longer stem units, not more
of them (Table 2). In contrast, spraying NAA in the same way
inhibited shoot elongation in Scots pine at a concentration of
0.05% (P = 0.0001) and in white spruce at 0.005 and 0.05%
(P = 0.0001), whereas a concentration of 0.0005% did not affect longitudinal growth in either species (data not shown).
Shoot elongation and stem unit length were increased in
Scots pine by injecting the stem with GA1 or GA 3, but not with
GA5 or GA20 (Table 3). Injecting GA 4 also increased stem unit
length, but simultaneously decreased stem unit number; thus,
shoot length was unaffected. The inhibitory effect of GA 4 injection on stem unit number, together with the observation that
the stem was visibly injured at the GA 4 injection sites, indicates that the concentration used was supra-optimal for this
GA. Injection of GA9 promoted shoot elongation at P = 0.11.
However, neither shoot elongation nor stem unit length nor
stem unit number was altered by injecting 0.2 µg of IAA or
NAA (data not shown).
Terminal bud development
In a first bud-drench experiment, terminal bud size and the
number of stem units in the shoot resulting from the bud were
increased by GA1 and GA 4, and by GA1, GA3 and GA 4, respectively, in white spruce (Figure 4), but not by any of these
GAs in Scots pine (data not shown). In white spruce, GA 4 was
more effective at a lower concentration (10 µg) than GA1 or
GA3. The highest concentration (100 µg) of GA1, GA3 and
GA 4 also increased resulting shoot length, but only the effect
of GA1 was significant (data not shown). Terminal bud size in
white spruce was also increased by drenching with auxin,
NAA being active at a lower concentration than IAA (Figures 5A and 5C); however, resulting shoot stem unit number
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76
LITTLE AND MACDONALD
Figure 1. Epicotyl elongation in Scots pine seedlings whose shoot tips
were drenched eight times at weekly intervals with GA1 (A), GA3 (B),
GA 4 (C), GA5 (D), GA9 (E) or GA20 (F). For each GA, means ± SE
(n = 12) followed by the same letter are not significantly different at
P < 0.05, measured 1 week after the last application.
Figure 2. Epicotyl elongation in white spruce seedlings whose shoot
tips were drenched eight times at weekly intervals with GA1 (A), GA3
(B), GA 4 (C), GA5 (D), GA9 (E) or GA20 (F). For each GA, means ±
SE (n = 12) followed by the same letter are not significantly different
at P < 0.05, measured 1 week after the last application.
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HORMONAL CONTROL OF SHOOT AND BUD DEVELOPMENT
77
Table 1. Epicotyl length and stem unit number and length in Scots pine and white spruce seedlings whose shoot tips were drenched eight times at
weekly intervals with 0 (control) or 100 µg of gibberellin (GA). Within columns, means ± SE (n = 12) followed by the same letter are not significantly different at P < 0.05, measured 1 week after the last application.
Treatment
Scots pine
Epicotyl length (mm)
Control
GA1
GA3
GA 4
149 ± 9 a
190 ± 7 bc
177 ± 9 ab
215 ± 14 c
White spruce
Stem unit
Epicotyl length (mm)
Number
Length (mm)
171 ± 9 a
161 ± 4 a
151 ± 5 a
167 ± 9 a
0.89 ± 0.06 a
1.18 ± 0.04 b
1.18 ± 0.05 b
1.28 ± 0.06 b
was unaffected by IAA and reduced by NAA (Figures 5B and
5D). In Scots pine, the only effects of auxin drenches were inhibitory: the 100-µg NAA treatment decreased terminal bud
149 ± 5 a
205 ± 8 b
189 ± 12 b
189 ± 8 b
Stem unit
Number
Length (mm)
385 ± 13 a
431 ± 16 a
429 ± 26 a
443 ± 23 a
0.39 ± 0.01 a
0.48 ± 0.02 b
0.44 ± 0.02 b
0.43 ± 0.02 ab
size and the 10- and 100-µg NAA treatments reduced the number of stem units in the resulting shoot (P = 0.0001).
Stereomicroscopic examination of fresh longitudinal sections through terminal buds in the first bud-drench experiment
revealed that, in white spruce, IAA and NAA, as well as high
concentrations of GA1 and GA 4, increased the width of the
bud-scale receptacle. This increase was associated with proliferation of parenchyma in the shoot immediately below the
developing bud. The enlarged bud-scale receptacle laterally
displaced the bud scales, resulting in a loosening of the typical
tightly packed bud-scale complex. At inhibitory concentrations, NAA resulted in death of the preformed shoot in white
spruce and of the entire bud in Scots pine.
In a subsequent bud-drench experiment involving only
Table 2. Epicotyl length and stem unit number and length in Scots
pine seedlings sprayed twice weekly for 8 weeks with GA3. Within
columns, means ± SE (n = 24) followed by the same letter are not significantly different at P < 0.05, measured 1 week after the last application.
% GA3
0
0.1
Epicotyl length (mm)
126 ± 4 a
149 ± 4 b
Stem unit
Number
Length (mm)
146 ± 5 a
137 ± 4 a
0.87 ± 0.02 a
1.10 ± 0.04 b
Table 3. Epicotyl length and stem unit number and length in Scots
pine seedlings injected six times at weekly intervals with 0 (control)
or 20 µg of gibberellin (GA). Within columns, means ± SE (n = 8) followed by the same letter are not significantly different at P < 0.05,
measured 1 week after the last application.
Treatment
Figure 3. Epicotyl length of white spruce seedlings whose shoot tips
were drenched five times at weekly intervals with GA1 (A), GA3 (B)
or GA 4 (C). Length was measured on July 20, 1 week after the last GA
application, and on October 23, after epicotyl elongation ended. For
each GA, means ± SE (n = 10) followed by the same letter are not significantly different at P < 0.05, measured on July 20 or October 23.
Control
GA1
GA3
GA 4
GA5
GA 9
GA20
Epicotyl length (mm)
85 ± 5 a
110 ± 5 c
103 ± 7 bc
80 ± 6 a
88 ± 5 ab
98 ± 8 abc
90 ± 6 ab
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Stem unit
Number
Length (mm)
148 ± 14 b
125 ± 5 ab
130 ± 6 ab
109 ± 9 a
130 ± 6 ab
144 ± 9 b
149 ± 7 b
0.57 ± 0.02 a
0.89 ± 0.04 c
0.80 ± 0.05 bc
0.75 ± 0.06 bc
0.68 ± 0.05 ab
0.69 ± 0.04 ab
0.61 ± 0.05 a
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LITTLE AND MACDONALD
Figure 4. Terminal bud width (A–C) and stem unit number in the resulting shoot (D–F) in white spruce seedlings whose shoot tips were drenched
five times at weekly intervals with GA1 (A, D), GA3 (B, E) or GA 4 (C, F) during the period of terminal bud development. For each GA and parameter, means ± SE (n = 10) followed by the same letter are not significantly different at P < 0.05, measured after terminal bud development ceased in
the autumn (bud width) or after the shoot produced by the terminal bud ceased elongating the next spring (stem unit number).
Scots pine, 100 µg of GA1, GA3, GA 4, GA5, GA9, GA20 or
IAA, or 10 µg of NAA, were applied for twice the number of
weeks during the bud development period as in the first
bud-drench experiment. The only effective GA was GA 4. This
GA treatment increased terminal long bud total length and
resulting shoot length (P = 0.0789) and stem unit number
without altering stem unit length (Figure 6). Exogenous GA 4
increased the total length of the terminal long bud, primarily
by stimulating longitudinal growth in the elongated portion
(Figure 6A). The GA 4-induced increase in stem unit number
was caused by a larger number of both sterile and fertile cataphylls (Figure 6C). The two auxin treatments did not affect terminal bud size and decreased resulting shoot length (P =
0.0015) and fertile stem unit number (P = 0.0017).
Injecting Scots pine stems with GA 4 and GA 9, but not with
GA1, GA3, GA5 or GA20, increased terminal long bud total
length (P = 0.0001) by stimulating longitudinal growth in the
elongated portion (Figure 7A). The GA 4 also increased the
length of the resulting shoot (Figure 7B). The number of fertile
stem units in the resulting shoot was increased by GA 4 and
GA9, and decreased by GA20 (Figure 7C). Resulting shoot
stem unit length was not altered by injection of any GA (Figure 7D). Stem injections with 20 µg of IAA or 2 µg of NAA decreased terminal bud size, resulting shoot length and resulting
shoot fertile stem unit number (P < 0.0005).
Spraying the foliage of Scots pine with GA3 increased the
total length of the terminal long bud (P = 0.002) by increasing
the length of the closed portion (Figure 8A). In addition, this
treatment increased resulting shoot length and fertile stem unit
number, without affecting stem unit length (Figures 8B–8D).
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79
Figure 5. Terminal bud width (A, B) and stem unit number in the resulting shoot (C, D) in white spruce seedlings whose shoot tips were drenched
five times at weekly intervals with indole-3-acetic acid (IAA; A, C) or naphthaleneacetic acid (NAA; B, D) during the period of terminal bud development. For each auxin and parameter, means ± SE (n = 10) followed by the same letter are not significantly different at P < 0.05, measured after terminal bud development ceased in the autumn (bud width) or after the shoot produced by the terminal bud ceased elongating the next spring
(stem unit number).
Discussion
Neoformed shoot elongation in first-year Scots pine and white
spruce seedlings was promoted by exogenous GA1, GA3 and
GA 4 (Figures 1–3), confirming results obtained in investigations of many conifer species (Little and Pharis 1995, Moritz
1995, Wang et al. 1997). Exogenous GA9 also increased neoformed growth in white spruce (Figure 2), but less obviously
in Scots pine (Table 3, P = 0.11). We show, for the first time,
that the GA-induced promotion of neoformed growth in conifer shoots was caused by an increase in stem unit length, rather
than in stem unit number (Tables 1–3). This finding is consistent with the conclusion that GA promotes shoot elongation
primarily by stimulating the activity of the subapical meristem
(Sachs 1965, Hansen et al. 1999). Exogenous GA appeared to
act mainly by increasing the rate of subapical meristematic activity in white spruce (Figure 2) and by extending the duration
of rapid subapical meristematic activity in Scots pine (Figure 1). However, GA application could not prevent the eventual cessation of subapical meristem activity in Scots pine
(Figure 1). As neoformed growth continued long after the end
of the GA application period in white spruce (Figure 3), it is
possible that exogenous GA could increase both stem unit
length and number under environmental conditions that markedly extend the duration of growth.
The development of vegetative terminal buds, as reflected in
bud size and the number of stem units in the shoot resulting
from the bud, was promoted by the application of GA1, GA3
and GA 4 in white spruce (Figure 4) and GA3, GA 4 and GA9 in
Scots pine (Figures 6–8). Exogenous GA increased bud width
in white spruce by enlarging the bud-scale receptacle, which
resulted in lateral displacement of the bud scales. In Scots
pine, GA application increased the total length of the terminal
long bud by stimulating longitudinal growth in the elongated
portion in some experiments (Figures 6A and 7A) and increasing the size of the closed portion in other experiments (Figure 8A). The GA-induced increase in the number of preformed
stem units explains the associated increase in the length of the
resulting shoot in Scots pine (Figures 6B, 7B and 8B). The
GA-induced increase in preformed stem unit number in Scots
pine was manifested primarily in sterile stem units (cataphylls
without axillary needle fascicles) in one experiment (Figure 6C) and in fertile stem units (cataphylls with axillary needle fascicles) in another experiment (Figure 7C). This difference in response presumably reflects the time at which an
effective amount of GA reached the apical meristem, i.e.,
mainly before or after the apical meristem had switched initiation from sterile cataphyll to fertile cataphyll. A GA-induced
increase in the number of fertile stem units in the bud would
enhance the photosynthetic capacity of the resulting shoot,
and this enhancement could thus contribute indirectly to the
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80
LITTLE AND MACDONALD
Figure 6. Length of elongated and closed portions of the terminal long
bud (A), length of resulting shoot (B) and number of sterile and fertile
stem units (C) and stem unit length (D) in the resulting shoot of Scots
pine seedlings whose shoot tips were drenched ten times at weekly intervals with 0 (control) or 100 µg of GA 4 during the bud development
period. For each parameter, means ± SE (n = 10) followed by the same
letter are not significantly different at P < 0.05, measured after terminal bud development ceased in the autumn (bud length) or after the
shoot produced by the terminal bud ceased elongating the next spring
(shoot length, stem unit number and length).
Figure 7. Length of elongated and closed portions of the terminal long
bud (A), length of resulting shoot (B) and number of sterile and fertile
stem units (C) and stem unit length (D) in the resulting shoot of Scots
pine seedlings whose stems were injected ten times at weekly intervals with 0 (control) or 20 µg of gibberellin (GA) during the bud development period. For each GA and parameter, means ± SE (n = 12)
followed by the same letter are not significantly different at P < 0.05,
measured after terminal bud development ceased in the autumn (bud
length) or after the shoot produced by the terminal bud ceased elongating the next spring (shoot length, stem unit number and length).
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HORMONAL CONTROL OF SHOOT AND BUD DEVELOPMENT
Figure 8. Length of elongated and closed portions of the terminal long
bud (A), length of resulting shoot (B), and stem unit number (C) and
length (D) in the resulting shoot of Scots pine seedlings sprayed twice
weekly with 0 (control) or 0.1% GA3 for 5 weeks during the bud development period. For each parameter, means ± SE (n = 13) followed
by the same letter are not significantly different at P < 0.05, measured
after terminal bud development ceased in the autumn (bud length) or
after the shoot produced by the terminal bud ceased elongating the
next spring (shoot length, stem unit number and length).
GA-induced promotion of resulting shoot length. The positive
relationship between bud length and resulting shoot length
found in GA-treated Scots pine (Figures 6–8) has also been
81
observed in Pinus contorta shoots injected with GA 4/7 (Longman 1982), as well as in untreated shoots of Pinus spp. (e.g.,
Norgren et al. 1996).
Based on the percentage increases in neoformed growth and
preformed stem unit number induced by each applied GA, as
well as the minimum effective GA concentration, it is apparent
that GA1 and GA 4 were the most active GAs for stimulating
subapical and apical meristematic activity, particularly GA 4 in
Scots pine. It is not known if the superior activity of exogenous
GA 4 in Scots pine is related to its uptake, translocation, metabolism or action, or if exogenous GA 4 is active per se or only after conversion to GA1 (Moritz et al. 1989, Moritz and Odén
1990, Odén et al. 1995, Wang et al. 1995b, 1996). We note that
applying GA 4 or GA 4/7 to conifer shoots has also been observed to promote cone bud production (cited in Pharis 1991,
Smith 1998) and the activity of the vascular cambium (Wang
et al. 1995a, 1995b, 1997).
The finding that exogenous GA9, in addition to GA1 and
GA 4, can promote neoformed growth and terminal bud development (Figures 1, 2 and 6; Table 3) is consistent with the view
that GA9 → GA 4 and possibly GA 4 → GA1 are key steps in the
GA biosynthetic pathway in species of the Pinaceae (Moritz et
al. 1989, Moritz and Odén 1990, Odén et al. 1995, Wang et al.
1995b, 1996). The relative inactivity of exogenous GA20, the
major precursor of GA1 in angiosperms (Olsen et al. 1995,
Hedden and Kamiya 1997), provides additional support for the
conclusion that GA20 → GA1 conversion is not a major route to
GA1 in Pinaceae species. Moritz (1995) found that neoformed
growth in Picea abies (L.) Karst. grown in a short photoperiod
(10 h) was promoted by applying GA 4 or GA1, but not GA9 or
GA20. On the basis of this and other evidence, Moritz (1995)
concluded that 3β-hydroxylation of GA9 to GA 4 and of GA20
to GA1 is under photoperiodic control.
The metabolic pathway leading to endogenous GA3 in conifer shoots has yet to be identified (Wang et al. 1995b, 1996).
However, the possibility that GA5 is its precursor (Wang et al.
1996) is not supported by the finding that exogenous GA5 invariably failed to promote neoformed growth or terminal bud
development (Figures 1, 2, 7; Table 3).
The three methods used to apply GA3 (shoot-tip drench,
foliar spray and stem injection) promoted neoformed growth
in Scots pine to a similar extent on a percentage basis (Tables 1
to 3). They also all increased the number of stem units formed
during terminal bud development, although only the effect of
spraying was significant (Figures 7 and 8, and data not shown).
Accordingly, the foliar spray application method is being used
in current experiments aimed at elucidating how GA3 promotes stem unit initiation within developing buds.
Our finding that exogenous IAA and NAA either did not affect or inhibited neoformed growth and terminal bud stem unit
number in Scots pine and white spruce is typical of the few
investigations concerning the effect of auxin application on
shoot elongation in the Pinaceae (Ross et al. 1983, Little and
Pharis 1995, Wang et al. 1997). This suggests that shoot elongation in such species is not normally limited by the supply of
auxin and that endogenous concentrations are likely optimal
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82
LITTLE AND MACDONALD
or supra-optimal. Thus, assessment of the effects of applied
auxin on stem unit number and length requires the development of a procedure that on the one hand decreases the endogenous auxin supply, e.g., by decapitation or defoliation (Wang
et al. 1997), and on the other hand supplies auxin to the subapical and apical meristems in an effective manner, e.g., for an
extended period via a wick (Bhatnagar and Talwar 1978, Yang
et al. 1993).
Acknowledgments
We thank Dr. P.C. Odén for valuable discussions, D. Kolotelo for
white spruce seed and J. Hacking, R. Hudson, J. Letourneau, J. Lewis,
R. Scagel, R. West and L. Yeates for technical assistance.
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