Plant Growth Regul (2008) 55:241–248
DOI 10.1007/s10725-008-9280-9
ORIGINAL PAPER
Gibberellin-mediated changes in carbohydrate metabolism
during flower stalk elongation in tulips
Anil P. Ranwala Æ William B. Miller
Received: 19 September 2007 / Accepted: 13 April 2008 / Published online: 15 May 2008
Ó Springer Science+Business Media B.V. 2008
Abstract We investigated the effects of a gibber-
ellin synthesis inhibitor (ancymidol) and gibberellin
(GA4+7) on carbohydrate metabolism and elongation
in internodes of the tulip (Tulipa gesneriana L.)
flower stalk during greenhouse growth. During the
initial stages of flower stalk growth, the lowermost
internode was mainly responsible for total flower
stalk length, whereas the uppermost internode mostly
contributed to total length during later stages. High
concentrations of hexose sugar (mainly glucose) and
increased activity of acid invertase were observed
when internodes were rapidly elongating. Inhibition
of gibberellin biosynthesis with ancymidol reduced
the elongation rate of internodes, and inhibited the
hexose sugar accumulation and acid invertase activ-
ity. Application of GA4+7 to ancymidol-treated plants
reversed these effects. The degree of response to
ancymidol and GA4+7 was, however, different in
different internodes such that the lowermost inter-
node was most responsive and the uppermost
internode was least responsive. The results indicate
that de novo biosynthesis of gibberellins is a
A. P. Ranwala
W. B. Miller (&)
Department of Horticulture, Cornell University,
134 Plant Science Bldg., Ithaca, NY 14853, USA
e-mail: wbm8@cornell.edu
Present Address:
A. P. Ranwala
Floralife Inc., 751 Thunderbolt Drive, Walterboro,
SC 29488, USA
requirement for expression of high acid invertase
activity during the rapid elongation phase in tulip
internodes which enables cleavage of imported
sucrose to hexoses that can be readily utilized in
elongating cells.
Keywords Ancymidol
Gibberellin

Internode elongation
Invertase

Soluble carbohydrates
Tulipa gesneriana
Introduction
After flower initiation, tulip (Tulipa gesneriana L.)
bulbs must be exposed to a period of low temperature
for proper elongation of the flower stalk and normal
flower development during subsequent growth at
warmer temperatures. Molecular regulatory mecha-
nisms of the low temperature effect on flower stalk
elongation in tulips are not fully understood, but it is
known that both gibberellins (Hanks 1982; Rebers
et al. 1994; Saniewski 1989; Shoub and De Hertogh
1974) and auxins (Rietveld et al. 2000) are involved
in this process. The inhibition of gibberellin biosyn-
thesis in properly cooled intact bulbs (Saniewski
1989; Shoub and De Hertogh 1974) or isolated
sprouts (Rebers et al. 1994) with gibberellin synthesis
inhibitors substantially reduced the rate of flower
stalk elongation, suggesting that de novo biosynthe-
sis of gibberellins is an essential factor for tulip
flower stalk elongation. In another line of research,
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242
exogenous gibberellins partially substituted for the
cold requirement for flower stalk elongation, also
implicating the involvement of this plant hormone in
the process (Hanks 1982).
During rapid flower stalk growth in tulips, elon-
gation occurs mainly due to cell expansion (Gilford
and Rees 1973). Cell expansion requires readily
available hexose substrates used for increased metab-
olism and biosynthesis of new material such as cell
walls. In properly cooled tulip bulbs, dynamic
changes in carbohydrate metabolism take place
during flower stalk elongation (Lambrechts et al.
1994; Lambrechts and Kolloffel 1993). The period of
rapid elongation in individual internodes of tulip
flower stalk is marked by high concentrations of
hexoses (especially glucose) and low concentrations
of sucrose and starch (Lambrechts and Kolloffel
1993; Lambrechts et al. 1994). Among the enzymes
associated with sucrose catabolism (soluble invertase,
insoluble invertase and sucrose synthase), only sol-
uble acid invertase exhibited significant changes
during stalk elongation where its activity increased
remarkably in parallel with rapid internode elonga-
tion (Balk and de Boer 1999; Lambrechts et al.1994;
Lambrechts and Kolloffel 1993).
Gibberellins have been shown to influence the
carbohydrate status in many plant species (Cano-
Medrano and Darnell 1997; Chen et al. 1994; Miyam-
oto et al. 1993; Morris and Arthur 1985; Yim et al.
1997). In elongating tissues, a common response to
exogenous gibberellin is an increase in acid invertase
activity (Kaufman et al. 1968; Kaufman et al. 1973;
Miyamoto et al. 1993; Morris and Arthur 1985; Wu
et al. 1993). Given the involvement of gibberellins in
flower stalk elongation in tulips and the dynamic
changes in carbohydrates profiles that take place
during rapid stalk elongation, investigation of gibber-
ellin-mediated changes in carbohydrate metabolism
will provide useful information for better understand-
ing the flower stalk elongation process in tulips.
In this study, we investigated changes in carbohy-
drate metabolism in tulip flower stalk caused by the
sequential application of a gibberellin synthesis
inhibitor (ancymidol) and gibberellin (GA4+7) during
forcing of fully cooled bulbs. The changes in the
length of each of four internodes of the flower stalk
were compared with the changes in non-structural
carbohydrate profiles and the activity of soluble acid
invertase.
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Plant Growth Regul (2008) 55:241–248
Materials and methods
Plant material and growth regulator treatments
Tulip (Tulipa gesneriana L. cv. Apeldoorn) bulbs that
were sufficiently developed to begin low temperature
treatment (‘‘stage G’’ plus 6 weeks) were obtained
from a commercial source. The low-temperature
requirement of the bulbs was satisfied by storing
them at 4°C for 12 weeks. At the end of cold storage,
a group of bulbs was treated with ancymidol
(gibberellin synthesis inhibitor, SePRO Corp, Car-
mel, IN, USA) by soaking the bulbs for 1 h in a
50 mg l-1 solution at 20°C. Bulbs soaked in distilled
water for the same duration served as controls. Bulbs
were then planted individually in pots (10 cm diam-
eter) containing soil-less media (Metro Mix-360;
Scotts-Sierra Horticultural Products Co., Marysville,
OH, USA), and grown in a greenhouse with a set
point temperature of 22/18°C (vent/heat), and under
full natural sunlight. Fifteen days after planting, a
group of ancymidol-treated plants was treated with
GA4+7. Each plant was supplied with 25 mg of GA4+7
(Abbott laboratories, North Chicago, IL, USA) as a
drench application (60 ml) to the planting media. Our
objective was to provide sufficient gibberellin to
counter the previous inhibition of gibberellin synthe-
sis by ancymidol, and to not model or quantify
gibberellin uptake or partitioning in the plant. Earlier
research on tulip (Shoub and De Hertogh 1974) and
lily (De Hertogh and Blakely 1972; Ranwala et al.
2003) demonstrated the effectiveness of soil-applied
gibberellin.
Plants were harvested at 5-day intervals, and the
flower stalk was carefully separated from other
tissues. The flower stalk was divided into the four
internodes, and the length and fresh weight of each
internode measured. Tissues were then frozen in
liquid nitrogen and stored at -80°C for carbohydrate
analysis and enzyme assays. Frozen tissues were
freeze-dried to obtain dry weight measurements.
Analysis of non-structural carbohydrates
Frozen tissues were freeze-dried and ground to a
fine powder. Tissue samples (50 mg DW) were ex-
tracted at 70°C with 80% ethanol (three extractions of
3 ml each, 1 h per extraction). Tissue suspensions
were centrifuged at 3,000g for 10 min after each
Plant Growth Regul (2008) 55:241–248
extraction, and the supernatants were combined. The
extracts were passed through ion exchange columns
consisting of 1 ml each of Amberlite IRA-67 (acetate
form) and Dowex 50W (hydrogen form) (Sigma, St.
Louis, MO, USA) to remove charged material. The
extracts were then evaporated to dryness under
reduced pressure at 55°C, and dissolved in 10 ml
distilled water. After appropriate dilution, samples
were subjected to high-performance anion exchange
chromatography with pulsed amperometric detection
(HPAE-PAD) using a Dionex DX-500 series chro-
matograph, equipped with a Carbopac PA-1 column,
a pulsed amperometric detector and a gold electrode
(Dionex, Sunnyvale, CA, USA). Carbohydrates were
eluted with 200 mM NaOH for 15 min at a flow rate
of 1.0 ml min-1. The amounts of glucose, fructose,
and sucrose were determined by comparison with
calibration curves derived from standard authentic
sugars purchased from Sigma.
For starch determination, the residue after ethanol
extraction was dried in an oven at 55°C overnight.
For each sample, 4 ml of 100 mM Na-acetate buffer
(pH 4.5) was added and boiled for 30 min to
gelatinize starch. After cooling, 50 units of amylo-
glucosidase (Sigma) dissolved in 1 ml of 100 mM
Na-acetate buffer (pH 4.5) was added to each sample
and incubated for 24 h at 55°C. The amount of
glucose released was determined by HPAE-PAD (as
above) using an aliquot of the digested sample. The
amount of starch was estimated according to the
amounts of glucose released.
Enzyme extraction and assay
All operations were carried out in a cold room at 4°C
or on ice. Tissues were homogenized (5 ml buffer per
g fresh weight) in 100 mM Hepes–NaOH (pH 7.0)
containing 2.5 mM DTT and 1 mM PMSF using a
Polytron homogenizer (Brinkmann Instruments,
Westbury, NY, USA) at full speed for two 20-s
intervals. The homogenate was squeezed through a
layer of moistened Miracloth, then the filtrate was
centrifuged at 13,800g for 20 min. The supernatant
was desalted with pre-packed columns filled with
Sephadex G-25 gel filtration medium (PD-10;
Pharmacia, Uppsala, Sweden). Proteins were eluted
with 10 mM Hepes/NaOH (pH 7.0) containing 1 mM
DTT, and the eluted fraction was used for enzyme
assay.
243
The reaction mixture for acid invertase assay
contained 40 mM Na-acetate buffer (pH 4.5), 50 mM
sucrose and enzyme in a total volume of 1.0 ml.
Buffer and enzyme were pre-incubated in a water
bath at 30°C for 2 min prior to substrate addition.
Standard reaction time was 30 min at 30°C, and the
assay was linear within standard time and enzyme
volume used. Addition of alkaline copper reagent
terminated the reaction, and the production of glucose
and fructose was quantified by completing the
Somogyi method (1952). Controls for the assay
consisted of reactions carried out with inactivated
(boiled for 5 min) enzyme. One unit of enzyme
activity was defined as the amount of enzyme
catalyzing the cleavage of 1 lmol of sucrose per
min at 30°C.
Results
Growth of flower stalk
In control plants, the elongation of the lowermost
internode was evident 5 days after planting (DAP) and
continued until 20 DAP (Fig. 1). The most rapid
elongation of the lowermost internode occurred
between 10 and 20 DAP. The second and third
internodes began elongating after the first internode,
and rapid elongation occurred between 15 and 20
DAP. The uppermost internode entered its rapid
elongation phase 20 DAP and continued until 30 DAP.
Ancymidol treatment reduced the elongation rate
in all internodes. The inhibitory effect, however, was
less pronounced in upper internodes than lower
internodes. For example, the uppermost internode of
ancymidol-treated plants reached a length close to
that of control plants by 30 DAP. Application of
GA4+7 to ancymidol-treated plants at 15 DAP
reversed the inhibitory effects of ancymidol on
internode elongation. This reversal effect was most
prominent in the lowermost internode, where its
length surpassed that of control plants.
The changes in dry weight of individual internodes
followed the same pattern as the internode length
(Fig. 2). While the weight of the uppermost internode
was similar to the lower internode, it was longer, and,
therefore, thinner. On average, the control, ancymidol
and ancymidol + GA4+7 treated plants reached
anthesis at 22, 24, 23 DAP, respectively.
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244 Plant Growth Regul (2008) 55:241–248

Fig. 1 Lengths of four

INTERNODE 1 INTERNODE 2
internodes of tulip flower 4
stalk during the growth. Just 7.5 Control

Internode length (cm)

Internode length (cm)

before planting, tulip bulbs Ancymidol
were treated (soaked) with Ancymidol 3
gibberellin synthesis + GA4+7
5
inhibitor (ancymidol) or
2
water (control). At 15 days
after planting, a group of
2.5
ancymidol-treated plants 1
were treated with
gibberellin (GA4+7).
Internodes are numbered 0 0
from bottom (1) to top (4). 0 5 10 15 20 25 30 0 5 10 15 20 25 30
Values are mean ± SE of
five plants
6 INTERNODE 3 25
INTERNODE 4
Internode length (cm)

Internode length (cm)

20

4
15

10
2

0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Time after planting (days)

Fig. 2 Dry weights of four

0.8 INTERNODE 1 0.3 INTERNODE 2
internodes of tulip flower
stalk during the growth. Just Control
before planting, tulip bulbs
Dry weight (g)

0.6 Ancymidol
Dry weight (g)

were treated (soaked) with Ancymidol 0.2

gibberellin synthesis + GA4+7
inhibitor (ancymidol) or 0.4
water (control). At 15 days
after planting, a group of 0.1
ancymidol-treated plants 0.2
were treated with
gibberellin (GA4+7).
Internodes are numbered 0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
from bottom (1) to top (4).
Values are mean ± SE of
five plants 0.3 0.8
INTERNODE 3 INTERNODE 4
Dry weight (g)

Dry weight (g)

0.6
0.2

0.4

0.1
0.2

0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Time after planting (days)

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Plant Growth Regul (2008) 55:241–248 245

Changes in carbohydrates
Glucose, fructose, sucrose and starch were the major
non-structural carbohydrates detected in the tulip
flower stalk. At planting, all internodes contained
about 250–350 mg g-1 DW starch and about
200 mg g-1 DW sucrose. Very little glucose or
fructose was present in any internode at the time of
planting.
In the lowermost internode, the concentration of
glucose and fructose began to increase with onset of
rapid internode elongation and continued until 20
DAP (Fig 3). The concentration of glucose increased
more rapidly than fructose, reaching about
250 mg g-1 DW on 20 DAP compared to about
35 mg g-1 DW fructose. Sucrose concentration
dropped about 2-fold by 5 DAP, remained fairly
constant until 25 DAP, and increased slightly by 30
DAP. Starch concentration continued to decrease
during elongation.
The rate of increase in glucose and fructose
concentrations was lower in ancymidol-treated plants
compared with controls. For example, on 15 DAP,
there was more than 2-fold difference in the glucose
concentration between control and ancymidol-treated
plants. The glucose concentration in ancymidol-
treated lowermost internodes, however, reached lev-
els comparable to those in controls at later stages (30
DAP). Starch breakdown was also slower in ancym-
idol-treated lowermost internodes compared with
control internodes. Gibberellin given to the ancym-
idol-treated plants on 15 DAP reversed these effects
as evidenced by sharp increase in glucose and
fructose concentrations, and decrease in sucrose and
starch concentrations 5 days after GA application
(Fig. 3).
The patterns of the changes in non-structural
carbohydrate profiles in the second and third inter-
nodes during the elongation and in response to
ancymidol and GA4+7 were similar to those of the
lowermost internode. However, the degree of ancym-
idol effect in second and third internodes was slightly
less than those in the lowermost internode (data not
shown). The least effect of ancymidol was observed
in the uppermost internode (Fig 4). The rapid elon-
gation phase in this internode was also marked by

Fig. 3 Concentrations of Control

non-structural 300 Ancymidol 60
carbohydrates in the first Ancymidol
Fructose (mg/g DW)

(lowest) internode of tulip

Glucose (mg/g DW)

+ GA4+7
flower stalk during the 45
growth. Values are 200
mean ± SE of three
30
replicates. Each replicate
consisted of tissues from at 100
least three plants 15

0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30

200 400
Starch (mg/g DW)
Sucrose (mg/g DW)

150 300

100 200

50 100

0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Time after planting (days)

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246 Plant Growth Regul (2008) 55:241–248

Fig. 4 Concentrations of
120
non-structural Control
carbohydrates in the fourth 300 Ancymidol

Fructose (mg/g DW)

(uppermost) internode of

Glucose (mg/g DW)

Ancymidol 90
tulip flower stalk during the + GA4+7
growth. Values are
200
mean ± SE of three 60
replicates. Each replicate
consisted of tissues from at
least three plants 100 30

0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30

250
200
Sucrose (mg/g DW)

200

Starch (mg/g DW)

150
150

100
100

50 50

0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Time after planting (days)

increased levels of glucose and fructose. Ancymidol Discussion

treatments caused slight slow down of this effect.
Acid invertase activity
Soluble acid invertase activity was barely detectable
at the time of planting in any internode. In the
lowermost internode, acid invertase activity increased
after 5 DAP, peaked at 15 DAP, then gradually
decreased (Fig. 5). Ancymidol treatment reduced
invertase activity compared with controls but activity
continued to increase until 20 DAP. Gibberellin
treatment increased acid invertase activity more than
2-fold in the 5 days after GA application to ancym-
idol-treated plants.
In the uppermost internode, there was a very sharp
increase in activity from 15 to 20 DAP in control plants
followed by a plateau. Internodes of ancymidol-treated
plants instead showed continuous increase in activity
from 15 to 25 DAP. Gibberellin treatment increased
the acid invertase activity in ancymidol-treated plants
on 20 DAP to levels comparable to control plants. In
all treatments, the activity decreased from 25 to 30
DAP.
The elongation of the tulip flower stalk is a result of
sequential elongation of individual internodes begin-
ning from the lowermost internode and progressing
upwards. During the initial stages of growth, the
lowermost internode is the most important contributor
to the total length of the tulip flower stalk, whereas
the uppermost internode is most important during later
growth stages (e.g. during flower opening, and
immediately thereafter). Increased concentrations of
hexose sugars (mainly glucose) and increased acid
invertase activity were the major characteristics
observed during the rapid elongation phase of all
internodes. These observations are consistent with
previously published results (Lambrechts et al. 1994;
Lambrechts and Kolloffel 1993), and indicate that
cleavage of imported sucrose may be an important
prerequisite for internode elongation in tulips.
The influence of gibberellins on acid invertase
activity in tulip internodes was clearly evident in our
study. Inhibition of gibberellin biosynthesis with
ancymidol prevented the normal rise in acid invertase
activity during the rapid elongation phase, and

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Plant Growth Regul (2008) 55:241–248 247

Fig. 5 Activity of acid INTERNODE 1 INTERNODE 4

invertase in the first

Invertase activity (µmoles/hr/g FW)

Invertase activity (µmoles/hr/g FW)

(lowest) and fourth 35 35
(uppermost) internodes of Control
30 Ancymidol 30
tulip flower stalk during the
growth. Values are 25 Ancymidol
25
+ GA4+7
mean ± SE of three
replicates. Each replicate 20 20
consisted of tissues from at
15 15
least three plants
10 10

5 5

0 0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Time after planting (days)

application of exogenous gibberellin reversed this also tested both GA3 and GA4+7 in tulips in
effect, which was evident by sharp increases in preliminary experiments, and found GA4+7 to be
activity immediately following the gibberellin treat- more effective than GA3 in internode elongation (data
ment. An increase in acid invertase activity in not shown).
response to exogenous gibberellin has been observed The effects of exogenous gibberellins (or inhibi-
in many elongating plant tissues such as Avena tion of gibberellin synthesis) on internode length,
internodes (Kaufman et al. 1968, 1973), bean (Phase- hexose sugar levels and acid invertase activity were
olus vulgaris) internodes (Morris and Arthur 1985), more pronounced in lower internodes than upper
and elongating dwarf pea (Pisum sativum L.) shoots internodes. There are two possibilities for this effect.
(Wu et al. 1993). Increased acid invertase activity in First, the effect of ancymidol (gibberellin synthesis
response to gibberellins in these tissues was also inhibitor) that is applied at the time of planting may
accompanied by increased hexose concentrations and wear out at later stages of development so that lesser
rapid elongation growth. Wu et al. (1993) showed an effects are seen in upper internodes where elongation
increase in acid invertase mRNA levels within 4 h occurs later than lower internodes. Second, factors
after gibberellin treatment in dwarf pea seedlings other than gibberellins may regulate the elongation of
indicating that enhanced invertase expression is an upper internodes. The involvement of auxins in tulip
initial response to gibberellins causing increased flower stalk elongation (especially upper internodes)
hexose sugar availability and elongation growth. has been shown in several studies. In those studies,
Several studies have focused on the activity of removal of the flower bud and leaves (both major
different gibberellins during the growth of tulip sources of auxins) reduced the flower stalk elongation
flower stalk (Shoub and De Hertogh 1974; Rebers considerably, and application of auxin (IAA)
et al. 1994). Shoub and De Hertogh (1974) showed reversed this effect (Saniewski and de Munk 1981;
that when applied as a soil drench to planted tulip Okubo and Uemoto 1985; Saniewski 1989). On the
bulbs, GA4+7 is more effective in promoting stem other hand, auxin-induced floral stalk elongation is
elongation than GA3. Rebers et al. (1994) compared inhibited by paclobutrazol, a gibberellin biosynthesis
exogenous GA1, GA4 and GA9 on in vitro grown inhibitor, suggesting that gibberellin biosynthesis is
isolated sprouts, and concluded that all three stimu- also necessary for auxin induced floral stalk elonga-
lated stalk elongation, but GA4 was the most tion (Saniewski 1989). In a study using isolated tulip
effective. The significance of GA4 in tulip flower internodes grown in tissue culture medium, Rietveld
stalk was further evident from the findings of Rebers et al. (2000) proposed that tulip internode elonga-
et al. (1995) where GA4 was identified as is the most tion is mainly due to the response to auxin, and low
abundant endogenous gibberellin in the sprout at the temperature prior to planting increases tissue res-
time of planting and during elongation while GA1, ponsiveness to auxin. This study, however, also
GA9 and GA34 were present in lower amounts. We suggested that the presence of a certain amount of

123
248
gibberellin was necessary for proper internode elon-
gation, whereby gibberellins positively enhance the
auxin response. In pea, auxin is responsible for
maintaining transcript levels of PsGA3ox1, allowing
formation of the bioactive GA1 from the inactive
precursor, GA20 (Ross et al. 2000). Additional work
would be necessary to demonstrate this model in
tulip, but the clear involvement of auxin with GA
responses suggests that commercial techniques for
reducing elongation of tulip (desirable when plants
are grown in containers) might in the future focus on
auxin inhibition rather than GA biosynthesis per se.
In conclusion, our data confirm that de novo
biosynthesis of gibberellins during the growth of
properly cooled tulip bulbs is necessary for proper
flower stalk elongation. Gibberellins play an impor-
tant role in expressing higher acid invertase activity
during the rapid elongation phase, enabling the
cleavage of imported sucrose to hexoses that are
utilized in elongating cells.
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