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Relationship between gibberellins, height, and stress tolerance in barley(Hordeum vulgare L.) seedlings

Sunita Sarkar1, Michel R. Perras1, Duane E. Falk2, Ruichuan Zhang3,Richard P. Pharis3 and R. Austin Fletcher1,*1Department of Environmental Biology, 2Department of Plant Agriculture, University of Guelph, Guelph,

Ont., Canada, N1G 2W1; 3Department of Biological Sciences, University of Calgary, Calgary, Alta,

Canada, T2N 1N4; *Author for correspondence (e-mail: rfletche@uoguelph.ca;

phone: (519) 824 4120 ext. 2678; fax: (519) 837 0442)

Received 24 March 2003; accepted in revised form 4 November 2003

Key words: Gibberellins, Mutants, Paclobutrazol, Stress tolerance

Abstract

The relationship between gibberellin (GA) levels, height, and stress tolerance was investigated using barley

(Hordeum vulgare L.) seedlings, which were exposed to heat stress (50 �C for 3 h) and a free radical generator

(Paraquat). Barley cv. Perth seedlings were treated with GA-inhibitors, either a triazole or one of two

acylcyclohexanediones. They were tested along with four mutants that were either responsive (MC96 and

dwf1) or non-responsive (MC90 and Dwf2) to applied growth-active GAs. Analyses of seedlings, whether

mutants, or treated with GA-inhibitors, gave a negative correlation between height and tolerance. We also

observed that the shorter seedlings with the least amount of growth active GAs were the most tolerant tostress. The overall results of this study suggest that reduced GA levels or sensitivity to GA, with a

concomitant reduction in height are important for induction of stress tolerance.

Abbreviations: ABA – Abscisic acid; DAP – Days after planting; GA – Gibberellin

Introduction

Plants treated with gibberellin (GA)-biosynthesisinhibitors are shorter and usually more tolerant to a

range of environmental stresses (Rademacher 1997;

Vettakkorumakankav et al. 1999). Paclobutrazol is

one of the number of triazoles that enhance toler-

ance to various environmental stresses in several

plant species (Fletcher et al. 2000). Triazoles act as

GA-biosynthesis-inhibitors and fungicides by

blocking cytochrome P450-mediated oxidationreactions (see reviews by Davis et al. 1988;

Fletcher et al. 2000). In higher plants the triazoles

reduce growth by interfering with the conversion of

ent-kaurene to ent-kaurenoic acid, an early step in

GA biosynthesis (Rademacher 2000). Triazole-

application also triggers other hormonal changes

including an increase in cytokinins, a transient risein abscisic acid (ABA), and a reduction in ethylene

levels (Fletcher and Hofstra 1988). Furthermore,

they increase the levels of antioxidants

(�-tocopherol and ascorbic acid) and antioxidant

enzymes (e.g. superoxide dismutase and glutathione

reductase) in treated plants (Kraus et al. 1995). It

has been proposed that the shift in the plants’

endogenous hormonal balance coupled with theincrease in antioxidant and antioxidant enzyme

levels mediate the stress-protective effects of the

triazoles (Kraus et al. 1995; Fletcher et al. 2000).

A newer generation of GA-biosynthesis inhibitors,

1252004 Kluwer Academic Publishers. Printed in the Netherlands.

Plant Growth Regulation 125–135, 2004.42:

the acylcyclohexanediones, including Trinexapac-

ethyl and Prohexadione-Ca, inhibit late-stage bio-

synthetic steps, particularly the conversion of the

inactive 3-deoxy GA, GA20 to the growth-active

3b-hydroxylated GA, GA1 (Rademacher 2000).The acylcyclohexanediones have been reported

to increase ABA levels (Adams et al. 1992), reduce

ethylene levels (Rademacher 2000), and increase

stress tolerance in plants (Rademacher 1995).

However, at maximally effective doses Paclo-

butrazol generally results in shorter plants than

the acylcyclohexanediones (Yelverton and Isgrigg

1997).Gilley and Fletcher (1998) reported that applica-

tion of GA3 counters both the growth inhibitory

and stress-protective effects of the triazoles.

Furthermore, a recent study using near-isogenic

lines of normal and dwarf-mutant (GA-responsive)

barley (Hordeum vulgare L.) seedlings showed that

tolerance to heat, chilling, and drought could be

enhanced in seedlings of the normal genotype byapplication of Paclobutrazol and reduced in the

GA-responsive dwarf seedlings by treatment with

GA3 (Vettakkorumakankav et al. 1999). The above

studies thus suggest that modulation of endogen-

ous GA levels is crucial for enhancing stress toler-

ance in plants. An interesting question then arises,

is a reduction in both endogenous GA levels and

shoot height essential for enhancing stress toler-ance in plants? We have attempted to answer this

question by examining stress tolerance in relation

to endogenous GA levels and shoot height in barley

cv. Perth seedlings treated with either a triazole, or

an acylcyclohexanedione, and in GA-responsive

and GA-non-responsive dwarf mutant lines of

barley.

Materials and methods

Plant materials

Barley (H. vulgare L.) cv. Perth was the control.

The other barley lines, GA-responsive dwf1 and

MC96, and GA-non-responsive Dwf2 and MC90,

have been described by Falk (1995), Falk and

Kasha (1982), Borner et al. (1999). The Dwf2 muta-tion occurred as a single dwarf tiller on a sponta-

neously haploid plant generated from anther

culture and the gene for this mutation has been

mapped on the short arm of chromosome 4H.

The GA-responsive mutants respond to GA3 appli-

cations over the same concentration range as the

wild type, and are proposed to have low levels of

endogenously physiologically active GAs. The GAnon-responsive mutants, however, are a bit more

complicated because a lack of response to GA in a

mutant does not necessarily mean a deficiency in

signaling, but rather could indicate deficiency in

another factor that regulates growth such as the

brassinosteroids (Chandler and Robertson 1999).

Chemicals

Paclobutrazol ((2RS,3RS)-1-(4-chlorophenyl)-4,

4-dimethyl-2-(1,2,4-triazoyl)-pentan-3-ol) and

Paraquat (1,10-dimethyl-4,40-bipyridinium dichlor-

ide) were obtained from ICI-Zeneca, Canada, andTrinexapac-ethyl (4-(cyclopropyl-�-hydroxy-methl

yene)-3,5-dioxo-cyclohexanecarboxylic acid methyl

ester) from Novartis Inc., Canada. Prohexadione-

Ca (calcium 3-oxido-4-propionyl-5-oxo-3-cyclo-

hexenecarboxylate) was provided by BASF Corp.,

USA.

Seed treatment and growing conditions

Seeds of barley cv. Perth were soaked in either

distilled water (control) or aqueous solutions of

100 mg/l (0.34 M) Paclobutrazol, 150 mg/l (0.59 M)

Trinexapac-ethyl, or 250 mg/l (1.09 M)Prohexadione-Ca, for 18 h at room temperature.

A preliminary experiment showed that these con-

centrations provided maximum growth inhibition.

Germination was delayed but not reduced (results

not shown). The seeds were then rinsed with dis-

tilled water, air-dried for 1 day, planted in Promix

BX (Plant Products, Canada) and grown in a

greenhouse with day/night temperatures of 24/18 ±4 �C (Fletcher and Hofstra 1990). The growth

regulatory and stress protective properties of the

different GA-biosynthesis-inhibitors were assessed

at day 10.

The stress tolerance properties of the barley

mutants MC90, Dwf2, MC96, and dwf1 were com-

pared to those of cv. Perth of normal height (con-

trol). Seeds of each genotype were planted in amixture of soil : peat : Turface (3 : 2 : 1) and grown in

a growth-room with 350 �mols light intensity, 60–

70% RH, 20/15 ± 2 �C day/night temperature and

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a 16-h photoperiod. Control and MC90 seedlings

were assessed 10 day after planting (DAP), whereas

for the slower-growing dwarf-mutant lines, Dwf2,

MC96, and dwf1, assessments were made 12 DAP.

All measurements for comparisons among theGA-inhibitors and of the mutants with the control

were accomplished using seedlings at a similar

morphological stage based on the appearance and

development of the first and second leaves. Shoot

height was measured as the total length from the

surface of the soil to the tip of the longest leaf when

extended. For both comparisons one set of 24 seed-

lings was maintained in the greenhouse, the otherset was exposed to stress as described below.

Heat stress

Seedlings were exposed to 50 ± 1 �C for 3 h at

60–70% RH under low light intensity (20 �mols),

then returned to the greenhouse. Various param-

eters were analyzed as described below (see also

Vettakkorumakankav et al. 1999).

Free-radical stress

The bi-pyridinium herbicide Paraquat was applied

as described in Gilley and Fletcher (1998). Leafsegments (1 cm) from the middle of the first leaf

were pre-incubated in 0, 2, and 5 �M Paraquat for

1 h in the dark, then exposed to 1000 �mols of light

provided by 400-W high-pressure sodium lamps

(P. L. Light Systems, Canada) for 6–8 h. Total

chlorophyll was measured as described below.

Stress parameters

Ion leakage, an indicator of membrane integrity,

was measured using the procedure described byGilley and Fletcher (1998). The whole shoot taken

from either a heat-stressed or unstressed plant was

immersed in 15 ml of distilled water and mixed in a

vortex for 15 s. Conductivity (Co) was measured 1 h

later using a Model-32 conductance meter (Yellow

Springs Instrument Co., Inc. Ohio, USA.). The

tubes were then left for 24 h at room temperature

and initial conductivity (Ci) measured. Finally, thetubes were placed in a boiling water bath for 30

min, cooled to room temperature and final conduc-

tivity (Cf) measured. The percent conductivity was

calculated as follows:

% Ion Leakage ¼ [(Ci�Co)/(Cf�Co)] � 100%

Chlorophyll fluorescence, a non-invasive method

for rapid identification of photosynthetic injury

to leaves, was estimated as described by

Vettakkorumakankav et al. (1999). The ratio of

variable to maximal fluorescence (Fv/Fm) was

measured using a Hansatech fluorescence meter(Model-FMS 2, Hansatech, Ltd., UK) with a

30-min dark adaptation period. Measurements

were made 24 h after heat stress on the first leaf

of stressed and unstressed plants.

Photosynthetic pigments were extracted from leaf

tissue in 80% aqueous ethanol using a Polytron

homogenizer (Brinkmann Instruments, Canada).

The homogenates were clarified by centrifugationat 2500 � g for 10 min, and absorbance read using

a Beckman DU-65 Spectrophotometer (Beckman

Inc., USA) at 663, 647, and 470 nm wavelengths.

Total chlorophyll (a + b) and carotene levels were

estimated as described in Gilley and Fletcher (1998).

Endogenous GA measurements

Control and Trinexapac-ethyl-treated samples were

harvested 10 DAP while dwf1 and Dwf2 were

harvested 12 DAP. Each sample consisted of 150

randomly selected seedlings all at the two-leaf stage.

The inner-half of the second leaf was dissected,

frozen in liquid N2, freeze-dried, pulverized anddivided into two sub-samples. The samples (1 g)

were then extracted with 80% aqueous metha-

nol (MeOH) with 20 ng each of [17, 17-2H2]

GA1/3/4/5/7/8/9/15/19/20/24/44/53 added as internal

standards.

The 80% MeOH extracts were then purified with

a C18 preparative column (C18-PC) made of a

syringe barrel (inside diameter (i.d.) 2 cm) filledwith 3 g of C18 preparative reversed-phase material

(Waters Associates), with the eluate being passed

through an ion-exchange column made of AG

1-X8 Resin (Bio-Rad Laboratories). The GAs

were then eluted with 5% acetic acid in absolute

MeOH. The GA-containing fractions were taken

to dryness in vacuo at 35 �C and then loaded onto a

SiO2 partition column (SiO2-PC) (column i.d. ¼1.5 cm) made of 5 g of Woelm SiO2 (32–100 mesh)

deactivated by equilibration with 20% water by

weight (Koshioka et al. 1983). The SiO2-PC was

127

eluted with ethyl acetate : hexane (95 : 5, v/v;

saturated with 0.5 M formic acid). The GA-contain-

ing fractions were taken to dryness in vacuo at 35 �C

and further purified by high performance liquid

chromatography (HPLC). The HPLC was aWaters Associates liquid chromatography appara-

tus with two model M-45 pumps, a model 680

automated gradient controller, and a model 7125

Rheodyne injector. The solvents were, pump A:

10% MeOH in 1% acetic acid (H2O : MeOH : acetic

acid ¼ 89 : 10 : 1, (v/v)), and pump B: 100% MeOH.

A Waters Associates reversed phase C18 Radial-

PAK �-Bondapak column (8 mm � 10 cm) wasused with a 10–73% linear gradient program at a

flow rate of 2 mL/min. The manually implemented

10–73% linear gradient program was 0–10 min

(pump A, 100%; pump B, 0%), 10–40 min (pump

A, 100–30%; pump B, 0–70%), and 40–50 min

(pump A, 30%; pump B, 70%). The HPLC fractions

(1 min, 2 ml fractions) were taken to dryness in vacuo.

The C18 HPLC fractions were further purified withan Alltech Associates Nucleosil N(CH3)2 HPLC

column (4.6 mm � 15 cm) eluted with 99.9%

MeOH in 0.1% acetic acid, the 1 min, 1 ml fractions

being taken to dryness in vacuo.

The GA-containing fractions, based on retention

times (Rts) of external [3H]-labeled external stan-

dards, eluted from the Nucleosil N(CH3)2 HPLC

were methylated by ethereal CH2N2. The methy-lated sample was then silylated by BSTFA with 1%

TMCS (Pierce Chemical Co.). Identification and

quantification of GAs was carried out by gas

chromatography-mass spectrometry (GC-MS) in

selected ion monitoring (SIM) mode. The deriva-

tized samples were injected onto a capillary column

installed in a Hewlett-Packard 5890 GC with a

capillary direct interface to a HP 5970 MassSelective Detector (MSD). The capillary column

was a 0.25 �m film thickness, 0.25 mm internal

diameter, 15 m DB1–15 N column (J & W

Scientific, Inc.). The capillary head pressure was

4 psi with a He carrier gas flow rate of 1.1 mL/min.

The GC temperature program was as follows:

0.1 min at 60 �C, climbing to 200 �C at 20 �C per

min, then to 250 �C at 4 �C per min and finally to300 �C at 25 �C per min, staying at 300 �C for

5 min, then returning to 60 �C. The interface tem-

perature was maintained at 300 �C and the MSD

was operated with the electron multiplier at 1600 V.

Three m/z ions for each deuterated and endogenous

GA were monitored. The dwell time was 10 s and

the data were processed using Hewlett Packard’s

G1034C MS ChemStation Software. Based on the

capillary GC Rts and relative abundance of the

characteristic m/z ions monitored, endogenousGAs were identified and amounts estimated based

on the isotope dilution technique described by

Fujioka et al. (1988).

Experimental design and statistical analyses

All experiments except GA analysis were rando-

mized complete blocks replicated three times, with

three sub-samples per block. Statistical analyses

were conducted using SAS version 6.12 (SASInstitute, USA). Proc MIXED (mixed model) ana-

lyses with contrast statements and multiple-means

comparisons were used to test the effect of treatment

with GA-biosynthesis inhibitors and the inherent

difference between the mutant lines. Pearson’s corre-

lation and regression in ANOVA with the variation

due to treatment partitioned into linear, quadratic,

and lack-of-fit components was conducted to deter-mine the nature of the relationship between height

and stress tolerance. Residuals were tested for ran-

domness, homogeneity, normal distribution, a mean

of zero, and a common variance. All hypotheses

were tested at a Type I error rate (p) of 0.05. The

GA analysis experiment was repeated once on the

sub-samples to determine accuracy and both times

the results were similar. No further statisticalanalyses could be conducted.

Results and discussion

Paclobutrazol and Trinexapac-ethyl delayed

germination of barley seedlings, which was expectedsince de novo synthesis of GAs in barley is neces-

sary for the mobilization of seed reserves and

embryo growth (Jacobsen et al. 1995). However,

Prohexadione-Ca had no effect on germination or

height of barley seedlings in our experimental con-

ditions (Figure 1A). This could be attributable to

poor uptake or rapid degradation (Rademacher

2000). Hence only the results from Paclobutrazoland Trinexapac-ethyl will be discussed further.

Ten days after planting, seedlings from all treat-

ments including the control, had emerged and had

128

a similar number of leaves. The leaves of

Paclobutrazol-treated plants appeared greener,

though the amount of total chlorophyll per seed-

ling was not significantly different (results not

shown), and more twisted than the controls, a

morphological effect that has been reported in

other species (Davis et al. 1988). Treatment withPaclobutrazol was the most effective, reducing

seedling height by 67% compared to control while

Trinexapac-ethyl treated seedlings were only 28%

shorter than controls (Figure 1A). This observation

is similar to that of Yelverton and Isgrigg (1997)

who studied the comparative effects of these che-

micals on Poa annua.

Following exposure to 50 �C for 3 h,

Paclobutrazol-treated seedlings showed no signs

of heat stress, such as leaf-tip burn and flaccidity,

whereas Trinexapac-ethyl-treated seedlings showed

minimal symptoms of heat stress (Figure 1B).Control seedlings, however, were severely damaged

(Figure 1B). The visual symptoms of stress were

corroborated by measurements of physiological

parameters. Ion leakage, an indicator of membrane

integrity, in both control and treated seedlings

Figure 1. Appearance of 10 day-old barley cv. Perth seedlings (A) before, and (B) 24-h after exposure to 50 ± 1 �C for 3 h. From left to right,

seedlings (two pots per treatment) from seeds imbibed in, water (control), Prohexadione-Ca, Trinexapac-ethyl, and Paclobutrazol. Their

mean heights were 115 (*), 114 (*), 84 (**), and 38 (***) mm respectively, with a standard error of 0.033, and n ¼ 144. Different number of

(*) symbols indicate a significant difference between the means at a Type I error rate of 0.05.

129

prior to heat stress were similar (approx. 10%).

However, after exposure to heat stress ion leakage

values increased to 52% and 25% in the controlsand Trinexapac-ethyl-treated plants, respectively

(Figure 2A) revealing some cellular damage.

There was no significant difference between the

values for heat-stressed and unstressed Paclobutrazol-

treated seedlings showing that Paclobutrazol

provided complete protection from heat stress.

Chlorophyll fluorescence was used to determine

the efficiency of Photosystem II before and afterheat stress. The ratio of variable to maximal fluor-

escence (Fv/Fm) for a normally functioning leaf

ranges from 0.75 to 0.85 and a decline in this ratio

is indicative of photoinhibitory damage (DeEll

et al. 1999). The Fv/Fm ratios measured 24 h after

heat stress revealed extensive damage to control

seedlings with a value of 0.06, whereas Trinexapac-

ethyl- and Paclobutrazol-treated plants maintainedfluorescence ratios of 0.28 and 0.41, respectively

(Figure 2A) indicating appreciably less damage to

their photosynthetic apparatus.

When leaf segments were treated with a high

concentration of Paraquat (5 �M), control tissues

were severely damaged, with only 16% of the chlor-

ophyll remaining after 6 h in light (Figure 2B).

Paclobutrazol-treated tissues retained two timesmore chlorophyll than control while Trinexapac-

ethyl-treated tissues showed intermediate results.

At the lower concentration of Paraquat, Trinexapac-

ethyl was the least damaged. Once again, however,

Trinexapac-ethyl- and Paclobutrazol- treated tissues

were not as severely damaged as the control showing

the protective effects of these GA biosynthesis

inhibitors against stress.In an earlier study by Kraus and Fletcher (1994),

it was suggested that the damage caused by heat

and Paraquat is in part due to increased generation

of active oxygen and that Paclobutrazol protects

plants by maintaining increased antioxidant

enzyme activities. Fletcher et al. (2000), state that

the stress-protective effect of triazoles, including

Uniconazole and Paclobutrazol, is mediated by anincrease in antioxidants (vitamins C and E) and

antioxidant enzyme activity. Hence, the ability of

the triazoles to maintain high levels of antioxidants

could be an important factor in maintaining cell

integrity under free radical stress. We are not aware

of any direct evidence that the acylcyclohexane-

diones increases antioxidants. However, similar to

the triazoles (Fletcher et al. 2000), the acylcyclo-hexanediones also increase ABA levels (Adams

et al. 1992) and decrease ethylene (Rademacher

2000). It is therefore not unlikely that their overall

protective action is also mediated via an effect on

antioxidant levels.

The combined physical and biochemical evi-

dence from control and GA-inhibitor-treated seed-

lings revealed that the shortest barley seedlings,those that were treated with Paclobutrazol, were

also the most stress tolerant. Trinexapac-ethyl-

treated seedlings showed intermediary height and

stress tolerance. Using ion leakage measurements,

from control and treated cv. Perth seedlings, as

Figure 2. Stress parameters for leaves from control, Trinexapac-

ethyl (TrixE)-, and Paclobutrazol (Pac)-treated barley cv. Perth

seedlings. (A) Ion leakage and chlorophyll fluorescence (Fv /Fm)

in leaves after exposure of seedlings to 50 ± 1 �C for 3 h

(standard error of the means for ion leakage ¼ 5.6, and

chlorophyll fluorescence ¼ 1.75, n ¼ 36). Average values for

ion leakage and chlorophyll fluorescence in all unstressed plants

were 10% and 0.8, respectively; (B) Total chlorophyll (a + b) in

leaf-segments floated in 2 or 5 �M Paraquat and exposed to

1000 �mols of light, for 6 h, expressed as a percentage of the

unstressed tissue. Different letters indicate a significant

difference between the means for the corresponding parameter

at a Type I error rate of 0.05.

130

an indicator of stress tolerance (Figure 2A),

correlation and linear regression analyses indicated

a significant relationship between shoot height and

tolerance to heat stress (r ¼ 0.97, R2 ¼ 0.94, p <0.05) (Figure 3).

To further clarify the apparent relationship

between height and stress tolerance, two GA-

responsive (dwf1 and MC96), and two GA-non-responsive (Dwf2 and MC90) mutant lines of barley

were studied. After 12 days of growth the height and

phenotype (dark green, twisted leaves) of the two

GA-responsive mutant lines MC96 and dwf1 were

similar and comparable to cv. Perth seedlings

treated with Paclobutrazol (Figures 1A and 4A).

Thus, reduced levels of growth-active GAs, whether

caused by mutation or by application ofPaclobutrazol, yield a similar morphological state.

Seedlings from the GA-non-responsive dwarf line

MC90 were somewhat similar in size and phenotype

to cv. Perth seedlings treated with Trinexapac-ethyl.

The other GA-non-responsive line, Dwf2, produced

seedlings of intermediate size (Figure 4A).

After exposure to heat stress, cv. Perth and

MC90 seedlings were severely damaged, whileseedlings of dwf1, the shortest mutant, appeared

visually unaffected, showing a high degree of stress

tolerance (Figures 4B and 5). The inherent heat

tolerance of dwf1 also allowed this mutant to main-

tain Fv/Fm ratios over 0.5, whereas the control

had the lowest Fv/Fm ratio and MC90, Dwf2, and

MC96 showed intermediate albeit severe damage

(Fv/Fm < 0.2). Measurement of ion leakage showeda gradual increase in protection against heat stress

as the height of the seedlings decreased. The tallest

mutant, similar in height to the control, showed

little tolerance while the shortest mutant (dwf1)

showed least signs of damage (Figure 4). Once

again correlation (r ¼ 0.91, p < 0.05) and regres-

sion analysis indicated that there was a significant

positive correlation between height and amount ofion leakage, with 82% of the variance in ion leakage

being due to height (R2 ¼ 0.82, p < 0.05) (Figure 6).

Leaf segments of the GA-responsive mutants,

dwf1 and MC96, that were exposed to the lower

concentration of 2 �M Paraquat retained 97 % and

99% of their origin chlorophyll levels, respectively

(Figure 5B). In contrast, the GA-non-responsive

mutants, Dwf2 and MC90, retained only 77% and79% of their chlorophyll, respectively. These latter

mutants were thus significantly more susceptible

to damage from free radicals than the two GA-

responsive dwarfs (Figure 5B). However, at the

highest concentration of Paraquat (5 �M), MC96

was the only mutant that was significantly pro-

tected from free radical damage (Figure 5B).

Further studies on MC96 are thus warranted.The relationship between height and endogenous

levels of growth-active GAs is well established

(Hedden 1999). Moreover, Croker et al. (1990) in

his study of the inhibitory effect of Paclobutrazol

on GA biosynthesis in barley, found that levels

of GA1 and GA3 were reduced by 93% and 73%,

respectively, and that levels of GA8, GA19 and

GA20 were also decreased, but to a lesser extent.It therefore seems likely that the significant corre-

lation between height and stress tolerance that we

have observed in this study is in fact a relationship

between reduced GA levels and stress protection as

suggested by Vettakkorumakankav et al. (1999).

In order to clarify the relationship between GA

content, seedling height, and stress protection, we

determined and compared GA levels in control,GA-inhibitor-treated seedlings, and two mutant

lines of barley. Endogenous GA levels in the

actively growing inner-portion of the second leaf

indicated that both the early 13-hydroxylation

and early non-13-hydroxylation pathways are

Figure 3. Relationship between shoot height and ion leakage

after exposure to 50 ± 1 �C for 3 h (shown by the smooth line)

of barley cv. Perth seedlings treated with water or the GA-

biosynthesis inhibitors. Each symbol (�) represents from left to

right, the mean height and ion leakage of seedlings (n ¼ 36)

treated with Paclobutrazol, Trinexapac-ethyl, Prohexadione-

Ca, or water. There was a significant positive correlation with

r ¼ 0.97 (p < 0.05), and regression (R2 ¼ 0.94, p < 0.05).

131

functional in barley cv. Perth (Table 1) confirming

earlier reports (Grosselindemann et al. 1992;

Hedden and Kamiya 1997). As expected, inTrinexapac-ethyl-treated seedlings the reduction

in shoot height was associated with drastic reduc-

tion in the growth-active 3�-hydroxylated GAs,

GA1, GA4, and GA3 (Table 1), along with

increased levels of the 3-deoxy GA, GA20, but not

of 3-deoxy GA9. Furthermore, the level of GA8,

the inactive 2�-hydroxylated metabolite of GA1,

was reduced by 78%, a finding that is also consis-tent with the mode of action of Trinexapac-ethyl

(Rademacher 2000). Similar results have also been

obtained previously in barley with another acylcy-

clohexanedione, LAB 236735 (Hedden 1991). The

pronounced reduction in growth active GAs inTrinexapac-ethyl-treated seedlings as well as in

Paclobutrazol treated seedlings (Croker et al.

1990) tend to confirm the relationship between

reduced GA levels, height, and increased stress

protection.

Analysis of the mutant dwf1 showed a dramatic

reduction of growth-active GAs, GA1, GA3, and

GA4 (Table 1). Thus, dwf1 can correctly be labeleda GA-responsive (grd) mutant as per the definitions

presented by Chandler and Robertson (1999). The

Figure 4. Appearance of barley seedlings from left to right, cv. Perth, GA-non-responsive MC90 and Dwf2, and GA-responsive MC96

and dwf1 dwarf mutants (A) before, and (B) 24-h after exposure to heat stress at 50 ± 1 �C for 3 h. Their mean heights were 133 (*), 98

(**), 56 (***), 51 (****), and 43 (*****) mm respectively, with a standard error of 1.5, n ¼ 36. Different number of (*) symbols indicate a

significant difference between the means at a Type I error rate of 0.05.

132

level of C19-GAs, including the growth active GAs,

was higher in Dwf2 than in the control (Table 1).

This confirms that Dwf2 is a GA-non-responsivemutant or gse, which has reduced sensitivity to GA

signaling that results in an accumulation of

C19-GAs including the growth active GAs (see

Chandler and Robertson 1999). More importantly

our study shows that dwf1, which had only one-

tenth the level of growth-active 3�-hydroxylated

GAs normally found in control barley seedlings,

was more tolerant to stress than the control. Dwf2,which had the highest levels of growth-active GAs,

was also more tolerant to stress than the control

but was less tolerant than dwf1.

It can therefore be hypothesized that reduced

sensitivity to GA may in fact have the same effect

as lowering active GA levels in terms of growth,morphology, physiology, and in turn stress toler-

ance of the plant . This then explains the intermedi-

ate stress tolerance observed in Dwf2 in spite of the

high GA levels and supports the hypothesis that

Figure 5. Stress parameters for leaves from barley seedlings of

cv. Perth, GA-non-responsive MC90 and Dwf2, and GA-

responsive MC96 and dwf1 dwarf mutants. (A) Ion leakage

and chlorophyll fluorescence in leaves after exposure of the

seedlings to heat stress at 50 ± 1 �C for 3 h (standard error of

the means for ion leakage ¼ 0.14, and chlorophyll fluorescence¼0.099, n ¼ 9). Average values for ion leakage and chlorophyll

fluorescence in all unstressed plants were 10% and 0.8,

respectively; (B) Total chlorophyll (a + b) in leaf segments

expressed as a percent of unstressed tissue. The leaf segments

were exposed to 2 and 5 �M of Paraquat under 1000 �mols of

light, for 8 h. Different letters indicate a significant difference

between treatment means at a Type I error rate of 0.05.

Figure 6. Relationship between shoot height and ion leakage

(shown by the smooth line) for control, GA-responsive and non-

responsive mutant lines of barley after exposure to 50 ± 1 �C for

3 h. Each symbol (�) represents from left to right, the mean

height and ion leakage of seedlings (n ¼ 9) of GA-responsive

dwf1 and MC96, GA-non-responsive Dwf2 and MC90, and cv.

Perth. There was a significant positive correlation with r ¼ 0.91

( p < 0.05), and regression (R2 ¼ 0.82, p < 0.05).

Table 1. Endogenous GA content (ng/g dry weight) in the inner

portion of the second leaf of 10 day-old control and Trinexapac-

ethyl-treated (TrixE) barley cv. Perth seedlings and 12 day-old

GA-responsive, dwf1 and GA-non-responsive Dwf2 barley

mutants. Extraction and quantification was repeated once with

similar results. Two 1-g sub-samples derived from 150 randomly

selected seedlings were used in each case (treatment/mutant).

GA Control TrixE dwf1 Dwf2

GA1* 6.47 3.63 ND 19.93

GA3* 35.69 6.87 ND 22.96

GA4* 8.55 1.67 5.80 9.22

GA5 NDa – ND –

GA7* ND ND ND 2.77

GA8 36.79 8.14 ND 57.65

GA9 13.43 8.00 41.19 6.53

GA15 ND ND ND ND

GA19 –b 76.00 – 6.54

GA20 3.68 44.06 35.56 10.95

GA24 ND ND ND ND

GA44 22.30 31.20 7.84 6.20

GA53 – Trace ND ND

aEndogenous GA not detected; [2H2] GA detected.b[2H2] GA not detectable.

*Growth-active GA.

133

both GA levels (or sensitivity) and height are

important factors for stress tolerance in barley.

However, altering the hormonal balance in plants

induces morphological as well as physiological

changes such as alterations in antioxidant levelsand membrane properties (Kraus et al. 1995),

which may be the key factors in increasing toler-

ance to various environmental stresses. Studies on

the relationships of other hormones including ABA

and brassinosteroids to GA, height and stress tol-

erance would be extremely interesting. However

further discussion on the role of these and other

hormones on stress tolerance is beyond the scope ofthis paper.

From our study we can conclude that in barley

the shorter the plant and the lower its GA levels the

better its inherent stress tolerance. Hence, GA-

inhibitors are useful candidates to protect plants

from environmental stresses, as is the use of GA-

deficient mutants. Plants are subjected to various

forms of stress during growth and development andthese can lead to a significant reduction in crop

yield and quality (Paliyath and Fletcher 1995). It

is therefore proposed that lowering growth active

GAs or rendering them inactive or less efficient at

the physiological and molecular levels is a means of

increasing stress tolerance in plants.

Acknowledgements

This work was supported by research grants from

the Natural Sciences and Engineering Research

Council of Canada to R.A.F., D.E.F., and

R.P.P., and an Ontario Graduate Scholarship to SS.

We wish to thank Zeneca Corp., BASF Inc.,

and Novartis Inc. for generous supplies of

Paclobutrazol, Prohexadione-Ca, and Trinexapac-

ethyl, respectively, and D. J. Hamilton and ShaunaCake for assistance with photography.

References

Adams R., Kerber E., Pfister K. and Weiler E.W. 1992. Studies

on the action of the new growth retardant CGA 163’935

(Cimectacarb). In: Karsen C.M., van Loon L.C. and

Vreugdenhil D. (eds), Progress Plant Growth Regulation.

14th Annual International Conference, July 1991,

Amsterdam, The Netherlands, pp. 818–827.

Borner A., Korzun V., Malyshev S., Ivandic V. and Graner A.

1999. Molecular mapping of two dwarfing genes differing in

their GA response on chromosome 2H of barley. Theor.

Appl. Genet. 99: 670–675.

Chandler P.M. and Robertson M. 1999. Gibberellin dose-

response curves and the characterization of dwarf mutants

of barley. Plant Physiol. 120: 623–632.

Croker S.J., Hedden P., Lenton J.R. and Stoddart J.L. 1990.

Comparison of gibberellins in normal and slender barley seed-

lings. Plant Physiol. 94: 194–200.

Davis T.D., Steffens G.L. and Sankhla N. 1988. Triazole plant

growth regulators. Hort. Rev. 10: 63–105.

DeEll J.R., van Kooten O., Prange R.K. and Murr D.P. 1999.

Applications of chlorophyll fluorescence techniques in post-

harvest physiology. Hort. Rev. 23: 69–107.

Falk D.E. 1995. New dominant dwarfing gene (Dwf2) in barley.

Barley Genet. Newslett. 24: 87–89.

Falk D.E. and Kasha K.J. 1982. A vegetative dwarf mutant in

barley. Barley Genet. Newslett. 12: 69–72.

Fletcher R.A., Gilley A., Sankhla N. and Davis T.D. 2000.

Triazoles as plant growth regulators and stress protectants.

Hort. Rev. 24: 55–138.

Fletcher R.A. and Hofstra G. 1988. Triazoles as potential plant

protectants. In: Berg D. and Plempel M. (eds), Sterol

Biosynthesis Inhibitors: Pharmaceutical and Agrochemical

Aspects, Ellis Horwood Ltd., Cambridge, UK, pp. 321–331.

Fletcher R.A. and Hofstra G. 1990. Improvement of unicona-

zole-induced protection in wheat seedlings. J. Plant Growth

Regul. 9: 207–212.

Fujioka S., Yamane H., Spray C.R., Gaskin P., MacMillan J.,

Phinney B.O. and Takahashi N. 1988. Qualitative and quan-

titative analysis of gibberellins in vegetative shoots of normal ,

dwarf-1, dwarf-2, dwarf-3, and dwarf-5 seedlings of Zea mays L.

Plant Physiol. 88: 1367–1372.

Gilley A. and Fletcher R.A. 1998. Gibberellin antagonizes

paclobutrazol-induced stress protection in wheat seedlings.

J. Plant Physiol. 103: 200–207.

Grosselindemann E., Lewis M.J., Hedden P. and Graebe J.E.

1992. Gibberellin biosynthesis from A12-aldehyde in a cell-

free system from germinating barley (Hordeum vulgare L., cv.

Himalaya) embryos. Planta. 188: 252–257.

Hedden P. 1991. Gibberellin biosynthetic enzymes and the

regulation of gibberellin concentration. In: Takahashi N.,

Phinney B.O. and MacMillan J. (eds), Gibberellins,

Springer-Verlag Inc., NY, pp. 94–105.

Hedden P. 1999. Recent advances in gibberellin biosynthesis.

J. Exp. Bot. 50(334): 553–563.

Hedden P. and Kamiya Y. 1997. Gibberellin biosynthesis:

Enzymes, genes and their regulation. Annu. Rev. Plant

Physiol. Plant Mol. Biol. 48: 431–460.

Jacobsen J.V., Gubler F. and Chandler P.M. 1995. Gibberellin

action in germinated cereal grains. In: Davies P.J. (ed.), Plant

Hormones, Kluwer Academic Publishers, The Netherlands,

pp. 246–271.

Koshioka M., Takeno K., Beall F.D. and Pharis R.P. 1983.

Purification and separation of gibberellin precursors, gibber-

ellins and gibberellin glucosyl conjugates from higher plants

using C18 reverse phase material. Plant Physiol. 73: 398–406.

Kraus T.E. and Fletcher R.A. 1994. Paclobutrazol

protects wheat seedlings from heat and Paraquat injury.

Is detoxification of active oxygen involved? Plant Cell

Physiol. 35: 45–52.

134

Kraus T.E., McKersie B.D. and Fletcher R.A. 1995.

Paclobutrazol-induced tolerance of wheat leaves to Paraquat

may involve increased antioxidant enzyme activity. J. Plant

Physiol. 145: 570–576.

Paliyath G. and Fletcher R.A. 1995. Paclobutrazol treatment

alters peroxidase and catalase activities in heat-stressed maize

coleoptiles. Physiol. Mol. Biol. Plants 1: 171–178.

Rademacher W. 1995. Growth retardants: Biochemical features

and applications in horticulture. Acta Horticulturae 394:

57–73.

Rademacher W. 1997. Bioregulation in crop plants with

inhibitors of gibberellin biosynthesis. In: Latimer J.G. (ed.),

Proceedings of the 24th Annual Meeting of Plant Growth

Regulation Society of America, Atlanta, Georgia, pp. 27–31.

Rademacher W. 2000. Growth retardants: Effects on gibberellin

biosynthesis and other metabolic pathways. Annu. Rev. Plant

Physiol. Plant Mol. Biol. 51: 501–531.

Vettakkorumakankav N.N., Falk D., Saxena P. and

Fletcher R.A. 1999. A crucial role for gibberellins in stress

protection of plants. Plant Cell Physiol. 40(5): 542–548.

Yelverton F.H. and Isgrigg J. 1997. Effects of plant growth

regulators on suppression of Poa annua sub. reptans in bent-

grass greens. In: Proceedings of the South Weed Science

Society, 50th Annual Meeting, pp. 68–69.

135