Effects of exogenous abscisic acid and gibberellic acidon pre-maturity a-amylase formation in wheat grains

K. R. Kondhare • P. S. Kettlewell • A. D. Farrell •

P. Hedden • J. M. Monaghan

Received: 7 July 2011 / Accepted: 21 April 2012 / Published online: 11 May 2012

� Springer Science+Business Media B.V. 2012

Abstract Effects of in situ and in vitro applied

abscisic acid (ABA) and gibberellic acid (GA3) on

pre-maturity a-amylase (PMA) were investigated

in glasshouse experiments under cool-temperature

shock-induced and non-induced conditions using UK

winter wheat varieties, Spark (low PMA susceptible)

and Rialto (high PMA susceptible). In the in situ study,

hormone solutions (ABA [100 lM], GA3 [50 lM] and

ABA ? GA3 [100 ? 50 lM]) were applied during

mid-grain development to intact, developing grains

of induced and non-induced plants. Alpha-amylase

activity was measured in embryoless half-grains at

maturity by a modified Phadebas assay. In the in situ

study under non-induced conditions, applied ABA had

no significant effect on a-amylase in either variety

whereas applied GA3 significantly increased a-amy-

lase in Rialto, but not in Spark. In the in situ study

under induced conditions, applied ABA and GA3

produced no significant effect on a-amylase in Spark;

however in Rialto, applied ABA produced a small but

significant decrease in a-amylase whereas GA3 sig-

nificantly increased a-amylase under induced condi-

tions. In the in vitro study, spikes were harvested from

induced/non-induced plants at 44/40 days after anthe-

sis. Embryoless half-grains were incubated in hor-

mone solutions at 25 �C and a-amylase activity was

measured. Results similar to the in situ study were

observed in the in vitro study for both varieties and

conditions, except GA3 treatment which significantly

increased a-amylase under non-induced conditions in

Spark. From these exogenous hormone studies, it

appears that GA-response is a major factor during

PMA induction by a cool-temperature shock whereas

ABA-response seems to be of less importance.

Keywords a-Amylase � Amylazyme substrate �Days after anthesis � Gibberellins � In situ � In vitro

Introduction

A cool-temperature shock during a window of sensi-

tivity, i.e. 26–30 days after anthesis (DAA), can

induce pre-maturity a-amylase (PMA) in later stages

of grain filling in susceptible wheat varieties (Triticum

aestivum, L) (Mares and Mrva 2008; Farrell and

Kettlewell 2008). PMA is also referred to as LMA

(late maturity a-amylase) or LMEA (late maturity

endosperm a-amylase) (Lunn et al. 2001). PMA

K. R. Kondhare � P. S. Kettlewell (&) � J. M. Monaghan

Crop and Environment Sciences, Harper Adams

University College, Newport TF10 8NB, UK

e-mail: pskettlewell@harper-adams.ac.uk

A. D. Farrell

Department of Life Sciences, The University of the West

Indies, St. Augustine, Trinidad, West Indies

P. Hedden

Plant Science Department, Rothamsted Research,

Harpenden, Hertfordshire AL5 2JQ, UK

123

Euphytica (2012) 188:51–60

DOI 10.1007/s10681-012-0706-0

represents the second major cause, following pre-

harvest sprouting (PHS), of a high level of high

isoelectric point (pI) a-amylase in mature grains. High

levels of a-amylase in sound grains result in a low

Hagberg Falling Number (HFN) and render them

unsuitable for bread making (Mares and Mrva 2008).

Several factors are involved in regulating PMA

synthesis in developing grains, including genotype

(e.g. the presence of PMA quantitative trait loci, GA-

insensitive dwarfing alleles or the 1B/1R transloca-

tion), agronomy, and environmental conditions (e.g.

grain drying rate, light, moisture stress, disease and

cool or warm temperatures) (Mrva et al. 2006; Farrell

and Kettlewell 2008; Farrell and Kettlewell 2009).

The occurrence of PMA is constitutive in some

genotypes and sporadic and unpredictable in others

even under inductive conditions (Mares and Mrva

2008). PMA is a major concern in UK winter wheat

varieties, where cool and wet periods during the

summer are common.

Abscisic acid (ABA) and gibberellins (GA) influ-

ence various aspects of grain development. Research

in Arabidopsis (Debeaujon and Koornneef 2000) and

maize (White et al. 2000) has shown that a high level

of GA (and low level of ABA) early in grain

development is required for the embryo development

and pattern formation, whereas a high level of ABA

(and reduced GA levels) late in grain development is

required not only for the embryo maturation but also

for the induction of seed dormancy. Thus, a high

endogenous ABA/GA ratio is essential during grain

development for repressing a-amylase synthesis.

Similarly, in cereals during PHS and germination,

both endogenous levels of ABA/GA and ABA or GA

sensitivity of the embryo regulate a-amylase activity

to regulate grain dormancy (Nyachiro et al. 2002). In

germinating grains, applied ABA inhibits whereas

applied GA stimulates a-amylase synthesis in the

embryo and aleurone (Chandler et al. 1984; Gold

1991; Hader et al. 2003). A similar role by ABA and

GA would therefore be anticipated in intact, develop-

ing grains in the induction of PMA by a cool-

temperature shock.

The presence of GA-insensitive dwarfing alleles,

e.g. Rht1 [Rht-B1b], Rht2 [Rht-D1b] and Rht3 [Rht-

B1c], significantly reduced PMA in the UK wheat

variety Maris Huntsman (Gold and Duffus 1993) and

Australian wheat varieties such as Spica and Lerma 52

(Mrva and Mares 1996). These findings suggested the

involvement of GA sensitivity in PMA induction

(Mares and Mrva 2008). The occurrence of bioactive

GA late in grain development is of considerable

relevance to PMA formation (Rejowski 1964; Radley

1976; Dathe and Sembdner 1978). However, several

studies demonstrated no correlation between the late

peaks of new GA and subsequent alpha-amylase

synthesis (Radley 1976; Gale and Lenton 1987; Mares

and Mrva 2008). Similarly, previous attempts to

demonstrate altered GA sensitivity of the aleurone

from ripening grains of Australian PMA susceptible

varieties have not been successful (Mares and Mrva

2008). In vitro studies with detached grains in cereals

by Cornford et al. (1987), Gold (1991) and Bethke

et al. (1997) investigated the response of the aleurone

tissue to applied ABA. They showed that applied

ABA inhibited high pI a-amylase synthesis. However,

the mode of ABA action as an endogenous regulator

for a-amylase synthesis in attached, developing wheat

grains still remains unknown.

Normally, during wheat grain development and

maturation, peak levels of GA occur around 15–20

DAA and that of ABA occurs across a wider range, i.e.

25–40 DAA (McWha 1975; Slominski et al. 1979;

Gutam et al. 2008). The occurrence of high ABA and

low bioactive GA levels in later stages of grain

maturation should ensure that mature grains have low

levels of a-amylase. A high level of a-amylase

produced during grain maturation in PMA inducible

varieties in response to cool temperatures is predicted

to occur through either altered ABA/GA levels (ABA/

GA biosynthesis) or altered aleurone sensitivity to

ABA or GA or both (Mares and Mrva 2008; Farrell

and Kettlewell 2008).

Yamauchi et al. (2004) investigated the effect of

cold stratification on the endogenous content of

bioactive GA in Arabidopsis seeds. They found that

the expression of GA biosynthesis genes (i.e. GA20ox

and GA3ox) was stimulated by cold stratification,

whereas the expression of a GA deactivating gene (i.e.

GA2ox) was down-regulated. As a result, the endog-

enous content of bioactive GA (especially GA1 and

GA4) in the cold stratified seeds was increased

significantly from those in the non-stratified seeds.

Therefore, Yamauchi et al. (2004) suggested an

important role for GA in the cold temperature

mediated signalling. Microarray analyses of Arabid-

opsis seeds have demonstrated that a quarter of GA

regulated genes were the cold responsive (Yamaguchi

52 Euphytica (2012) 188:51–60

123

2008). It has been suggested that environmental

factors such as light and cold treatment can promote

germination in Arabidopsis seeds by inducing GA

biosynthesis (Yamaguchi et al. 1998) or by increasing

GA sensitivity of the embryo (Karssen and Lacka

1986). Moreover, cold stratification not only induces a

decline in the endogenous ABA content but also

affects the ABA sensitivity by inhibiting the expres-

sion of several ABA signalling transcription factors

(e.g. ABI3, ABI4, ABI5, PYL6 and PP2C5) (Weitbr-

echt et al. 2011). Thus, the combined effect of

endogenous ABA/GA and ABA/GA sensitivity regu-

late cold-induced germination in Arabidopsis. Hence,

there is a possibility that a similar phenomenon exists

during grain development in cereals.

In this study, the response of intact and detached,

developing grains to applied ABA and GA3 under

cool-temperature shock-induced and non-induced

conditions, measured as a-amylase activity, was

studied using UK winter wheat varieties, Spark and

Rialto. In the 1990s both Spark and Rialto were

recommended for bread-making purposes in the UK

(Anon. 1997). Spark has performed well and produced

reasonably high HFN and good quality bread. How-

ever, Rialto did not perform well under cool and wet

summer conditions of the UK, and often produced low

HFN, which contributed to poor quality bread. Later, it

was found in field trials that Rialto was more

susceptible to PMA under inductive conditions. In

the last decade, these two varieties have been used in

the UK for PMA related studies, where Spark has been

referred to as a non-inducible genotype and Rialto as

an inducible genotype (Farrell and Kettlewell 2008;

Flintham et al. 2011). These two varieties differ in

their Rht-D1 genotype. Spark has the GA-sensitive tall

(Rht-D1a) allele, whereas Rialto has a GA-insensitive

semi-dwarf (Rht-D1b) allele (Flintham et al. 1997).

The key hypothesis examined in this study was that

a cool-temperature shock applied mid-way through

grain development to induce PMA will alter ABA and/

or GA sensitivity of the aleurone. To test this

hypothesis, two glasshouse experiments involving in

situ and in vitro studies were carried out with a low

PMA susceptible variety, Spark and a high PMA

susceptible variety, Rialto. The in situ study investi-

gated the effect of four treatments (10 % ethanol

[untreated], ABA [100 lM], GA3 [50 lM] and

ABA ? GA3 [100 ? 50 lM]) applied to intact,

developing grains during a window of sensitivity on

a-amylase synthesis under cool-temperature shock-

induced and non-induced conditions. The in vitro

study investigated the sensitivity of the aleurone of

detached, developing grains to applied ABA and GA3

at 44 and 40 DAA under cool-temperature shock-

induced and non-induced conditions respectively.

Materials and methods

Seeds of UK winter wheat varieties, Spark (low PMA

susceptible) and Rialto (high PMA susceptible) (Far-

rell and Kettlewell 2008), were obtained from the John

Innes Centre (Norwich, UK). Spark and Rialto seeds

were planted on 5 October 2009 in a glasshouse in trays

filled with John Innes No. 2 Compost (Keith Single-

ton’s Seaview Nurseries, Cumbria, UK). Each variety

was grown separately on glasshouse benches. Follow-

ing germination and emergence, plants were vernalised

at 4 �C for 8 weeks in a cold room and then transferred

in a glasshouse to 1.3 L pots containing John Innes

No. 2 Compost (one plant/pot, pot dimensions: 11 9

12 9 18 cm). Plants were grown at minimum day/

night temperatures of 15/5 �C for 1 month. This was

mainly to prevent plants from getting devernalised by

the warm temperatures used later (i.e. 25/15 �C) and

also to simulate field conditions. After 1 month,

minimum temperatures were then gradually increased

over 5 weeks by 2 �C per week to 25/15 �C. Plants

were watered automatically with capillary matting

wetted three times a day (for 10 min every time).

Supplementary light (high pressure sodium, 400 W)

provided a minimum of 16 h day length throughout the

experiments. The protocol for growing plants is shown

in Fig. 1 (Farrell and Kettlewell 2008). Excess side

tillers were removed periodically to leave the main

shoot plus four secondary shoots. Fertilisers and

fungicides were used whenever required between

Zadoks Growth Stages 10 (ZGS10) and 69 (ZGS69).

The main spike on each plant was tagged with coloured

tape at early anthesis, i.e. ZGS61 (Zadoks et al. 1974).

No fertilisers and fungicides were applied following

ZGS71. The experimental design was completely

randomised, where each variety was grown on separate

benches in a glasshouse. Two temperature condi-

tions used in this experiment were cool-temperature

shock-induced and non-induced. Hormone treatments

applied were the four combinations as shown in

Table 1. Therefore, the four combinations of hormonal

Euphytica (2012) 188:51–60 53

123

treatments were arranged in a 2 9 2 factorial analysis

of variance (ANOVA) with the two hormones (ABA

and GA3) as the two factors and the two concentrations

as the two levels of each factor. As the ABA and GA3

powders (purchased from Sigma-Aldrich, UK) were

both dissolved in 10 % ethanol, the 0 lM ABA

plus 0 lM GA3 treatments involved the application

of 10 % ethanol. Data from induced and non-induced

conditions were analysed separately for either variety.

There were ten replicate plants for each hormone

treatment.

The cool-temperature shock-induction

The cool-temperature shock was given at 26 DAA by

transferring plants to an air-conditioned glasshouse

cooled to provide a constant temperature of 12 �C for

8 days (Farrell and Kettlewell 2008). It was assumed

that when plants were transferred to an air-conditioned

glasshouse for the cool-temperature shock induction, a

cool-temperature will have major effects whereas any

other environmental differences will have minor

effects on plants as the possible a-amylase influencing

conditions such as light, water and nutrient application

to plants were kept the same under both induced and

non-induced conditions. A cool-temperature shock-

induction was conducted during mid-March to early

April 2010.

The in situ study: Studying the ABA and GA3 sensitivity

of intact, developing grains under cool-temperature

shock-induced and non-induced conditions.

The in situ hormone application

ABA or GA3 solution (10 ll) was applied directly by a

pipette onto the crease region of two outermost grains

(spikelet 7 or 9; counting acropetally) of the main

shoot on each plant by gently pulling back the lemma

with forceps in cool-temperature shock-induced and

non-induced plants. Hormone application was carried

out in three doses (1st dose- 26 DAA, 2nd dose- 29

DAA and 3rd dose- 32 DAA) during a window of

Fig. 1 The PMA induction protocol for winter wheat varieties

Table 1 Treatment design

Factor 1

ABA treatment

Level 1 Level 2

Not applied Applied

Factor 2

GA3 treatment

Level 1 No ABA 100 lM ABA

Not applied No GA3 No GA3

Level 2 No ABA 100 lM ABA

Applied 50 lM GA3 50 lM GA3

54 Euphytica (2012) 188:51–60

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sensitivity (Mares and Mrva 2008). This period

corresponded with ZGS75–77 (Zadoks et al. 1974).

The cool-temperature shock-induced plants were

returned to a warm glasshouse heated to 25/15 �C,

where they remained until maturity. Plants were then

harvested depending upon treatments given i.e. non-

induced plants at 60 DAA and induced plants at 64

DAA. The different time point for harvesting induced

and non-induced plants was calculated from the

difference in the degree days accumulated during the

cool-temperature shock-induction.

Measurement of a-amylase activity by a modified

Phadebas assay

This assay followed the protocol of Yongfang Wan

(Personal communication) using the commercial amy-

lazyme substrate (Megazyme International Ireland Ltd.,

Ireland). This substrate consists of azurine-crosslinked

amylose, and is prepared by dyeing and cross-linking

high amylose maize starch. Hormone-treated grains (two

grains per replicate plant) were removed from harvested

spikes. Distal half-grains were obtained by excising the

embryos. Half-grains (single half-grain per well) were

ground against the force exerted by the stainless steel

balls (one ball per well) in a 96 well block (Appleton

Woods, UK) using a TissueLyser II (Qiagen, UK) at

2 9 30 Hz for 4 min. Following grinding, the stainless

steel balls were poured off and 300 ll of extraction

buffer (300 mM maleic acid, 500 mM NaOH, 6 mM

CaCl2, and 30 mM NaCl, pH 6) was added to each well.

The contents were mixed by shaking the plate 4–5 times

by hand before placing the plate on a shaker for 1 h at

300 rpm at room temperature. The plate was centrifuged

at 1233 g (3,500 rpm) for 15 min. Alpha-amylase

activity in the supernatant was assayed by a modified

Phadebas assay. The amylazyme substrate solution

(30 ll of one tablet in 1 ml extraction buffer) was

incubated with the enzyme extract (30 ll) at 37 �C for

15 min. During the incubation, the plate was shaken by

hand every 5 min. The enzyme-substrate reaction was

stopped by the addition of 840 ll stopping buffer (1 M

NaOH, extraction buffer [1:2 diluted with distilled

water], final pH 12). The plate was centrifuged at 1233 g

(3500 rpm) for 10 min and 200 ll of the supernatant

was taken to measure the absorbance at 620 nm using a

microplate reader [Bio-Rad (Benchmark), UK]. These

absorbance readings were considered as the measure-

ment of a-amylase activity (OD units).

The in vitro study: Studying the sensitivity of

detached, developing grains to applied ABA and GA3

at 44 and 40 DAA under cool-temperature shock-

induced and non-induced conditions respectively.

Spikes from the main shoot of non-induced and

induced plants were harvested at 40 DAA and 44 DAA

respectively, and were taken directly to the laboratory

on the same day. Forty grains were pooled from the

middle region of harvested spikes. Grains were surface

sterilised with 70 % ethanol for 1 min, followed

by 5 % sodium hypochlorite (available chlorine

10–15 %, Sigma-Aldrich, UK) for 30 min and then

rinsed five times with distilled water. After 1 h of

soaking, grains were cut into equal halves using a

scalpel (sterilised with 70 % ethanol) and embryo

halves were discarded. Forty embryoless half-grains

from each replicate spike were distributed equally into

four eppendorf tubes (1.5 ml) for four different

treatments, i.e. 10 embryoless half-grains/treatment.

Embryoless half-grains were then incubated in 50 ll

of hormone solutions (i.e. 10 % ethanol [untreated],

ABA [100 lM], GA3 [50 lM] and ABA ? GA3

[100 ? 50 lM]) with 950 ll of incubation medium

(citric acid-sodium citrate buffer, 10 mM, pH 5 ?

CaCl2, 0.2 M) for 72 h at 25 �C in the dark.

Incubated samples were homogenised in 1 ml of

extraction buffer (50 mM Tris–HCl, 10 mM CaCl2,

pH 7.5) on ice and centrifuged at 9,660 g

(12,000 rpm) for 10 min (Hader et al. 2003). Alpha-

amylase activity in the supernatant was then assayed

by a modified Phadebas assay (as described above).

Statistical analysis and presentation

Alpha-amylase activity data were analysed statisti-

cally by a 2 9 2 factorial ANOVA at P \ 0.05 with

Genstat 13th edition (VSN International Ltd., Hemel

Hempstead, UK). For the in situ study, the analysed

a-amylase activity data represents the mean of

a-amylase activity measured from two outermost

grains (spikelet 7 or 9; counting acropetally) of the

main spike per ten replicate plants (i.e. n = 10). For

the in vitro study, the analysed a-amylase activity data

represents the mean of a-amylase activity measured

from ten embryoless half-grains obtained from middle

region of the main spike with ten replicate plants (i.e.

n = 10). Data from induced and non-induced condi-

tions were analysed separately for either variety. Each

Euphytica (2012) 188:51–60 55

123

variety was also analysed separately for both exper-

iments. There were two reasons behind analysing each

variety separately. Firstly, each variety was grown on

separate benches in a glasshouse. Secondly, there was

a huge variation in coefficient of variation (CV) values

for a-amylase activity data between the two varieties

used. In order to reduce variance heterogeneity, alpha-

amylase activity data were transformed using a power

(i.e. x-2) transformation prior to ANOVA. Variance

heterogeneity and normality of the data were satisfac-

tory after transformation. For the simplicity of under-

standing and to avoid the distortion of data caused

by a power transformation, means of back trans-

formed treatments were used for presenting the results

(Table 1; Figs. 2, 3).

Results

In situ study

Under non-induced conditions

In non-induced grains, the overall effect of applied

ABA on a-amylase activity at maturity was not signif-

icant in either variety; Spark (P = 0.414) and Rialto

(P = 0.207). Overall, applied GA3 gave a significant

increase in a-amylase activity at maturity in non-

induced grains in Rialto (P = 0.007), but not in Spark

(P = 0.125). The two way interaction between ABA

and GA3 was not significant in either variety; Spark

(P = 0.299) and Rialto (P = 0.106) (Table 2; Fig. 2).

Under cool-temperature shock-induced conditions

In general, applied ABA produced no significant effect

on a-amylase activity at maturity in cool-temperature

shock-induced grains of Spark (P = 0.572), but the

effect was marginally significant in induced grains of

Rialto (P = 0.061). Similar to non-induced condi-

tions, applied GA3 overall resulted in a significant

increase in grain a-amylase activity at maturity under

cool-temperature shock-induced conditions in Rialto

(P = 0.005), but not in Spark (P = 0.219). The

ABA 9 GA3 interaction was not significant in either

variety; Spark (P = 0.761) and Rialto (P = 0.385)

(Tables 2, 3; Fig. 2).

In vitro study

Under non-induced conditions

The effect of applied ABA on a-amylase activity in

mature grains was not significant overall under non-

induced conditions in either variety; Spark (P = 0.101)

and Rialto (P = 0.297). In contrast to applied ABA,

applied GA3 significantly increased a-amylase activity

in mature grains under non-induced conditions in both

Fig. 2 The effect of the in situ applied hormones (10 % ethanol

[untreated], ABA [100 lM], GA3 [50 lM] and ABA ? GA3

[100 ? 50 lM]) on PMA under cool-temperature shock-

induced and non-induced conditions in two winter wheat

varieties, a. Spark and b. Rialto. Standard Error of Mean

(SEM) values were back-transformed to OD units. Spark: Non-

induced conditions—SEM = 0.028 and CV = 35.9 %;

Induced conditions—SEM = 0.032 and CV = 38.5 %. Rialto:

Non-induced conditions—SEM = 0.097 and CV = 51.4 %;

Induced conditions—SEM = 0.159 and CV = 85.6 %

56 Euphytica (2012) 188:51–60

123

varieties; Spark (P \ 0.001) and Rialto (P = 0.041).

The two way interaction between ABA and GA3 was

significant in Spark (P \ 0.001); however not in Rialto

(P = 0.186) (Tables 2, 4; Fig. 3).

Under cool-temperature shock-induced conditions

In general, applied ABA had no significant effect on

a-amylase activity in mature grains under cool-temper-

ature shock-induced conditions in Spark (P = 0.759),

but the effect was significant in Rialto (P = 0.039).

Applied GA3 gave a significant increase in grain

a-amylase activity at maturity overall under cool-

temperature shock-induced conditions in Rialto (P =

0.012); however not in Spark (P = 0.812). The two way

ABA 9 GA3 interaction was not significant in either

variety; Spark (P = 0.219) and Rialto (P = 0.204)

(Tables 1, 3; Fig. 3).

Discussion

Singh and Paleg (1984a, b) conducted a study using

isolated aleurone layers and detached, embryoless

Fig. 3 The sensitivity of developing grains to the in vitro

applied hormones (10 % ethanol [untreated], ABA [100 lM],

GA3 [50 lM] and ABA ? GA3 [100 ? 50 lM]) using

detached embryoless half-grains under cool-temperature

shock-induced and non-induced conditions in two winter wheat

varieties, a. Spark and b. Rialto. SEM values were back-

transformed to OD units. Spark: Non-induced conditions—

SEM = 0.005 and CV = 9.6 %; Induced conditions—SEM =

0.018 and CV = 25.5 %. Rialto: Non-induced conditions—

SEM = 0.09 and CV = 61.8 %; Induced conditions—SEM =

0.147 and CV = 82.9 %

Table 2 Means of a-amylase activity in hormone treated

(ABA [100 lM], GA3 [50 lM] and ABA ? GA3 [100 ?

50 lM]) grains under cool-temperature shock-induced and

non-induced conditions in the in situ and in vitro studies are

shown in two winter wheat varieties, Spark and Rialto

Study Variety Temperature

treatment

Mean a-amylase activity (OD units/distal half grain)

Untreated ABA GA3 ABA ? GA3

In situ study Spark NI 204.08 (0.070) 187.65 (0.073) 111.50 (0.095) 164.36 (0.078)

CSI 124.84 (0.090) 117.63 (0.092) 185.10 (0.074) 148.72 (0.082)

Rialto NI 300.36 (0.058) 229.56 (0.066) 110.80 (0.095) 025.53 (0.198)

CSI 080.72 (0.111) 121.02 (0.091) 010.64 (0.307) 023.70 (0.205)

In vitro study Spark NI 409.77 (0.049) 365.59 (0.052) 234.51 (0.065) 348.07 (0.054)

CSI 202.34 (0.070) 304.57 (0.057) 172.22 (0.076) 222.10 (0.067)

Rialto NI 162.27 (0.079) 130.31 (0.088) 028.99 (0.186) 082.64 (0.110)

CSI 041.89 (0.155) 078.31 (0.113) 008.00 (0.353) 030.38 (0.182)

The x-2 transformed means are shown with corresponding back transformed means in brackets for each treatment

NI Non-induced conditions; CSI Cool-temperature shock-induced conditions

Euphytica (2012) 188:51–60 57

123

half-grains with wheat varieties having at least one of

the GA-insensitive dwarfing alleles (i.e. Rht-B1b, Rht-

D1b and Rht-B1c). They showed that a cool-temper-

ature treatment (i.e. 5–10 �C), prior to the addition of

GA3, induced GA sensitivity in the aleurone tissue.

Worldwide, many commercial high-yielding wheat

varieties have one of these dwarfing alleles, and are

classified as ‘dwarf’ or ‘semi-dwarf’. In both studies in

this work, the higher a-amylase activity was observed

in induced grains of Rialto in response to applied GA3,

which suggested that GA sensitivity was induced in

intact, developing grains under cool-temperature

shock-induced conditions. Thus, it may be that

induced GA sensitivity was retained during later

stages of grain maturation producing a high level of

a-amylase activity. This finding is supported by Gold

and Duffus (1995), who suggested that an environ-

mental trigger during the early stages of development

might switch on GA sensitivity in the aleurone tissue

of developing grains. In the in vitro study in both

varieties, the ABA ? GA3 treatment (although not

significant) produced an intermediate level of a-amy-

lase activity compared to ABA and GA3 treatments

alone. These results were consistent with the findings

of Gold (1991). Gold (1991) studied the sensitivity of

isolated aleurone tissue to applied ABA, GA3 and

ABA ? GA3 in UK winter wheat varieties, Fenman

and Maris Huntsman. Gold (1991) observed an

intermediate level of a-amylase activity for the

ABA ? GA3 treatment between GA3 and ABA incu-

bations alone. This study by Gold (1991) found a high

PMA level in glasshouse grown plants of these

varieties, although the study did not include a cool-

temperature shock-induction.

Similar patterns of results were observed for both

varieties between the two sensitivity studies, i.e. in situ

with embryos and in vitro without embryos, except in

Spark in the in situ study. This confirms the fact that

PMA was mainly induced in the aleurone tissue, and

not in the embryo. This finding is consistent with Mrva

et al. (2006), who observed that the PMA induction

process was aleurone dependent (and independent of

the embryo). The results obtained from the in vitro

sensitivity study suggest that a cool-temperature shock

treatment induced GA sensitivity in the aleurone of

developing grains in both varieties. This effect was

long lasting in both varieties as a cool-temperature

shock (26–34 DAA) was conducted 10 days before the

in vitro sensitivity was tested (44 DAA). These results

differ from those of Singh and Paleg (1984a) where

Table 3 ANOVA

comparison of hormonal

treatments for cool-

temperature shock-induced

and non-induced conditions

in the in situ study

Source of variation Degrees of

freedom (d.f.)

Under non-induced

conditions

Under cool-temperature

shock-induced conditions

Spark Rialto Spark Rialto

P values P values P values P values

ABA 1 0.414 0.207 0.572 0.061

GA3 1 0.125 0.007 0.219 0.005

ABA 9 GA3 1 0.299 0.106 0.761 0.385

Residual 36

Total 39

Table 4 ANOVA

comparison of hormonal

treatments for cool-

temperature shock-induced

and non-induced conditions

in the in vitro study

Source

of variation

Degrees of

freedom (d.f.)

Under non-induced conditions Under cool-temperature

shock-induced conditions

Spark Rialto Spark Rialto

P values P values P values P values

ABA 1 0.101 0.297 0.759 0.039

GA3 1 \0.001 0.041 0.812 0.012

ABA 9 GA3 1 \0.001 0.186 0.219 0.204

Residual 36

Total 39

58 Euphytica (2012) 188:51–60

123

induced GA sensitivity was temporary. A possible

explanation for this discrepancy may be a difference in

behaviour between intact (attached) and detached

grains. The GA sensitivity induced by a cool-temper-

ature shock in in situ and in vitro studies in Rialto may

involve either an increase in the number of GA

receptors on/in the aleurone cells or an increase in the

expression of GA signalling genes, which leads to

increased synthesis and secretion of a-amylase by

aleurone cells. However, considering the possibility of

an increase in GA receptors, it appeared that the

ultimate production of a-amylase after GA sensitivity

is induced in induced grains will depend on the

endogenous GA available at that stage of grain

development. Thus, it appears that the combination

of a sufficient level of endogenous GA and GA

sensitivity (induced by a cool-temperature shock

treatment) is an important factor in PMA induction.

In both studies, applied ABA had no significant

effect on a-amylase activity in mature grains under

non-induced conditions in either variety. This non-

significant effect of applied ABA on grain a-amylase at

maturity in non-induced grains was expected in both

varieties as a minimum level of a-amylase (back-

ground a-amylase and not true PMA) is present in all

grains no matter how much ABA is applied. In contrast,

applied ABA produced a significant effect on grain a-

amylase activity at maturity under cool-temperature

shock-induced conditions in Rialto, but not in Spark.

This was possibly because induced untreated (10 %

ethanol treated) grains in Rialto have significantly

higher a-amylase for ABA to inhibit compared to non-

induced untreated (10 % ethanol treated) grains. For

both varieties, the level of a-amylase activity observed

in ABA treated non-induced grains was similar to

untreated (10 % ethanol treated) non-induced grains.

Garcia-Maya et al. (1990) found that the synthesis of

low pI a-amylase by the embryo and aleurone tissue

was independent of the in vitro applied ABA and the

synthesis of high pI a-amylase was ABA dependent. In

contrast, an in vitro applied GA3 produces both low and

high pI a-amylases (Cornford et al. 1987). Thus, there

is a possibility that the a-amylase activity observed in

ABA and untreated (10 % ethanol treated) grains

represent low pI a-amylase. Moreover, the amylazyme

substrate used in this study is not specific to particular

a-amylase isoenzymes and detects both low and

high pI a-amylases. Thus, in future experiments, the

isoelectric separation of both a-amylase isoenzymes on

agarose gel will help to answer this question.

The two experiments described in this paper have

used exogenous hormones. The confirmation of the

role of ABA and GA in PMA induction needs further

research to investigate the effect of a cool-temperature

shock-induction on endogenous levels of ABA and

GA in developing grains. Moreover, using in situ and

in vitro experiments to look at the ABA and GA

sensitivity across Spark 9 Rialto lines differing in the

Rht-D1b allele will be useful in understanding the role

of GA-insensitive semi-dwarfing allele Rht-D1b dur-

ing PMA induction by a cool-temperature shock in

wheat grains.

Conclusions

Under cool-temperature shock-induced conditions,

GA sensitivity (measured as a-amylase synthesis in

response to applied GA3) was induced in developing

grains of a high PMA susceptible variety (Rialto), but

not in a low PMA susceptible variety (Spark). There

was little evidence for a change in ABA sensitivity as a

result of the cool-temperature shock-induction in

either variety. However, applied ABA significantly

inhibited PMA under induced conditions in Rialto

mostly because there was significantly higher a-amy-

lase for ABA to inhibit in induced untreated grains

compared to non-induced untreated grains.

Acknowledgments The authors are grateful to all the staff in

the Crop and Environment Research Centre and Princess

Margaret Laboratories for their kind support and help

throughout the work. This work was funded by Harper Adams

University College and Home-Grown Cereals Authority.

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