ACTA PHYSIOLOGIAE

PLANTARUM Vol. 25. No. 2. 2003:151-161

ABA and gibberellin-like substances during prehardening, cold acelimation, de- and reacclimation of oilseed rape

Marcin Rapacz 1, Piotr Waligdrski 2, Franciszek Janowiak 2

1 Corresponding author, Department of Plant Physiology, Faculty of Agriculture and Economics Agricultural University of Cracow, e-mail: rrrapacz@cyf-kr.edu.pl

2 The Franciszek G6rski Department of Plant Physiology, Polish Academy of Sciences, Podtu2na 3, 30-239 Krak6w, Poland

Key words: abscisic acid, Brassica napus var. oleifera, cold acclimation, cold deacclimation, gib- berellins, growth rate, prehardening, PSII excita- tion pressure

Abstract

Previously published results showed that high relative reduc- tion state of PSII (PSII excitation pressure) during both early seedling growth (prehardening) as well as cold deacclimation caused significant changes in growth pattern. The differences in elongation growth rate were related to the cold acclimation of photosynthetic apparatus and to frost resistance. To study changes in the hormonal balance connected with alterations in elongation growth rate observed during prehardening and deacclimation under different PSII excitation pressure (modu- lated by day-temperatures), endogenous concentration of ABA, GA3 and GA-like substances (GAs) were analysed. Analyses were also performed during cold acclimation and reacclilnation of plants characterized by different elongation growth rate triggered by prehardening or deacclimation under different day-temperatures. Growth under high PSII excitation pressure (prehardening) resulted in a significant increase in ABA and a considerable decrease in GAs contents. On the other hand, different ABA content played almost no role in controlling growth rate during cold deacclimation and subse- quent reacclimation, when the induction of elongation growth was connected with the changes in concentration of GAs in- cluding GA3. The possible rote of ABA and GAs in controlling prehardening, cold acclimation and deacclimation is dis- cussed.

List o f abbreviations: ABA, abscisic acid; CE-MS, capillary electrophoresis-mass spectroscopy; DW, dry weight; ELISA, enzyme-linked immuno- sorbent assay; FW, fresh weight; GAs, gibberellin-like substances; qp, photochemi- cal quenching of chlorophyll a fluorescence; 1-qp, PSII excitation pressure

Introduction

Changes in environmental conditions such as light intensity or temperature result in an imbalance be- tween the light energy absorbed through photo- chemistry and the energy utilized through photo- synthetic metabolism. Such energy imbalance af- fects the relative reduction state of PSII (Huner et al. 1998). The relative changes in the reduction state of PSII may be expressed as PSII excitation pressure (Adams et al. 1995). Exposure to high PSII excitation pressure during both early seedling growth (prehardening) as well as cold acclimation caused signif icant changes in growth patterns (Huner et al. 1998, Rapacz 1998 a,c). The decrease in elongation growth rate under elevated PSII exci- tation pressure (low temperature/or high light in- tensity) were accompanied by adaptation of the

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photosynthetic apparatus to cold and by increased frost resistance (Huner et al. 1998, Rapacz 1998 a, b,c). Recent results suggest that during cold accli- mation and deacclimation PSII excitation pressure may affect frost resistance indirectly via regulation of elongation growth rate (Rapacz 1998 c, Rapacz 2002 a,b). Huner et aL (1998) suggested that the ef- fect of elevated PSII excitation pressure on com- pact plant morphology may be a result of some link- age between growth regulators and a PSII-depen- dent environmental temperature sensing mecha- nism. Growth rate of plants is determined by the balance of the inhibitor (i.e. ABA) and a promoter (i.e. GA) of growth (Kende and Zeevaart 1997). During shoot growth and development of oilseed rape a 300-fold increase in the concentration of GAs was observed by the time of floral initiation and stem elongation (Rood et al. 1989). At the same time a dramatic decrease in frost resistance was re- ported by Rapacz (2002 a). Both ABA and GAs are also involved in the regulation of cold acclimation and deacclimation. Many genes induced by low temperature during cold acclimation are also in- duced by ABA. Also, ABA acts synergistically with cold to express some other genes involved in the increase of frost resistance (Shinozaki et al. 2000, Knight and Knight 2001). GAs are consid- ered as an important plant hormone group in cold deacclimation (Carter and Brenner 1985) and de- creased frost resistance caused by gibberellins is, at least in part, the result of stimulated growth (Junt- tila 1997). GAs are reported to increase stress sus- ceptibility of plants and they may interfere with ABA as a stress-hormone (Vettakkorumakankav et al. 1999). ABA may affect frost resistance also via modification of plant growth pattern and decreased cell expansion rate, which were observed during prehardening or cold acclimation (Rapacz 1998 a, b,c, Kacperska and Szaniawski 1993, Stefanowska et al. 1999).

A B A and GAs may also regulate the acclimation of the photosynthetic apparatus to cold, which, in turn, may affect PSII-related temperature sensing. Treat- ment with exogenous ABA, similarly as growth un- der high PSII excitation pressure increased resis- tance of PSII to photoinduced inactivation at low temperature (Ivanov et al. 1995, Saradhi et al. 2000, Rapacz 2002c), whereas gibberellin A 3

(GA3) increased susceptibility to low temperature photoinhibition (Rapacz 2002 c). Moreover, the in- crease in PSII excitation pressure, like ABA, re- stricts elongation rate (Rapacz 1998 a,b, 2002 a), whereas a decrease in PSII excitation pressure ac- celerates elongation similarly to GAs (Rapacz 2002a).

The above-described results suggest that growth under different PSII excitation pressure during prehardening and cold-deacclimation may cause changes in ABA or (and) GAs concentrations. The aim of the present experiments was to examine the role of ABA and GAs in the control of elongation growth rate, frost resistance and acclimation of photosynthetic apparatus to cold observed in previ- ous experiments (Rapacz 1998 a,b,c, Rapacz and Janowiak 1998, Rapacz 2002 a,b, Rapacz and Hura 2002).

Materials and methods

Plant mater ia l and experimental design

Seedlings of winter oilseed rape (Brassica napus L. var. oleifera f. bienae cv. G6rczafiski; MHR-HBP, Poland), used in all experiments and spring oilseed rape (Brassica napus L. var. oleifera f. annua cv. Star; DLF Trifolium, Denmark), used in experi- ments 2 and 3, were grown in groups of 8 plants per 20-cm pot containing loam soil: sand: peat (1:1:1; v:v:v). Seeds were germinated under con- trolled-environment conditions: photosynthetic photon flux density (PPFD) 300 pmol-m-2.s -1 (so- dium light, 'Agro', Philips), 20 °C (day and night), photoperiod 12 h. When about 50 % of plants emerged, the temperature was set to the desired growth temperature with the same condition of daylength and PPFD. Three experiments with dif- ferent regimes of temperature and time were per- formed. The objective of this complicated experi- mental design was to simulate the complex, natural autumn and early spring conditions of rape growth in field. In experiment 1, growth temperatures of 20/20, 20/12, 12/20 and 12/12 °C were used to sim- ulate the autumn conditions of growth, whereas in experiments 2 and 3 plants were grown before cold acclimation at 12/12 °C, only. Plants grown at 12/20 and 12/12 are defined as prehardened plants

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ABA, GIBBERELLINS AND FROST RESISTANCE OF RAPE

(Rapacz 1998 a,b). After 5 weeks, plants were sub- jected for 4 weeks to cold acclimation at 2 °C with 10-h photoperiod and PPFD of 250 pmol(quan- tum).m2-s -1. Experiment 1 was terminated at that time, whereas plants in experiments 2 and 3 were subjected to cold deacclimation under various tem- perature conditions. Conditions of deacclimation: 2/2 (control), 2/12, 12/2, 12/12 °C (experiment 2, warm breaks during winter) and: 12/12, 12/20, 20/12, 20/20 °C (experiment 3, early spring condi- tions). Twelve-hour photoperiods were maintained throughout. 12/2, 2/12 and 20/12, 12/20°C are con- ditions with the same sum of temperature and dif- ferent PSII-excitation pressure (day temperature). Plants subjected to fluctuating temperatures were moved between growth chambers of different tem- peratures within 2 mins. Deacclimation lasted 3 (experiments 2) or 4 weeks (experiment 3). After deacclimation plants were transferred back to 2 °C (10-h photoperiod and PPFD of 250 lamol(quan- tum)-m-2.s -1) to reacclimate. Reacclimation lasted 3 (experiments 2) or 4 weeks (experiment 3). Dur- ing each experiment, water and nutrients were sup- plied as half-diluted Hoagland's solution.

All the reagents, unless specified, were purchased from Sigma-Aldrich Co Ltd. (affiliate Poznan, Po- land).

Measurements of PSll-excitation pressure, growth and developmental analysis

PSII excitation pressure was measured by means of chlorophyll fluorescence (Adams et al. 1995). Chlorophyll a fluorescence was measured on the youngest but fully expanded leaves. Measures were taken using a pulse modulated portable fluorometer FMS2 (Hansatech Instruments Ltd., King's Lynn, UK) during the day at the growth temperature, usu- ally in 10 replicates. Measurements were done after four weeks of prehardening and after one week of cold deacclimation. Leaves were dark adapted at the measuring temperature for 1 h prior to measure- ment. The second measurement (light acclimated leaf) was done after 15 min at 300 pmol.m2.s -1 PPFD. PSII excitation pressure was calculated as 1-qe and photochemical quenching coefficients (qp) were calculated according to Havaux et al. (1991).

During experiments 2 and 3 measurements of elon- gation growth rate (stem and leaf area) were done in 10 replicates, as described elsewhere (Rapacz 2002b). In the present paper only data concerning the week in which the intense elongation growth was observed in every studied plants is presented. The observations of generative development were done on each plant grown during the experiments. The week of flower bud formation or of flowering was the week in which over 95 % of plants had flower buds or flowers formed, respectively.

Sample collection

Samples for analysis of growth regulators were collected in all experiments after initial plant growth and cold-acclimation, and then after 1, 3 (ABA only) or 1, 2 (ABA only) and 4 (ABA only) week(s) of deacclimation (experiment 2 and 3, re- spectively), and also after reacclimation (in experi- ment 3 samples for ABA measurements were col- lected also after 2 weeks of rcacclimation). Sam- ples were collected from leaf blades of two youn- gest fully expanded leaves on which elongation growth rate and properties of photosynthetic appa- ratus had previously been measured (Rapacz 2002a,b, Rapacz and Hura 2002). Inexperimcnts 1 and 2 all samples were collected from leaves that were fully developed before cold acclimation. In experiment 3, samples were collected after pre- hardening (early growth at 12/12°C), cold acclima- tion and 1 and 2 weeks of deacclimation from leaves developed during prehardening. After 4 weeks of deacclimation and after rcacclimation, samples were collected from leaves that developed during deacclimation. Leaves were cut out, weighed, immediately frozen in liquid nitrogen and stored at -80 °C. From each cultivar and treatment three samples (biological replications) of about 5 g each (FW) consisting of segments from 3-6 leaves were prepared. Samples of shoot apexes (about 500 rag) were collected from the winter cultivar 'G6rczafiski' for additional ABA analysis after deacclimation (4 th week).

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M. RAPACZ, P. WALIGORSKI & F. JANOWIAK

Determination of GA 3 and GA-like substances

Extraction and purification

Two samples were analysed, the third one was ana- lysed only if the results obtained for two samples differed by more than 10 %. Gibberellins were ex- tracted and purified using a method modified from Rood et al. (1989). Leaves were frozen in liquid ni- trogen and ground with a mortar and pestle. Pow- dered samples were placed in 90 % cold methanol to extract gibberellins. After overnight extraction at 4 °C, samples were centrifuged at 12,000 rpm for 30 minutes at 4 °C. Supernatant was collected and methanol was evaporated in vacuo. Then pH was raised to 8.5 with KOH and the aqueous extract was washed three times with diethyl ether. The pH was decreased to 2.5 with HCI and GAs were extracted by ethyl acetate and divided in two parts. The or- ganic phase was evaporated to dryness in vacuo. Then the first part of the sample (for CE-MS analy- sis) was dissolved in 0.2 cm 3 methanol and the sec- ond (for bioassay) in appropriate volumes (1 cm 3 per 0.5 g of the fresh weight of the sample) of ace- tate buffer (pH 4.8).

CE-MS analysis of GA 3

The extract was diluted 1:1 by 1% NH4aq and fil- tered through 0.2 gm polyvinyl disposable syringe filters. GA 3 content was determined according to Yeo at al. (1992), using CE-MS system (Hewlett Packard HPCE3d Capillary Electrophoresis Sys- tem combined with Agillent Technologies MS Sys- tem 1100 Series LC/MSD). Samples were injected into the CE system for 25 s under pressure of 50 mbar, voltage of 20 kV was applied. CE buffer was 40 mM ammonium acetate, pH 8.5. We used bare fused silica capillary 64.5 cm total length and 50 6m id. Temperature was set to 25 °C. MS was set to neg- ative po]arisation, at SIM (single ion monitoring) 345 D - for deprotonised form of GA 3. Peak ofGA 3 was observed at 4th minute of the analysis. Analy- sis was made in at least 3 replicates (instrumental replications).

B(oas~,.flaY f l f GA-~ke substances

The bioassay was performed using a lettuce hypocotyl assay (Frankland and Wareing 1960) with modifications by Okagami (1971). The lettuce

variety Arctic was used. Bioassays were performed in 5 replications per each sample, using four con- centrations of GA3 (5, 50, 250, 500 ng/cm 3) as a standard.

Determination of ABA content

Two samples were taken for analyses, the third one was analysed if the results obtained for the two samples differed by more than 10 %. After freeze- -drying the plant material was ground in a ho- mogeniser (Retch, Kroll, Germany) and cold dis- tilled water was added (50 : 1 H20 : DW of sample). The samples were shaken overnight at 4 °C. On the following day the extracts were centrifuged in a cool centrifuge at 16,000 g (Heraeus .Biofuge, Ger- many). From the supernatant 0.3 cm 3 were taken for ABA determination by an indirect ELISA ac- cording to the protocol of Walker-Simmons (1987), modified by Hansen and Drrffling (1999), using the antibody MAC 252 obtained from Dr. Steve Quarrie, John Innes Centre, Norwich, UK. Cross- -reaction tests for the determination of non-specific binding were performed in crude extracts. The lin- ear relationship between exogenous ABA concen- tration in water and the extracts indicated that cross reactivity was minimal. For each sample three mea- surements in independent ELISA plates, each with three replicates, were performed.

Results

Growth temperature, PS I I excitation pressure and growth characteristics o f plants

In the all experiments plants growing at higher day temperature were characterize by louver PSII exci- tation pressure (Table 1). Plants grown at higher PSII excitation pressure during prehardening were characterized with compact plant morphology (Ta- ble 1, see also Rapacz 1998a). Cold deacclimation under very low PSII excitation pressure (experi- ment 3, day temperature 20 °C) resulted in promo- tion of elongation growth in winter plants. Spring plants irrespective of deacclimation conditions started to elongate shoots and to expand leaf area. The date in which the acceleration of elongation growth was observed was proportional to the PSII excitation pressure and plants deacclimated under

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ABA, GIBBERELLINS AND FROST RESISTANCE OF RAPE

Table 1. PSII excitation pressure, growth and developmental characteristics of plants studied in the experiments. Relative reduction state of PSII measured by means of chlorophyll a fluorescence (PSII excitation pressure, l-qp) after four weeks of prehardening (experiment 1) and after one week of cold deacclimation of oilseed rape (experiments 2 and 3). Means of 10 measurements + Se. Values within the experiment and cultivar denoted with the same letter do not differ significantly at P=0.05 (Duncan's multiple range test). Growth characteristic of plants during prehardening (experiment 1) are presented according to Rapacz et al. 1998a. In experiments 2 and 3 the week in which the intense elongation growth (stem elongation and leaf area expansion rate) was observed in all studied plants (0 - the beginning of cold acclimation). The week of flower bud formation or of flowering was the week in which over 95 % of plants had flower buds or flowers formed, respectively (0 - the beginning of cold acclimation).

Temperature PSII excitation pressure Growth characteristics Flower buds Flowering

during: (week) (week)

Cultivar

(day/night, °C) winter spring winter spring spring spring

prehardening (experiment 1) Morphology

- Elongated - - -

- Elongated - - -

- Compact - - -

- Compact - - -

20/20 O. 190 b + 0.022

20/12 0.208 b 4- 0.033

12/20 0.381" 4- 0.041

12/12 0.411 ~ 4- 0.036

cold deacclimation (experiment 2) Promotion of elongat ion

growth (week)

12/12 0.299b + 0.028 0.178b + 0.031 Not observed 7 10

12/2 0.31 lb + 0.041 0.169b + 0.022 Not observed 7 -

2/12 0.501 ~ + 0.037 0.334 ~ + 0.024 Not observed 10 -

2/2 (control) 0.483 • 4- 0.029 0.326 a 4- 0.030 Not observed 10 -

cold deaccl imation (experiment 3) Promotion o f elongat ion

growth (week)

20/20 0.122 b 4- 0.032 0.091~ + 0.021 8 5 8 11

20/12 0.134hi 0 .021 0.082b + 0.019 8 5 5 6

12/20 0.312" 4- 0.035 0.145 a 4- 0.016 Not observed 8 6 6

12/12 0.306a + 0.028 0.136a 4- 0.022 Not observed 8 10 -

h igher 1-qp started to e longa te later. D u r i n g the sec-

ond and third expe r imen t spr ing plants started to

f lower. In the second expe r imen t f lower bud forma-

t ion was observed on ly in plants de -acc l ima t ing un-

der cond i t ions of 12/12°C (Table 1). Buds were

formed dur ing the last week of the expe r imen t on

not fully e longa ted f lower stems. In the third exper-

imen t all spr ing plants fo rmed f lower buds and on ly

12/12°C - deacc l imated plants did not m a n a g e d to

f lower unt i l the end of the exper iment . The se-

quence of f lower bud fo rmat ion and f lower ing were

as fo l lows : 20/12 , 12/20, 20 /20- d e a c c l i m a t e d

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M. RAPACZ, P. WALIGORSKI & F. JANOWIAK

Table 2. Content ofABA, GA-like substances and GA3 during prehardening and subsequent cold acclimation (4 weeks at 2 °C) in leaves of winter oilseed-rape cv. G6rczatiski (experiment t). Presented values are the means (-+ Se) of 3 independent ELIS A mea- surements with 3 replicates (ABA), 5 measurements (GAs, bioassay) and 3 measurements (CE-MS) for each of the 3 samples (about 5 g FW collected from 4-6 plants, each). Values within columns denoted with the same letter do not differ significantly at P=0.05 (Duncan's multiple range test).

Conditions of prehardening ABA GA-like substances in bioassay GAs in CE-MS analysis

(day/night, °C) (nmol g4DW) (rig GA3 equivalent g-i DW) (rig g-i DW)

20/20

20/12

12/20

12/12

Before cold After cold. Before cold After cold Before cold After cold

acclimation acclimation acclimation acclimation acclimation acclimation

3.58 ~ ± 0.428 2.11" ± 0.314 12" • I 9 ' ± 2 4,53" ± 0.75 3,86 ' ± 0.6

4.22 b ± 0.532 1.66 ~ ± 0,222 14' ± 1.5 10 ' ± 1.5 4.52 ' ± 0.45 3.83 ~ ± 0.6

7.02 ° ± 0.834 2.08 ~ ± 0.231 8 b ± 1.5 5 b ± I 2.71 b ± 0.55 2.11 b ± 0.45

6.51°± 0,953 1.86"± 0.271 9b± 1,5 4b± 1 2.43b ± l , I 2,87b ± 0.65

plants. The delay of f lowering observed in 20/20-deacclimated plants was caused by the plant transfer to 5 °C (week 9).

Changes in ABA content

During early seedling growth enhanced levels of ABA were observed in prehardened plants, i.e.

plants growing at low day temperature (elevated PSII excitation pressure) (Table 2). Leaves of both winter and spring plants grown at 12 °C contained comparable amounts of ABA before cold acclima- tion (Figs. 1 and 2). During four weeks of cold ac- climation a similar decrease in ABA content was observed in both cultivars. Following a further pe- riod of cold, ABA concentrations remained un- changed (Fig. 1, plants grown at 2/2°C during deacclimation). Cold deacclimation resulted in a slight increase in ABA content. A clear difference between the cultivars was observed during this pe- riod. In the winter cultivar G6rczafiski the highest amounts of ABA were observed after one (experi- ment 2) or two (experiment 3) weeks of deaccli- mat±on in plants deacclimated at 2/12 °C (Fig. I A) and 20/20 °C (Fig. 2A). During further deacclima- tion the ABA content decreased with the exception of 12/20 °C-deacclimated plants and as a result, the highest ABA level in the end of deacclimation was observed in 2/12, 12/20 and 20/20 °C-deacclimated plants of winter cultivar. In the case of spring cultivar the highest level of ABA was observed af- ter cold deacclimation at 12/12 and 12/2 (Fig. 1 B) and 20/20 (Fig. 2B). Also the absolute values of ABA content there were higher than in winter cultivar. During cold reacclimation of winter plants

the highest ABA amounts were noticed in plants deacclimated at 2/12, 12/2 (Fig. IA) and 12/20, 20/i 2 (Fig. 2A) and only these values were slightly higher than those observed after cold acclimation. In spring plants ABA content after reacclimation was higher than in winter plants and in most cases close to the values observed before cold acclima- tion (Figs. 1B and 2B). The highest values were ob- served in plants deacclimated at 12/12, 12/2 (Fig. 1 B) and 20/20, 12/20 and 20/12 °C (Fig. 2B).

Changes in GA3 and GA-l ike substances content

In all experiments differences between treatments affirmed by means of bioassay (GA-like sub- stances) were convergent with those observed us- ing GA3 analysis with CE-MS system. In this con- nection the results of both analyses are described together as the results of GAs analysis.

In experiment 1 a lower level of GAs was observed in prehardened plants of winter oilseed-rape than in non-prehardened (Table 2). During cold acclima- tion the amounts of GAs decreased slightly irre- spective of growth conditions before cold acclima- tion.

During the first week of deacclimation of both the winter and spring cultivar, performed in experiment 2, an increase in GAs content was observed regard- less of the deacclimation regime (Fig. 3A,C). The GAs level observed in non-deacclimated plants (2/2 plants, the controls) was almost four times higher in spring than in winter plants. After reaccli- mat±on of winter plants GAs level reached the level of the non-deacclimated plants (2/2 °C) with the ex-

156

ABA, GIBBERELLINS AND FROST RESISTANCE OF RAPE

A.

./~ .......... i ....... ;

i

! B.

2 ~ 4 s s T s 9 1o Weeks

Condi~ons of growth during deacctimation ( 'C, day/night, 1241 photofleriod):

• - 0 - 1 2 / 1 2 - - ~ - 12/2 " -~- -2 /12 - - O - 2 / 2

Fig. 1. The effect of cold acclimation (2 °C), deacclimation and reacclimation (2 °C) on ABA content in leaves of winter (A) and spring (B) oil- seed rape (experiment 2). Before cold acclimation plants were grown at 12 °C. Each point is the mean (+ Se) of 3 independent ELISA measurements with 3 replicates for each of at least 2 samples (about 5 g FW collected from 4~6 plants each).

2 3 4 s s 7 o i 1o ~1 weeks

. . . . i i B I

. . . . J i . . . . •

Conditions of growlh during deaccltmation ( 'C, day/nigM, 12-h pholopedod):

- -0 -12 /12 --k ' - 12/20 --,~-.20/12 - 0 - 2 0 / 2 0

Fig. 2. The effect of cold acclimation (2 °C), deacclimation and reacclimation (2 °C) on ABA content in leaves of winter (A) and spring (B) oil- seed rape (experiment 3). Before cold acclimation plants were grown at 12 °C. Each point is the mean (-+ Se) of 3 independent ELISA measurements with 3 replicates for each of at least 2 samples (about 5 g FW collected from 4-6 plants each).

ception to 12/12 °C -deacclimated plants in which a slightly higher (of about 4 ng.g -1 DW) content was observed (Fig. 3B). During reacclimation of spring plants a further increase in GA content was ob- served in all plants including the control and the fi- nal concentrations were as follows: the highest in 12/12 °C -deacclim/lted plants, then in 2/12 and 12/2-deacclimated, and the lowest in the control (Fig. 3D).

Af ter 1 week of deacclimation (experiment 3) an elevated GAs content was observed in the winter cultivar at higher day-temperature: 20/12 and 20/20 (lower PSII excitation pressure) (Fig. 4A). The higher content of GAs was visible in these treat- ments even after reacclimation (Fig. 4B). During deacclimation at 12/12 and 12/20, the level of GAs was similar to those observed before cold acclima- tion (Fig. 4A, Table 2) and decreased back after fur- ther reacclimation (Fig. 4B). In spring plants the level of GAs increased during the first week of deacclimation about ten times more than in winter plants and the fastest increase was observed also in plants deacclimated at higher day-temperature (Fig. 4C). During reacclimation the content of GAs increased to a greater extend in 12/12 °C -deaccli- mated plants (Fig. 4D). GAs increased also in the

case of 12/20 °C -deacclimated plants, whereas in the case of 20/12 and 20/20 °C -deacclimated some decrease in GAs was observed, but also in these cases the content was about five-times higher than in winter plants (Fig. 4B,D).

Discussion

A B A a n d GAs as the factors regulating elongation growth rate in response to different PSH excita-

tion p r e s s u r e

The results of the present paper show that differ- ences in elongation growth rate during preharde- ning, i.e. growth before cold acclimation under high PSII excitation pressure, reported by Rapacz (1998 a,b,c), correlated with higher ABA and lower gibberellin-like substances contents. These results suggest that during seedling growth in early au- tumn, changes in hormonal balance leading to the cessation of growth (Kacperska-Palacz 1978) may be triggered by increasing PSII excitation pressure. The decrease in GA-like substances and the in- crease in ABA during this period were not con- nected with any detectable changes in water bal- ance (Rapacz 1998 c), which had been suggested so

157

M. RAPACZ, P. WALIGORSKI & F. JANOWIAK

After subsequent cold Directly after cold deacclimation reacclimation at 2 *C (3 weeks)

300 C . Spdng cultivsr

o m . ~ . m . E B • , ,

Coadl~o~s of deacclimat~on (*C - daylight)

Fig. 3. The content of GA-like substances and GA3 in leaves of winter (A, B) and spring (C, D) oilseed rape after the first week of deacclimation (A, C) and after cold reacclimation (B, D) as affected by conditions of deacclimation (experiment 2). Be- fore cold acclimation plants were grown at 12 °C. Each point is the mean (_+ Se) of 5 measurements (lettuce hypocotyl bioassay of GA-like substances) or of 3 analyses (CE-MS analysis of GA3) for each of 3 samples (about 5 g FW collected from 4-6 plants each).

far as the possible triggering factor (Kacperska- Palacz 1978).

Changes in ABA content observed during deaccli- mation and reacclimation were distinct from those associated with both elongation rate and adaptation of photosynthetic apparatus to cold reported previ- ously (Rapacz 2002 a,b, Rapacz and Hura 2002). A general increase in ABA content was observed dur- ing deacclimation, confirming the observations of Dt~rffling et al. (1990). However, in the spring cultivar, the highest ABA concentrations after reacclimation occurred in plants with more rapid flower initiation and flowering (experiment 3: 20/12 and 12/20 °C-deacclimated plants), whereas the slowest elongation rate was measured in 12/20 and 12/12 °C -deacclimated plants (see also Rapacz 2002 b). This may be caused, for instance, by accel- erated leaf senescence in faster growing oilseed rape, which was reported previously (Rapacz 1998 c). Similar relationships were also observed in ex- periment 2.

O n the other hand, changes in both elongation growth rate and frost resistance reported previously in plants deacclimated under the same conditions (Rapacz 2002 a,b) were very similar to those re- ported here in the content of GAs. Plants with higher foliar GAs concentrations were not only faster-growing but they deacclimated more rapidly,

Directly after cold deacdimation

: t A . ~h~ cul~var 5o [ 17 GA-~e substances

.

After subsequent cold reacclimation at 2 *C (4 weeks)

~ i . ~ .

Conailions ot deaCclim~on ('C - dayknight)

Fig. 4. The content of GA-like substances and GA3 in leaves of winter (A, B) and spring (C, D) oilseed rape after the first week of deacclimation (A, C) and after cold reacclimation (B, D) as affected by conditions of deacclimation (experiment 3). Be- fore cold acclimation plants were grown at 12 °C. Each point is the mean (_+ Se) of 5 measurements (lettuce hypocotyl bioassay of GA-like substances) or of 3 analyses (CE-MS analysis of GA3) for each of 3 samples (about 5 g FW collected from 4-6 plants each).

were unable to adapt the photosynthetic apparatus to cold and they had lower capacity to reacclimate (Rapacz 2002 a,b). The changes in GAs content during the first week of deacclimation and during reacclimation may be triggered by both PSII excita- tion pressure during deacclimation (experiment 3), the beginning of generative development in spring plants (experiment 2, including the control) and also by flower bud formation and development (ex- periment 3, spring oilseed-rape). The question how does PSII excitation pressure regulate GA biosyn- thesis requires further investigations. Similarly as during prehardening, observed alterations to GAs content were not triggered by changes in water bal- ance of the plants (Rapacz 2002 b). Changes in GAs content during generative development in spring rape were similar to those observed by Rood et al.

(1989); a decrease in GAs content was observed at the time of flower bud formation and early flower- ing (12/20, 20/12 and 20/20 °C-deacclimated plants of spring rape after reacclimation). At the same time a decrease in elongation growth rate, an in- crease in cold acclimation abilities and better ad- justment of photosynthetic apparatus to cold were observed previously (Rapacz 2002 a). During pro- longed cold-stay of spring rape (experiment 2, the control) an increasing GAs content was also ac- companied by a decrease in frost resistance and photosynthetic activity in cold as well as by an in-

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ABA, GIBBERELLINS AND FROST RESISTANCE OF RAPE

crease in elongation growth rate (Rapacz et al.

2001).

The possible role o f A B A level during cold accli-

mat ion

The present results suggest that ABA concentra- tion has little impact in regulation of both frost re- sistance and acclimation of photosynthetic appara- tus to cold during cold deacclimation and reaccli- mation.

A B A is considered to be a main growth regulator involved in cold acclimation. Recent studies show that, at least in the model plant Arab idops i s

thaliana, cold- and ABA-triggered signal trans- duction pathways run partly parallel and partly in- tersect (Pearce 1999, Shinozaki et al. 2000). Thus ABA is considered as an alternative factor affecting expression of some but not all cold-regulated genes. In some cases, both cold and ABA are needed for full expression (Knight and Knight 2001). On the other hand, ABA protects the photosynthetic apparatus against cold-induced photoinactivation (Rapacz 2002 c) and decreases the rate of elongation growth (Kende and Zeevart 1997). Both actions are also required for proper cold acclimation (Levitt 1972, Huner et al. 1993, Rapacz 1998 a,b,c, Rapacz and Janowiak 1998). The signalling role of ABA during cold-acclima- tion seems to be connected with a transient increase in ABA content observed in a few hours or days af- ter temperature change (Drrffiing et al. 1990, Smolefiska-Sym et al. 1995). Such increases may be involved in low-temperature gene expression and connected with calcium signalling (Knight and Knight 2001, Plieth 2001). It is also possible that a triggering factor for the observed increase in ABA content may be the decrease in osmotic potential re- ported after transfer to cold-acclimating tempera- tures (Drrffling et al. 1990, Smolefiska-Sym et al. 1995). Further acclimation results in a decrease in ABA content, which was observed also in the pres- ent study (Drrffling et al. 1990, Dallaire et al. 1994, Smolefiska-Sym et al. 1995).

Some role of higher ABA concentrations may be proposed for prehardening. The elevated ABA level during this period may lead both to a modifi- cation of the growth pattern during prehardening

and subsequent cold acclimation and may also con- tribute to lower susceptibility of PSII to photoin- duced inactivation observed in prehardened plants (Rapacz and Janowiak 1998, Rapacz 2002 c).

The possible role of gibbereUins level during cold accl imation

Observed changes in elongation growth rate, which are closely related to changes in both frost re- sistance and acclimation of photosynthetic appara- tus to cold during prehardening, cold acclimation, deacclimation and reacclimation (Rapacz 2002 a,b, Rapacz and Hura 2002), seem to be closely related to the content of GAs observed in the present exper- iments. GAs have been earlier reported to increase stress susceptibility of plants (Vettakkorumakan- kav et al. 1999). These authors suggested that GAs might disturb the ABA function as a stress hor- mone, i.e. as a factor affecting gene expression. Ac- cording to our results, in the case of cold acclima- tion/deacclimation GAs probably act antagonisti- cally to ABA mainly in the regulation of elongation growth and not in the signalling role of ABA. GAs accelerating elongation growth rate triggers the loss of cold acclimation abilities caused by repro- gramming of plant development from cold accli- mation to growth (Rapacz and Chilmonik 2000, Rapacz et al. 2001, Rapacz 2002b). Previous re- suits suggest also that treatment with exogenous GA 3 may also disturb cold acclimation increasing the susceptibility of the photosynthetic apparatus to cold-induced photoinactivation (Rapacz 2002 c).

It must also be mentioned that not only ABA and GAs may be involved in the processes studied in the present paper. For instance, plant growth regulators other than GAs, mainly cytokinins are involved in the formation of generative organs (Rood et al. 1988, Chang et al. 1999) and cytokinins together with ABA also seems to be involved as antagonists in controlling cold acclimation under field condi- tions as reported for winter wheat (Taylor et aL

1989).

Acknowledgements

This research was supported by Polish State Com- mittee for Scientific Research (KBN) grant 5 P06A 032 18. The technical support of Dorota Kowalew-

159

M. RAPACZ, P. WALIG()RSK1 & F. JANOWIAK

ska-Rapacz is gratefully acknowledged. CE-MS analyses were possible by courtesy of Dr. Jan Pietrag (Academy of National Defense, Warsaw, Poland).

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Received August 11, 2002; accepted December 01, 2002 edited by: W. Filek

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