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Phenylpropanoid deficiency affects the course of plant acclimation to
cold
Danuta Solecka and Alina Kacperska*
Department of Plant Resistance, Institute of Plant Experimental Biology, Warsaw University, Miecznikowa 1, 02–096 Warsaw, Poland*Corresponding author, e-mail: [email protected]
Received 21 March 2003; revised 14 April 2003
The effects of phenylpropanoid deficiency on plant growth,
photosynthetic efficiency of the photosystem II and freezingtolerance of leaves were studied during acclimation of winter
oilseed rape plants (Brassica napus L. var. oleifera L. cv
Jantar) at low temperature. Application of 2-amino-
2-indanophosphonic acid inhibited phenylalanine ammonia-lyase (E.C. 4.3.1.5) activity by about 90%. This was followed
by a marked reduction of soluble phenolics (in particular
hydroxycinnamic acids) and anthocyanins in leaves. Inhibition
of the cold-promoted incorporation of ferulic acid into cell
walls was also observed. The reduction of phenylpropanoid
contents was associated with: (1) partial abrogation of thecold-induced growth effects, such as inhibition of leaf fresh
weight increments and accumulation of dry matter, proteins
and cell walls; (2) decreased photochemical efficiency of
photosystem II in low temperature-affected leaves; and (3)decreased ability of leaves to develop tolerance to the extra-
cellular formation of ice. These findings are discussed in terms
of phenylpropanoids’ role in plant responses to cold (> 0�C)
and freezing temperatures.
Introduction
Stress factors increase the activity of phenylalanineammonia-lyase (PAL, E.C. 4.3.1.5), a key enzyme inphenylpropanoid biosynthesis (Hahlbrock and Scheel1989). This effect is due to the stress-promoted transcrip-tion or translation of PAL protein (e.g. Bolwell et al.1986, Bolwell 1992, Durner et al. 1998) or to its slowedturnover (Crosby and Vayda 1991). As a consequence ofthe increased PAL activity, phenylpropanoid derivativesaccumulate in the stress-affected tissues and are thoughtto protect plants against various biotic and certain abio-tic stressors (Dixon and Paiva 1995). Phenylpropanoidderivatives serve a specific role in pathogen defence,antiherbivory, ultraviolet screening, energy dissipationand radical scavenging, as well as structural componentsof cell walls (Grace and Logan 2000). The increase inPAL activity and accumulation of phenylpropanoidswere also observed in plants subjected to low non-
freezing temperatures (e.g. Chalker-Scott et al. 1989,Parra et al. 1990, Christie et al. 1994, Leyva et al. 1995,Solecka and Kacperska 1995, Solecka et al. 1999, Janaset al. 2000, 2002). However, the role played by phenolicsin low temperature-affected cells is far from being under-stood: depending on the tissue studied, phenolics havebeen proposed to be involved in cell wall reinforcement(Chalker-Scott et al. 1989), in cell protection againstexcess of radiation (Christie et al. 1994) or against oxi-dative stress (Grace and Logan 2000). In previous studies(Solecka and Kacperska 1995, Solecka et al. 1999) wehave shown that the increase of PAL activity as well asthe rate of accumulation of different phenolics in winteroilseed rape leaves depended on the range of lowtemperatures to which the plants were subjected: a brieffreezing treatment was more effective than extendedexposure of plants to chilling (. 0�C). As a result of
PHYSIOLOGIA PLANTARUM 119: 253–262. 2003 Copyright# Physiologia Plantarum 2003
Printed in Denmark – all rights reserved ISSN 0031-9317
Abbreviations – AIP, 2-amino-2-indanophosphonic acid; AOS, active oxygen species; CAn, cold-acclimated leaves sampled after 2, 8, 21, 23 or28 days of cold acclimation; CA21Fn, cold-acclimated leaves sampled 1, 2 or 7 days after freezing; DTT, dithiothreitol; DM, dry matter; F0, Fv,Fm, initial, variable and maximum fluorescence of photosystem II, respectively; FM, fresh matter; GC-MS, gas chromatography–massspectrometry; HCA, hydroxycinnamic acids; HPLC, high-pressure liquid chromatography; IAA, indole-3-acetic acid; LT50, the temperatureof 50% injury; NAn, non-acclimated leaves sampled after 2, 8, 21, or 28 days of growth; PAL, phenylalanine ammonia-lyase; PAR,photosyntetically active radiation; PMSF, phenylmethylsulphofluoride; PSII, photosystem II.
Physiol. Plant. 119, 2003 253
these observations we proposed that the role of phenyl-propanoids might differ in the first and the second stageof plant acclimation to low temperature (Solecka et al.1999). The first stage, occurring at temperatures above0�C, leads to growth and metabolic adjustments to coldand to some increase in freezing resistance. The secondstage, induced by brief sublethal freezing, results mainlyin increased cell tolerance to freezing (Kacperska 1989).
The aim of the present work was to determine the roleof phenylpropanoids in the first and the second stages ofplant acclimation to low temperature. We have examinedthe effects of PAL inhibition on the content and comp-osition of phenylpropanoids in the course of leaf growthunder acclimating and non-acclimating conditions andstudied the consequences of the reduced phenylpropa-noid levels for low temperature-dependent modificationsin growth, photosynthetic efficiency of photosystem II(PSII) and freezing tolerance of leaves. To inhibit PALwe used 2-amino-2-indanophosphonic acid (AIP), thecompetitive inhibitor of the enzyme (Zon and Amrhein1992). The inhibitor appeared to be a powerful tool instudies of the functional aspects and the biologicalimportance of phenylpropanoids in different plant sys-tems (Reuber et al. 1993, Chen et al. 1998, Gitz et al.1998, Cvikrova et al. 1999, Janas et al. 2002).
Materials and methods
Plant material and cold-acclimation conditions
Winter oilseed rape plants (Brassica napus L. var. oleiferaL. cv Jantar) were grown for 21 days in a mixture of sandand peat (1 : 1, v/v) under a 16-h photoperiod and 20/15�C (day/night) temperatures for 3 weeks, as describedpreviously (Maciejewska and Kacperska 1987). The levelof photosynthetically active radiation was approximately200mmol m�2 s�1. After 3 weeks the plants were exposedto different temperature treatments (Fig. 1, the ‘0’ day ofthe experiment). Half of the plants were continuouslygrown at 20/15�C (non-acclimated plants, NA) whereasthe other half were transferred to an acclimation cham-ber and kept at 2 (±1)�C for 4 weeks with the lightconditions unchanged (cold-acclimated plants, CA).After 3 weeks of cold acclimation, some of the CA plantswere subjected to freezing at �5�C for 18 h in darkness ina freezing chamber (Dual Program Illuminated Incuba-tor 818; Precision Scientific, Dublin, OH, USA) andallowed to recover at 2�C for 6 h in darkness (CA21F1
plants). This treatment increases the freezing tolerance ofCA leaves by 3�C and increases the ability of leaf bladeto expand (Kacperska and Kulesza 1987). Then, thefrost-pretreated plants were grown under a 16-h photo-period at 2�C for a further 1 (CA21F2 plants) or 7 days(CA21F7 plants).
At the ‘0’ day of the experiment, 3-week-old plantswere fed AIP solution through debladed petioles of thethird leaves from the plant base (Fig. 1, filled symbols).The petioles were placed in the tubes containing 30 mMAIP solution or distilled water (control). In preliminary
experiments this dose was found most effective in inhi-biting PAL activity and showed no toxic effects onpetiole functioning. During the next 28 days the volumesof liquids in the tubes were measured each 2–3 days andthe losses were made up with the appropriate freshmedium. All types of plants (NA, CA and CAF) weresubjected to inhibitor treatment.
All analyses were performed on young blades of thefourth leaf, counted from the plant base, full expansionof which was taking place at different temperatures butequal levels of PAR. Preliminary experiments showedthat low temperature- or inhibitor-induced modificationsobserved in the blades of the fourth leaves representadequately those noted in other plant tissues.
Leaf blades, trimmed of main and lateral veins, weresampled from the plants and used fresh to determinatePAL activity, photochemical efficiency and freezing tol-erance or they were immediately frozen in liquid nitrogenand used later to extract and identify soluble phenolicsand anthocyanins.
Growth characteristics
At each sampling day the fresh weight of the leaf and thecontents of dry matter (1 g of leaf blades dried at 105�C for2 h), cell walls, and proteins (see below) in the tissues weredetermined. All these parameters were taken as indicativeof the treatment-dependent modifications in leaf growth.
Soluble protein extraction
The leaf blade samples (1 g FM each) were homogenized in0.1M Tris-HCl buffer (pH 7.2), containing 1 mM PMSFand 1 mM DTT. Extracts were centrifuged at 14 000� g for20 min and the supernatants were used for protein andenzyme assays. All procedures were carried out at 4�C.
–5
–10
0
5
10
15
20
25
0 14 21 287
Days
Tem
pera
ture
(°C
)
NA
CA
CAF
NA + AIP
CA + AIP
CAF + AIP
Fig. 1. Scheme of the experimental design used in the present work.At the day ‘0’, 3-week-old plants were subjected to differenttreatments, depicted by the symbols: (*), non-acclimated (NA)plants; (&), cold-acclimated (CA) plants; broken lines, plantssubjected to a transient freezing; filled symbols, plants treated withAIP.
254 Physiol. Plant. 119, 2003
Protein concentration in crude extracts was determined bythe method of Bradford (1976) using bovine serum albumin(Sigma, Munich, Germany) as a standard.
Cell wall preparation
Preparation of cell wall was performed according toMacheix (1974), as described previously (Solecka et al.1999). The amount of cell walls, obtained after four-stepextraction of leaf samples (3 g FM each), was determinedby weighing after air-drying.
Determination of chlorophyll fluorescence for estimation
of PSII efficiency
Chlorophyll a fluorescence was measured with PSMMark II Plant Stress Meter (BioStress Monitor SCI;AB, Umea, Sweden) as described by Oquist and Wass(1988). The NA, CA and CAF plants were dark adaptedfor 1 h at 20�C, after which the initial fluorescence (F0)and maximum (Fm) fluorescence from PSII was deter-mined using an actinic photon flux density of400mmol m�2 s�1. The variable fluorescence (Fv) wascalculated as Fm�F0. The Fv/Fm ratio indicates themaximal photochemical efficiency of PSII. In accordancewith Oquist and Wass (1988), similar values of Fv/Fm
were obtained using actinic irradiance ranging from 200to 400 mmol m�2 s�1. All measurements (at least 40 foreach treatment) were performed on the apical parts ofthe leaves attached to plants.
Determination of freezing tolerance
The frost killing temperature (LT50) was determinedaccording to Kacperska and Kulesza (1987). Leaf discs(15 mm diameter) were frozen in cryostats at desiredtemperatures for 2 h (cooling rate 0.2�C min�1). Onepart of the sample was seeded with ice at �1�C toprovoke extracellular ice formation. The other part wascooled in the absence of exogenous ice to allow somesupercooling, which then resulted in a sudden freezing ofwater at temperatures below the threshold supercoolingtemperature (Sakai and Larcher 1987). After freezing,leaf samples were thawed (at the rate of 0.5�C min�1)and allowed to recover from reversible injury for 20 hat 12�C in darkness. Next, leakage of intracellular elec-trolytes from the discs was determined using electricconductivity (N 572 conductometer; MERA ELWRO,Poland) and taken as a measure of freezing injury. Theindex of injury was calculated according to Flint et al.(1967) and plotted as a function of temperature to esti-mation of the LT50. All measurements were performedwith 10 replicates for each freezing temperature, eachreplicate consisted of 10 leaf discs sampled at random.
Determination of PAL activity
The reaction mixture (3 ml) contained 0.016 mML-phenylalanine, 0.15 mM borate buffer (pH 8.8), 3.6 mM
NaCl and 0.5 mg of extracted proteins. Incubation wasperformed at 37�C for 3 h. Production of cinnamicacid was measured as an increase in absorbancy at290 nm (Havir and Hanson 1968). The enzyme activitywas calculated per unit weight of the dry matter orprotein.
Preparation of soluble and wall-bound phenolics
Preparation of soluble and wall-bound phenolics wasperformed according to Macheix (1974), as describedearlier (Solecka et al. 1999). The cytoplasmic fractionwas subjected to acid hydrolysis (for determination oftotal phenolics) or to alkaline hydrolysis (for determina-tion of esterified forms of phenolics). Cell wall-boundphenolics were liberated from cell wall preparations byalkaline hydrolysis. The phenolic contents were deter-mined spectrophotometrically at 750 nm, using theFolin method (Forrest and Bendall 1969) and ferulicacid (Sigma) as a standard. Total lignin content in cellwall preparations was determined spectrophotometri-cally at 280 nm (as a complex with acetyl bromide),according to Johnson et al. (1961).
Separation and determination of phenolic acid contents
Free phenolic acids in soluble and wall-bound phenolicfractions were separated by HPLC (Solecka et al. 1999).Phenolic acids were detected at 330 nm using a VWM2141 (Pharmacia LKB, Uppsala, Sweden) spectrophoto-meter detector and identified by comparison with authenticstandards (Sigma). The contents of phenolic acids weredetermined from the peak areas of each compound usingCHROMA (v. 3.2, POL-LAB, Warsaw, Poland) computerintegrator. Identification of phenolic acids was previouslyconfirmed (Solecka et al. 1999) by GC–MS analysis, usingauthentic standards (Sigma).
Anthocyanin extraction
Anthocyanins were extracted from frozen leaf samples(1 g FM each) with 1% HCl in methanol, centrifuged at10 000� g for 20 min and determined spectrophoto-metrically at 570 nm, as described by Mori and Sakurai(1995).
Statistics
All experiments were repeated at least three times. Threeindependent samples were collected at each samplingday, unless indicated otherwise. The presented valuesare the means of at least nine determinations ±SD. Theeffects of the treatments were tested by one-way analysisof variance (ANOVA). Means were compared between thetreatments by least significant difference (LSD) at the 0.05probability level using Tukey’s test. Means marked withthe same letter do not differ significantly at this prob-ability level.
Physiol. Plant. 119, 2003 255
Results
PAL activity and phenylpropanoid contents as affected the
AIP treatment
The rate of AIP uptake by the whole plant was higher inNA than in CA plants (data not shown). However,2 days after the beginning of the experiment the contentsof inhibitor in NA and CA plants reached similar level ifcalculated per dry matter unit (about 200 nmol g�1 DM).
One week after the beginning of the experiment, thetotal PAL activity in the AIP-treated plants decreased byabout 80% in both NA and CA leaves, compared to therespective controls (Fig. 2). Specific activity of theenzyme decreased by about 90% in both NA and CAleaves during the first week of the treatment (data notshown). The administration of AIP eliminated the frost-induced increase of the PAL activity (Fig. 2).
Treatment with the inhibitor resulted in a pronounced,time-dependent decrease of phenolics in NA and CAleaves, to a level about three times lower than that inthe respective controls (Fig. 3A). It also prevented thefreezing-induced accumulation of phenylpropanoids inCA leaves. Accumulation of soluble hydroxycinnamicacids in CA leaves was also inhibited by the administra-tion of AIP (Fig. 3B). The comparison of changes in totalsoluble phenolic contents with those in total solubleHCA contents, indicate that AIP diminished thefreezing-induced accumulation of phenolics other thanhydroxycinnamic acids (compare the respective data inFig. 3 A and 3B). The inhibitor treatment also resulted ina small decrease of anthocyanins’ content in NA leaves(Fig. 3C) and effectively prevented the accumulation ofanthocyanins in CA tissues. The anthocyanin level inthese leaves was not affected by the transient freezingor by inhibitor.
Lignin content in the tissues, deprived of the mainveins was very low (about 18 mg g�1 cell wall) and wasnot affected by cold acclimation, freezing treatment orthe application of AIP (data not shown).
HPLC analysis revealed four hydroxycinnamic acidsin the soluble phenolic fractions (Fig. 4). The inhibitortreatment decreased HCA contents in NA tissues and
00
a
a
b
c
a
40
30
20
10
14 21 287
Days
PA
L ac
tivity
(nm
ol g
–1 D
M)
Fig. 2. Changes in PAL activity during plant growth at differenttemperatures: (*), non-acclimated (NA) plants; (&), cold-acclimated (CA) plants; broken lines, plants subjected to atransient freezing; filled symbols, plants treated with AIP. Barsindicate±SD.
0
1
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Sol
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nolic
s co
nten
t (m
g g–1
DM
)H
CA
con
tent
(m
g g–1
DM
)A
ntho
cyan
in c
onte
nt (
mg
g–1 D
M)
16
12
0 7 14 21 28
Days
Fig. 3. Changes in soluble phenolic (A), hydroxycinnamic acid (B)and anthocyanin contents in leaves during plant growth at differenttemperatures: (*), non-acclimated (NA) plants; (&), cold-acclimated (CA) plants; broken lines, plants subjected to atransient freezing; filled symbols, plants treated with AIP. Barsindicate±SD.
256 Physiol. Plant. 119, 2003
effectively prevented the accumulation of HCAs in CAand CAF leaves. In CA leaves, the most pronounced AIPeffects were observed for ferulic, sinapic and p-coumaricacids, all of which had very high accumulation rates. Theinhibitor prevented cold- and freeze-dependent esterifica-tion of phenolic acids with the content of the esterifiedforms remaining at 20–60%, depending on the acid,whereas in the non-treated tissues, 100% of HCAs weresubjected to esterification (Fig. 4).
Among the hydroxycinnamic acids, only ferulic acidwas found in cell walls. Its content remained unchangedin cell walls from NA tissues, increased by about 25% inCA leaves and transiently decreased in CA tissuesexposed to a short freezing event (Table 1). The inhibitoreliminated the cold-dependent accumulation of ferulicacid in the cell walls.
Physiological consequences of phenylpropanoid deficiency
In NA plants, the fresh weight of leaves increased six-foldduring the 4-week experiment (Fig. 5A). Cold acclimationinhibited leaf fresh weight increase, and this effect wasreversed to a large extent by application of AIP(Fig. 5A). However, in CA leaves there was a significantaccumulation of dry matter, which was reduced by treat-ment with AIP (Fig. 5B). In NA leaves the inhibitor had noeffect on the dry matter content, which decreased by about30% in the course of the experiment.
The 4-week acclimation of plants in cold caused three-and two-fold increase in protein and cell wall contents,
respectively (Fig. 6A and B). The cold-dependent accu-mulation of these compounds was markedly inhibited byAIP. In NA leaves there was no accumulation of pro-teins, and no increase in cell wall contents. No effects ofAIP on these parameters were observed.
The determination of chlorophyll fluorescence para-meters revealed that photochemical efficiency of PSII, asindicated by the Fv/Fm ratio, was equally high in NA28
and CA28 leaves (Fig. 7A). However, the ratio markedlydecreased 2 days after the beginning of the cold treat-ment. The effect was associated with an increase in F0
(Fig. 7B). Transient freezing decreased markedly the Fv/Fm ratio but the system was able to recover 7 days afterthe treatment. The administration of AIP prevented PSIIrecovery in leaves subjected to acclimation in cold formore than 2 weeks and in the frost–thawed CA tissues. It
600
p-coumaric acid (pCA) caffeic acid (CA)
ferulic acid (FA)
Treatments Treatments
sinapic acid (SA)
500
400
300
200
100
0
600
500
400
300
200
100
0
NA0 NA28 CA28 CAF1 CAF7
NA0 NA28 CA28 CAF1 CAF7
600
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100
0NA0 NA28 CA28 CAF1 CAF7
600
500
400
300
200
100
0NA0 NA28 CA28 CAF1 CAF7
pCA
con
tent
(%
)F
A c
onte
nt (
%)
CA
con
tent
(%
)S
A c
onte
nt (
%)
25 25 3525
50
35
80
35
100
45
50 6045
95
100
100
40 45 45
55 60
45
90
45
100
60
100
60
25
20
40
25
65
30
90
35
Fig. 4. Changes in the contents ofparticular hydroxycinnamic acidsin the course of plant growth atdifferent temperature inpercentage of their contentsregistered at the at the day ‘0’ ofthe experiment, taken as 100%.At that time, the contents ofpCA, CA, FA and SA were 154,150, 175, and 312mg g�1,respectively. Open and filledcolumns indicate phenolic acidscontents in the leaves that wereeither non-treated or treated withAIP, respectively. Numbersabove the columns show thepercentage of the esterified formin the total pool of the indicatedphenolic acid. Bars indicate±SD.
Table 1. The content of cell wall-bound ferulic acid in NA and CAleaves. Means marked with the various letters differ significantly at0.05 probability level.
Ferulic acid (mg g�1 cell wall)
Plant –AIP 1AIP
NA0 13.6a –NA21 14.0a 13.2a
NA28 14.3a 13.4a
CA8 14.2a 13.9a
CA21 16.3b 14.1a
CA28 16.9b 14.2a
CA21F1 14.7a 13.8a
CA21F2 15.9b 14.1a
CA21F7 16.7b 14.0a
Physiol. Plant. 119, 2003 257
did not affect PSII photochemical efficiency in NAleaves.
In NA plants, freezing tolerance of leaves was main-tained at a low level (LT50 value¼�4�C) and was notaffected by the inhibitor (Fig. 8). In CA plants, toleranceof leaves to extracellular ice (determined in the presenceof ice crystals seeded on the samples, see Methods)increased by about 5�C (Fig. 8A). A further increase of3�C was noted in leaves of CA plants exposed to brieffreezing at �5�C. The administration of AIP reduced theeffectiveness of the cold-dependent step in the develop-ment of freezing tolerance and had practically no effecton the frost-dependent one. When freezing tests wereperformed in the absence of extracellular ice, the inhibi-tor had no effect on development of freezing tolerance inCA leaves (Fig. 8B).
Discussion
Low temperature- and AIP-induced modifications in PAL
activity and phenylpropanoid metabolism
The results of these experiments are in line with ourearlier observations (Solecka and Kacperska 1995,
Solecka et al. 1999) which indicates that induction ofphenylalanine ammonia-lyase activity and the accumula-tion of soluble phenolics, hydroxycinnamic acids andanthocyanins in leaves of chilling-resistant plantsdepends on the range of temperature to which theseplants are exposed. AIP appeared to be a very effectiveinhibitor of PAL. It reduced the enzyme activity byabout 80%, in both NA and CA leaves. In CA tissuesAIP almost completely eliminated the frost-dependentincrease of PAL activity (Fig. 2). Application of the inhibi-tor allowed us to minimize problems related to stresseffects on the synthesis and turnover of PAL protein (e.g.Manvandad et al. 1990) and we did not encounter diffi-culties related to a large variation in PAL and phenyl-propanoid levels that have been observed in transgenicPAL-silenced lines (Korth et al. 2001). Inhibition of PALwas followed by: (1) a marked reduction of soluble phen-olic contents in both NA and CA leaves and inhibitionof freeze-induced accumulation of soluble phenolics inCA tissues (Fig. 3A); (2) decreased amounts of solubleHCAs in NA and CA leaves (Fig. 3B); (3) inhibition ofthe freeze-induced accumulation and esterification ofHCAs (Fig. 4); (d) inhibition of cold-promoted ferulicacid incorporation into cell walls (Table 1); (5) inhibition
0
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10
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b
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00
a
c
b
B
Leaf
fres
h w
eigh
t (g)
200
150
100
50
Dry
mat
ter
cont
ent (
mg
g–1 F
M)
Days
7 14 21 28
Fig. 5. Changes in fresh weight of a leaf (A) and dry matter content(B) during plant growth at different temperatures: (*), non-acclimated (NA) plants; (&), cold-acclimated (CA) plants; brokenlines, plants subjected to a transient freezing; filled symbols, plantstreated with AIP. Bars indicate±SD.
014 21 2870
B
a
a
b
c
0
A
a
b
c
Pro
tein
con
tent
(m
g g–1
DM
)C
ell w
all c
onte
nt (
mg
g–1 D
M)
Days
300
200
100
300
400
500
600
200
100
Fig. 6. Changes in protein (A) and cell wall (B) contents duringplant growth at different temperatures: (*), non-acclimated (NA)plants; (&), cold-acclimated (CA) plants; broken lines, plantssubjected to a transient freezing; filled symbols, plants treated withAIP. Bars indicate±SD.
258 Physiol. Plant. 119, 2003
of cold-dependent accumulation of anthocyanins(Fig. 3C). These results are in agreement with the obser-vations of other investigators who found that treatmentwith AIP reduced anthocyanins in buckwheat and redcabbage (Zon and Amrhein 1992, Gitz et al. 1998),reduced HCAs esters, flavonoids and anthocyanins inrye and soybean roots (Reuber et al. 1993, Janas et al.2000, 2002), and reduced the wall-bound HCAs in maizeand alfalfa cell suspension cultures (Grabber et al. 1995,Cvikrova et al. 1999, Hrubcova et al. 2000) and in radishseedlings (Chen et al. 1998).
Physiological effects of the AIP-induced phenylpropanoid
deficiency in non-acclimated and cold-acclimated winter
oilseed rape leaves
The AIP-dependent reduction in phenylpropanoids inwinter oilseed rape leaves was associated with: (1) apartial abrogation of the commonly observed cold-induced growth effects, such as inhibition of leaf growth(Fig. 5), accumulation of soluble proteins and increased
cell wall content (Fig. 6); (2) decreased PSII photochem-ical efficiency in the cold-acclimated and in the freeze-pretreated leaves (Fig. 7); and (3) a decreased ability ofleaves to tolerate extracellular freezing (Fig. 8A).
The slight acceleration of growth by AIP was pre-viously noted for cotyledons of red cabbage seedlings(Gitz et al. 1998) and hypocotyls of radish seedlings(Chen et al. 1998). Complex modifications of leaf growthand development were also observed in transgenictobacco plants with repressed hydroxycinnamic andmonolignol metabolism (Tamagnone et al. 1998) orwith silenced PAL gene (Korth et al. 2001). It seemsthat independently of the phenolics’ role in control ofIAA (Lee et al. 1980, Brown et al. 2001) or ethylene(Cvikrova et al. 1999) levels, the growth-promoting effectof the inhibitor may be due to the prevention of ferulicacid accumulation in the cell walls of cold-acclimatedleaves (Table 1). Feryolation of matrix polysaccharides(pectins and hemicelluloses) is known to increase cellwall stiffness and limits cell wall extensibility (Fry 1986,Grabber et al. 1995). The cold-dependent inhibition ofcell growth is commonly associated with increased accu-mulation of proteins (Kacperska-Palacz et al. 1977, Guy
00 14 21 28
B
a
b
aa
0
1A
b
aa
c
0.8
0.6
0.4
0.4
0.3
0.2
0.1
0.2
Days
7
F0
Fv /F
m
Fig. 7. Changes in apparent quantum yield (Fv/Fm, A) and theinitial fluorescence (F0, B) in leaves subjected to differenttemperature and inhibitor treatments: (*), non-acclimated (NA)plants; (&), cold-acclimated (CA) plants; broken lines, plantssubjected to a transient freezing; filled symbols, plants treated withAIP. Bars indicate±SD.
0
ab
c
d
0
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A
–14
–12
–10
–8
–6
–4
–2
–14
–12
–10
–8
–6
–4
–2
LT50
(°C
)LT
50 (
°C)
Days
14 21 287
Fig. 8. Changes in leaf tolerance to freezing (expressed as LT50)estimated in the presence (A) and absence (B) of extracellular ice.(*), non-acclimated (NA) plants; (&), cold-acclimated (CA)plants; broken lines, plants subjected to a transient freezing; filledsymbols, plants treated with AIP.
Physiol. Plant. 119, 2003 259
1990). Therefore, the decreased accumulation of proteinsin the AIP-treated cold-acclimated leaves (Fig. 4A) seemsto be the consequence of the growth-promoting effect ofthe inhibitor. The possibility that the increased free phe-nylalanine pool in the PAL-inhibited cells will promoteprotein accumulation in CA leaves is unlikely becauseonly 10–15% of phenylalanine is used for protein synth-esis (Hall and Yeoman 1991).
Independently of the involvement of phenolic com-pounds in the control of leaf growth, the light screeningand/or antioxidant properties of these compounds (Rice-Evans et al. 1997) should be also taken into considera-tion. In leaves grown in cold under moderate lightintensity, the energy imbalance sensed by increasedPSII excitation pressure (Huner et al. 1996) leads tophotoinhibition (Osmond 1994). This, in turn, may resultin the formation of damaging reactive oxygen species(Asada 1996). In the present study, photoinhibition isseen as a decrease in the ratio of variable to maximumfluorescence (Fv/Fm) and increased initial fluorescence(F0) in leaves that were either exposed to cold for1 week or pretreated with frost (Fig. 7A and B). Theincreased F0 may indicate an impaired energy trappingefficiency in the PSII reaction centres or a partial dis-connection of the antennae from the centres (Somersaloand Krause 1990). In agreement with the results ofothers (Somersalo and Krause 1990, Maciejewska andBauer 1992), cold-acclimated leaves were observed torecover from the cold- and freezing-induced photo-inhibition (Fig. 5). However, recovery was inhibited inAIP-treated leaves. This observation suggests that phenyl-propanoids protect PSII in cold-acclimated leavesagainst light-induced damage. The compounds mayserve as screening agents, direct radical scavengers or asreducing substrates for guaiacol peroxidase, and the phenyl-propanoid pathway may provide an alternative routefor photon use under conditions of carbohydrate accu-mulation and/or excess light absorption (Grace andLogan 2000). The comparison of cold-, frost- and AIP-induced changes in the levels of different phenylpropa-noids (Fig. 3) indicates that protection of PSII during thefirst (cold-dependent) and the second (frost-induced)stage of plant acclimation may be due to different phen-olic compounds. The observation that the inhibition ofanthocyanins accumulation in AIP-treated cold-grownleaves (Fig. 3C) resulted in the loss of recovery fromphotoinhibition (Fig. 7) indicates that during the firststage of plant acclimation to low temperature anthocya-nins may play a prominent role serving as screeningpigments (Chalker-Scott 1999) or antioxidants(Yamasaki et al. 1996). Their rapid synthesis may alsoprovide an additional carbon sink under excess lightconditions (Grace and Logan 2000). The link betweenphotosynthesis and phenolic metabolism has been shownfor Arabidopsis mutants in which the import of phospho-enolpyruvate into the chloroplasts is blocked (Streatfieldet al. 1999). In the mutants, anthocyanin synthesis wasimpaired and the electron transport rate of PSIIdecreased. On the other hand, in leaves subjected to
transient freezing, the marked reduction of photo-chemical efficiency of the PSII was observed both inanthocyanin deficient (treated with AIP) and in antho-cyanin-accumulating (non-treated with AIP) tissues(Fig. 8). This indicates that anthocyanins were probablynot involved in protection of PSII against the freezing-induced stress. The ability to recover from the freezing-induced photoinhibition was lost in CA leaves not ableto accumulate hydroxycinnamoyl esters (compare data inFigs 4 and 7). Hydroxycinnamic esters have a high anti-oxidant activity (Rice-Evans et al. 1997, Grace andLogan 2000). They can be used as substrates for vacuolarperoxidases in the peroxidase/phenolic/ascorbate system,scavenging hydrogen peroxide diffusing across mem-branes to vacuoles (Takahama and Oniki 1997,Yamasaki et al. 1997). Therefore in leaves subjected tothe second, frost-dependent stage of acclimation, animportant role for hydroxycinnamoyl esters in protectionof PSII against the freezing-induced-oxidative stressneeds to be taken into consideration.
Our results show that AIP-dependent phenylpropa-noid deficiency in winter rape leaves impaired the devel-opment of tolerance to extracellular freezing during thefirst, cold-dependent stage of plant acclimation (Fig. 8A).The tolerance of plants to extracellular freezing dependson avoidance and/or tolerance of freezing-induced celldehydration and on the protection of cells against exces-sive shrinkage (see Sakai and Larcher 1987 for a review).It appears that the AIP treatment resulted in decreasedwater retention in CA leaves because it prevented thecold-dependent accumulation of hydrophilic proteins inthe cytoplasm (Fig. 6A) and hydrocinnamates andanthocyanins (Fig. 3) in the vacuoles. The osmotic roleof anthocyanins in the cold hardening of plants wassuggested by Chalker-Scott (1999). However, her sugges-tion that the role of these compounds rely on theincreased ability of anthocyanin-containing cells toavoid freezing through increased water supercoolingability is not supported by the results of our studies:the AIP-dependent decrease in anthocyanin level hadno influence on tolerance of freezing by supercooledleaves (Fig. 8B). The results of Leyva et al. (1995) alsoshowed that anthocyanins were not required for thesuccessful development of freezing tolerance in twoArabidopsis mutants unable to accumulate the pigments.Basing on the results of our studies we propose that thedecreased content of ferulic acid in cell walls (Table 1)and the reduction of the cell wall content (Fig. 6B) inAIP-treated CA leaves resulted in decreased cell wallrigidity. This, in turn, decreased the tissue ability tocounteract the injurious effects of freezing-induced dehy-dration and cell shrinkage (Rajashekar and Lafta 1996).
In conclusion, the use of the competitive inhibitor ofPAL, 2-aminoindano-2-phosphonic acid (AIP) enabledus to examine the role of phenylpropanoids in the cold-acclimation processes. We propose that during the firststage of acclimation, anthocyanins shield mesophyll tis-sues from excess of photosynthetically active radiation,preventing leaves from becoming photoinhibited. The
260 Physiol. Plant. 119, 2003
accumulation of ferulic acid in the cell walls during thisstage may result in increased cell wall rigidity and cellresistance to mechanical stress brought about by extra-cellular freezing of water. An increase in ferulic acid incell wall limits the expansion of leaf cells and may be oneof the reasons that growth is reduced in the cold. Duringthe second, frost-induced stage of plant acclimation,esterified hydroxycinnamic acids localized in vacuolesmay protect mesophyll cells against the frost-inducedoxidative stress, scavenging hydrogen peroxide diffusingacross membranes to vacuoles.
Acknowledgements – This work was financially supported by grantno. 6P04C 07812 from the State Committee for Scientific Researchof Poland. The authors are grateful to Dr Jerzy Zon (Institute ofOrganic Chemistry, Biochemistry and Biotechnology, TechnicalUniversity of Wrocław, Wrocław, Poland) for the generous gift ofAIP. We also thank Mrs Barbara Dobrska for her skilful technicalassistance.
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