Vol. 32 No 3 2009

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Fou­di­g Editor D. GAL, Hu­g­ry

Editor-i­-Chief S. K. IVANOV, Bu­g­ri­

Editors M. I. BONEVA, Bu­g­ri­ Zh. D. KALITCHIN, Bu­g­ri­

Editori­­ Bo­rd R. L. A­G­STINE, ­SA S. W. BENSON, ­SA S. BO­RBIGOT, Fr­­ce D. BRADLEY, ­K A. M. BRA­N, Germ­­y E. B. B­RLAKOVA, Russi­ J. DAHLMANN, Germ­­y Sh. GAO, Chi­­ N. GETOFF, Austri­ V. K. G­­TA, I­di­ J. HA­­EL, ­SA J. A. HOWARD, C­­­d­ F. MARTA, Hu­g­ry M. F. R. M­LCAHY, Austr­­i­ A. NEMETH, Hu­g­ry E. NIKI, J­p­­ K. OHK­BO, J­p­­ R. A. SHELDON, The Nether­­­ds G. E. ZAIKOV, Russi­ J. J. ZIOLKOWSKI, ­o­­­d Z­WEI Xi, Chi­­

or­nj bl­ckor­nj bl­ck

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Ltd., Co.

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* For correspondence.

Oxidation Communications 32, No 3, 685–696 (2009)

antioxiDant asPect of therMal harDening of Maize

seeDlings

R. BACZEK-KWINTA*, M. ZACZYNSKI

Plant Physiology Department, Faculty of Agriculture and Economics, Agricultural

University, 3 Podluzna Street, 30 239 Krakow, Poland E-mail: rrbaczek@syf-kr.edu.pl

abStRact

The effect of 7-day thermal hardening (15ºC) of seedlings of maize (Zea mays l. ssp.

indentata) hybrids on foliar H2o

2 content and anti-oxidative response was studied

during chilling (7 days at 7ºC). H2o

2 was assayed spectroluorimetrically, whereas

the measurements of catalase (CAT) and ascorbate peroxidase (APX) activities were performed spectrophotometrically. Hardening treatment diminished the H

2o

2 con-

tent in chilled plants and altered the CAT and APX activity, although these activities were dependent on the protein pool, which were ca. 2–3-folds higher in the leaves of hardened seedlings, suggesting that the increment in CAT and APX activity was caused mainly by the synthesis of new proteins. Leaf area and necroses together with electrolyte leakage (assayed conductometrically) were diminished by hardening. The hardened plants of the cultivar which were less injured during chilling, had the high-

est activities of H2o

2 scavengers among the studied genotypes. Irrespective of the

similar supply source of all cultivars, their response to chill differed and this probably involved in discrepancies in the signalling processes.

Keywords: ascorbate peroxidase, catalase, chilling stress, growth, hydrogen perox-

ide.

aimS anD backGRounD

The aim of the work was to establish the inluence of hardening on the conditions of maize (Zea mays L.) seedlings during subsequent chilling and the key enzymes involved in H

2o

2 scavenging. In the studies on chill-sensitive plants maize is often

considered as a model species1,2. It is also the world’s third most common species in terms of area cultivated (141 mln ha). Therefore, the physiological, biochemical and agronomic aspects were designed.

Maize is an annual grass, grown mainly in tropical and sub-tropical climate re-

gions. In the breeding process, both inbreds and hybrids are used, although the culti-

686

vation of hybrids is mainly of interest because of the high yield and better adjustment to mechanical harvesting (www.fao.org, faostat – agriculture). Maize requires a long and warm vegetative period to form the kernels in the cob. It belongs to the species sensitive to a strong chill (the temperature of 0 to 10°C), which occurs mainly in early spring. The photosynthetic organs are extremely chill-sensitive3. In many countries it is dificult to avoid this environmental stress, because when maize is sown too late, it does not develop its cobs, and may be grown only for silage.

Chilling stress is very dangerous when accompanied with high light intensity, because the leaves are provided with more photochemical energy than they can use in photosynthesis3. The excess of energy leads to perturbations in the photosynthetic and respiratory electron chains, and induces generation of reactive oxygen species (RoS), e.g. H

2o

2, which results in accelerated ageing of the leaves4. However, as a

charge-lacking substance, H2o

2 is relatively easily transported from cell to cell, and

this predisposes it to be an excellent signalling molecule, inducing various biochemi-

cal and physiological responses in plant cells5. to maintain the H2o

2 pool on a non-

toxic, signalling level, the cells are equipped with key enzymes which catalyse its decomposition through catalase (CAT) and ascorbate peroxidase (APX), and other scavenging systems6.

Catalase may be a good indicator of chill-dependent oxidative stress. Its activity is suppressed by light, and the chill accelerates this photoinactivation7,8. Environmental

factors may improve the condition of seedlings, and the diminished CAT inhibition contributes to the acquisition of chilling tolerance9,10.

Ascorbate peroxidase seizures the role of CAT in those cell compartments where CAT is not present, mainly in plastids. Its afinity to H

2o

2 is superior to that of cat

(Refs 11 and 12), although there are the reports that in case of chilling stress, APX activity is depleted due to the shortage of its electron donor, ascorbate. This results from a restricted transport process between the mesophyll and bundle sheath13,14.

the hardening of plants occurs in the natural environment, and in controlled

conditions is made possible through the application of various methods. However, the most obvious and natural hardening treatment is the sub-optimal temperature, ranged

from 10 to 15°C (Refs 15–18). Although such conditions may inhibit photosynthetic activity when compared with plants grown at 25ºC (Ref. 19), numerous adaptive biochemical alterations occur in leaves at the time. One of them is the temporary increase of H

2o

2 concentration

. This is considered a very important chemical signal

inducing the nuclear and plastidic genes20,21. the induction of catalase and peroxidase

genes were also reported. On the contrary, during strong continuous chilling without pre-chilling (acclimation) in mild low temperatures, H

2o

2 concentration increases

rapidly acting cytotoxically13,14.

687

EXPERIMENTAL

Plant material. Caryopses of maize (Zea mays l. ssp. indentata) hybrid cultivars of the ‘PR39G12’, ‘PR39H32’ and ‘PR39T68’, FAO 210-230 (Pioneer Hi-Breed Po-

land), commercially grown for the grain and silage were used. Seeds were sown into plastic pots (volume 5 dm3) with a mixture of peat/organic soil/sand (3/2/1, v/v/v) of pH ca. 6.0.

Vegetation before chilling. Plants (10 in a pot) were grown for 14 days at 25/20°C (day/night), relative humidity (RH) ca. 50%, and natural photoperiod (16/8 h). The minimal value of photosynthetic photon lux density (PPFD) was kept at the level of 400 mmol(quantum) m–2 s–1 during all treatments.

Hardening and chilling treatments. Plants were divided into control and hardened groups. Control plants were transferred into a vegetative chamber and kept at 25/20°c.

Hardened plants were kept in another chamber at 15/13°C and RH 50–60% for 7 days. Then both groups were transferred into a 7/5°C and kept for 7 days. After chilling, plants were recovered at 25/20°C for 7 days in a vegetative chamber. RH, photoperiod and PPFD were maintained as before.

Chilling injury. Electrolyte leakage (EL) was assayed on 4 leaf discs with an area of 0.75 cm2 each with a conductometer with automatic temperature compensation (CC-315 ELMETRON, Poland). Samples were taken from the middle, necroses-free part of the 3rd leaf. Plant material was washed with deionised water, closed in tubes with 15 cm3 of deionised water and shaken for 24 h. Conductivity was measured, then samples were boiled at 100 °C for 15 min, shaken for 24 h and then the assay was repeated. To eliminate the effect of the boiling on the tubes, the conductivity of deionised water was also measured, both at the beginning of analysis and during the second analysis of plant samples. Electrolyte leakage was calculated as a percentage of total electrolyte content.

For leaf area, the leaves were scanned as black and white drawings and the im-

ages were processed.

H2O

2 content. Plant material was prepared according to Refs 22 and 23. Samples (the

3rd leaf of individual plant) were cut, immediately submerged in liquid nitrogen, and then stored at –80°C until preparation. Tissue was crushed in liquid N

2 and ho-

mogenised in a mortar and a pestle, in 0.25 M HCl (5 ml; Sigma Aldrich). Then the samples were centrifuged at 12 000 g and 4°C, for 5 min. To remove the pigments, the supernatant was mixed with 10% (w/v) suspension of activated charcoal, and vortexed for 1 min, at room temperature. Then, aliquots were centrifuged twice for 5 min, at 12 000 g and 4°C. The last preparation step was the neutralisation with TRIS/HCl (pH 7.5, 0.5 M; Sigma Aldrich) in 1:10 ratio (v/v; TRIS/HCl: sample). Measurements were performed using the procedure developed in Ref. 24, with a spectroluorimeter Perkin Elmer (USA). The reaction mixture contained 2.89 ml HEPES (50 mM, pH=7.5; Fluka), 30 ml of homovanilic acid (4-hydroxy-3-metoxy-phenylacetic acid;

688

Sigma Aldrich) dissolved in HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid; TRIS, 2-amino-2-hydroxymethyl-1,3-propanediol; 50mM, pH=7.5 pH=7.5), 50 ml sample aliquot and 30 ml horseradish peroxidase (4 mM; Sigma Aldrich). Samples were excited with λ=315 nm, when emission measurements were at λ=425 nm. The results were related to standard curve performed for H

2o

2 in the concentration range

0.1–10 nM. Measurements were performed in 5 biological replicates, and each of them in 3 analytical replicates.

Enzyme extraction and activity, and protein content. Catalase activity (CAT; EC 1.11.1.6) was determined on dialysed crude extracts of leaves. Samples (the 3rd leaf of individual plant) were cut, their area was fast measured, then they were submerged in liquid nitrogen, and then stored at –80°C until preparation. Tissue was crushed in liquid N

2 and homogenised in an electric homogeniser (ultraturrax, ika labortechnik,

Germany), in a pre-cooled extraction buffer (50 mM potassium phosphate, pH 7.0; POCh Poland) containing 0.1 mM of EDTA (SIGMA-ALDRICH) and 0.5% of BSA (bovine serum albumine; SIGMA-ALDRICH). The homogenate was centrifuged at 10 000 g for 3 min, at 4°C. The supernatant was dialysed at 4°C for 6 h (in darkness), in a dialysis tubing cellulose membrane, against a potassium phosphate buffer (50 mM, pH 7.0) consisting of 0.1 mM of EDTA. Dialysates were kept in the Eppendorf type tubes on ice, in darkness. The analytical procedure relied on the monitoring the disappearance of H

2o

2 (SIGMA-ALDRICH) at 240 nm (Ref. 25); (a spectrophotometer

LKB Biochrom Ultrospec II, UK). Activity of the enzyme was expressed as mmol of its

substrate (H2o

2) decomposed in 1 min in an analytical mixture, in a quartz cuvette.

Ascorbate peroxidase activity (APX; EC 1.11.1.11) was assayed on dialysed crude extracts of leaves according to Ref. 26 with minor modiications. The dialysing buffer contained 0.5 mM ascorbic acid (AsA; SIGMA-ALDRICH) to prevent the loss of enzyme activity. In the analytical procedure, the oxidation rate of AsA at 290 nm (spectrophotometer LKB Biochrom Ultrospec II, UK) was measured. Activity of the enzyme was expressed as mmol of AsA decomposed in 1 min in analytical mixture, in a quartz cuvette. Corrections were made for low, non-enzymic oxidation by H

2o

2

and for the oxidation of ascorbate in the absence of H2o

2.

Soluble protein content was measured spectrophotometrically at 595 nm ac-

cording to Ref. 27 using bovine serum albumine (BSA; SIGMA-ALDRICH) for the calibration curve.

The statistical signiicance of differences was evaluated by variance analysis, followed by the Student t-test for the comparison of two means of a particular geno-

type and the Duncan test for comparison of means among the genotypes, obtained at various stages of the experiment. Measurement and analyses were performed on the 3rd leaf, where one leaf was one biological replicate. The number of biological replicates was 6–8, and analytical replicates 2–3.

689

RESultS anD DiScuSSion

Chilling injury and leaf growth. Thermal hardening (15°C) diminished the electrolyte leakage (EL) of plants of all cultivars during subsequent chilling (7-day at 7°C; Fig. 1a). The effect was especially visible on the ‘PR39T68’ plants, which were the most severely injured (Fig. 2). On the contrary, leaves of ‘PR39H32’ seedlings were less injured when compared to other genotypes (Figs 1a and 2).

fig. 1. Foliar electrolyte leakage (EL) (a) and leaf area (b) of hardened (7 days at 15°C) and non-hard-

ened maize seedlings. Values shown are the means ± SE; n=6–8. Differences among the cultivar are signiicant according to the Student t-test: p=0.05

Hardening inhibited chloroses and necroses formation, as well as the wilting of all leaves developed during the experiment (from the 1st to the 3rd; Fig. 2). This was seen on all cultivars, but especially on cv. ‘PR39T68’. The leaves of hardened plants of all cultivars revealed some chlorotic areas, mainly on the top of the leaf, and/or were more yellow but less purple-dyed and desiccated to a lesser extent than the control specimens.

690

fig. 2. Leaves (1st, 2nd and 3rd) of hardened (7 days at 15°C) and non-hardened maize seedlings. Samples are representative from 6–8 plants of each treatment

Theories explaining chilling injury imply an increment in membrane permeability leading to ion lux28. On the other hand, at a given temperature, the susceptibility of individual species to such conditions varies upon the genetic formula. in this paper, it

was noted that the EL was increased in case of cv. ‘PR39T68’, and it was accompanied by a visual estimation of the necroses and wilting of leaves of different age. There-

fore, this genotype seems to be the most sensitive to chilling stress. However, its EL values of 8–16% obtained during chilling are very low when compared to the EL of genotypes grown several years ago9. Low injury is probably the consequence of the breeding progress which adapts maize to mass production in the temperate climatic zones. Similar values were obtained on chill-resistant maize and rice18,29. We also have to remember that samples for the EL assay were taken from the middle part of each leaf, and this section was free from necroses. When taking the sample partially from the top, from the middle part and the leaf basis, the EL of chilled plants may reach up to 80% of total electrolyte leakage9. However, the procedure of taking samples was uniied for all cultivars used in this experiment, and therefore we can estimate their chilling sensitivity by comparison with the obtained results18.

It is known that chilling stress directly depletes leaf growth. This is inluenced by the increase in osmotic potential and decrease in membrane plasticity30. Low temperature also triggers increased ion and water lux from the cells, and causes physiological drought observed as wilting9. Hardening applied in this experiment

counteracted growth inhibition, and this effect was visible on leaves of different ages. Hardened plants had larger leaves than that of the control (Fig. 1b). When comparing

691

the leaf area of individual cultivars, the biggest was that of cv. ‘PR39G12’ (49–55 cm2) and it was impossible to show the whole leaf lamina on (Fig. 2). the smallest

leaves were of cv. ‘PR39T68’ (37–40 cm2). This was in agreement with the cultivar characteristics made by the supplier (www.pioneer.info.pl).

H2O

2 content. It was related to the total leaf area and leaf pool of soluble proteins (Fig.

3). Since the leaf area of cultivars was different and this parameter was dependent on the treatment, we considered that the H

2o

2 content and enzymatic activity together

with the protein content should be recalculated to different bases. It is noteworthy that maize leaf area is rather big (in the described experiment it was 30–50 cm2) when compared to leaf lamina thickness (ca. 10–200 mm; Ref. 31). this ratio suggests,

that the calculation of other parameters to leaf area characterises the pattern of their

changes far better than the relation to fresh weight, which changes during the stress due to the different water content in leaves32. Additionally, as H

2o

2 is dissolved in

cellular water phase and both H2o

2 and its enzymatic scavengers are assayed in liquid

homogenate prepared from an individual leaf, this basis used for recalculation, seems

to be superior than dry weight containing the debris of various substances.

fig. 3. H2o

2 contents in leaves of hardened (7 days at 15°C) (a) and non-hardened maize seedlings at

the chilling stage (7 days at 7°C) (b). bars represent the means ± SE; n = 6–8. Differences among the cultivar are signiicant according to the Student t-test: p = 0.05; n.s. – differences not signiicant

692

in leaves, the H2o

2 content was signiicantly lower in hardened seedlings the

‘PR39T68’ cultivar than in the control ones (Fig. 3a). its control plants also revealed

the highest H2o

2 content among the cultivars used in the experiment. Interestingly,

this cultivar was considered the most susceptible to chill. When analysed the H2o

2

pool related to protein content in the leaf, the effect of hardening was visible on plants of all cultivars as the lower means were obtained from hardened plants (Fig. 3b). ad-

ditionally, the differences between them and the control seedlings were bigger than in the previously described case. Hardened plants of ‘PR39T68’ had ca. 50% less H

2o

2

than the control ones (3.29 and 1.61 mmol H2o

2 g (protein)–1, respectively). Similarly

to the previous observations, these seedlings also had the highest values among all

the studied specimens. Thus, the biggest chilling sensitivity of ‘PR39T68’ cv. may be linked to the highest H

2o

2 content. Similar results were obtained on rice29. How-

ever, H2o

2 is under the control of various peroxidases and catalases. their activities

are considered as one of the key factors involved in chilling resistance, because they prevent H

2o

2 accumulation reaching the toxic level, but allow the signalling amount

to be maintained15,16. Therefore CAT and APX activities were studied.

Catalase and ascorbate peroxidase activity, and protein content. Leaf CAT activity in hardened plants of all cultivars was higher than that from the control plants (Fig. 4a).

The biggest, 13-fold difference between means was noted in the most chill-resistant ‘PR39H32’ cv. Interestingly, its control plants had the lowest mean (14.5 mmol H

2o

2

decomposed in 1 min), some 10-times lower than the controls of other genotypes, whereas the hardened specimens had the highest (ca. 200 mmol). Analysis of CAT activity calculated on protein concentration (Fig. 4a) retained the previous observa-

tion that hardened seedlings of this genotype had greater values than the control ones, although the difference was not as big as in the previous case. Additionally, the ratio of speciic CAT activity in the hardened and control seedling of ‘PR39H32’ cv. was similar to the ratio in protein content, and the correlation between these parameters was signiicant (Table 1).

The pattern of changes in APX activity was also similar to that of CAT activity (Figs 5a, b), and hardened plants also had higher values when compared to the control ones. However, the differences varied upon the genotype and the basis of calculation. Also similar to the CAT activity, the lowest APX activity was obtained in ‘PR39H32’ cv. control seedlings. The difference in leaf APX of this genotype was similar to that in the protein content, and these parameters were correlated (Table 1). Protein content itself was in general stimulated by hardening in all the cultivars.

693

fig. 4. catalase (cat) (a, b) and ascorbate peroxidase (APX) (c, d) activity in leaves of hardened (7 days at 15°C) and non-hardened maize seedlings at the chilling stage (7 days at 7°C). Bars represent the means ± SE; n=6–8. Differences among the cultivar are signiicant according to the Student t-test:

p=0.05 (*), 0.01 (**), and 0.001 (***); n.s. – differences not signiicant

table 1. Protein content in leaves of hardened (7 days at 15°C) and non-hardened maize seedlings at the chilling stage (7 days at 7°C)

cultivarProtein content (mg (protein) leaf–1)

hardened control

‘PR39G12’ 5.68±0.21(184%)

3.09±0.25*

(100%)r

cat/prot. = 0.78**

rAPX/prot. = 0.52 n.s.‘PR39H32’ 4.68±0.21

(380%)1.23±0.16*

(100%)r

cat/prot. = 0.61**

rAPX/prot. = 0.71**

‘PR39T68’ 3.67±0.28(171%)

2.15±0.09*

(100%)r

cat/prot. = 0.53 n.s.

rAPX/prot. = 0.53 n.s.

Values shown are the means ± SE; n = 6–8. Differences among the cultivar are signiicant according to the Student t-test: p=0.001*; r

cat, rAPX – correlation coeficients between protein content and catalase

(CAT) or ascorbate peroxidase (APX) activity, respectively; signiicant** if p < 0.05, n.s. – correlation not signiicant.

694

Chilling conditions must have involved partially, the photoinactivation of catalase and also the depletion in its recovery7, but hardening treatment increased enzyme activity and diminished H

2o

2 concentrations. This effect may be explained by the

adaptive alterations on the nuclear level allowing the plants to make the turnover of enzymatic proteins20,21. Diminished CAT proteolysis triggered by hardening should also be taken into consideration, as protein content was higher in hardened plants than in the control ones. Increased afinity of the enzyme to its substrate seems to be unlikely, as the chilling stage was long (7 days) and the temperature was also low enough to affect the conformation of individual cat molecules, both in the control

and hardening treatments.

The fact that hardened plants, especially that of ‘PR39H32’ cv., had APX activ-

ity higher than the control, means two explanations may exist. Firstly, the increment in activity is caused mainly by the synthesis of new molecules of various APX isoforms. Alternatively, the process of transport of APX electron-donor ascorbate is improved13,14.

Various genotypes often reveal different antioxidant characteristics and stress re-

sistance32–34. Interestingly, ‘PR39H32’ cv. which was the most resistant to chill, had the lowest APX and CAT activities and protein content in non-hardened plants, although the H

2o

2 content was similar to other genotypes. That suggests the different response

of non-speciic peroxidases in its leaves than in other genotypes. This is supported by the intense growth of ‘PR39H32’ leaves in chilling, as cell elongation involves numerous biochemical reactions in which H

2o

2 is required and scavenged6. on the

other hand, we notice the maximal differences in APX activity between control and hardened plants of this cultivar and, the highest values of its hardened plants among

all genotypes. That suggests the effective scavenging of excessive H2o

2 (which is not

decomposed by CAT) by APX isoforms11,12, and improved plants condition during

severe chilling. Similarly in Ref. 35 is reported that maize seedlings with elevated APX activity were characterised by better vigour. This also implies the hypothesis involved in the distinct signalling response in the ‘PR39H32’ cultivar than the other two, although their common source of supply suggest similar selection tools are used during their breeding.

concluSionS

From the overall data obtained in this work, it can be surmised that the thermal harden-

ing of maize seedlings diminishes the chilling injury suffered by leaves and acceler-ates their growth during subsequent chilling. The acquisition to chilling tolerance is linked with the anti-oxidative effect; namely, the large increase in foliar CAT and APX activities allows the scavenging of excessive H

2o

2 up to 50%. However, irrespective

of the common supply source of cultivars, their response to chilling differs and this probably involves in discrepancies in the signalling processes.

695

ACKNOWLEDGEMENTS

The technical assistance of Msc. Agnieszka Adamska (AU Krakow), is gratefully acknowledged.

REFEREncES

1. A. MASSACCI, A. IANNELLI, F. PIETRINI, F. LORETO: The Effect of Growth at Low Tempera-

ture on Photosynthetic Characteristics and Mechanisms of Photoprotection of Maize Leaves. J. Exp. bot., 48, 119 (1995).

2. R. BACZEK-KWINTA, E. NIEWIADOMSKA, Z. MISZALSKI: Physiological Role of Reactive Oxygen Species in Chill-sensitive Plants. Phyton – Ann. Rei Bot., 45, 25 (2005).

3. A. H. KINGSTON-SMITH, C. H. FOYER: Bundle Sheath Proteins Are More Sensitive to Oxidative Damage than Those of the Mesophyll in Maize Leaves Exposed to Paraquat or Low Temperatures. J. Exp. bot., 342, 123 (2000).

4. M. J. FRYER, J. R. ANDREWS, K. OXBOROUGH, D. A. BLOWERS, N. R. BAKER: Relation Between CO

2 Assimilation, Photosynthetic Electron Transport and Active O

2 metabolism in leaves

of Maize in the Field during Periods of Low Temperature. Plant Physiol., 116, 571 (1998). 5. R. DESIKAN, M.-K. CHEUNG, A. CLARKE, S. GOLDING, M. SAGI, R. FLUHR, Ch. ROCK,

J. HANCOCK, S. J. NEILL: Hydrogen Peroxide is a Common Signal for Darkness- and ABA-in-

duced Stomatal closure in Pisum sativum. Funct. Plant Biol., 31, 913 (2004). 6. N. SMIRNOFF (Ed.): Antioxidants and Reactive Oxygen Species in Plants. Blackwell Publishing

Ltd., 2005. 7. J. FEIERABEND, C. SCHAAN, B. HERTWIG: Photoinactivation of Catalase Occurs under Both

High- and Low-Temperature Stress Conditions and Accompanies Photoinhibition of Photosystem II. Plant Physiol., 100, 1554 (1992).

8. Ch.-K. AUH, J. G. SCANDALIOS: Spatial and Temporal Responses of the Maize Catalases to Low Temperature. Physiol. Plantarum, 101, 149 (1997).

9. G. SKRUDLIK, R. BACZEK-KWINTA, J. KOSCIELNIAK: The Effect of Short Warm Breaks during Chilling on Photosynthesis and the Activity of Antioxidant Enzymes in Plants Sensitive to chilling. J. agron.& crop Sci., 184, 233 (2000).

10. R. BACZEK-KWINTA, J. KOSCIELNIAK: Anti-oxidative Effect of Elevated CO2 concentration

in the Air on Maize Hybrids Subjected to Severe Chill. Photosynthetica, 41, 161 (2003).11. H. WILLEKENS, S. CHAMNONGPOL, M. DAVEY, M. SCHRAUDNER, C. LANGEBARTELS,

M. van MONTAGU, D. INZE, W. van CAMP: Catalase Is a Sink for H2o

2 and is indispensable for

Stress Defence in C3 Plants. EMBO J., 16, 4806 (1997).12. A. FATH, P. C. BETHKE, R. L. JONES: Enzymes that Scavenge Reactive Oxygen Species Are

Down-regulated Prior to Gibberelic Acid-induced Programmed Cell Death in Barley Aleurone. Plant Physiol., 126, 156 (2001).

13. A. G. DOULIS, N. DEBIAN, A. H. KINGSTON-SMITH, Ch. H. FOYER: Differential Localization of Antioxidants in Maize Leaves. Plant Physiol., 114, 1031 (1997).

14. G. M. PASTORI, Ch. H. FOYER, P. M. MULLINEAUX: Low Temperature-induced Changes in the Distribution of H

2o

2 and Antioxidants between the Bundle Sheath and Mesophyll Cells of Maize

leaves. J. Exp. bot., 51, 107 (2000).15. T. K. PRASAD, M. B. ANDERSON, C. R. STEWART: Acclimation, Hydrogen Peroxide and Abscisic

Acid Protect Mitochondria against Irreversible Chilling Injury in Maize Seedlings. Plant Physiol., 105, 619 (1994a).

16. T. K. PRASAD, M. B. ANDERSON, B. A. MARTIN, C. R. STEWART: Evidence for Chilling-induced Oxidative Stress in Maize and Regulatory Role for Hydrogen Peroxide. Plant Cell, 6, 65 (1994b).

696

17. J. LEIPNER, Y. FRACHEBOUD, P. STAMP: Acclimation by Suboptimal Growth Temperature Diminishes Photooxidative Damage in Maize Leaves. Plant Cell Environ., 20, 366 (1997).

18. F. JANOWIAK, E. LUCK, K. DÖRFFLING: Chilling Tolerance of Maize Seedlings in the Field dur-ing Cold Periods in Spring Is Related to Chilling-induced Increase in Abscisic Acid Level. J. Agron. crop Sci., 189, 156 (2003).

19. G.-Y. NIE, S. P. LONG, N. R. BAKER: The Effects of Development at Sub-optimal Growth Tem-

peratures on Photosynthetic Capacity and Susceptibility to Chilling-dependent Photoinhibition in Zea mays. Physiol. Plantarum, 85, 554 (1992).

20. Y. KOVTUN, W. L. CHIU, G. TENA, J. SHEEN: Functional Analysis of Oxidative Stress-activated Mitogen-activated Protein Kinase Cascade in Plants. Proc. Natl. Sci. USA, 97, 2940 (2000).

21. T. PFANNSCHMIDT: Chloroplast Redox Signals: How Photosynthesis Controls Its Own Genes? Trends Plant Sci., 8, 33 (2003).

22. G. CREISSEN, J. FIRMIN, M. FRYER, B. KULAR, N. LEYLAND, H. REYNOLDS, G. PASTORI, F. WELLBURN, N. BAKER, A. WELLBURN, P. MULLINEAUX: Elevated Glutathione Biosyn-

thetic Capacity in the Chloroplasts of Transgenic Tobacco Plants Paradoxically Causes Increased Oxidative Stress. Plant Cell, 11, 1277 (1999).

23. M. MEINHARD, P. L. RODRIGUEZ, E. GRILL: The Sensitivity of ABI2 to Hydrogen Peroxide Links the Abscisic Acid-response Regulator to Redox Signaling. Planta, 214, 775 (2002).

24. G. G. GUILBAUT, D. N. KRAMER, E. HACHLEY: A New Substrate for Fluorometric Determina-

tion of Oxidative Enzymes. Anal. Chem., 39, 271 (1967).

25. H. AEBI: Catalase in vitro. Meth. Enzymol., 105, 121 (1984).

26. Y. NAKANO, K. ASADA: Hydrogen Peroxide Is Scavenged by Ascorbate-speciic Peroxidase in Spinach chloroplasts. Plant Cell Physiol., 22, 867 (1981).

27. m. bRaDFoRD: a Rapid and Sensitive method for the Quantitation of microgram Quantities of

Protein-dye Binding. Anal. Biochem., 72, 248 (1976).

28. R. G. PINHERO, G. PALIYATH, R. Y. YADA, D. P. MUR: Chloroplast Membrane Organization in Chilling-tolerant and Chilling-sensitive Maize Seedlings. J. Plant Physiol., 155, 691 (1999).

29. Z. GUO, W. OU, S. LU, Q. ZHONG: Differential Response of Antioxidative System to Chilling and Drought in Four Rice Cultivars Differing in Sensitivity. Plant Physiol. Biochem., 44, 828 (2006).

30. E. D. SCHULZE: Whole Plant Responses to Drought. Aust. J. Plant Physiol., 13, 127 (1986).

31. D. STOYANOVA, E. TCHAKALOVA, I. YORDANOV: Inluence of Different Soil Moisture on Anatomy of Maize Leaves and Ultrastructure of Chloroplasts. Bulg. J. Plant Physiol., 28, 11

(2002).32. R. BACZEK-KWINTA, J. KOSCIELNIAK: The Mitigating Role of Environmental Factors in Seed-

ling Injury and Chill-dependent Depression of Catalase Activity in Maize Leaves. Biol. Plantarum, 2008 (in press).

33. D. StaJnER, m. kRalJEvic-balalic, m. miloSEvic, m. vuJakovic, m. zlokolica:

Stress Resistance in Seeds. oxid. commun., 25, 596 (2002).34. D. StaJnER, n. mimica-Dukic, m. miloSEvic, m. zlokolica: Herbicide Stress and its

Effect on Scavenging System of Vegetable Sorts. Oxid. Commun., 25, 456 (2002).35. L. de GARA, C. PACIOLLA, M. C. TULLIO, M. MOTTO, O. ARRIGONI: Ascorbate-depend-

ent Hydrogen Peroxide Detoxiication and Ascorbate Regeneration during Germination of Highly Productive Maize Hybrid: Evidence of an Improved Detoxiication Mechanism against Reactive Oxygen Species. Physiol. Plantarum, 109, 7 (2000).

Received 11 April 2008

Revised 3 May 2008