ozone and UV-B - Sodininkystė ir daržininkystė

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ozone and UV-B - Sodininkystė ir daržininkystė

SCIENTIFIC WORKS OF THE LITHUANIAN INSTITUTE OF

HORTICULTURE AND LITHUANIAN UNIVERSITY OF

AGRICULTURE. SODININKYSTĖ IR DARŽININKYSTĖ. 2008. 27(2).

Reaction of model plant Crepis capillaris to

stress-inducing factors – ozone and UV-B

Vida Rančelienė, Regina Vyšniauskienė

Institute of Botany, Žaliųjų ežerų str. 49, Vilnius LT-08406, Lithuania

E-mail: vida.ranceliene@botanika.lt

Stress inducing effect of ozone and UV-B on plants is well known. However, despite the

different molecular mechanisms of their action, both factors have common mechanisms, such

as affected photosynthesis and synthesis of photosynthetic pigments, induction of oxidative

burst. Action of different doses of both factors was compared on the same plant material of

Crepis capillaris in experimentally changed temperature (21/14 °C, 25/16 °C) and CO 2 (350

and 700 ppm) conditions. The doses of UV-B were 0; 2; 4 kJm -2 and of O 3 – 20; 40; 80 ppb.

The most sensitive for UV-B was the leaf area, which decreased proportionally to the dose of

UV-B. C. capillaris has primarily developed mechanisms of adaptation to alterations of the

tested environmental conditions. That was shown by increased activity of SOD (superoxide

dismutase), relatively stable level of pigment synthesis and only slow alteration of protein content

in leaves. However, the observed effects depended on temperature and CO 2 conditions.

Key words: Crepis capillaris, complex action, elevated CO 2 , temperature, ozone,

UV-B.

Introduction. Rising concentrations of CO 2, tropospheric ozone (O 3 ) and surface

level of UV radiation as a result of increasing industrial activity (IPCC, 2001; Karnosky

et al., 2005; Hidema, Kumagai, 2006) alter plant performance in both natural and

managed ecosystems. Enriched CO 2 and O 3 typically have opposing effects on plant

productivity, resistance to pathogens and other physiological features, including activity

of photosynthesis (Allen et al., 1997; Krupa, 2003; Ashmore, 2005; Rämö et al., 2006;

Qaderi et al., 2007; Awmack et al., 2007; Plessl et al., 2007). However, joint action of

those factors is investigated insufficiently and depends upon plant species, genotype,

geographic location and many uncontrolled environmental conditions (Feder, Shrier,

1990; Allen et al., 1997; Krupa, 2003; Karnosky et al., 2005; Rämö et al., 2006; Koti

et al., 2006; Tegelberg et al., 2008).

Our many-year experience shows that model plant Crepis capillaris (L.) Wallr.

is sensitive enough to action of UV-B and O 3, and that effects of both environmental

factors depend upon plant parameters. The most sensitive parameters are fresh weight

and leaf area (Rančelienė et al., 2005; 2006). The use of model plant allows escaping

uncontrolled environmental conditions and revealing affect pure effect of the interaction

of investigated factors.

The aim of our investigation was to analyze complex action of UV-B, CO 2 and O 3

on various features of model plant Crepis capillaris (L.) Wallr., representing different

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aspects of plant response to the examined environmental factors: integrative trait – leaf

area; protein content in leaves, expressing synthetic capacity of plant and destructive

processes. On the other hand, concentration of chlorophylls and carotenoids in leaves,

partially expressing photosynthetic activity of leaves; activity of superoxide dismutase

in leaves, expressing alterations of the protective systems of plant.

Object, methods and conditions. Seeds of Crepis capillaris were harvested at the

Laboratory of Cell Engineering of the Institute of Botany (Vilnius). For experiments

with ozone, plants were grown for 21 days, for other experiments – 28 days in the same

conditions as for seed harvesting in the growth chamber in controlled conditions. Thus

conditions for preliminary preparation of plants were the same in all our experiments

with 12/12 h dark cycle (21 ± 2 °C) in pots filled with soil and watered daily with tap

water. In each pot five plants were grown. For illumination lamps OSRAM L36/77

Flora (PAR 53 µm m -2 s -1 ) were used.

During the second stage, the preliminary grown plants were transferred to the

growth chambers of the Lithuanian Institute of Horticulture (Babtai, Kaunas district).

Illumination conditions in Babtai were different from those in Vilnius: lamps SON-T

Agro (PAR – 100 µm m - І s - №) were used with 16/8 h photoperiod. Experiments were

conducted only after 2 days adaptation in a new regime.

Irradiation with UV-B was made with UV-B lamps TL 40W/12 RS (Philips).

Doses were measured with radiometer VLX-3 (Vilber-Lourmat, France) equipped with

a 312 nm probe. The UV-B doses were 0; 2; 4 kJ m - І d - №. The ozone concentrations

were maintained by ozone generator OSR-8 (Ozone Solutions, Inc.) 7 h a day, 5 days

a week. The ozone level was determined by portable ozone sensor OMC-1108 (Ozone

Solutions, Inc.) Ozone concentrations were 20, 40 and 80 ppb daily. Different CO 2

regime was created with an automatic gas system in a phytotron chamber and monitored

by a CO 2 controller (Regin, Sweden).

An integrated regime of both factors, CO 2 and temperature, on the investigated

plants was analyzed in two variants of treatment: at normal temperature and

CO 2 (and as control) conditions of CO 2 concentration were 350 ppm at 21 °C –

day /14 °C – night, and elevated temperature and CO 2 conditions were 700 ppm CO 2

at 25 °C – day /19 °C – night.

All experiments were made in three replications. The position of pots was

randomized. Plants were examined the next day after treatment. For comparison of

different features, tested in our experiments, all results are expressed in percentage

to control plants. The leaf area of three–five plants taken from different pots in each

sample was measured. Leaves were scanned and analyzed with the Sigma Scan Pro.

The same plants were used for determination of fresh and dry weight.

Concentrations of chlorophylls and carotenoids were determined in 100 % acetone

by method of Wettshtein (1957) with spectrophotometer at 662, 644 and 440.5 nm for

chlorophyll a, chlorophyll b and carotenoids, respectively.

For determination of SOD (SOD, EC 1.15. 1.1) activity and protein content in

leaves, leaf material was grounded with an extraction buffer in 0.05 M Na-K phosphate

buffer pH 7.8 at 4 °C. Supernatants were used as a crude extract for SOD assays by

Beyer (Beyer, Fridovich, 1987) and for soluble protein determination according to

Bradford (1976). Conditions are described in Rančelienė et al. (2005).

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Data analysis was carried out employing the package of statistical analysis tools

of MS Excel 2003 (Microsoft Corporation) program. We considered treatment effects

at P = 0.05 level.

Results. An action of ozone and UV-B on Crepis capillaris plants is contradictory

and very clearly depends upon the plant parameter used for the evaluation of effect.

That peculiarity of UV-B or ozone action is revealed very clearly if both stress-inducing

factors are employed to affect several distinct features of the same plants. So, according

to our previous work on C. capillaris (Rančelienė et al., 2006), it was expected that the

leaf area is the most sensitive parameter for evaluation of UV-B and ozone action. At

the present series of investigation, it was true only for UV-B. The leaf area decreased

proportionally to the UV-B dose (Fig. 1).

Fig. 1. Effects of UV-B (A) and ozone (B) on leaf area of Crepis capillaris plants

depending on temperature and CO 2 concentration. Conditions of plant irradiation

with UV-B or ozone (O 3 ) treatment: UVB and O 3 – at 21/14 °C day/night and

CO 2 – 350 ppm; UVB + t° – at elevated temperature 25/19 °C and CO 2 350 ppm;

UVB + t° + CO 2 and O 3 + t° + CO 2 – at 25/19 °C and CO 2 – 700 ppm

1 pav. UV-B (A) ir ozono (B) poveikis Crepis capillaris augalų lapų plotui,

priklausomai nuo temperatūros ir CO 2 koncentracijos. Poveikio UV-B ir ozonu (O 3 ) sąlygos:

UVB ir O 3 – temperatūra dieną/naktį 21/14 °C, CO 2 – 350 ppm;

UVB + t° – 25/19 °C, CO 2 – 350 ppm; UVB + t° + CO 2 ir

O 3 + t° + CO 2 – 25/19 °C ir CO 2 – 700 ppm

However, action of ozone in the normal environment or in conditions of elevated

temperature and CO 2 on leaf area was insufficient or even slightly stimulating (Fig. 1).

The reason for such discrepancy becomes clear when action of UV-B is tested in

conditions of increased (25/16 °C) temperature. It was unexpected, but negative effect

of UV-B was convincingly restored namely if C. capillaris was treated with UV-B

in conditions of increased temperature. Concentration of CO 2 did not have impact

on restoration effect, because expression of observed effect was about the same,

independently of elevated CO 2 concentration as additional factor (Fig. 1). Slight positive

effect on leaf area was observed also for ozone in conditions of elevated temperature

and CO 2 in treated plants environment (Fig. 1).

Manipulation with temperature and CO 2 conditions allowed us to show negative

effect of UV-B on concentrations of carotenoids and chlorophyll a and b in leaves

(Fig. 2).

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Fig. 2. Effects of UV-B (A) and ozone (B) on content of photosynthetic pigments

in leaves of Crepis capillaris depending on temperature and CO 2 concentration.

Conditions of plant irradiation with UV-B or ozone (O 3 ) treatment: UVB and

O 3 – at 21/14 °C day/night and CO 2 – 350 ppm; UVB + t° – at elevated temperature

25/19 °C and CO 2 – 350 ppm; UVB + t° + CO 2 and

O 3 + t° + CO 2 – at 25/19 °C and CO 2 – 700 ppm.

2 pav. UV-B (A) ir ozono (B) poveikis fotosintezės pigmentų kiekiui Crepis capillaris

augalų lapuose, priklausomai nuo temperatūros ir CO 2 koncentracijos.

Poveikio UV-B ir ozonu (O 3 ) sąlygos: UVB ir O 3 – temperatūra dieną/naktį 21/14 °C,

CO 2 – 350 ppm; UVB + t° – 25/19 °C, CO 2 – 350 ppm;

UVB + t° + CO 2 ir O 3 + t° + CO 2 – 25/19 °C ir CO 2 – 700 ppm.

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However, clearer negative effect was observed under conditions of both modifying

factors, i. e. temperature and CO 2, being elevated. Concentration of pigments in ozone

treated plants altered insufficiently.

Content of soluble proteins in leaves of C. capillarisafter treatment with different

doses of UV-B or ozone was about the same as in leaves of the control, untreated

plants (Fig. 3).

Fig. 3. Effects of UV-B (A) and ozone (B) on soluble protein content

in leaves of Crepis capillaris depending on temperature and CO 2 concentration.

Conditions of plant irradiation with UV-B or ozone (O 3 ) treatment:

UVB and O 3 – at 21/14 °C day/night and CO 2 – 350 ppm;

UVB + t° – at elevated temperature 25/19 °C and CO 2 – 350 ppm;

UVB + t° + CO 2 and O 3 + t° + CO 2 – at 25/19 °C and CO 2 – 700 ppm

3 pav. UV-B (A) ir ozono (B) poveikis tirpių baltymų kiekiui Crepis capillaris

augalų lapuose, priklausomai nuo temperatūros ir CO 2 koncentracijos.

Poveikio UV-B ir ozonu (O 3 ) sąlygos: UVB ir O 3 – temperatūra dieną/naktį 21/14 °C, CO 2 –

350 ppm; UVB + t° –25/19 °C, CO 2 – 350 ppm;

UVB + t° + CO 2 ir O 3 + t° + CO 2 – 25/19 °C ir CO 2 – 700 ppm.

Manipulation of temperature or temperature and CO 2 conditions did not alter

that relation.

Very appreciable effect of UV-B and especially ozone on activity of SOD in

leaves was observed (Fig. 4). After treatment with ozone, activity of SOD increased

about 3.5 times, and the effect was proportional to increasing concentration of ozone.

UV-B treatment also increased activity of SOD in leaves but not so strongly (about

1.5 times) and not so proportionally to UV-B dose.

It is worth noticing that under conditions of elevated temperature and CO 2 activity

of SOD in leaves of UV-B treated plants was lower, but differences between doses

of UV-B remained: the activity of SOD in leaves decreased proportionally to dose of

UV-B. However, opposite effect for UV-B and ozone in those conditions of treatment

became obvious (Fig. 4).

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Fig. 4. Effects of UV-B (A) and ozone (B) on superoxide dismutase (SOD) activity

in leaves of Crepis capillaris depending on temperature and CO 2 concentration.

Conditions of plant irradiation with UV-B or ozone (O 3 )

treatment: UVB and O 3 – at 21/14 °C day/night and CO 2 – 350 ppm;

UVB + t° – at elevated temperature 25/19 °C and CO 2 – 350 ppm;

UVB + t° + CO 2 and O 3 + t° + CO 2 – at 25/19 °C and CO 2 – 700 ppm.

4 pav. UV-B (A) ir ozono (B) poveikis superoksido dismutazės (SOD)

aktyvumui Crepis capillaris augalų lapuose, priklausomai nuo temperatūros ir

CO 2 koncentracijos. Poveikio UV-B ir ozonu (O 3 ) sąlygos:

UVB ir O 3 – temperatūra dieną/naktį 21/14 °C, CO 2 – 350 ppm;

UVB + t° – 25/19 °C, CO 2 – 350 ppm;

UVB + t° + CO 2 ir O 3 + t° + CO 2 – 25/19 °C ir CO 2 – 700 ppm.

Discussion. Results of the present work very convincingly show that action of

UV-B and ozone noticeably depends on environmental conditions of plant treatment.

According to many investigators (Krupa, 2003; Frohnmeyer, Staiger, 2003; Kakani

et al., 2003; Busotti et al., 2005; Brazaitytė et al., 2006), the most sensitive feature of

UV-B and ozone action is concentration of photosynthetic pigments in leaves. In our

previous works the most sensitive parameters to ozone or UV-B action were the leaf

area and plant fresh weight (Rančelienė et al., 2005; 2006). During the present work,

the leaf area decreased significantly only after irradiation with UV-B, but significant

negative effect was also observed on photosynthetic pigment concentration in plants

treated with UV-B in environment of elevated temperature and CO 2 .

On the other hand, this investigation confirms previous conclusion that C. capillaris

plants have well expressed means against UV-B and ozone action. One of such means

may be significantly increasing SOD activity in the leaves of treated plants. For ozone

treatment this effect was revealed independently of environmental conditions. Effects

of UV-B on SOD activity depended on temperature and CO 2 concentration. Under

elevated conditions of temperature and CO 2 , SOD activity in leaves was even slightly

lower.

Increasing SOD activity also confirms the conclusion of many investigators that

one of the main and common mechanisms of both stress-inducing factors, UV-B and

ozone, is oxidative burst (Krupa, 2003; Frohnmeyer, Staiger, 2003; Rančelienė et al.,

2005; Mittler, 2006).

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Conclusions. 1. Effects of UV-B and ozone on Crepis capillaris plants depend

on plant feature, used for evaluation of the effect, and on environmental conditions of

treatment (temperature and concentration of CO 2 ).

2. The most informative features are leaf area and activity of superoxide

dismutase.

3. Crepis capillaris plants have natural systems to avoid negative action of UV-B

and ozone.

Acknowledgements. This research was supported by the Lithuanian State Science

and Studies Foundation programme “APLIKOM”. The authors gratefully acknowledge

the Lithuanian Institute of Horticulture for possibility to perform our experiments in

growth chambers of the Institute.

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SODININKYSTĖ IR DARŽININKYSTĖ. MOKSLO DARBAI. 2008. 27(2).

Modelinio augalo Crepis capillaris reakcija į stresą sukeliančius

veiksnius – ozoną ir UV-B

V. Rančelienė, R. Vyšniauskienė

Santrauka

Stresą sukeliantis ozono ir UV-B poveikis augalams yra pripažintas reiškinys, tačiau

nepaisant skirtingų molekulinių veikimo mechanizmų, abiem stresą sukeliantiems veiksniams

būdingos ir bendros savybės: neigiamas poveikis fotosintezei, oksidacinis „sprogimas“ ir kt.

Šiame darbe abiejų veiksnių poveikis ištirtas priklausomai nuo temperatūros (21/14 °C) ir

(25/16 °C) ir CO 2 koncentracijos (350 ir 700 ppm). UV-B dozės buvo 0; 2; 4; kJ/m 2 , o O 3 – 20;

40; 80 ppb. Poveikis priklausė nuo UV-B tyrimo sąlygų ir nuo pasirinkto tyrimams požymio.

Vienas svarbiausių požymių, ypač UV-B, buvo lapų plotas. Fotosintezės pigmentų (chlorofilo

a ir b, karotinoidų) koncentracija lapuose pakito priklausomai nuo poveikio UV-B sąlygų.

Labai padidėjo superoksido dismutazės (SOD) aktyvumas. UV-B ir ozono poveikis pastebimai

priklausė nuo temperatūros ir CO 2 koncentracijos.

Reikšminiai žodžiai: Crepis capillaris, kompleksinis poveikis, ozonas, padidėjusi

temperatūra, CO 2 , UV-B.

137

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