Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2007
Volume 10
Issue 1
Topic:
Environmental Development
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Borowiak K. , Drzewiecka K. , Goliński P. , Zbierska J. 2007. PHYSIOLOGICAL REACTION OF TOBACCO PLANTS TO AMBIENT AIR POLLUTION WITH TROPOSPHERIC OZONE – PRELIMINARY STUDIES, EJPAU 10(1), #06.
Available Online: http://www.ejpau.media.pl/volume10/issue1/art-06.html

PHYSIOLOGICAL REACTION OF TOBACCO PLANTS TO AMBIENT AIR POLLUTION WITH TROPOSPHERIC OZONE – PRELIMINARY STUDIES

Klaudia Borowiak1, Kinga Drzewiecka2, Piotr Goliński2, Janina Zbierska1
1 Department of Ecology and Environmental Protection, August Cieszkowski Agricultural University of Poznan, Poland
2 Department of Chemistry, August Cieszkowski Agricultural University of Poznan, Poland

 

ABSTRACT

The tropospheric ozone is recently reported to be one of the most important air pollutants. Many methods and experiments have been designed to measure the tropospheric ozone concentration and to determine its influence on living organisms. The most sensitive bioindicator of ozone is the tobacco plant (Nicotiana tabacum L.). The physiological reaction of this and other plants to ozone are recently the subject of intensive studies. During experiments with tobacco, plants of two different cultivars were in two series exposed to ambient air for a time period of 14 days. After exposure the following parameters were measured: daily growth of plants, percentage of leaf injury, dry matter, chlorophyll and salicylic acid content. This method of ozone biomonitoring based on two tobacco cultivars of different susceptibility to ozone not only gives the unique opportunity to measure a visible effect on plants, but also to establish the mechanism of physiological reaction. Plants used for testing showed visible leaf injury, decreased in growth and chlorophyll content, increased in dry matter and salicylic acid content as the result of reaction to higher ozone concentration.

Key words: tobacco plant, tropospheric ozone, bioindication, chlorophyll, salicylic acid.

INTRODUCTION

The tropospheric ozone, because of its high phytotoxicity and still increasing concentration is lately reported as one of the most important air pollutants. Ozone is a well known atmospheric constituent, named by Schönbein [18] in the nineteenth century, indicating characteristic flavour induced during electrical discharge. Several methods and experiments have been designed to measure the tropospheric ozone concentration and to determine its influence on living organisms.

Since the beginning of the last century the concentration of ozone in the air has doubled [4]. The physiological reaction of plants to ozone is recently the subject of intensive studies [2, 3, 21].

In several countries, plants are commonly used as bioindicators for assessing ozone [7, 8, 10]. The results achieved in such studies indicate real influence of air pollutants on plants and makes it possible to measure symptoms. Plants can be used as bioindicators in areas where it is difficult and/or impossible to locate analytic equipment. The most sensitive bioindicator for ozone is the tobacco plant (Nicotiana tabacum L.) used for the first time in the 1950s [10]. Since then, tobacco plants have been used in numerous biomonitoring programs throughout Europe and North America [7, 8, 9]. Physiological reactions of tobacco plants are used as an indicator of the presence of ozone in the air and ozone induced oxidative stress. Experiments in open top chambers (OTC) under controlled conditions combined with field exposure allows to estimate the impact of air pollutants on plants, also taking into account climatic factors.

The ozone sensitive tobacco cultivar Bel-W3 not only exhibits visible symptoms of ozone influence, but also reveals intracellular reactions of oxidative stress by increase of chemical biomarkers, enhanced activity of antioxidants and higher accumulation of signalling and defence substances.

Ozone (O3), being a very reactive three atom allotrope of oxygen, can react with plants in the solid, gas and liquid phase causing visible changes on the leaf surface as the result of damage of lipids, proteins and other cellular components. It was previously suggested, that ozone does not penetrate deeply into the leaf cells, but rather reacts with surface cell constituents of the wall and plasma membrane. Reactive oxygen species (ROS), such as the super oxide anion and hydrogen peroxide, as well as the effect of O3 dissociation in aqueous solution were suggested by Weiss [23]. On the other hand O3 reacts with lipid molecules generating aldehydes and H2O2 while *O2- and H2O2 form hydroxyl radicals (OH*) with transition metals. Furthermore, O3 reacts with thiol groups, amines and/or phenolic compounds exacerbating formation of OH* and singlet oxygen (1O2) [11, 15].

Fig. 1. Salicylic acid and its conjugates

Increased ROS concentration at the cell surface may cause changes in plasma membrane permeability resulting in changes of ion fluxes, especially the raise of Ca2+ influx. This reaction follows ROS overproduction and accumulation called “oxidative burst” [11]. According to the high reactivity of ROS, rapid oxidation of membrane lipids, proteins and other cellular components, followed by their dysfunction and induced cell death manifested by the appearance of necrotic lesion in and around the location of leaf surface penetration by O3 was observed. The above reaction is recognized as the hypersensitive response (HR) [11, 16] and ROS are also suggested as HR regulators due to their ability to activate complex defence mechanisms, gene expression and synthesis of several different defence and signalling compounds [11]. Hydrogen peroxide is well known as a compound activating the phenylpropanoid pathway and biosynthesis of salicylic acid (SA) [9, 12, 18]. SA was found in tobacco leaf tissue as a free acid, as volatile methyl ester and as SA-glucoside (Fig. 1.). The latter compound was found only in and around necrotic lesions and works probably as a SA storage metabolite slowly releasing free salicylic acid when needed [5, 17].

Recent studies indicate that SA can reduce the activity of catalase (CAT) and ascorbate peroxidase (APx), two enzymes of the antioxidative complex detoxifying ROS, leading to elevated hypersensitive response (Fig. 2.) [17].

Fig. 2. Salicylic acid biosynthesis pathway and its function in plant cell

MATERIALS AND METHODS

The field experiment with tobacco plants (Nicotiana tabacum L.) was performed in the densely populated city of Poznań (Central-West, Poland). Four exposure sites were located across the city, including one site in the town centre, where the car traffic is most intense. One site was located 30 km out of the city in the surrounding county side (Fig. 3). On each site five plants of the sensitive cultivar (Bel-W3) and one of the resistant cultivar (Bel-B) were placed. The number of plants at exposure site was planned according to Giordani [6]. Plants were cultivated at controlled conditions for 8 weeks, and then exposed to ambient air conditions in different sites. In each location exposed plants were in the same stage of growth. Plants were held 1 m above the ground level and watered by glass fibre wicks, which conveyed water from a water tank located below. Shading fabric was used to prevent plants from strong sun operation. During the experiment plants were two times exposed to an ambient air for 14 days (May 26 till June 8 and from June 23 till July 6, 2003).

Fig. 3. Localization of exposure sits at Poznań city area and rural area

Two weeks after exposure to ozone the following parameters of plant and leaf growth were measured: plants’ daily growth, percentage of leaf injury, dry matter, chlorophyll and salicylic acid content. The percentage of leaf injury and chemical parameters were analysed for the 5th leaf from the bottom of the plants. The percentage of ozone induced injury in plants was measured by visual estimation, still the best and fastest method of quantitative analysis [12]. Daily growth was evaluated using metrical methods. Dry matter was measured according to the method described earlier by Ostrowska et al. [14]. After extraction with dimethyl sulphoxide (DMSO) the chlorophyll content was analysed according to the method of Shoaf and Lium (5 ml of DMSO per 200 mg of leaf tissue, after 20 min. of incubation) [20]. Extracts were assayed immediately or stored at 0-4 °C till colorimetric analysis at wavelengths 645, 652 and 663 nm. A Spekol Karl Zeiss spectrophotometer and the following equations were used to determine chlorophyll contents (in mg per g of fresh matter) [1]: (12.7 D663 – D645)·(V/1000·W) for chlorophyll a or (22.9 D645 – D663)·(V/1000·W) for chlorophyll b or 27.8 D652·(V/1000·W) for chlorophyll a+b, where D is absorbance for wave length; V – total extract volume [cm3] and W – weight of sample [g].

Salicylic acid content in tobacco leaf tissue was measured by modified method described by Yalpani [22]. Leaf tissue samples (0.5 g of fresh weight), were instantly frozen in liquid nitrogen and ground to a fine powder in a mortar. SA was extracted with 90 % methanol aq. followed by straight solvent. Combined organic fractions were mixed, divided into two equal parts, evaporated to dryness under a stream of nitrogen and reconstituted with 5 % TCA. For total SA measurement, pellets were first resuspended with β-glucosidase solution and incubated for 90 minutes at 37 °C, SA was partitioned three times with the extraction medium containing ethyl acetate : cyclopentane : isopropanol (100:99:1 v/v/v). Organic phases were combined and evaporated to dryness, dissolved in the mobile phase (0.2 M sodium acetate water solution, pH 5.0) and after separation on a C18 analytical HPLC column SA concentration was determined by fluorescence intensity measurement. The recovery for the spiked samples was 86-91 %.

The results obtained were analysed statistically using of STATISTICA package, and the distribution of values was analysed by the Shapiro-Wilk test, the correlation of measured parameters was tested by Pearson's index [19].

RESULTS

The ozone-induced leaf injury was observed only for the sensitive cultivar, which proved the medium ozone concentrations (80-100 µg·m-3). The highest leaf injury for ozone sensitive cultivars reached 55 %, the lowest one 0 % (Tab. 1). A greater leaf injury was observed at almost all of the sites in the first exposure series (May 26 till June 8, 2004), when higher ozone concentrations occurred due to meteorological conditions supporting their generation (high solar radiation, temperature and air humidity). The exception was the site number two, where leaf injury on plants might be due to local emission of air pollutants (ozone precursors). There were not any necrotic lesions on tobacco leaves in the exposure site number 4 localized in the city centre. The highest leaf injury was observed for plants at suburban sites, what confirms the relationship between ozone generation and trans-border transport of its precursors.

Table 1. The percentage of leaf injury, leaf dry matter, plant daily growth and salicylic acid content in tobacco leaf tissue

Exposure site

Time of exopsure series*

leaf injury
[%]

dry matter
[%]

daily growth [cm·day-1]

salicylic acid [ng·g-1]

fresh matter

dry matter

sen

sen**

res**

sen

res

sen

res

sen

res

1

1

10

17.4

8.2

3.73

3.92

403

19

2317

229

2

1

8.8

6.8

2.43

3.05

30

19

339

284

2

1

3

6.5

4.7

4.12

4.60

217

12

3347

265

2

10

6.1

4.5

2.63

2.79

251

14

4139

299

3

1

12

20.2

8.6

2.42

2.66

242

20

1195

226

2

8

6.9

6.8

1.99

1.83

-

-

-

-

4

1

0

6.1

5.5

2.68

3.20

14

14

232

259

2

0

7.7

8.6

1.96

2.00

18

20

240

235

5

1

55

12.6

10.1

2.85

3.30

533

16

4232

162

2

10

7.3

8.0

1.68

2.20

256

15

3500

190

Control

1

0

8.2

7.0

-

-

9

8

107

110

2

0

8.2

7.0

-

-

10

8

117

115

*) 1 – 26/05-9/06/2003; 2 – 23/06-6/07/2003
**) sen – sensitive; res – resistant cultivar

In most cases the percentage of dry matter was higher in samples from plants located in suburban and rural sites than in those from control plants. In plants exposed in the city centre and in the Old Town the percentage of dry matter was lower than in control ones. This was valid for both, resistant and sensitive cultivars.

Daily growth of plants was higher for the resistant cultivar Bel-B than for the sensitive cultivar Bel-W3 almost at all analysed sites in both exposure terms.

As a result of leaf injury a decreased concentration of total chlorophyll in the leaf tissue and an increase of dry matter was observed. The reduction of chlorophyll a concentration was similar to that of chlorophyll b. Chlorophyll content in dry matter was usually lower in the resistant cultivar grown under controlled conditions, but in the field experiment it was valid only for lower leaf injury values. For higher leaf injuries chlorophyll concentration in dry matter was higher in sensitive plants (Tab. 2). The higher correlation was found rather for the chlorophyll content in dry matter and the percentage of leaf injury than for chlorophyll content in fresh matter.

Table 2. Chlorophyll content in tissue of tobacco leaves

exposure site

time of exposure series*

chlorophyll a+b
[mg·g-1 of fresh matter]

chlorophyll a+b
[mg·g-1 of dry matter]

chlorophyll a
[mg·g-1 of fresh matter]

chlorophyll a
[mg·g-1 of dry matter]

chlorophyll b
[mg·g-1 of fresh matter]

chlorophyll b
[mg·g-1 of dry matter]

sen**

res**

sen

res

sen

res

sen

res

sen

res

sen

res

1

1

0.29

0.40

1.69

4.93

0.18

0.27

1.03

3.30

0.09

0.11

0.52

1.35

2

0.23

0.48

2.56

7.06

0.15

0.32

1.70

4.71

0.07

0.14

0.79

2.06

2

1

0.57

0.78

8.74

16.52

0.55

0.58

8.48

12.33

0.14

0.15

2.16

3.19

2

0.41

0.56

6.76

12.29

0.25

0.37

4.13

8.17

0.13

0.16

2.15

3.53

3

1

0.36

0.28

1.80

3.29

0.26

0.19

1.28

2.20

0.10

0.08

0.49

0.93

2

0.22

0.48

3.15

7.13

0.15

0.33

2.19

4.88

0.06

0.13

0.87

1.92

4

1

0.46

0.39

7.57

7.07

0.39

0.27

6.37

4.92

0.09

0.09

1.47

1.64

2

0.52

0.45

6.75

5.15

0.32

0.29

4.15

3.35

0.16

0.12

2.07

1.39

5

1

0.29

0.36

2.30

3.58

0.18

0.28

1.43

2.76

0.08

0.07

0.64

0.69

2

0.29

0.25

3.93

3.08

0.17

0.17

2.32

2.12

0.09

0.06

1.23

0.75

control

-

0.66

0.49

8.11

5.83

0.44

0.39

4.97

3.51

0.20

0.11

2.70

2.45

*) 1 – 26/05-9/06/2003; 2 – 23/06-6/07/2003
**) sen – sensitive; res – resistant cultivar

Salicylic acid content showed significant differences between plants with the highest value of 4232 ng per gram of dry matter in site number 5. The increased SA concentration due to the plant's physiological reaction to ozone was observed before visible leaf injury (site number 4).

The stronger plant reaction to ozone (higher injury of leaves) was positively correlated with salicylic acid content in fresh leaf tissue, its concentration in dry matter was close to be significantly correlated with leaf injury.

Table 3. Pearson linear correlations coefficient of measured parameters

Variable

injury

dm
sen

dm
res

growth sen

growth res

ch a+b sen

ch a+b res

ch a+b dm sen

ch a+b dm
res

ch a
sen

ch a
res

ch a
dm sen

ch a
dm
res

ch b
sen

ch b
res

ch b
dm sen

ch b
dm
res

S.A.
sen

S.A.
res

S.A.
dm
sen

S.A.
dm
res

Injury

1.00

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

dm sen

0.33

1.00

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

dm res

0.59

0.61

1.00

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

growth sen

0.01

0.26

-0.10

1.00

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

growth res

0.19

0.25

-0.21

0.88*

1.00

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ch a+b sen

-0.33

-0.27

-0.39

0.48

0.43

1.00

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ch a+b res

-0.28

-0.46

-0.69*

0.49

0.41

0.49

1.00

 

 

 

 

 

 

 

 

 

 

 

 

 

 

ch a+b dm sen

-0.43

-0.71*

-0.69*

0.23

0.24

0.85*

0.62

1.00

 

 

 

 

 

 

 

 

 

 

 

 

 

ch a+b dm res

-0.34

-0.52

-0.85*

0.40

0.44

0.54

0.95*

0.72*

1.00

 

 

 

 

 

 

 

 

 

 

 

 

ch a sen

-0.34

-0.29

-0.52

0.60

0.57

0.92*

0.62

0.83*

0.68*

1.00

 

 

 

 

 

 

 

 

 

 

 

ch a res

-0.18

-0.44

-0.64*

0.55

0.48

0.49

0.99*

0.61

0.93*

0.66*

1.00

 

 

 

 

 

 

 

 

 

 

ch a dm sen

-0.41

-0.61

-0.71*

0.41

0.42

0.84*

0.69*

0.95*

0.78*

0.93*

0.71*

1.00

 

 

 

 

 

 

 

 

 

ch a dm res

-0.30

-0.50

-0.82*

0.45

0.48

0.55

0.95*

0.71*

0.99*

0.71*

0.95*

0.79*

1.00

 

 

 

 

 

 

 

 

ch b sen

-0.29

-0.20

-0.23

0.32

0.22

0.87*

0.43

0.69*

0.44

0.66*

0.40

0.58

0.43

1.00

 

 

 

 

 

 

 

ch b res

-0.47

-0.41

-0.70*

0.16

0.06

0.25

0.85*

0.43

0.80*

0.28

0.76*

0.40

0.75*

0.35

1.00

 

 

 

 

 

 

ch b dm sen

-0.41

-0.72*

-0.62

0.09

0.08

0.79*

0.62

0.94

0.69*

0.67*

0.58

0.81*

0.67*

0.81*

0.53

1.00

 

 

 

 

 

ch b dm res

-0.41

-0.52

-0.89*

0.17

0.23

0.39

0.87*

0.62

0.94*

0.46

0.81*

0.61

0.90*

0.39

0.91*

0.67*

1.00

 

 

 

 

SA sen

0.81*

0.51

0.44

0.31

0.50

-0.24

-0.18

-0.43

-0.18

-0.25

-0.09

-0.38

-0.14

-0.14

-0.36

-0.38

-0.26

1.00

 

 

 

SA res

-0.07

-0.15

-0.01

-0.25

-0.49

-0.46

0.04

-0.25

-0.06

-0.35

0.02

-0.23

-0.07

-0.46

0.17

-0.24

0.01

-0.23

1.00

 

 

SA dm sen

0.57

0.02

0.00

0.19

0.48

0.07

0.13

0.06

0.23

0.04

0.20

0.05

0.26

0.19

-0.09

0.16

0.16

0.79*

-0.43

1.00

 

SA dm res

-0.44

-0.11

-0.52

0.17

0.33

0.51

0.35

0.46

0.46

0.44

0.30

0.42

0.43

0.51

0.44

0.47

0.53

-0.25

-0.74*

0.08

1.00

Abbrevations:
Dm – dry matter; res – resistant cultivar; sen. – sensitive cultivar; growth – daily growth; ch a+b – chlorophyll a+b; ch a – chlorophyll a; ch b – chlorophyll b; SA – salicylic acid

The statistical analysis revealed no correlation (at alpha level 0.05) between ozone induced leaf injury and other parameters. The chlorophyll content was negatively correlated (at alpha level 0.05) with dry matter and also negatively but not significantly correlated with leaf injury (Tab. 3).

CONCLUSIONS

  1. The experiment of the ozone concentration biomonitoring using two tobacco cultivars of different susceptibility gives not only the unique opportunity to measure a visible effect of ozone on plants, but also to assess the mechanism of physiological reaction of plants to ozone.

  2. Test plants showed visible leaf injury, decrease in growth and chlorophyll content, increase of dry matter and salicylic acid content as the result of their reaction to higher ozone concentration in the ambient air. The meteorological conditions influenced the injury of leaves due to the impact on ozone generation. Compared to the fresh matter the higher correlation was observed between the chlorophyll content in dry matter and the leaf injury.

  3. Significant correlation between the leaf injury and salicylic acid concentration in the tissue was found. Next to role of SA – major signalling compound in plant reaction to oxidative stress in plants are expected to elucidate complex intracellular mechanism of plant response to ozone, including antioxidants and enzymes activity measurements.


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Accepted for print: 18.01.2007


Klaudia Borowiak
Department of Ecology and Environmental Protection,
August Cieszkowski Agricultural University of Poznan, Poland
Piatkowska 94C, 61-691 Poznan, Poland
email: klaudine@au.poznan.pl

Kinga Drzewiecka
Department of Chemistry,
August Cieszkowski Agricultural University of Poznan, Poland
Wojska Polskiego 75, 60-625 Poznan, Poland
email: kingad@au.poznan.pl

Piotr Goliński
Department of Chemistry,
August Cieszkowski Agricultural University of Poznan, Poland
Wojska Polskiego 75, 60-625 Poznan, Poland
email: piotrg@au.poznan.pl

Janina Zbierska
Department of Ecology and Environmental Protection,
August Cieszkowski Agricultural University of Poznan, Poland
Piatkowska 94C, 61-691 Poznan, Poland
email: jzbier@au.poznan.pl

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