Effects of Low SO2 Levels on Superoxide Dismutase and Peroxidase Isoenzymes in Two Different Wheat Cultivars

Effects of Low SO2 Levels on Superoxide Dismutase and Peroxidase Isoenzymes in Two Different Wheat Cultivars

Biochem. Physiol. Pflanzen 188, 67-71 (1992) Gustav Fischer Verlag Jena Short Communication Effects of Low S02 Levels on Superoxide Dismutase and Pe...

373KB Sizes 2 Downloads 48 Views

Biochem. Physiol. Pflanzen 188, 67-71 (1992) Gustav Fischer Verlag Jena

Short Communication

Effects of Low S02 Levels on Superoxide Dismutase and Peroxidase Isoenzymes in Two Different Wheat Cultivars ANNAMARIA RANIERI!) MAURO DURANTE 2), ARMANDO VOLTERRANI!), GIACOMO LORENZINI 3 ) and GIAN FRANCO SOLDATINI 1) Istituto di Chimica Agrarial); Sezione di Genetica del Dipartimento di Biologia delle Piante Agrarie 2); Sezione di Patologia Vegetale del Dipartimento di Coltivazione e Difesa delle Specie Legnose 3 ); Universita di Pisa, Italy Key Term Index: Peroxidase, S02 pollution, superoxide dismutase; Triticum aestivum

Summary The isoenzyme patterns of peroxidase (POD) and superoxide dismutase (SOD) were studied in the leaves of two different cultivars of wheat (Triticum aestivum cv. Chiarano and Mec) exposed to low concentrations of S02 (35, 75, 120 nl I-I). After the S02 treatments quantitative differences in the profile of the peroxidase bands were found, but no new isoforms were detected. An increase in the isoforms showing SOD activity was seen, this effect being more pronounced in cv. Chiarano than in cv. Mec. Our data show that the two wheat cultivars differ in their susceptibility to S02 pollution, cv. Chiarano exhibiting a higher resistance.

In S02-treated plants the destruction of chlorophyll and the production of peroxydative products of unsaturated fatty acids have been shown to be caused by O 2 and 102 , respectively (SHIMAZAKI et al. 1980). If S02 toxicity is, even in part, due to oxygen toxicity, plants might protect themselves against SOz-induced oxygen toxicity by using scavengers for active oxygen such as superoxide dismutase (SOD) and peroxidase (POD) (TANAKA and SUGAHARA 1980). FRIDOVICH and HANDLER (1961) first suggested that when hydrogen peroxide is available, sulphite may be oxydized to sulphate in the presence of POD; this would explain the observed increase in POD activity in plant cells exposed to S02. To date there is little information on the effects of permanent low levels of S02 pollution, as may commonly be found in rural areas, on the isoenzyme polymorphism of leaf SOD and POD in crop plants. This work is aimed at evaluating the responses of two ""heat cultivars with different sensitivities to S02 (LORENZINI et al. 1990) through a comparison of the isoenzyme polymorphism of the protective enzymes SOD and POD. Abbreviations: TRIS, tris(hydroxymethyl)aminomethane; PVP, polyvinylpyrrolidone; EDTA, ethylenediaminetetraacetic acid; TEMED, N, N, N', N'-tetramethylethylenediamine

5*

BPP 188 (1992) 1

67

Plant Material

Commercial caryopses of two cultivars of Triticum aestivum L. (Mec and Chiarano) were germinated and grown in perforated black polyethylene bags containing 900 cm3 of a fertilized compost. After seven days, ten bags were placed for four months in S02 fumigation chambers that were ventilated with filtered air. To each chamber a known concentration of S02 was added: 35 ± 7.9, 75 ± 15.2 and 120 ± 17.5 nil-i. For comparison another group of ten bags was placed in chambers with charcoal filtered air (LORENZINI et al. 1990) for the same period. Each treatment was replicated two times. At the end of the growing period there were an average of 3 shoots per bag. The flag leaf of eacp shoot was collected and stored at - 80°C. The plants were regularly subirrigated via a bed of perlite. The chambers were placed inside a greenhouse under normal environmental conditions. The incoming photosynthetically active solar radiation was monitored by spot measurements (LI-COR 185B quantum radiometer) and was found to be about 70% of the ambient values. Soluble protein and enzyme extraction Leaf samples (2 g) were crushed in liquid N2 and then homogenized in a mortar at 4 °C in 0.2 M Tris HCI (PH 7.6) buffer containing 0.1 % insoluble PVP and I mM EDTA. After centrifugation of the homogenate at 24,000 x g for 10 min at 5°C, the supernatant yielded a soluble protein fraction suitable for the protein determination and electrophoretic enzyme assays after dialysis against water (CHAHAL et aI. 1988; LEIDI et aI. 1989).

Electrophoresis

Polyacrylamide gel electrophoresis was performed in a vertical slab apparatus on 7 % acrylamide gels to separate the SOD (EC 1.15.1.1) isoenzymes. Electrophoresis was carried out at 4 °C with a constant voltage of 140 V in 0.1 M Tris-glycine (PH 8.3) buffer. The POD isoenzymes were separated by horizontal slab isoelectric focusing using the LKB PAGE plate pH range 3.5-9.5. Samples of 20 f-lg of total protein were dissolved in I % glycine, absorbed on rectangular pieces of filter paper (Whatman 3 MM) and applied to the gel surface. The IEF was carried out at 25 W for I h 30 min. Densitometric readings were performed by a VERNON P.I.6 scanner. The relative amount of protein corresponding to each band was calculated by taking the percentage of each peak area over the total area of the electrophoretogram. Staining procedure

A Coomassie blue staining procedure was used to detect the soluble proteins. The POD banding patterns were visualized with benzidine dihydrochloride by the method of KUHNS and FREK (1978). The SOD isoenzymes were visualized by the negatively stained photochemical procedure of BEAUCHAMP and FRIDOVICH (1971). All the data presented are the means of three replicates.

Previous studies on growth parameters have reported cv. Mec to be more sensitive to S02 fumigation than the cv. Chiarano (LORENZINI et al. 1990). These data have been

confirmed by measurements of "in vivo" protein synthesis, determined by the incorporation of labelled methionine. With this method a more pronounced fall in amino acid labelled incorporation was seen in the cv. Mec plants subjected to S02 pollution than in the cv. Chiarano plants (BERNARDI et al. 1990). The different responses to S02 of these two cvs. were observed in this work, as the patterns of protective enzymes are concerned. 68

BPP 188 (1992) 1

Peroxidase isoenzyme patterns The isoenzymes of total leaf POD were identified by isoelectrofocusing analysis. Many bands with POD activity were detected in both wheat cultivars. The isoelectric points of these bands ranged from pH 3.1 to 9.2. Differences in the intensity of single bands between the cultivars and among the S02 treatments were observed. At pI 4.8 two well separated bands of POD activity were clearly evident in the Mec plants subjected to S02' In the control plants this region was marked by a diffuse and weakly stained area which was not separated into distinct bands. A very different pattern was shown by cv. Chiarano; at pI 4.8 a single, very clear band was apparent in the control and in the sample treated with 35 nll- i of S02, while at the higher S02 concentrations this band tended to become indistinct. The gel densitometric tracings are reported in Fig. 1.

1 41

o c:

o

CtI

..0

...

o

35

..0

75

III

<

120

9.2 8.6 7.9 7.4 7.0 6.4 5.3 4.8

3.1

9.2 8.6

pi Fig. 1. IEF densitometric tracings of leaf peroxidase isoenzymes from leaves of Triticum aestivum cv. Chiarano (A) and cv. Mec (B). The numbers on the right indicate the S02 treatments (nIl-I). The experimental pI values are reported at the bottom.

POD is one of the most widely studied of those plant enzymes which serve as indicators of the metabolic disturbances caused by chronic fumigation with air pollutants. Increases in POD activity after exposure to 0 3 or S02 have been observed in the leaves of ornamental trees, bean and Scots pine, increases which were not accompanied by the synthesis of new isozymes of POD (CURTIS and HOWELL 1971; KIELISZEWSKA-RoKICKA 1979). In our experiments as well no new protein bands with POD activity were detected in the fumigated leaves. Superoxide dismutase isoenzyme pattern The SOD patterns were identified by PAGE analysis. The negative staining technique revealed three bands with SOD activity. The first band (Rf = 0.92) after densitometric BPP 188 (1992) 1

69

)--__ +'/1

A 250 ~

1 1 1 1

>. III

200

c:

...c:

I

Q)

I

150

>

a::

/

.J.,

/

I

I /

1 . 1/

aI

Q)

I

1

Q)

...

B

----r

100

I

/

"~'-'-'~.-.-.~ 50~----~----~----~~----~----~----~

o

35

75

120

35

75

120

802 (nl·r')

Fig. 2. Relative staining intensities of the three SOD bands: 1, (e); 2, (0); 3, (A) of the two wheat cvs: Chiarano (A) and Mec (B). The bars indicate the confidence limits at P = 0.05.

analysis (Fig. 2) showed a higher intensity in the S02 treated samples in both cultivars. The second band (Rf = 0.28) of SOD appeared less dense in the control plants in comparison to the treated plants, especially in cv. Chiarano where this band showed an increase in staining intensity of up to 150 % with the treatments. The behaviour of the third band (Rf = 0.068) with the S02 treatment was similar to the others in cv. Chiarano; on the contrary in cv. Mec this band showed a tendency to decrease slightly in the SOztreated samples in comparison to the control. The amount of SOD found in the leaves is thought to correlate with plant resistance to S02 toxicity. TANAKA and SUGAHARA (1980) found that poplar leaves which had acquired a high level of SOD activity after fumigation with low levels of S02 (0.1 nIl-I) were more resistant to the toxicity of a higher concentration of S02 (2.0 nIl-I). A marked increase in the band staining of all the SOD isoenzymes with S02 treatments was observed in the cv. Chiarano. The cv. Mec showed a lower increase in two band and even a decrease in the 3rd band. This could indicate a higher capacity on the part of cv. Chiarano to detoxify harmful superoxide radicals. This cultivar appears to have a higher resistance than cv. Mec to S02 pollution in spite of its greater sulphur uptake (BERNARDI et al. 1990). Probably these plants possess a mechanism of tolerance which serves to mitigate any potential damage caused by the pollutant. Our results add to the as yet all too sparse literature concerning experiments with low levels of S02 fumigation and show that alterations in plant isoenzyme patterns may occur in absence of visible injury, as reported by VARSHNEY and VARSHNEY (1984). Moreover our data show that the monitoring of superoxide dis mutase isoenzyme patterns provides a 70

BPP 188 (1992) 1

more efficient tool than the much more complex POD isoenzyme to evaluate the different susceptibilities to S02 treatment of different plant cultivars. As reprorted by QUEIROZ (1988), alterations in the isoenzyme patterns in response to S02 pollution could indicate differences in gene expression. To further improve our understanding of the effects of S02 on plant enzymes, more precise information is needed on the gene-protein process and those steps which are affected by the pollutant. Acknowledgements This study has been financially supported by the Ministero Universita Ricerca Scientifica e Tecnologica, Rome (40 % Project).

References BEAUCHAMP, C., and FRlDOVICH, l.: Superoxide dismutase : improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276-287 (1971). BERNARDI, R., RANIERI, A., VOLTERRANI, A., and SOLDATINI, G. F.: Effect of S02 concentrations on "in vivo" protein synthesis in wheat plants. Giornale Bot. It. 124, 155-156 (1990). CHAHAL, S. S., KUMAR, R., SIDHU , 1. S., and MINOCHA, 1. L.: Peroxidase isoenzyme pattern in pearl millet lines resistant and susceptible to downy mildew. Plant Breeding 101, 256-259

(1988).

CURTIS, c. R., and HOWELL, R. K.: Increase in peroxidase isoenzyme activity in bean leaves exposed to low doses of ozone. Phytopathol. 61 , 1306-l307 (1971) . FRIDOVICH , I., and HANDLER, P .: Detection of free radicals generated during enzymatic oxidations by the initiation of sulphite oxidation . 1. BioI. Chern. 236,1834-1840 (1961). KIELlSZEWSKA-RoKICKA, B .: Peroxidase activity in varieties of Weigela and Pinus sylvestris resistant and susceptible to S02' Arboretum Kornickie 24,313-320 (1979). KUHNS, J., and FRETZ, T. A.: Distinguishing rose cultivars by polyacrylamide gel electrophoresis . Isoenzyme variation among cultivars. J. Am. Soc. Hort. Sci. 103, 509-516 (1980). LEIDI, E. 0., GOMEZ, M., and DEL RIO , L. A.: Peroxidase isozyme patterns developed by soybean genotypes in response to manganese and iron stress. Biochem. Physiol. Pflanzen 185,

391-396 (1989). LoRENZINI, G., PANICUCCI , A., and GUIDI.: Growth dynamics of wheat (Triticum aestivum L.) exposed to sulfur dioxide polJution . Bull. Environ. Contam . Toxicol. 45 , 408-414 (1990). QUEIROZ , 0. : Air pollution, gene expression and post-translational enzyme modifications, In : Air Pollution and Plant Metabolism (Eds. SCHULTE-HoSTEDE, S ., DARRAL , N . M. ,BLANK, L. W ., WELLBURN, A . R.) pp . 238-254. Elsevier Applied Science, London and New York 1988. SHIMAZAKI, K., SAKAKI, T., and SUGAHARA, K.: Active oxygen participation in chlorophyll destruction and lipid peroxidation in SOz-fumigated leaves of spinach. In: Studies on the effects of air pollutants on plants and mechanisms of phytotoxicity. pp . 91-101. National Institute for Environmental Studies,Tsukuba , Japan 1980. TANAKA, K., and SUGAHARA, K. : Role of superoxide dismutase in defense against S02 toxicity and an increase in superoxide dismutase activity with S02 fumigation. Plant & Cell Physiol. 21 ,

606-611 (1980). VARSHNEY , S. R . K. , and VARSHNEY , C. K .: Effect of low levels of S02 on glutamate dehydrogenase isoenzymes in crop plants . Biochem. Physiol. Pflanzen 179, 433-437 (1984) .

Received April 2, 1991; revised form accepted July 4, 1991 Authors' address: GIAN FRANCO SOLDATINI, 1st. Chimica agraria, Universita di Pisa, Via S. Michele degli Scalzi 2, 56124 Pisa, Italy.

BPP 188 (1992) 1

71