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Antioxidants and growth regulators counter the effects of O3 and SO2 in crop plants
..Igrwulture, Ecosystems and Environment, 38 ( 1992 ) 99- ! 06 Elsevier Science Publishers B.V., Amsterdam
99
Antioxidants and growth regulators counter the effects of O3 and SO2 in crop plants E.H. Leea, G.F. Kramera, R.A. Rowland a and M. Agrawalb ~('iimate Stress Laboratory, USDA-ARS, Beltsville, MD 20705-2350, USA ~'Dept. o~Botan.l'. Barnaras ltindu Universit),. Varasnasi-221005, India (Accepted 25 May 1991 )
ABSTRACT Lee, E.H., Kramer, G.F., Rowland, R.A. and Agrawai, M., 1992. Antioxidants and growth regulators counter the effects ofO~ and SO,, in crop plants. Agric. ifcos.w'tems Environ., 38: 99-106. Antioxidant compounds such as cthylenediurea (EDU); N- [ 2-(2-oxo-I-imidazolidinyl ) ethyl ]-N'phenylurea have been found to be effecti~ e in suppressing O3-induced leaf injury. Gibberellic acid (GA) inhibitors such as paclobutrazol, tetcyclaics and flurprimidoi suppress visible SO2-induced injury. Antioxidant compounds are relatively ineffective in protecting plants from SO2-induced injury, while GA inhibitors are ineffective in protecting plants from 03 injury. EDU and GA inhibitors do not alter the stoma~.al behavior, however they can induce resistance to air pollutants stress. Furthermore. EDU and paclobutrazol (PP,33) do not alter foliar membrane lipid composition. EDU does not alter polyamine composition. This suggests that the mechanism of plant tolerance to air pollutant exposure is probably biochemical rather than biophysical. Free radical scavenging enzymes or endogenous antioxidant compounds are more important than stomatal restriction of pollutant diffusic.n to the sensitive leaf mesophyll sites.
INTRODUCTION
Interest in the usefulness of antioxidants for cellular protection against oxidative stress in plants has increased dramatically (Larson, 1988; Rao and Dubey, 1990). Ethylenediurea (EDU); N-[2-(2-oxo-l-imidazolidinyl) ethyl ]-N'-phenylurea has been used to determine the effects of ambient ozone (O3) on plants (Carnahan et al., 1978; Brennan et al., 1987 ). While EDU has shown induction of cellular defenses against oxidative stress (Lee and Bennett, 1982 ), the exact mechanism of EDU-induced resistance to air pollutants still remains unclear. A close interrelationship between EDU-induced resistance and oxidative stress is generally thought to result from free radical reactions (Lee and Bennett, 1982 ). Recent work has shown that increased plant tolerance to environmental stress may be achieved by means of plant growth regulators and antioxidant treatments (Lee et al., 1987; Hausladen and Kunet* 1990). An understanding of the biochemical events providing protec© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-8809/92/$05.00
100
E.H. LEE ET AL.
tion against oxidative damage is essential for the development of crop plants more resistant to a growing number of environmental stresses (Monk et ai., 1989). Our objectives were to determine the effects of soil applications of GA inhibitors and antioxidants on plant sensitivity to SO: and 03 and to evaluate the nature of susceptibility to these air pollutants. MATERIALS AND M E T H O D S
Clwmical modification of plants and air pollution treatments Two cultivars of snapbean (Phaseolus vulgaris L. ) and soybean (Glycine max ( L. ) Merr ) shown previously to differ in their response to 03 stress were used in this study. Pollutant-sensitive (S) cultivars used were 'Bush Blue Lake290' (BBL-290) snapbean and 'Hark' soybean; O.rresistant (R) cultivars used were 'Astro' snapbean and 'Hood' soybean (Lee et al., 1984). Plants were germinated in 15 cm plastic pots containing Jiffy Mix potting mixture (Jiffy Products of America Inc., Chicago, IL, USA), and cultured in a charcoalfiltered (CF) air greenhouse as previously described ( Lee and Bennett, 1982 ). Treatments began when the first trifoliate leaves were 80% fully expanded. At this time, EDU or GA inhibitors were applied as a soil drench at equal dosages (Tables 1 and 2 ). Only single chemical applications were made. Control plants were watered with distilled water at the same time. All experiments were conducted as a split plot randomized experimental design. Plants were grown in CF air greenhouses as described (Lee and Bennett, 1982). Two to tbur days alter treatment, tbur replicate pots (one plant per pot) were subfABLE I
Abaxial surface stomatal resistance tbr fully expanded leaves ot'O~-sensitive (S) and Orresistant ( R ) plants ~ith and without E i ) t l ( 500 mg I r+~) treatment and O~ stress
Snapbean cultivar
Stomatal resistance (s cm - ~)
Pro-exposure
During exposure ~
Post-exposure:
1.92" 2.05"
3.2 I" 3.35"
3.43" 3.55"
- EDtl
2.15"
3.41"
4,15"
+ EDtl
2.30'
3.40"
4.25 h
"Bush Blue Lake-290" (S) - EDI, I + EDI, I '-~st ro" ( R )
'Planl,~ ~vcre exposed to 350 Ill ! ' O~ t't~r I-2 h. "Plants were exposed to the same concentration as above for 3 h. Values ill each column followed by the same superscript arc not significanlly different at the 0.05 level of signiticance using Duncan's multiple range lest.
ANTIOXIDANTS AND GROWTH REGULATORS AFFECT 02 AND SO, SENSITIVITY
10 !
TABLE 2 Stomatal resistance in fully expanded leaves of soybean (Glycine m a x (L.) Merr) cuiti, c "Hark' treated with selected GA inhibitors and exposed to SO2 at 1500 nl !- ' for 3 h Treatment
Injury score
Concentration (rag pot-~ )
Control Paclobutrazol Tetcyclacis Flurprimidol
0.00 0.20 2.50 0.25
Stomatal resistance (s cm- ' )
6.8" 0.0" 0.5 a 0.2 a
Pre-exposure
During exposure
Post-exposure
1.92" 1.89~ 2.04 ~b 2.38 h
4.10" 4.26" 4.12" 4.85"
4.54 a 4.76" 3.85" 5.2u"
Means represent four replicatiens/treatment. Within a column, any values having a superscript in common are not significantly different at the 5% level by the Duncan's multiple range tesl.
1 h exposure
~100
p
mp
50-
250 nl I-~
I I G)
/
80
0
E
I
40-
I I 30-
6o
a 40
ASTRO
j
13 ID 20 _.1
:-'
~_,I~-,iw
0
--
~ t
"~
I
200
i
40:,
nl
_
1
600
I --1 03
t
/
20 -
ASTRO
,, ,. "
x4" m
i!1//
10 -
1
800
0 !
1000
0
i
i
i
i
i
1
2
3
4
5
Exposure Time (h)
Fig. !. Dose-response curves showing eultivar differences in 03 sensitivity. Right: leaf damage at 250 nl i-~ 03 after 1-5 h of exposure. Left: leaf damage as ~ function of O3 concentration. Plants were rated for foliar 03 injury at 48 h after fumigation. The first trifoliate leaves were assessed for the percentage of the surface showing injury.
jected to SO., or 03 fumigation. Test plants were fumigated at concentrations as shown in Fig. 1 and Table 2. Ozone and SO2 fumigations were conducted in two separate environmentally controlled growth chambers (Controlled Environments Inc., Model PGW 36 ) as previously described (Lee and Bennett, 1982; Lee et al., 1984). Treated plants with or without air pollution stress were sampled for later polyamine and lipid analysis. After pollutant exposure, remaining plants were returned to the CF air greenhouse for 03 i,jury scoring after 48 h. Leaf injury from 0 to 10 was determined on fully expanded leaves (0, no injury; i O, severe injury ).
102
E.H. LEE ET AL.
Stomatal resistance measurement Stomatal resistance (s cm- t ) for the lower surfaces of fully expanded leaves were measured with a LI- 1600 steady state diffusion porometer (LI-COR Inc., Lincoln, NE). Prior to measurement, plants were equilibrated in the growth chambers for ' - 2 h. Porometer measurements were taken I h before fumigation and 1.5 h during, and 3 h after fumigation.
Biochemical ph,~spholipidand polyamine analysis Extraction and quantitative analyses oftotal leaf phospholipid contents were conducted on th(: first trifoliate leaf samples for all treatments. The methods employed have ht:en previously described (Lee et al., 1987; Whitaker et al., 1990). Membraae phospholipids were analyzed by GC or HPLC (Lee et al., 1987, Kendall ~:nd McKersie, 1989). Free polyamines were determined by methods similal to those reported by Kramer and Wang (1989).
Statistical analysis An analysis of variance (ANOVA) was performed on all data of leaf injury scores, phospholipid, and stomatal resistance to determine significant differences, followed by a Duncan's multiple range test to separate means. RESU LTS AN D D! S C U S I O N S
Crop plants response to O~ stress Figure 1 shows graphically the comparative dose-response curves for 03 injury in the both the snapbean cultivars as a function of O3 concentration and duration of' O~ exposure. The O3-sensitive cultivar BBL-290 exhibited foliar injury at a low concentration while much higher 03 levels were required to injure the Orresistant cultivar 'Astro'.
Characterization of stomatai resistance Leaf stomatal resistance measurements were made on the abaxial side of the leaves. Results given in Table 1 show that the stomatal resistance of O3-R and OrS plants was comparable within the pre-exposure, and during 03 exposure. Resistance for both O r S and O3-R cultivars increased from pre-exposure to post-exposure. However, no significant differences were noted for O3-S and O r R plants except for post-exposure. 'Astro' showed a significantly higher stomatal resistance level than O3-S snapbean during the post-exposure
ANTIOXIDANTS AND GROWTH REGULATORSAFFECT02 AND SO:,SENSITIVITY
103
period. Table 1 indicates no statistical differences between EDU-treated and untreated plants for stomatal diffusion. Resistance to air pollutant injury can be largely due to stomatal closure (Heath, 1988 ). Differences in stomatal frequency in leaves can be an important factor in determining pollutant susceptibility. However, in this study, measurements of leaf diffusive resistance in EDU-treated (Table l ) or GA inhibitor-treated and non-treated plants (Table 2) did not show differences in stomatal resistance sufficient to account for air pollutant exclusion from the leaf mesophyll. This result suggests EDU- and GA inhibitor-induced tolerance to air pollutant exposure probably is biochemical at leaf mesophyll sites rather than exclusion by physical means.
Polyamines and EDU treatment Polyamines have been shown to be antioxidants and to protect membranes from peroxidation (Bors et al., 1989). Polyamine content of fully expanded leaves of EDU-treated and control plants were compared before and after 03 exposure (Table 3 ). EDU does not alter the polyamine composition of snapbean leaves. However, with 03 stress alone (-EDU), significant increases of total polyamine were observed. This difference in the total polyamine content was due to increases in both putrescine (Put) and spermidine (Spd) while spermine (Spn) remained unchanged. The total polyamine content was unaffected by 03 stress in the presence of EDU. The antiozonant activity of EDU does not appear to involve the enhancement of polyamine levels. TABLE 3 Polyamine levels in levels in fully expanded leaves of snapbean cultivar BBL-290 subjected to O~ stress with or without EDU ( 500 mg I- ~) treatment Treatment
Control (no ozone ) -EDU +EDU Ozone treated" -EDU +EDU
Polyamine j (nmol g FW-~ )
Total polyamine
Put (SD)
Spd (SD)
Spn (SD)
15.9 (I.7) a" 14.3 (!.6) a
164 (14.6) a 176 (25.3) a
70.9 (6.3) a 66.4 (6.8) a
250.8 a 256.7 a
26.5 (5.9) b 15.4 ( l . 4 ) a
193 (5.4) b 144 (7.2) a
62.7 (3.7) a 62.4 (3.3) a
282.2 b 221.8a
' Put = putrescine, Spd = spermidine, Spn = spermine. -'Plants were exposed to the same concentration as above for 3 h. Values in each column followed by the same letter are not significantly different at the 0.05 level of significance using Duncan's multiple range test.
104
E.H. LEE ET AL.
GA inhibitor and protection against SO., injuo' The effects of the GA inhibitors, paclobutrazol (PP333), tetcyclacis and flurprimidol, on visible SO2-induced injury and stomatal resistance are shown in Table 2. Leaves treated with PP333, tetcyclacis and flurprimidol showed a significant reduction in SO., injury. In addition, stomatal resistance of fully expanded leaves of GA inhibitor-treated and control plants were compared before, during, and after SO., exposure. With the exception of flurprimidol, stomatal resistance of soybean plants increased during and after SO., exposure but was unaffected by GA inhibitor treatment. Stomatal closure is not involved tn GA inhibitor-induced resistance to SO:. In previous studies (Lee at al., 1987), we have shown that in the absence of a SO, stress, no perceptible change occurred in the total lipid content of leaves from PP~.~-treated plants. When plants were subjected to SO., stress with 1500 nl i-~ SO: for 3 h, both PP333-treated and control plants showed a slight but non-significant decrease in total lipid content. Although the PP333 ;reatment (without SO, fumigation) did not alter the total lipid content of the leaves, it did induce an increase in the ratio of non-polar to polar lipids. GA inhibitors appear to be potential agents to induce SO, resistance, but the major constraint is the accompanying retardation of growth. Hence, attempts should be made to search for effective growth substances which will induce SO., resistance without affecting the growth of plants.
Dtil.'lD'entialair pollutant protection response to EDU and GA inhibitor Two to tbur days after soil drench with EDU or PP333, plants were exposed to two different types of air pollution stress. EDU provided good foliar protection of O~ stress, but this type of protection was not found in the PP333treated plants (data not shown). PP333 provided good foliar protection with increased resistance to SO,. However, soil application of PP333 at the same concentration which induced tolerance to SO,-stressed plants was ineffective for O.~ damage. Why does paciobutrazoi protect plants from SO:-induced injury, while EDU does not? GA inhibitors are capable of modifying the biosynthesis and action of GA in plant tissues through their affect on RNA and protein synthesis (Martin and Northcote, 1982 ). GA3, in particular, is involved in the synthesis ofmRNA and proteins in many plant organs. GA3 could promote changes in the levels ofcellular mRNA and protein/enzyme which are involved in the initiation of growth and regulate SO,, sensitivity. These results suggest that the mechanism of susceptibility of plants to 03 and SO2 are very different.
ANTIOXIDANTS AND GROWTH REGULATORS AFFECT O, AND SO, SENSITIVITY
105
EDU and 0.~ protection in maintaining integrity offofiar membranes Previous studies (Whitaker et al., 1990) have shown that snapbean treated with EDU did not alter leaf chlorophyll or carotenoid content, but did reduce the loss of these pigments during 03 exposure by about 12%. The ratio of chlorophyll to carotenoid did not change with EDU treatment or with exposure to 03. The chlorophyll a/b ratio was slightly higher in leaves of EDUtreated plants, but did not change significantly in either control or treated plants during 03 fumigation. The EDU does not appear to alter the foliar membrane chemistry of phospholipid composition. The ratio of 18: 2/18: 3 decreased with 03 exposure in untreated controls, but increased with exposure in EDU-treated plants. Thus, EDU could play an important role in maintaining integrity of the foliar membranes. CONCLUSION
Plant resistance to air pollution stress is often complex and involves a wide array of plant responses from the molecular, cellular, and whole-plant level. Antioxidant-induced O3-tolerant plants and GA inhibitor-induced SO2-tolerant plants have been used to study the mode of action of chemical protection. The results suggest EDU and GA inhibitor-induced tolerance to air pollutant exposure probably is biochemical at leaf mesophyll sites rather than exclusion by physical means. The protective nature of EDU on O3 stress is not related to alteration of either membrane phospholipids or to polyamine levels within the leaf tissue. These results suggest that other characteristics such as free radical scavenging enzymes or endogenous antioxidant compounds are responsible. ACKNOWLEDGMENTS
The author thanks S.J. Wilding and J. Collins for their assistance in carrying out analytical work and data analyses. I am very grateful to H.E. Heggestad, and W.J. Manning and R. Pausch for their critical comments on this manuscript, and to Prof. C.L. Mulchi for his valuable advice.
REFERENCES Bors, W., Langebartels, C., Michel, C. and Sandermann, H. Jr., 1989. Polyamines as radical scavengers and protectants agaiost ozone damage. Phytochemistry, 28: ! 589-! 595. Brennan, E., Leone, i. and Clarke, B., 1987. EDU: a chemical for evaluating ozone foliar injury and yield reduction in field-grown crops. Int. J. Trop. Dis., 5: 35-42. Carnahan, J.E., Jenner, E.L. and War, E.K.W., 1978. Prevention of ozone injury to plants by a new protectant chemical. Phytopathology, 68:1225- ! 229.
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E.H. LEE ET AL.
Hausladen, A. and Kunert, K.J., 1990. Effects of artificially enhanced levels of ascorbate and glutathione on the enzymes monohydroascorbate reductase, hydroascorbate reductase, and glutathione reductase in spinach. Physiol. Plant., 79: 384-388. Heath, R.L., 1988. Biochemical mechanisms of pollutant stress, in: W.W. Heck, O.C. Taylor, and D.T. Tingey (Editors), Assessment of Crop Loss From Air Pollutants. Elsevier, London, New York, pp. 259-286. Kendall, E.J. and McKersie, B.D., 1989. Free radical and freezing inju, ~ to cell membranes of winter wheat. Physiol. Plant., 76: 86-94. Kramer, G.F. and Wang, C.Y., 1989. Correlation of reduced chilling injury and oxidative damage with increased polyamine levels in zucchini squash. Physiol. Plant., 76: 479-484. Larson, R.A., 1988. The antioxidants of higher plants. Phytochemistry, 27: 969-978. Lee, E.H. and Bennett, J.H., 1982. Superoxidc dismutase: A possible protective enzyme against ozone injury in snapbeans (Phaseolus vulgaris L. ). Plant Physiol., 69: ! 444-1449. Lee, E.H., Jersey, J.A., Gifford, C. and Bennett, J.H., 1984. Differential ozone tolerance in soybeans and snapbeans: Analysis of ascorbic acid in O~-susceptible and O.~-resistant cultivars by high performance liquid chromatography. Environ. Exp. Bot., 24: 331-341. Lee, E.H., Saftner, R.A., Wilding, S.J., Clark, H.D. and Rowland, R.A., 1987. Effects ofpacl~butrazol on GA biosynthesis and fatty acid composition. Proc. Plant Growth Reg. Soc. Am., 14: 295-302. Martin, C. and Northcote, D.H., 1982. The action of exogenous gibberellic acid on protein and m-RNA on germinating castor bean seeds. Planta, 154: i 68-173. Monk, L.S., Fagerstedt, K.V. and Crawford, R.M.M., 1989. Oxygen toxicity and superoxide dismutase as an antioxidant in physiological stress. Physiol. Plant., 76: 456-459. Rao, l~.V. and Dubey, P.S., 1990. Biochemical aspects (antioxidants) for development of tolerance in plants growing at different low levels of ambient air pollutants. Environ. Pollut., 64: 55-66. Whitaker, B.D., Lee, E,H. and Rowland, R.A., 1990. EDU and Ozone protection: Foliar glycerolipids and steryl iipids in snapbean exposed to O.~. Physiol. Plant., 80: 286-293.
99
Antioxidants and growth regulators counter the effects of O3 and SO2 in crop plants E.H. Leea, G.F. Kramera, R.A. Rowland a and M. Agrawalb ~('iimate Stress Laboratory, USDA-ARS, Beltsville, MD 20705-2350, USA ~'Dept. o~Botan.l'. Barnaras ltindu Universit),. Varasnasi-221005, India (Accepted 25 May 1991 )
ABSTRACT Lee, E.H., Kramer, G.F., Rowland, R.A. and Agrawai, M., 1992. Antioxidants and growth regulators counter the effects ofO~ and SO,, in crop plants. Agric. ifcos.w'tems Environ., 38: 99-106. Antioxidant compounds such as cthylenediurea (EDU); N- [ 2-(2-oxo-I-imidazolidinyl ) ethyl ]-N'phenylurea have been found to be effecti~ e in suppressing O3-induced leaf injury. Gibberellic acid (GA) inhibitors such as paclobutrazol, tetcyclaics and flurprimidoi suppress visible SO2-induced injury. Antioxidant compounds are relatively ineffective in protecting plants from SO2-induced injury, while GA inhibitors are ineffective in protecting plants from 03 injury. EDU and GA inhibitors do not alter the stoma~.al behavior, however they can induce resistance to air pollutants stress. Furthermore. EDU and paclobutrazol (PP,33) do not alter foliar membrane lipid composition. EDU does not alter polyamine composition. This suggests that the mechanism of plant tolerance to air pollutant exposure is probably biochemical rather than biophysical. Free radical scavenging enzymes or endogenous antioxidant compounds are more important than stomatal restriction of pollutant diffusic.n to the sensitive leaf mesophyll sites.
INTRODUCTION
Interest in the usefulness of antioxidants for cellular protection against oxidative stress in plants has increased dramatically (Larson, 1988; Rao and Dubey, 1990). Ethylenediurea (EDU); N-[2-(2-oxo-l-imidazolidinyl) ethyl ]-N'-phenylurea has been used to determine the effects of ambient ozone (O3) on plants (Carnahan et al., 1978; Brennan et al., 1987 ). While EDU has shown induction of cellular defenses against oxidative stress (Lee and Bennett, 1982 ), the exact mechanism of EDU-induced resistance to air pollutants still remains unclear. A close interrelationship between EDU-induced resistance and oxidative stress is generally thought to result from free radical reactions (Lee and Bennett, 1982 ). Recent work has shown that increased plant tolerance to environmental stress may be achieved by means of plant growth regulators and antioxidant treatments (Lee et al., 1987; Hausladen and Kunet* 1990). An understanding of the biochemical events providing protec© 1992 Elsevier Science Publishers B.V. All rights reserved 0167-8809/92/$05.00
100
E.H. LEE ET AL.
tion against oxidative damage is essential for the development of crop plants more resistant to a growing number of environmental stresses (Monk et ai., 1989). Our objectives were to determine the effects of soil applications of GA inhibitors and antioxidants on plant sensitivity to SO: and 03 and to evaluate the nature of susceptibility to these air pollutants. MATERIALS AND M E T H O D S
Clwmical modification of plants and air pollution treatments Two cultivars of snapbean (Phaseolus vulgaris L. ) and soybean (Glycine max ( L. ) Merr ) shown previously to differ in their response to 03 stress were used in this study. Pollutant-sensitive (S) cultivars used were 'Bush Blue Lake290' (BBL-290) snapbean and 'Hark' soybean; O.rresistant (R) cultivars used were 'Astro' snapbean and 'Hood' soybean (Lee et al., 1984). Plants were germinated in 15 cm plastic pots containing Jiffy Mix potting mixture (Jiffy Products of America Inc., Chicago, IL, USA), and cultured in a charcoalfiltered (CF) air greenhouse as previously described ( Lee and Bennett, 1982 ). Treatments began when the first trifoliate leaves were 80% fully expanded. At this time, EDU or GA inhibitors were applied as a soil drench at equal dosages (Tables 1 and 2 ). Only single chemical applications were made. Control plants were watered with distilled water at the same time. All experiments were conducted as a split plot randomized experimental design. Plants were grown in CF air greenhouses as described (Lee and Bennett, 1982). Two to tbur days alter treatment, tbur replicate pots (one plant per pot) were subfABLE I
Abaxial surface stomatal resistance tbr fully expanded leaves ot'O~-sensitive (S) and Orresistant ( R ) plants ~ith and without E i ) t l ( 500 mg I r+~) treatment and O~ stress
Snapbean cultivar
Stomatal resistance (s cm - ~)
Pro-exposure
During exposure ~
Post-exposure:
1.92" 2.05"
3.2 I" 3.35"
3.43" 3.55"
- EDtl
2.15"
3.41"
4,15"
+ EDtl
2.30'
3.40"
4.25 h
"Bush Blue Lake-290" (S) - EDI, I + EDI, I '-~st ro" ( R )
'Planl,~ ~vcre exposed to 350 Ill ! ' O~ t't~r I-2 h. "Plants were exposed to the same concentration as above for 3 h. Values ill each column followed by the same superscript arc not significanlly different at the 0.05 level of signiticance using Duncan's multiple range lest.
ANTIOXIDANTS AND GROWTH REGULATORS AFFECT 02 AND SO, SENSITIVITY
10 !
TABLE 2 Stomatal resistance in fully expanded leaves of soybean (Glycine m a x (L.) Merr) cuiti, c "Hark' treated with selected GA inhibitors and exposed to SO2 at 1500 nl !- ' for 3 h Treatment
Injury score
Concentration (rag pot-~ )
Control Paclobutrazol Tetcyclacis Flurprimidol
0.00 0.20 2.50 0.25
Stomatal resistance (s cm- ' )
6.8" 0.0" 0.5 a 0.2 a
Pre-exposure
During exposure
Post-exposure
1.92" 1.89~ 2.04 ~b 2.38 h
4.10" 4.26" 4.12" 4.85"
4.54 a 4.76" 3.85" 5.2u"
Means represent four replicatiens/treatment. Within a column, any values having a superscript in common are not significantly different at the 5% level by the Duncan's multiple range tesl.
1 h exposure
~100
p
mp
50-
250 nl I-~
I I G)
/
80
0
E
I
40-
I I 30-
6o
a 40
ASTRO
j
13 ID 20 _.1
:-'
~_,I~-,iw
0
--
~ t
"~
I
200
i
40:,
nl
_
1
600
I --1 03
t
/
20 -
ASTRO
,, ,. "
x4" m
i!1//
10 -
1
800
0 !
1000
0
i
i
i
i
i
1
2
3
4
5
Exposure Time (h)
Fig. !. Dose-response curves showing eultivar differences in 03 sensitivity. Right: leaf damage at 250 nl i-~ 03 after 1-5 h of exposure. Left: leaf damage as ~ function of O3 concentration. Plants were rated for foliar 03 injury at 48 h after fumigation. The first trifoliate leaves were assessed for the percentage of the surface showing injury.
jected to SO., or 03 fumigation. Test plants were fumigated at concentrations as shown in Fig. 1 and Table 2. Ozone and SO2 fumigations were conducted in two separate environmentally controlled growth chambers (Controlled Environments Inc., Model PGW 36 ) as previously described (Lee and Bennett, 1982; Lee et al., 1984). Treated plants with or without air pollution stress were sampled for later polyamine and lipid analysis. After pollutant exposure, remaining plants were returned to the CF air greenhouse for 03 i,jury scoring after 48 h. Leaf injury from 0 to 10 was determined on fully expanded leaves (0, no injury; i O, severe injury ).
102
E.H. LEE ET AL.
Stomatal resistance measurement Stomatal resistance (s cm- t ) for the lower surfaces of fully expanded leaves were measured with a LI- 1600 steady state diffusion porometer (LI-COR Inc., Lincoln, NE). Prior to measurement, plants were equilibrated in the growth chambers for ' - 2 h. Porometer measurements were taken I h before fumigation and 1.5 h during, and 3 h after fumigation.
Biochemical ph,~spholipidand polyamine analysis Extraction and quantitative analyses oftotal leaf phospholipid contents were conducted on th(: first trifoliate leaf samples for all treatments. The methods employed have ht:en previously described (Lee et al., 1987; Whitaker et al., 1990). Membraae phospholipids were analyzed by GC or HPLC (Lee et al., 1987, Kendall ~:nd McKersie, 1989). Free polyamines were determined by methods similal to those reported by Kramer and Wang (1989).
Statistical analysis An analysis of variance (ANOVA) was performed on all data of leaf injury scores, phospholipid, and stomatal resistance to determine significant differences, followed by a Duncan's multiple range test to separate means. RESU LTS AN D D! S C U S I O N S
Crop plants response to O~ stress Figure 1 shows graphically the comparative dose-response curves for 03 injury in the both the snapbean cultivars as a function of O3 concentration and duration of' O~ exposure. The O3-sensitive cultivar BBL-290 exhibited foliar injury at a low concentration while much higher 03 levels were required to injure the Orresistant cultivar 'Astro'.
Characterization of stomatai resistance Leaf stomatal resistance measurements were made on the abaxial side of the leaves. Results given in Table 1 show that the stomatal resistance of O3-R and OrS plants was comparable within the pre-exposure, and during 03 exposure. Resistance for both O r S and O3-R cultivars increased from pre-exposure to post-exposure. However, no significant differences were noted for O3-S and O r R plants except for post-exposure. 'Astro' showed a significantly higher stomatal resistance level than O3-S snapbean during the post-exposure
ANTIOXIDANTS AND GROWTH REGULATORSAFFECT02 AND SO:,SENSITIVITY
103
period. Table 1 indicates no statistical differences between EDU-treated and untreated plants for stomatal diffusion. Resistance to air pollutant injury can be largely due to stomatal closure (Heath, 1988 ). Differences in stomatal frequency in leaves can be an important factor in determining pollutant susceptibility. However, in this study, measurements of leaf diffusive resistance in EDU-treated (Table l ) or GA inhibitor-treated and non-treated plants (Table 2) did not show differences in stomatal resistance sufficient to account for air pollutant exclusion from the leaf mesophyll. This result suggests EDU- and GA inhibitor-induced tolerance to air pollutant exposure probably is biochemical at leaf mesophyll sites rather than exclusion by physical means.
Polyamines and EDU treatment Polyamines have been shown to be antioxidants and to protect membranes from peroxidation (Bors et al., 1989). Polyamine content of fully expanded leaves of EDU-treated and control plants were compared before and after 03 exposure (Table 3 ). EDU does not alter the polyamine composition of snapbean leaves. However, with 03 stress alone (-EDU), significant increases of total polyamine were observed. This difference in the total polyamine content was due to increases in both putrescine (Put) and spermidine (Spd) while spermine (Spn) remained unchanged. The total polyamine content was unaffected by 03 stress in the presence of EDU. The antiozonant activity of EDU does not appear to involve the enhancement of polyamine levels. TABLE 3 Polyamine levels in levels in fully expanded leaves of snapbean cultivar BBL-290 subjected to O~ stress with or without EDU ( 500 mg I- ~) treatment Treatment
Control (no ozone ) -EDU +EDU Ozone treated" -EDU +EDU
Polyamine j (nmol g FW-~ )
Total polyamine
Put (SD)
Spd (SD)
Spn (SD)
15.9 (I.7) a" 14.3 (!.6) a
164 (14.6) a 176 (25.3) a
70.9 (6.3) a 66.4 (6.8) a
250.8 a 256.7 a
26.5 (5.9) b 15.4 ( l . 4 ) a
193 (5.4) b 144 (7.2) a
62.7 (3.7) a 62.4 (3.3) a
282.2 b 221.8a
' Put = putrescine, Spd = spermidine, Spn = spermine. -'Plants were exposed to the same concentration as above for 3 h. Values in each column followed by the same letter are not significantly different at the 0.05 level of significance using Duncan's multiple range test.
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GA inhibitor and protection against SO., injuo' The effects of the GA inhibitors, paclobutrazol (PP333), tetcyclacis and flurprimidol, on visible SO2-induced injury and stomatal resistance are shown in Table 2. Leaves treated with PP333, tetcyclacis and flurprimidol showed a significant reduction in SO., injury. In addition, stomatal resistance of fully expanded leaves of GA inhibitor-treated and control plants were compared before, during, and after SO., exposure. With the exception of flurprimidol, stomatal resistance of soybean plants increased during and after SO., exposure but was unaffected by GA inhibitor treatment. Stomatal closure is not involved tn GA inhibitor-induced resistance to SO:. In previous studies (Lee at al., 1987), we have shown that in the absence of a SO, stress, no perceptible change occurred in the total lipid content of leaves from PP~.~-treated plants. When plants were subjected to SO., stress with 1500 nl i-~ SO: for 3 h, both PP333-treated and control plants showed a slight but non-significant decrease in total lipid content. Although the PP333 ;reatment (without SO, fumigation) did not alter the total lipid content of the leaves, it did induce an increase in the ratio of non-polar to polar lipids. GA inhibitors appear to be potential agents to induce SO, resistance, but the major constraint is the accompanying retardation of growth. Hence, attempts should be made to search for effective growth substances which will induce SO., resistance without affecting the growth of plants.
Dtil.'lD'entialair pollutant protection response to EDU and GA inhibitor Two to tbur days after soil drench with EDU or PP333, plants were exposed to two different types of air pollution stress. EDU provided good foliar protection of O~ stress, but this type of protection was not found in the PP333treated plants (data not shown). PP333 provided good foliar protection with increased resistance to SO,. However, soil application of PP333 at the same concentration which induced tolerance to SO,-stressed plants was ineffective for O.~ damage. Why does paciobutrazoi protect plants from SO:-induced injury, while EDU does not? GA inhibitors are capable of modifying the biosynthesis and action of GA in plant tissues through their affect on RNA and protein synthesis (Martin and Northcote, 1982 ). GA3, in particular, is involved in the synthesis ofmRNA and proteins in many plant organs. GA3 could promote changes in the levels ofcellular mRNA and protein/enzyme which are involved in the initiation of growth and regulate SO,, sensitivity. These results suggest that the mechanism of susceptibility of plants to 03 and SO2 are very different.
ANTIOXIDANTS AND GROWTH REGULATORS AFFECT O, AND SO, SENSITIVITY
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EDU and 0.~ protection in maintaining integrity offofiar membranes Previous studies (Whitaker et al., 1990) have shown that snapbean treated with EDU did not alter leaf chlorophyll or carotenoid content, but did reduce the loss of these pigments during 03 exposure by about 12%. The ratio of chlorophyll to carotenoid did not change with EDU treatment or with exposure to 03. The chlorophyll a/b ratio was slightly higher in leaves of EDUtreated plants, but did not change significantly in either control or treated plants during 03 fumigation. The EDU does not appear to alter the foliar membrane chemistry of phospholipid composition. The ratio of 18: 2/18: 3 decreased with 03 exposure in untreated controls, but increased with exposure in EDU-treated plants. Thus, EDU could play an important role in maintaining integrity of the foliar membranes. CONCLUSION
Plant resistance to air pollution stress is often complex and involves a wide array of plant responses from the molecular, cellular, and whole-plant level. Antioxidant-induced O3-tolerant plants and GA inhibitor-induced SO2-tolerant plants have been used to study the mode of action of chemical protection. The results suggest EDU and GA inhibitor-induced tolerance to air pollutant exposure probably is biochemical at leaf mesophyll sites rather than exclusion by physical means. The protective nature of EDU on O3 stress is not related to alteration of either membrane phospholipids or to polyamine levels within the leaf tissue. These results suggest that other characteristics such as free radical scavenging enzymes or endogenous antioxidant compounds are responsible. ACKNOWLEDGMENTS
The author thanks S.J. Wilding and J. Collins for their assistance in carrying out analytical work and data analyses. I am very grateful to H.E. Heggestad, and W.J. Manning and R. Pausch for their critical comments on this manuscript, and to Prof. C.L. Mulchi for his valuable advice.
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Hausladen, A. and Kunert, K.J., 1990. Effects of artificially enhanced levels of ascorbate and glutathione on the enzymes monohydroascorbate reductase, hydroascorbate reductase, and glutathione reductase in spinach. Physiol. Plant., 79: 384-388. Heath, R.L., 1988. Biochemical mechanisms of pollutant stress, in: W.W. Heck, O.C. Taylor, and D.T. Tingey (Editors), Assessment of Crop Loss From Air Pollutants. Elsevier, London, New York, pp. 259-286. Kendall, E.J. and McKersie, B.D., 1989. Free radical and freezing inju, ~ to cell membranes of winter wheat. Physiol. Plant., 76: 86-94. Kramer, G.F. and Wang, C.Y., 1989. Correlation of reduced chilling injury and oxidative damage with increased polyamine levels in zucchini squash. Physiol. Plant., 76: 479-484. Larson, R.A., 1988. The antioxidants of higher plants. Phytochemistry, 27: 969-978. Lee, E.H. and Bennett, J.H., 1982. Superoxidc dismutase: A possible protective enzyme against ozone injury in snapbeans (Phaseolus vulgaris L. ). Plant Physiol., 69: ! 444-1449. Lee, E.H., Jersey, J.A., Gifford, C. and Bennett, J.H., 1984. Differential ozone tolerance in soybeans and snapbeans: Analysis of ascorbic acid in O~-susceptible and O.~-resistant cultivars by high performance liquid chromatography. Environ. Exp. Bot., 24: 331-341. Lee, E.H., Saftner, R.A., Wilding, S.J., Clark, H.D. and Rowland, R.A., 1987. Effects ofpacl~butrazol on GA biosynthesis and fatty acid composition. Proc. Plant Growth Reg. Soc. Am., 14: 295-302. Martin, C. and Northcote, D.H., 1982. The action of exogenous gibberellic acid on protein and m-RNA on germinating castor bean seeds. Planta, 154: i 68-173. Monk, L.S., Fagerstedt, K.V. and Crawford, R.M.M., 1989. Oxygen toxicity and superoxide dismutase as an antioxidant in physiological stress. Physiol. Plant., 76: 456-459. Rao, l~.V. and Dubey, P.S., 1990. Biochemical aspects (antioxidants) for development of tolerance in plants growing at different low levels of ambient air pollutants. Environ. Pollut., 64: 55-66. Whitaker, B.D., Lee, E,H. and Rowland, R.A., 1990. EDU and Ozone protection: Foliar glycerolipids and steryl iipids in snapbean exposed to O.~. Physiol. Plant., 80: 286-293.