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Effects of Water Deficit on Proline Accumulation and Growth of two Cotton Genotypes of Different Drought Resistances
Originalarbeiten . Original Papers Departamento de Bioquimica e Biologia Molecular, Centro de Ci&ncias, Universidade Federal do Ceara, c.P. 1065 - 60.000 Fortaleza, Ceara, Brazil
Effects of Water Deficit on Proline Accumulation and Growth of two Cotton Genotypes of Different Drought Resistances Lurz
G.
R.
FERREIRA, JOSE G. DE SOUZA
and
JosE
T.
PRISCO
With 3 figures Received October 20,1978' Accepted November 16, 1978
Summary Cotton genotypes (Cruzeta Serid6-9193 and IAC-12.2) differing in drought resistance under dry land farming conditions were used in the present study. Seeds were sown in polythene bags containing sand-soil mixture, and placed on a greenhouse bench. After 27 days from sowing the plants were divided into a control group which was watered every other day, and a stressed group from which water was withheld for 7, 10, and 14 days. Rewatering was performed at the 14th day of stress. When water was withheld both genotypes exhibited progressive reduction in R WC, and 48 hours after rewatering their water status was recovered. Cruzeta Serid6-9193 plants always had higher RWC values than IAC-12.2. When plants were stressed both genotypes exhibited progressive increase on leaf free proline concentration, and 48 hours after rewatering the concentration of this imino acid almost reached the control level. The variations of free proline in the roots followed the same pattern as in the leaves, although reaching lower levels. IAC-12.2 plants had higher proline concentration values both in leaves and roots as compared to Cruzeta Serid6-9193. When plants were either watered (control) or stressed Cruzeta Serid6-9193 genotype always had lower shoot: root ratios than IAC-12.2. Net assimilation rate (NAR), relative growth rate (RGR), and compensation point determinations as a function of water status have indicated that Cruzeta Serid6-9193 genotype is more resistant to drought than IAC-12.2. The present results did not agree with the hypothesis that free proline accumulation is linked to a genetic characteristic that is responsible for drought resistance. At least for the two cotton genotypes investigated drought resistance is correlated with the capacity of the plants to absorb andlor retain water in their tissues.
Key words: water stress, proline accumulation, cotton.
Introduction
When plants are subjected to water stress there is a decrease in plant height 1964; SLATYER, 1969), leaf size, leaf number (SLATYER, 1967 and 1969; SILVA, 1973), flower primordia formation, as well as in root growth (SLATYER, 1969). (GATES,
Z. PJlanzenphysiol. Bd. 93. S. 189-199. 1979.
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L. G. R. FERREIRA,
J. G. DE SOUZA and]. T.
PRISCO
These morphological changes are simply the result of stress induced modifications of plant processes. Among these, cell growth appears to be the most sensitive (HSIAO, 1973; HSIAO and ACEVEDO, 1974; HSIAO et a1., 1976). However, when plants are exposed to a long period of water stress cell division may be as sensitive as cell elongation (HSIAO, 1973). Although photosynthesis is less sensitive to water stress than cell expansion it is also decreased (VAADIA et a1., 1961; BOYER, 1976). It is assumed that this response is mediated partly by way of impeded CO 2 supply following stomatal closure and partly by a direct effect of dehydration on the photosynthetic system (SLATYER, 1969; SILVA, 1973; BOYER, 1976). SHAH and LOOMIS (1965) found that both soluble and total protein contents of sugar beet leaves declined progressively in a matter of days when water was withheld. This decrease in protein concentration may occur as a result of decreased protein synthesis (BEN-ZIONI et a1., 1967; HSIAO, 1970) and increased protein degradation (SLATYER, 1969). Another frequently observed effect of stress is the appearance of high levels of free amino acids in leaves, especially proline, and ami des (SLATYER, 1969). KEMBLE and MACPHERSON (1954) observed that free proline increased several fold when Lolium. perenne plants were subjected to dehydration. The accumulation of free proline in leaves of different plant species (ROUTLEY, 1966; MORRIS et a1., 1969; MASCIOTTI, 1974; WALDREN et a1., 1974; MADRUGA, 1976) was also observed in roots of citrus (CHEN et a1., 1964), and barley (SINGH, PALEG, and ASPINALL, 1973) subjected to water stress. However, when excised plant tissues are subjected to dehydration, only leaves are able to accumulate proline (STEWART et a1., 1966; SINGH, ASPINALL, PALEG and BOGGESS, 1973). Knowing that proline accumulation was suggested to be correlated with drought resistance (SAUNIER et a1., 1968; PALFI and JUHAsz, 1971; SINGH et a1., 1972) the present investigation was set up to study the effects of water deficit on free proline accumulation in leaves and roots, as well as its effects on growth of two cotton cultivars of different drought sensitivities. The objective of this study was to identify physiological parameters that could be used in the selection of drought resistant cotton plants. Material and Methods Growth Conditions Cotton plants, Gossypium hirsutum, cultivars Cruzeta Serid6-9193 (G. hirsutum var. marie-galante HUTCH.) and IAC-12.2 (G. hirsutum var. latifolium HUTCH.) were chosen because they differ in drought resistance under dry land farming conditions. The perennial Cruzeta Serid6-9193 is known to be more resistant to water stress than the annual IAC-12.2 Seeds were treated with concentrated sulfuric acid for 40 min (PONTE, 1960), and washed with distilled water to remove the excess of acid. Then they were sown in polythene bags containing 10 kg of sand-soil mixture, and placed on a greenhouse bench. After thinning the plants were also grown in the greenhouse under natural daylight, watered each other day to field capacity, and fertilized weekly with half-strength Hoagland solution, modified by JOHNSON et al. (1957). The temperature in the greenhouse ranged from a low of 27°C during Z. Pflanzenphysiol. Bd. 93. S. 189-199. 1979.
Proline accumulation and drought resistance
191
the night to a hight of 29 °e during the day. Relative humidity fluctuated between 85 and 96 0/0, and the intensity of solar radiation ranged from a minimum of 132 to a maximum of 288 cal X cm- 2 X day-I. After 27 days from sowing the plants were divided into a control group which was watered every other day, and a stressed group from which water was withheld. Plants in both groups were sampled after 7, 10, and 14 days. When the plants showed the first signs of wilting (14 days of stress) they were rewatered. Samples for all determinations were taken at 600 a.m. (sunrise) on each sampling day. In all cases it was attempted to select leaves of the same physiological age by sampling only the fifth leaf from the top of the plants (WEATHERLEY, 1950). Water Status oj the Plants The water status of the plants was estimated by determining the Rwe of the leaves (BARRS and WEATHERLEY, 1962) in four replicates, each consisting of five discs (1. mm diameter) excised from leaf blades avoiding the major veins. Leaf discs were saturated by floating them abaxially on distilled water, at 25 ± 1 °e for four hours, under fluorescent lamps (690 lux), and dry weights were obtained after dehydration in an oven at 80 °e for 48 hours. Free Proline Free proline was determined in four replicates of both leaves and roots. Five leaf discs or 0.1 g fresh weight of root tips were collected, submerged in 2 ml of methanol: chloroform: water (12 : 5 : 1, v/v), and stored at -15 °e for further analysis. The extraction and determination of free proline were performed according to MESSER (1961) as modified by MASCIOTTI (1974). All proline concentrations were expressed on dry weight of plant tissue basis. Leaf R we and free proline concentration values for both leaves and roots were used to calculate correlation coefficients at 1 % significance level, and then adjusted to a linear regression equation (SNEDECOR, 1956). Growth Analysis and Drought Resistance Growth analysis was carried out by using indexes such as net assimilation rate (NAR), relative growth rate (RGR), and shoot: roOt ratio (RADFORD, 1967). In the determination of NAR and RGR the experimental period was divided into two phases: the first phase from 0 to 7 days, and the second one from 7 to 14 days. The tolerance of leaves to dehydration was determined by measuring colorimetric ally the compensation point (LIETH and ASHTON, 1961) as a function of the RWC. Three replicates of leaf strips (5 X 1 cm), collected from each of the two leaf samples were mounted in rubberstoppered test tubes (18 X 1.7 cm) containing 2 ml of cresol red indicator. The test tubes were placed for 3 hours under fluorescent-incandescent light bench so that the upper surface of the leaf strips received 4300 lux.
Results Plants of both cultivars exhibited progressive reduction in RWC when they were subjected to water stress, and 48 hours after rewatering their R WC reached values close to the control treatment (Figure 1 A). Fourteen days after the water was withheld plants of both cultivars showed signs of wilting, and the greatest reduction in RWC. Cruzeta Serid6-9193 always presented higher RWC values than IAC-12.2 The RWC of each one of the cultivars did not change to a great extent in the control treatment. Z. Pjlanzenphysiol. Bd. 93. S. 189-199. 1979.
192
L. G. R.
FERREIRA,
J. G. DE
SOUZA
and
J. T.
PRISCO
A
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y. REWATERli~ 7
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EXPERIMENTAL PERIOD (days)
14
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Proline accumulation and drought resistance
193
Figure 1 B shows changes in leaf free proline concentration for both control and water stressed plants. Proline concentration for both cultivars in the control treatment was close to zero, meaning that free proline levels in turgid cotton leaf tissues is very low. When plants were stressed for 7 days, a reduction in RWC of 5 0 /0 (IAC-12.2) or 6 Ufo (Cruzeta Serid6-9193) did not induce appreciable increase in free proline concentration. Ten days after withholding the water, when the reduction in R WC was 31 Ufo for Cruzeta Serid6-9193, and 34 Ufo for IAC-12.2, free proline increased 41 times in the former, and 70 times in the latter cultivar. By the end of the water stress period (14 days), the amount of free proline reached the highest level in relation to the control, that is, 107 times for Cruzeta Serid6-9193, and 111 times for IAC-12.2. Twenty-four hours after rewatering, there was an increase in water content (Figure 1 A) and a great reduction in free proline level, which continued to decrease, reaching almost the control level at the end of the experimental period (Figure 1 B). There was a negative correlation between RWC (Figure 1 A) and leaf proline concentration (Figure 1 B) of -0.94 for Cruzeta Serid6-9193, and -0.92 for IAC-12.2. The adjusted linear regression equation for Cruzeta Serid6-9193 was y = -2.30x + 201.06 (r2 = 0.92), and y = -2.33x + 199.39 (r2 = 0.96) for IAC-12.2 (Figure 2 A). The variations of free proline in the roots followed the same pattern as in the leaves, although not reaching levels as high as in the leaves (Figure 1 C). A negative correlation of -0.92 for Cruzeta Serid6-9193, and -0.93 for IAC-12.2 between leaf RWC (Figure 1 A) and root proline concentration (Figure 1 C) was observed. The adjusted linear regression equation for Cruzeta Serid6-9193 was y = -O.96x + 84.60 (r2 = 0.95), and y = -1.35x + 116.84 (r2 = 0.97) for IAC-12.2 (Figure 2 B). The variations in shoot: root ratio of both control and water stressed plants are shown in Table 1. When plants were either watered (control) or stressed IAC-12.2 genotype had always higher shoot: root ratios than Cruzeta Serid6-9193. When both cultivars were subjected to water stress there was no significant reduction in this parameter. The highest NAR for both control and water stressed plants was observed during the first phase of the experimental period (Table 2). When well watered, IAC-12.2 plants reached greater NAR values than Cruzeta Serid6-9193. However, when they were subjected to water stress, Cruzeta Serid6-9193 had always higher NAR than IAC-12.2. The highest RGR for both cultivars when they were either watered (control) or stressed was observed during the first phase of the experimental period (Table 3). In both control and stressed treatments. Cruzeta Serid6-9193 had higher RGR than IAC-12.2 during both phases. RGR of Cruzeta Serid6-9193 was less affected by Fig. 1: Variations in RWC (A), leaf free proline (B), and root free proline (C) of Cruzeta Serid6 - 9193 and IAC-12.2 cotton plants along the experimental period.
z. Pflanzenphysiol.
Bd. 93. S. 189-199. 1979.
194
L. G. R. FERREIRA,
J. G. DE SOUZA and J. T.
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PRISCO
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y=- 2.33 X + 199.39
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00 RWC (%) Fig. 2: Relationship between leaf free proline and leaf water status (A), and between root free proline and leaf water status (B) of Cruzeta Serid6 - 9193 and lAC - 12.2 cotton plants. Adjusted linear regression equations for each one of the cultivars are given.
z. PJlanzenphysiol. Bd. 93. S.
189-199. 1979.
Proline accumulation and drought resistance
195
Table 1: Mean shoot: root ratios of Cruzeta Serid6 - 9193 and lAC - 12.2 cotton plants subjected to water stress or not l ). Experimental Period (days)
Shoot: root Ratio Cruzeta Serid6 - 9193 lAC - 12.2 Control Stressed Control Stressed
0 7 10 14
1.15 1.44 1.41 1.67
1.15 1.29 1.25 1.26
2.31 2.08 2.04 1.83
2.31 1.98 1.97 1.74
1) Each value represents the mean of 4 replicates.
Table 2: Mean net assimilation rates (NAR) of Cruzeta Serid6 - 9193 and lAC - 12.2 cotton plants subjected to water stress or not l ). Experimental Period (phases)
Net Assimilation Rate (mg X dm-2 X day-l) Cruzeta Serid6 - 9193 lAC -12.2 Control Stressed Control Stressed
First Second
78.5 59.0
69.0 24.3
85.7 78.1
66.2 7.5
1) Each value represents the mean of 4 replicates.
Table 3: Mean relative growth rates (RGR) of Cruzeta Serid6 - 9193 and lAC - 12.2 cotton plants subjected to water stress or not l ). Experimental Period (phases)
Relative Growth Rate (mg X g-l X day-l) Cruzeta Serid6 - 9193 lAC - 12.2 Control Stressed Control Stressed
First Second
115.5 74.8
103.7 30.4
93.4 70.6
67.2 7.3
1) Each value represents the mean of 4 replicates.
water stress than RGR of IAC-12.2. This effect of water stress on RGR was more conspicuous during the second phase. Both cultivars reached their compensation points at low R WC levels (Figure 3). However, IAC-12.2 plants were less resistant to water stress than Cruzeta Serid6-9193. This was evidenced by the fact that when RWC dropped to 56 Ofo the majority of IAC-12.2 plants had reached their compensation point, while all Cruzeta Serid6-9193 plants were still photosynthesizing. Z. P/lanzenphysiol. Bd. 93. S. 189-199. 1979.
196
L. G. R. FERREIRA,
J. G. DE SOUZA and J. T. PRISCO
•
PLANTS PREDOMINANTLY PHOTOSYNTHESIZING
~
PLANTS AT THE COMPENSATION POINT
D
PLANTS PREDOMINANTLY RESPIRING
t\I
~ ,
o
ct
II II
R.w.C. (%) Fig. 3: Compensation point as a function of leaf water status of Cruzeta Serid6 - 9193 and lAC - 12.2 cotton plants.
Discussion When water was withheld both cottOn genotypes exhibited progressive reduction in R WC, and 48 hours after rewatering their water status was recovered (Figure 1 A). Similar results were observed for ladino clover (ROUTLEY, 1966), sorghum (BLUM and EBERCON, 1976), and rice (MADRUGA, 1976). The fact that Cruzeta Serid6-9193 always had higher RWC values than IAC-12.2 suggests differences in behavior of the two genotypes concerning the capacity to absorb and/or retain water. Assays of leaf and root extracts failed to detect the presence of proline in the well watered control plants (Figures 1 Band 1 C). These results differ from the ones found for both bean (MASCIOTTI, 1974), and rice (MADRUGA, 1976) leaves. However, these authors have used other species of plants, and they have assayed proline in excised leaf tissues kept in a high humidity environment, while in the present experiment this imino acid was assayed in intact leaves from well watered (control) plants. The accumulation of free proline in water stressed plants (Figure 1 Band 1 C) has also been observed for Lolium perenne (KEMBLE and MACPHERSON, 1954), citrus (CHEN et aI., 1964), ladino clover (ROUTLEY, 1966), barley (SINGH, PALEG, and ASPINALL, 1973),
z. PJlanzenphysiol.
Bd. 93. S. 189-199. 1979.
Proline accumulation and drought resistance
197
and for sorghum (BLUM and EBERCON, 1976). This increase in proline as a result of water stress may be due to its release from a storage form in the leaves and roots, its de novo synthesis (BARNETT and N AYLOR, 1966; THOMPSON et al., 1966; MORRIS et al., 1969), and/or inhibition of its oxidation (STEWART et al., 1977). As the plants recovered from water stress their proline level decreased, and 48 hours after rewatering the concentration of this imino acid had almost reached the control level (Figures 1 Band 1 C). This recuperation could be interpreted as resulting from its utilization (oxidation) as an energy source for the tissue (STEWART, 1973; RENA and SPLITTSTOESSER, 1974). Another explanation for the decrease in leaf free proline upon rewatering is that this substance would have been translocated to other parts of the plant, especially to the roots (MIZUSAKI et al., 1964). This appears not to be the case since in the present investigation its concentration also decreased in the roots, after rewatering (Figure 1 C). Cruzeta Serid6-9193 from both control and stress treatments always had lower shoot: root ratios than IAC-12.2 plants (Table 1). This indicates a relatively better root development of the former in comparison with the genotype IAC-12.2. It appears that this is the major reason for better water status of Cruzeta Serid6-9193 when compared with IAC-12.2 (Figure 1 A). During the first phase of the experimental period NAR (Table 2) and RGR (Table 3) for both control and water stressed plants were the highest. When well watered, IAC-12.2 had greater NAR than Cruzeta Serid6-9193. This was probably due to the fact that IAC-12.2 being an annual plant photosynthesized more intensively than the perennial Cruzeta Serid6-9193. When they were stressed NAR and RGR of IAC-12.2 plants were much more affected than those of Cruzeta Serid6-9193. It is felt that this is simply a result of the higher drought resistance presented by Cruzeta Serid6-9193 when compared with IAC-12.2 plants (Figure 3). Some investigators (PALFI and JUHASZ, 1971; SINGH et al., 1972) have suggested that proline accumulation was linked to a genetic characteristic that is responsible for drought resistance. The obtained results did not agree with this since free proline accumulation in both leaves and roots of cotton plants was not correlated with drought resistance (Figures 1 and 3). HANSON et al. (1977) have also observed that free proline accumulation is not a positive index of drought resistance suitable for cereal breeding programs. Since there was a negative correlation between proline levels in both leaves and roots and R WC of leaves (Figure 2) it indicates that proline accumulation reflects lowered water status. The present results suggests that, at least for the two cotton genotypes investigated, drought resistance is correlated with the capacity of the plants to absorb and/or retain water in their tissues. Therefore, proline could be possibly functioning as a source of respiratory energy and nitrogen during the rewatering phase (BLUM and EBERCON, 1976), as a neutralizing agent for the ammonia that is released as a result of proteolysis occurring during tissue dehydration (PROTSENKO et al., 1968), and/or be involved in osmoregulation (HELLEBUST, 1976). Z. Pjlanzenphysiol. Bd. 93. S. 189-199. 1979.
198
L. G. R. FERREIRA,
J. G. DE
SOUZA and
J. T.
PRISCO
Acknowledgement The authors are grateful to Dr. JAMES W. O'LEARY (University of Arizona) for revision of the manuscript. We wish to thank the National Council for Scientific and Technological Development (CNPq), and the Bank of Northeast Brazil (BNB) for supporting this research.
References BARNETT, N. M. and A. W. NAYLOR: Amino acid and protein metabolism in bermuda grass during water stress. Plant Physio!. 41, 1222-1230 (1966). BARRS, H. D. and P. E. WEATHERLEY: A re-examination of the relative turgidity technique for estimating water deficits in leaves. Aust. J. Bio!. Sci. 15, 413-248 (1962). BEN-ZIONI, A., C. ITAI, and Y. VAADIA: Water and salt stress, kinetin and protein synthesis in tobacco leaves. Plant Physio!. 42, 361-365 (1967). BLUM, A. and A. EBERCON: Genotypic responses in sorghum to drought stress. III. Free proline accumulation and drought resistance. Crop Sci. 16, 428-431 (1976). BOYER, J. S.: Photosynthesis at low water potentials. Phi!. Trans. R. Soc. Lond. 273, 501-512 (1976). CHEN, D., B. KESSLER, and S. P. MONSELISE: Studies on water regime and nitrogen metabolism of citrus seedlings grown under water stress. Plant Physio!. 39, 379-386 (1964). GATES, C. T.: The effect of water stress on plant growth. J. Aust. Inst. Agric. Sci. 30, 3-22 (1964). HANSON, A. D., C. E. NELSON, and E. H. EVERSON: Evaluation of free proline accumulation as an index of drought resistance using two barley cultivars. Crop Sci. 17, 720-726 (1977). HELLEBUST, J. A.: Osmoregulation. Ann. Rev. Plant Pysio!. 27, 485-505 (1976). HSIAO, T. C.: Rapide changes in levels of polyribosomes in Zea mays in response to water stress. Plant Physio!. 46, 281-285 (1970). - Plant responses to water stress. Ann. Rev. Plant Physio!. 24, 519-570 (1973). HSIAO, T. C. and E. ACEVEDO: Plant responses to water deficits, water-use efficiency, and drought resistance. Agric. Meteoro!' 14, 59-84 (1974). HSIAO, T. c., E. ACEVEDO, E. FERERES, and D. HENDERSON: Water stress, growth, and osmotic adjustment. Phi!. Trans. R. Soc. Lond. 273, 479-500 (1976). JOHNSON, C. M., P. R. STOUT, T. C. BROYER, and A. B. CARLTON: Comparative chlorine requirements of different plant species. Plant and Soil 8, 337-353 (1957). KEMPLE, A. R. and H. T. MACPHERSON: Liberation of amino acids in perennial rye grass during wilting. Biochem. J. 58, 46-49 (1954). LIETH, H. and D. H. ASHTON: The light compensation points of some herbaceous plants inside and outside deciduous woods in Germany. Can. J. Bot. 39, 1255-1259 (1961). MADRUGA, L. A. N.: Efeito do dUice hidrico sobre 0 metabolismo de aminoacidos livres e proteinas foliares de cinco cultivares de arroz (Oryza sativa L.). M.s. Thesis. Univ. Fed. Vi~osa, 44 pp. (1976). MASCIOTTI, G. Z.: Efeito do dUice hidrico sobre 0 metabolismo do nitrogenio e 0 crescimento de alguns cultivares de feijao (Phaseo/us vulgaris L.). M. S. Thesis. Univ. Fed. Vi~osa, 37 pp. (1974). MESSER, M.: Interference by amino acids and peptides with the photometric estimation of proline. Ana!' Biochem. 2,353-359 (1961). MIZUSAKI, S., M. NOGUCHI, and E. TAMAKI: Studies on nitrogen metabolism In tobacco plants. VI. Metabolism of glutamic acid, y-aminobutyric acid, and proline In tobacco leaves. Arch. Biochem. Biophys. 105, 599-605 (1964). MORRIS, C. J., J. F. THOMPSON, and C. M. JOHNSON: Metabolism of glutamic acid and N-acetylglutamic acid in leaf discs and cell-free extracts of higher plants. Plant Physio!. 44, 1023-1026 (1969). Z. Pjlanzenphysiol. Bd. 93. S. 189-199. 1979.
Proline accumulation and drought resistance
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PALFI, G. and J. JUHASZ: The theoretical basis and practical application of a new method of selection for determining water deficiency in plants. Plant and Soil 34, 503-507 (1971 ). PONTE, J. J.: Influ~ncia do acido sulfurico concentrado (densidade 1,84) sobre a germina,
JOSE, T. PRISCO, Departamento de Bioquimica e Biologia Molecular, Centro de Ci&ncias, Universidade Federal do Ceara, c.P. 1065 - 60.000 Fortaleza, Ceara, Brazi!.
z. PJlanzenphysiol.
Ed. 93. S. 189-199. 1979.
Effects of Water Deficit on Proline Accumulation and Growth of two Cotton Genotypes of Different Drought Resistances Lurz
G.
R.
FERREIRA, JOSE G. DE SOUZA
and
JosE
T.
PRISCO
With 3 figures Received October 20,1978' Accepted November 16, 1978
Summary Cotton genotypes (Cruzeta Serid6-9193 and IAC-12.2) differing in drought resistance under dry land farming conditions were used in the present study. Seeds were sown in polythene bags containing sand-soil mixture, and placed on a greenhouse bench. After 27 days from sowing the plants were divided into a control group which was watered every other day, and a stressed group from which water was withheld for 7, 10, and 14 days. Rewatering was performed at the 14th day of stress. When water was withheld both genotypes exhibited progressive reduction in R WC, and 48 hours after rewatering their water status was recovered. Cruzeta Serid6-9193 plants always had higher RWC values than IAC-12.2. When plants were stressed both genotypes exhibited progressive increase on leaf free proline concentration, and 48 hours after rewatering the concentration of this imino acid almost reached the control level. The variations of free proline in the roots followed the same pattern as in the leaves, although reaching lower levels. IAC-12.2 plants had higher proline concentration values both in leaves and roots as compared to Cruzeta Serid6-9193. When plants were either watered (control) or stressed Cruzeta Serid6-9193 genotype always had lower shoot: root ratios than IAC-12.2. Net assimilation rate (NAR), relative growth rate (RGR), and compensation point determinations as a function of water status have indicated that Cruzeta Serid6-9193 genotype is more resistant to drought than IAC-12.2. The present results did not agree with the hypothesis that free proline accumulation is linked to a genetic characteristic that is responsible for drought resistance. At least for the two cotton genotypes investigated drought resistance is correlated with the capacity of the plants to absorb andlor retain water in their tissues.
Key words: water stress, proline accumulation, cotton.
Introduction
When plants are subjected to water stress there is a decrease in plant height 1964; SLATYER, 1969), leaf size, leaf number (SLATYER, 1967 and 1969; SILVA, 1973), flower primordia formation, as well as in root growth (SLATYER, 1969). (GATES,
Z. PJlanzenphysiol. Bd. 93. S. 189-199. 1979.
190
L. G. R. FERREIRA,
J. G. DE SOUZA and]. T.
PRISCO
These morphological changes are simply the result of stress induced modifications of plant processes. Among these, cell growth appears to be the most sensitive (HSIAO, 1973; HSIAO and ACEVEDO, 1974; HSIAO et a1., 1976). However, when plants are exposed to a long period of water stress cell division may be as sensitive as cell elongation (HSIAO, 1973). Although photosynthesis is less sensitive to water stress than cell expansion it is also decreased (VAADIA et a1., 1961; BOYER, 1976). It is assumed that this response is mediated partly by way of impeded CO 2 supply following stomatal closure and partly by a direct effect of dehydration on the photosynthetic system (SLATYER, 1969; SILVA, 1973; BOYER, 1976). SHAH and LOOMIS (1965) found that both soluble and total protein contents of sugar beet leaves declined progressively in a matter of days when water was withheld. This decrease in protein concentration may occur as a result of decreased protein synthesis (BEN-ZIONI et a1., 1967; HSIAO, 1970) and increased protein degradation (SLATYER, 1969). Another frequently observed effect of stress is the appearance of high levels of free amino acids in leaves, especially proline, and ami des (SLATYER, 1969). KEMBLE and MACPHERSON (1954) observed that free proline increased several fold when Lolium. perenne plants were subjected to dehydration. The accumulation of free proline in leaves of different plant species (ROUTLEY, 1966; MORRIS et a1., 1969; MASCIOTTI, 1974; WALDREN et a1., 1974; MADRUGA, 1976) was also observed in roots of citrus (CHEN et a1., 1964), and barley (SINGH, PALEG, and ASPINALL, 1973) subjected to water stress. However, when excised plant tissues are subjected to dehydration, only leaves are able to accumulate proline (STEWART et a1., 1966; SINGH, ASPINALL, PALEG and BOGGESS, 1973). Knowing that proline accumulation was suggested to be correlated with drought resistance (SAUNIER et a1., 1968; PALFI and JUHAsz, 1971; SINGH et a1., 1972) the present investigation was set up to study the effects of water deficit on free proline accumulation in leaves and roots, as well as its effects on growth of two cotton cultivars of different drought sensitivities. The objective of this study was to identify physiological parameters that could be used in the selection of drought resistant cotton plants. Material and Methods Growth Conditions Cotton plants, Gossypium hirsutum, cultivars Cruzeta Serid6-9193 (G. hirsutum var. marie-galante HUTCH.) and IAC-12.2 (G. hirsutum var. latifolium HUTCH.) were chosen because they differ in drought resistance under dry land farming conditions. The perennial Cruzeta Serid6-9193 is known to be more resistant to water stress than the annual IAC-12.2 Seeds were treated with concentrated sulfuric acid for 40 min (PONTE, 1960), and washed with distilled water to remove the excess of acid. Then they were sown in polythene bags containing 10 kg of sand-soil mixture, and placed on a greenhouse bench. After thinning the plants were also grown in the greenhouse under natural daylight, watered each other day to field capacity, and fertilized weekly with half-strength Hoagland solution, modified by JOHNSON et al. (1957). The temperature in the greenhouse ranged from a low of 27°C during Z. Pflanzenphysiol. Bd. 93. S. 189-199. 1979.
Proline accumulation and drought resistance
191
the night to a hight of 29 °e during the day. Relative humidity fluctuated between 85 and 96 0/0, and the intensity of solar radiation ranged from a minimum of 132 to a maximum of 288 cal X cm- 2 X day-I. After 27 days from sowing the plants were divided into a control group which was watered every other day, and a stressed group from which water was withheld. Plants in both groups were sampled after 7, 10, and 14 days. When the plants showed the first signs of wilting (14 days of stress) they were rewatered. Samples for all determinations were taken at 600 a.m. (sunrise) on each sampling day. In all cases it was attempted to select leaves of the same physiological age by sampling only the fifth leaf from the top of the plants (WEATHERLEY, 1950). Water Status oj the Plants The water status of the plants was estimated by determining the Rwe of the leaves (BARRS and WEATHERLEY, 1962) in four replicates, each consisting of five discs (1. mm diameter) excised from leaf blades avoiding the major veins. Leaf discs were saturated by floating them abaxially on distilled water, at 25 ± 1 °e for four hours, under fluorescent lamps (690 lux), and dry weights were obtained after dehydration in an oven at 80 °e for 48 hours. Free Proline Free proline was determined in four replicates of both leaves and roots. Five leaf discs or 0.1 g fresh weight of root tips were collected, submerged in 2 ml of methanol: chloroform: water (12 : 5 : 1, v/v), and stored at -15 °e for further analysis. The extraction and determination of free proline were performed according to MESSER (1961) as modified by MASCIOTTI (1974). All proline concentrations were expressed on dry weight of plant tissue basis. Leaf R we and free proline concentration values for both leaves and roots were used to calculate correlation coefficients at 1 % significance level, and then adjusted to a linear regression equation (SNEDECOR, 1956). Growth Analysis and Drought Resistance Growth analysis was carried out by using indexes such as net assimilation rate (NAR), relative growth rate (RGR), and shoot: roOt ratio (RADFORD, 1967). In the determination of NAR and RGR the experimental period was divided into two phases: the first phase from 0 to 7 days, and the second one from 7 to 14 days. The tolerance of leaves to dehydration was determined by measuring colorimetric ally the compensation point (LIETH and ASHTON, 1961) as a function of the RWC. Three replicates of leaf strips (5 X 1 cm), collected from each of the two leaf samples were mounted in rubberstoppered test tubes (18 X 1.7 cm) containing 2 ml of cresol red indicator. The test tubes were placed for 3 hours under fluorescent-incandescent light bench so that the upper surface of the leaf strips received 4300 lux.
Results Plants of both cultivars exhibited progressive reduction in RWC when they were subjected to water stress, and 48 hours after rewatering their R WC reached values close to the control treatment (Figure 1 A). Fourteen days after the water was withheld plants of both cultivars showed signs of wilting, and the greatest reduction in RWC. Cruzeta Serid6-9193 always presented higher RWC values than IAC-12.2 The RWC of each one of the cultivars did not change to a great extent in the control treatment. Z. Pjlanzenphysiol. Bd. 93. S. 189-199. 1979.
192
L. G. R.
FERREIRA,
J. G. DE
SOUZA
and
J. T.
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A
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EXPERIMENTAL PERIOD (days)
14
_
15
16
Proline accumulation and drought resistance
193
Figure 1 B shows changes in leaf free proline concentration for both control and water stressed plants. Proline concentration for both cultivars in the control treatment was close to zero, meaning that free proline levels in turgid cotton leaf tissues is very low. When plants were stressed for 7 days, a reduction in RWC of 5 0 /0 (IAC-12.2) or 6 Ufo (Cruzeta Serid6-9193) did not induce appreciable increase in free proline concentration. Ten days after withholding the water, when the reduction in R WC was 31 Ufo for Cruzeta Serid6-9193, and 34 Ufo for IAC-12.2, free proline increased 41 times in the former, and 70 times in the latter cultivar. By the end of the water stress period (14 days), the amount of free proline reached the highest level in relation to the control, that is, 107 times for Cruzeta Serid6-9193, and 111 times for IAC-12.2. Twenty-four hours after rewatering, there was an increase in water content (Figure 1 A) and a great reduction in free proline level, which continued to decrease, reaching almost the control level at the end of the experimental period (Figure 1 B). There was a negative correlation between RWC (Figure 1 A) and leaf proline concentration (Figure 1 B) of -0.94 for Cruzeta Serid6-9193, and -0.92 for IAC-12.2. The adjusted linear regression equation for Cruzeta Serid6-9193 was y = -2.30x + 201.06 (r2 = 0.92), and y = -2.33x + 199.39 (r2 = 0.96) for IAC-12.2 (Figure 2 A). The variations of free proline in the roots followed the same pattern as in the leaves, although not reaching levels as high as in the leaves (Figure 1 C). A negative correlation of -0.92 for Cruzeta Serid6-9193, and -0.93 for IAC-12.2 between leaf RWC (Figure 1 A) and root proline concentration (Figure 1 C) was observed. The adjusted linear regression equation for Cruzeta Serid6-9193 was y = -O.96x + 84.60 (r2 = 0.95), and y = -1.35x + 116.84 (r2 = 0.97) for IAC-12.2 (Figure 2 B). The variations in shoot: root ratio of both control and water stressed plants are shown in Table 1. When plants were either watered (control) or stressed IAC-12.2 genotype had always higher shoot: root ratios than Cruzeta Serid6-9193. When both cultivars were subjected to water stress there was no significant reduction in this parameter. The highest NAR for both control and water stressed plants was observed during the first phase of the experimental period (Table 2). When well watered, IAC-12.2 plants reached greater NAR values than Cruzeta Serid6-9193. However, when they were subjected to water stress, Cruzeta Serid6-9193 had always higher NAR than IAC-12.2. The highest RGR for both cultivars when they were either watered (control) or stressed was observed during the first phase of the experimental period (Table 3). In both control and stressed treatments. Cruzeta Serid6-9193 had higher RGR than IAC-12.2 during both phases. RGR of Cruzeta Serid6-9193 was less affected by Fig. 1: Variations in RWC (A), leaf free proline (B), and root free proline (C) of Cruzeta Serid6 - 9193 and IAC-12.2 cotton plants along the experimental period.
z. Pflanzenphysiol.
Bd. 93. S. 189-199. 1979.
194
L. G. R. FERREIRA,
J. G. DE SOUZA and J. T.
~
A
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100
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.!! o
~
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~
60
lAC - 12.2'/
y=- 2.33 X + 199.39
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z :; 40 o
~\,
~
r2= 0.96
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~
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,
o 100
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ell
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.........
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.........
..........
..........
CRUZETA SERI06 - 9193
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...... ......
20
9=-0.96 X + S4.60 r2= 0.95
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00 RWC (%) Fig. 2: Relationship between leaf free proline and leaf water status (A), and between root free proline and leaf water status (B) of Cruzeta Serid6 - 9193 and lAC - 12.2 cotton plants. Adjusted linear regression equations for each one of the cultivars are given.
z. PJlanzenphysiol. Bd. 93. S.
189-199. 1979.
Proline accumulation and drought resistance
195
Table 1: Mean shoot: root ratios of Cruzeta Serid6 - 9193 and lAC - 12.2 cotton plants subjected to water stress or not l ). Experimental Period (days)
Shoot: root Ratio Cruzeta Serid6 - 9193 lAC - 12.2 Control Stressed Control Stressed
0 7 10 14
1.15 1.44 1.41 1.67
1.15 1.29 1.25 1.26
2.31 2.08 2.04 1.83
2.31 1.98 1.97 1.74
1) Each value represents the mean of 4 replicates.
Table 2: Mean net assimilation rates (NAR) of Cruzeta Serid6 - 9193 and lAC - 12.2 cotton plants subjected to water stress or not l ). Experimental Period (phases)
Net Assimilation Rate (mg X dm-2 X day-l) Cruzeta Serid6 - 9193 lAC -12.2 Control Stressed Control Stressed
First Second
78.5 59.0
69.0 24.3
85.7 78.1
66.2 7.5
1) Each value represents the mean of 4 replicates.
Table 3: Mean relative growth rates (RGR) of Cruzeta Serid6 - 9193 and lAC - 12.2 cotton plants subjected to water stress or not l ). Experimental Period (phases)
Relative Growth Rate (mg X g-l X day-l) Cruzeta Serid6 - 9193 lAC - 12.2 Control Stressed Control Stressed
First Second
115.5 74.8
103.7 30.4
93.4 70.6
67.2 7.3
1) Each value represents the mean of 4 replicates.
water stress than RGR of IAC-12.2. This effect of water stress on RGR was more conspicuous during the second phase. Both cultivars reached their compensation points at low R WC levels (Figure 3). However, IAC-12.2 plants were less resistant to water stress than Cruzeta Serid6-9193. This was evidenced by the fact that when RWC dropped to 56 Ofo the majority of IAC-12.2 plants had reached their compensation point, while all Cruzeta Serid6-9193 plants were still photosynthesizing. Z. P/lanzenphysiol. Bd. 93. S. 189-199. 1979.
196
L. G. R. FERREIRA,
J. G. DE SOUZA and J. T. PRISCO
•
PLANTS PREDOMINANTLY PHOTOSYNTHESIZING
~
PLANTS AT THE COMPENSATION POINT
D
PLANTS PREDOMINANTLY RESPIRING
t\I
~ ,
o
ct
II II
R.w.C. (%) Fig. 3: Compensation point as a function of leaf water status of Cruzeta Serid6 - 9193 and lAC - 12.2 cotton plants.
Discussion When water was withheld both cottOn genotypes exhibited progressive reduction in R WC, and 48 hours after rewatering their water status was recovered (Figure 1 A). Similar results were observed for ladino clover (ROUTLEY, 1966), sorghum (BLUM and EBERCON, 1976), and rice (MADRUGA, 1976). The fact that Cruzeta Serid6-9193 always had higher RWC values than IAC-12.2 suggests differences in behavior of the two genotypes concerning the capacity to absorb and/or retain water. Assays of leaf and root extracts failed to detect the presence of proline in the well watered control plants (Figures 1 Band 1 C). These results differ from the ones found for both bean (MASCIOTTI, 1974), and rice (MADRUGA, 1976) leaves. However, these authors have used other species of plants, and they have assayed proline in excised leaf tissues kept in a high humidity environment, while in the present experiment this imino acid was assayed in intact leaves from well watered (control) plants. The accumulation of free proline in water stressed plants (Figure 1 Band 1 C) has also been observed for Lolium perenne (KEMBLE and MACPHERSON, 1954), citrus (CHEN et aI., 1964), ladino clover (ROUTLEY, 1966), barley (SINGH, PALEG, and ASPINALL, 1973),
z. PJlanzenphysiol.
Bd. 93. S. 189-199. 1979.
Proline accumulation and drought resistance
197
and for sorghum (BLUM and EBERCON, 1976). This increase in proline as a result of water stress may be due to its release from a storage form in the leaves and roots, its de novo synthesis (BARNETT and N AYLOR, 1966; THOMPSON et al., 1966; MORRIS et al., 1969), and/or inhibition of its oxidation (STEWART et al., 1977). As the plants recovered from water stress their proline level decreased, and 48 hours after rewatering the concentration of this imino acid had almost reached the control level (Figures 1 Band 1 C). This recuperation could be interpreted as resulting from its utilization (oxidation) as an energy source for the tissue (STEWART, 1973; RENA and SPLITTSTOESSER, 1974). Another explanation for the decrease in leaf free proline upon rewatering is that this substance would have been translocated to other parts of the plant, especially to the roots (MIZUSAKI et al., 1964). This appears not to be the case since in the present investigation its concentration also decreased in the roots, after rewatering (Figure 1 C). Cruzeta Serid6-9193 from both control and stress treatments always had lower shoot: root ratios than IAC-12.2 plants (Table 1). This indicates a relatively better root development of the former in comparison with the genotype IAC-12.2. It appears that this is the major reason for better water status of Cruzeta Serid6-9193 when compared with IAC-12.2 (Figure 1 A). During the first phase of the experimental period NAR (Table 2) and RGR (Table 3) for both control and water stressed plants were the highest. When well watered, IAC-12.2 had greater NAR than Cruzeta Serid6-9193. This was probably due to the fact that IAC-12.2 being an annual plant photosynthesized more intensively than the perennial Cruzeta Serid6-9193. When they were stressed NAR and RGR of IAC-12.2 plants were much more affected than those of Cruzeta Serid6-9193. It is felt that this is simply a result of the higher drought resistance presented by Cruzeta Serid6-9193 when compared with IAC-12.2 plants (Figure 3). Some investigators (PALFI and JUHASZ, 1971; SINGH et al., 1972) have suggested that proline accumulation was linked to a genetic characteristic that is responsible for drought resistance. The obtained results did not agree with this since free proline accumulation in both leaves and roots of cotton plants was not correlated with drought resistance (Figures 1 and 3). HANSON et al. (1977) have also observed that free proline accumulation is not a positive index of drought resistance suitable for cereal breeding programs. Since there was a negative correlation between proline levels in both leaves and roots and R WC of leaves (Figure 2) it indicates that proline accumulation reflects lowered water status. The present results suggests that, at least for the two cotton genotypes investigated, drought resistance is correlated with the capacity of the plants to absorb and/or retain water in their tissues. Therefore, proline could be possibly functioning as a source of respiratory energy and nitrogen during the rewatering phase (BLUM and EBERCON, 1976), as a neutralizing agent for the ammonia that is released as a result of proteolysis occurring during tissue dehydration (PROTSENKO et al., 1968), and/or be involved in osmoregulation (HELLEBUST, 1976). Z. Pjlanzenphysiol. Bd. 93. S. 189-199. 1979.
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L. G. R. FERREIRA,
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SOUZA and
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PRISCO
Acknowledgement The authors are grateful to Dr. JAMES W. O'LEARY (University of Arizona) for revision of the manuscript. We wish to thank the National Council for Scientific and Technological Development (CNPq), and the Bank of Northeast Brazil (BNB) for supporting this research.
References BARNETT, N. M. and A. W. NAYLOR: Amino acid and protein metabolism in bermuda grass during water stress. Plant Physio!. 41, 1222-1230 (1966). BARRS, H. D. and P. E. WEATHERLEY: A re-examination of the relative turgidity technique for estimating water deficits in leaves. Aust. J. Bio!. Sci. 15, 413-248 (1962). BEN-ZIONI, A., C. ITAI, and Y. VAADIA: Water and salt stress, kinetin and protein synthesis in tobacco leaves. Plant Physio!. 42, 361-365 (1967). BLUM, A. and A. EBERCON: Genotypic responses in sorghum to drought stress. III. Free proline accumulation and drought resistance. Crop Sci. 16, 428-431 (1976). BOYER, J. S.: Photosynthesis at low water potentials. Phi!. Trans. R. Soc. Lond. 273, 501-512 (1976). CHEN, D., B. KESSLER, and S. P. MONSELISE: Studies on water regime and nitrogen metabolism of citrus seedlings grown under water stress. Plant Physio!. 39, 379-386 (1964). GATES, C. T.: The effect of water stress on plant growth. J. Aust. Inst. Agric. Sci. 30, 3-22 (1964). HANSON, A. D., C. E. NELSON, and E. H. EVERSON: Evaluation of free proline accumulation as an index of drought resistance using two barley cultivars. Crop Sci. 17, 720-726 (1977). HELLEBUST, J. A.: Osmoregulation. Ann. Rev. Plant Pysio!. 27, 485-505 (1976). HSIAO, T. C.: Rapide changes in levels of polyribosomes in Zea mays in response to water stress. Plant Physio!. 46, 281-285 (1970). - Plant responses to water stress. Ann. Rev. Plant Physio!. 24, 519-570 (1973). HSIAO, T. C. and E. ACEVEDO: Plant responses to water deficits, water-use efficiency, and drought resistance. Agric. Meteoro!' 14, 59-84 (1974). HSIAO, T. c., E. ACEVEDO, E. FERERES, and D. HENDERSON: Water stress, growth, and osmotic adjustment. Phi!. Trans. R. Soc. Lond. 273, 479-500 (1976). JOHNSON, C. M., P. R. STOUT, T. C. BROYER, and A. B. CARLTON: Comparative chlorine requirements of different plant species. Plant and Soil 8, 337-353 (1957). KEMPLE, A. R. and H. T. MACPHERSON: Liberation of amino acids in perennial rye grass during wilting. Biochem. J. 58, 46-49 (1954). LIETH, H. and D. H. ASHTON: The light compensation points of some herbaceous plants inside and outside deciduous woods in Germany. Can. J. Bot. 39, 1255-1259 (1961). MADRUGA, L. A. N.: Efeito do dUice hidrico sobre 0 metabolismo de aminoacidos livres e proteinas foliares de cinco cultivares de arroz (Oryza sativa L.). M.s. Thesis. Univ. Fed. Vi~osa, 44 pp. (1976). MASCIOTTI, G. Z.: Efeito do dUice hidrico sobre 0 metabolismo do nitrogenio e 0 crescimento de alguns cultivares de feijao (Phaseo/us vulgaris L.). M. S. Thesis. Univ. Fed. Vi~osa, 37 pp. (1974). MESSER, M.: Interference by amino acids and peptides with the photometric estimation of proline. Ana!' Biochem. 2,353-359 (1961). MIZUSAKI, S., M. NOGUCHI, and E. TAMAKI: Studies on nitrogen metabolism In tobacco plants. VI. Metabolism of glutamic acid, y-aminobutyric acid, and proline In tobacco leaves. Arch. Biochem. Biophys. 105, 599-605 (1964). MORRIS, C. J., J. F. THOMPSON, and C. M. JOHNSON: Metabolism of glutamic acid and N-acetylglutamic acid in leaf discs and cell-free extracts of higher plants. Plant Physio!. 44, 1023-1026 (1969). Z. Pjlanzenphysiol. Bd. 93. S. 189-199. 1979.
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PALFI, G. and J. JUHASZ: The theoretical basis and practical application of a new method of selection for determining water deficiency in plants. Plant and Soil 34, 503-507 (1971 ). PONTE, J. J.: Influ~ncia do acido sulfurico concentrado (densidade 1,84) sobre a germina,
JOSE, T. PRISCO, Departamento de Bioquimica e Biologia Molecular, Centro de Ci&ncias, Universidade Federal do Ceara, c.P. 1065 - 60.000 Fortaleza, Ceara, Brazi!.
z. PJlanzenphysiol.
Ed. 93. S. 189-199. 1979.