Bruno P. A. Cammue*2, Willem F. Broekaert2, Jan T. C. Kellens, Natasha V. Raikhel, and Willy J. Peumans. Laboratorium voor Plantenbiochemie K. U. Leuven, ...
Received for publication February 21, 1989 and in revised form July 24, 1989
Plant Physiol. (1989) 91, 1432-1435 0032-0889/89/91/1 432/04/$01 .00/0
Stress-induced Accumulation of Wheat Germ Agglutinin and Abscisic Acid in Roots of Wheat Seedlings1 Bruno P. A. Cammue*2, Willem F. Broekaert2, Jan T. C. Kellens, Natasha V. Raikhel, and Willy J. Peumans Laboratorium voor Plantenbiochemie K. U. Leuven, Willem De Croylaan, 42, B-3030 Leuven Belgium (B.P.A.C., W.F.B., J.T.C.K., W.J.P.), and MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824-1312 (N.V.R.) have been many investigations on stress-induced ABA accumulation in leaves, few studies have examined endogenous ABA levels in plant roots. This paper reports enhanced ABA levels in roots of water-stressed wheat seedlings. Since ABA reportedly stimulates WGA synthesis in wheat roots (16), we further describe (a) the increase of WGA content and synthesis in wheat seedling roots as a result of drought and osmotic stress, and (b) the inhibition of this stress-induced increase by fluridone, an inhibitor of ABA synthesis (2, 4, 5, 14).
ABSTRACT Wheat germ agglutinin (WGA) levels in roots of 2-day-old wheat seedlings increased up to three-fold when stressed by air-drying. Similar results were obtained when seedling roots were incubated either in 0.5 molar mannitol or 180 grams per liter polyethylene glycol 6000, with a peak level of WGA after 5 hours of stress. Longer periods of osmotic treatment resulted in a gradual decline of WGA in the roots. Since excised wheat roots incorporate more [35S]cysteine into WGA under stress conditions, the observed increase of lectin levels is due to de novo synthesis. Measurement of abscisic acid (ABA) levels in roots of control and stressed seedlings indicated a 10-fold increase upon air-drying. Similarly, a five- and seven-fold increase of ABA content of seedling roots was found after 2 hours of osmotic stress by polyethylene glycol 6000 and mannitol, respectively. Finally, the stress-induced increase of WGA in wheat roots could be inhibited by growing seedlings in the presence of fluridone, an inhibitor of ABA synthesis. These results indicate that roots of water-stressed wheat seedlings (a) contain more WGA as a result of an increased de novo synthesis of this lectin, and (b) exhibit higher ABA levels. The stress-induced increase of lectin accumulation seems to be under control of ABA.
MATERIALS AND METHODS Growth of Wheat Seedlings Wheat grains (Triticum aestivum L., cv Fidel) were surfacesterilized by consecutive treatments of 70% ethanol (3 min), sterile distilled water, 1% NaOCl (diluted commercial bleach, 30 min) and further washed in five changes of sterile distilled water. The grains were germinated under sterile conditions on moist filter paper in petri dishes (20 cm diameter), and incubated in the dark at 25C. Seedlings used for experiments were 2 d old and had three roots. Treatment of Wheat Seedlings
Wheat seedlings were osmotically stressed by transferring the roots into solutions containing mannitol or PEG 6000 as osmoticum. In drought stress experiments, wheat seedlings were air-dried for 3 h at 25°C at a relative humidity of 65%. This treatment resulted in loss of 84% of fresh weight. (±) ABA (Sigma) and fluridone (Eli Lilly) were treated as
Wheat germ agglutinin (WGA3) is an N-acetyl-glucosamine specific lectin, which was originally isolated from commercial wheat germ (1). Although it has for a long time been considered as a seed-specific, or more precisely as an embryo-specific lectin, evidence has accumulated during the past decade that it occurs also in vegetative tissues, including roots and coleoptiles of wheat plants (12). Moreover, experiments with ABA indicated that the synthesis of WGA is probably under control of this plant growth regulator, both in embryos ( 11) and adult plants (14, 16). ABA is known to play a central role as a stress hormone (for a recent review, see ref. 19). Although there
described previously (14). Determination of WGA and ABA Determination of WGA and ABA were performed on lots of 40 excised seedling roots (1.5-2 cm) using ELISA as previously described (12, 13).
'Supported in part by grants of the National Fund for Scientific Research (Belgium) and by a grant (0056/88) from the North Atlantic Treaty Organization for collaboration between N. V. R. and W. J. P. W. J. P. is Senior Research Associate and W. F. B. is Research Assistant of this fund. B. P. A. C. and J. T. C. K. receive a fellowship of the Belgian Instituut tot Aanmoediging van het Wetenschappelijk Onderzoek in Nijverheid en Landbouw (IWONL). 2Present address: F.A. Janssens Memorial Laboratory of Genetics K. U. Leuven, Willem De Croylaan 42, B-3030 Leuven, Belgium. I Abbreviations: WGA, wheat germ agglutinin.
In Vivo Lectin Synthesis
Roots of treated seedlings were carefully removed and incubated in 0.6 mL nutrient medium (containing 4% w/v sucrose, 4 mM CaC12, 5 mm KC1, 150 mm glutamine, and 0.05% w/v ampicillin, pH 6.1) supplemented with 1.8 MBq [35S]cysteine (43 TBq mmol-') for 3.5 h at 30°C. After labeling, the roots were washed extensively with ice-cold distilled 1432
STRESS-INDUCED ACCUMULATION OF WGA AND ABA IN WHEAT ROOTS
water. Extraction, affinity purification and determination of newly synthesized WGA were performed as previously described elsewhere (16). To ensure that the radioactivity recovered in the lectin fraction was really incorporated into lectin, the affinity-purified polypeptides were analyzed by SDS-PAGE and fluorography (16) (results not shown).
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RESULTS
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Effect of Drought and Osmotic Stress on WGA
Preliminary experiments in
our
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laboratory indicated that
extracts of drought-stressed wheat roots exhibited enhanced hemagglutinin activity as compared to nonstressed plants. A
much more sensitive WGA-detection technique (ELISA) was used in further experiments to quantify these stress effects. In the first experiment, 2-d-old seedlings were air-dried, and the WGA content of the roots was determined. As shown in Figure 1, drought-stressed roots contained nearly 3.5 times more lectin than unstressed roots. In the same experiment, wheat seedlings were also stressed 5 h with mannitol solutions of increasing concentration. Figure 1 shows a steady rise of WGA content in the roots with increasing severity of osmotic stress.
In a second experiment, wheat seedlings were exposed for increasing time periods to a solution of 0.5 M mannitol, equal to an osmotic potential of approximately -12 bars (7) (Fig. 2). After 2 h of this severe stress, WGA content of the roots started to rise with a peak level after 5 h. With longer periods of osmotic stress, WGA content eventually declined to a level equal to that of unstressed roots (after 10 h). The increase of WGA in the roots was not due to a specific effect of mannitol since similar results were obtained with PEG 6000 as osmoticum (data not shown). These results raised the question of whether the enhanced WGA level in roots of stressed wheat seedlings was due to an increased de novo synthesis in the roots or originated from other parts of the seedling. To address this question, wheat seedlings were air-dried or stressed with 0.5 M mannitol for different periods. After these treatments roots were removed and incubated for 3.5 h in the presence of [35S]cysteine. Both total protein and lectin synthesis were measured. As can be seen in Table I, both drought and osmotic stress increased de novo synthesis of WGA in roots of wheat seedlings. Moreover, when seedlings were osmotically stressed for increasing periods of time, the highest level of WGA synthesis was found in roots stressed for 3 h with 0.5 M mannitol (Table I). This maximal rate of WGA synthesis coincides with the commencement of the increase in total WGA content of the roots (Fig. 2).
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Treatment Figure 1. WGA levels in the roots of control and water-stressed wheat seedlings. Wheat seedlings were treated as follows. Treatment 1, seedling roots were incubated in distilled water for 5 h (control plants); treatment 2, seedlings were air-dried for 3 h at 25°C; treatments 3, 4, 5, 6, and 7, seedling roots were incubated for 5 h in 0.1, 0.2, 0.3, 0.5, and 1 M mannitol, respectively. Results are the average of three experiments. The ratios of standard deviation to average were less than 20%.
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Treatment time (h)
Effect of Drought and Osmotic Stress on ABA Since the synthesis of WGA seems to be under control of ABA, the ABA content of wheat roots under stress conditions (similar to those described above) was determined. Preliminary experiments indicated that roots of air-dried wheat seedlings contained about 10 times more ABA than unstressed roots (results not shown). A similar, but smaller increase was obtained when seedlings were osmotically stressed. Figure 2
Figure 2. Effect of osmotic stress on both WGA and ABA content of wheat seedling roots as a function of treatment time. Wheat seedlings were osmotically stressed by incubation of the roots for increasing time periods in either 0.5 M mannitol (@) or in 180 g/L PEG 6000 (A). Control plants were grown in distilled water (U). Both WGA (full curves) and ABA (dotted curves) content of the roots were determined. Results are the average of three experiments. The ratios of standard deviation to average were less than 18 and 24% in the WGA and ABA determinations, respectively.
Plant Physiol. Vol. 91,1989
CAMMUE ET AL.
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Table 1. [35S]Cysteine Incorporated into Total Protein and AffinityPurified WGA in Roots of 2-Day-Old Wheat Seedlings Treatment
Incorporation of [35S]Cysteine into Total Lectin protein cpm/root
Control Air drying 0.5 M Mannitol at h: 1 2 3 4 5
20
Relative Rate of WGA
Synthesis %
7473 7903
66 119
0.91 1.51
7581 7468 6934 7571 7613
64 59 111 79 72
0.85 0.79 1.60 1.07 0.95
shows the ABA levels in roots of wheat seedlings treated for increasing time periods with 0.5 M mannitol (severe stress; -12 bars) or 180 g/L PEG 6000 (mild stress; -4 bars [8]). In both treatments ABA levels started to rise after 1 h of stress. As compared to the controls, a seven- and five-fold increase of ABA was observed in roots stressed with mannitol or PEG, respectively. During longer periods of stress, ABA levels declined to a steady concentration of about 15 ng/root, which, however, was still twofold higher than in roots of unstressed seedlings. Effect of Changing Exogenous and Endogenous ABALevels in WGA
Earlier studies (14) showed that wheat seedlings grown for days in 10-' M ABA contained about two times more lectin in the distal portion of the roots than untreated plants. We observed a similar twofold increase in the roots of 2-dold wheat seedlings treated for 48 h with 10-' M ABA (results not shown). In another experiment, wheat seeds were germinated for 48 h in 10 mg/L fluridone, a herbicide which reportedly decreases endogenous ABA levels by inhibiting carotenoid synthesis (4, 5, 14). These 2-d-old seedlings were further treated for 8 h with 0.5 M mannitol, and the WGA content of roots was determined. Figure 3 shows that osmotic stress did not induce an increase of the WGA content in roots of fluridone-treated seedlings. In addition, germination of seeds in the presence of fluridone resulted in a lower WGA content ofthe roots, which is consistent with earlier reports ( 14).
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5 6 2 3 4 Treatment time (h)
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Figure 3. Effect of fluridone on the level of WGA in roots of control and osmotically stressed wheat seedlings. Wheat grains, germinated in the presence (dashed lines) or absence (solid lines) of 10 mg/L fluridone, were osmotically stressed by incubation of the roots for increasing time periods in 0.5 M mannitol (C). Unstressed plants were grown in distilled water (0). Results are the average of three experiments. The ratios of standard deviation to average were less than 18%.
11
DISCUSSION
Although WGA is one of the best characterized plant lectins, its physiological function still remains unclear. Earlier reports indicated that WGA may be regulated developmentally in different tissues of wheat. In embryos, for example, WGA accumulation seems to be highly correlated with embryogenesis (10, 17), while WGA synthesis in root tips of young wheat plants gradually declines as seedling development proceeds (9, 16). The present report is, to our knowledge, the first to indicate that WGA synthesis in wheat plants can be modulated by environmental factors, such as drought and
osmotic stress. Air-dried seedlings contained up to 3.5 times more WGA in the roots than control plants. When wheat roots were exposed to either 0.5 M mannitol or 180 g/L PEG 6000, WGA levels rapidly increased to a maximum after 5 h of treatment. In vivo synthesis experiments with excised wheat roots indicate that this increased WGA content resulted from stress-induced enhancement of WGA synthesis. When seedlings were osmotically stressed for periods longer than 5 h, WGA gradually declined to nonstressed levels. Recent experiments in our laboratory suggest that this decrease of WGA content is at least partially due to a release of WGA in the medium, rather than a degradation of WGA in the root. Moreover, we observed that air-dried seedlings do not exhibit such a decline of stress-induced WGA content (BPA Cammue, unpublished results). Under similar stress conditions, ABA levels also increased in roots of wheat seedlings. Air-dried roots contained 10 times more ABA, while treatment with 0.5 M mannitol or 180 g/L PEG 6000 caused a seven- or five-fold increase, respectively. Walton et al. (18) observed that treatment with 180 g/L PEG 6000 caused a 16-, 10- and 3-fold ABA increase in roots of bean, pea, and sunflower, respectively. Our results show that the response to water stress occurs rapidly as ABA levels increase by 1 h and reach a maximum after 2 h. Walton et al. (18) reported a similar, rapid increase of ABA in bean (Phaseolus vulgaris) roots. In contrast to their results, we
STRESS-INDUCED ACCUMULATION OF WGA AND ABA IN WHEAT ROOTS
observed that ABA levels in wheat roots dropped over the next 3 h of treatment to a steady state level two-fold above the control level. Possible reasons may be the transformation of ABA into conjugates not detectable by the assay or degradation to acidic metabolites. However, Lehmann and Schutte (6) found that drought treatment did not change the proportions of ABA and its metabolites in wheat seedlings. Another possible explanation for this rapid ABA decrease may be that ABA is released into the medium. Rivier et al. (15) reported that the capacity of Zea mays roots to release endogenous ABA increases after osmotic stress. Similar results on the exodiffusion of ABA were found in osmotically stressed roots of cocklebur (Xanthium strumarium) and tomato (Lycopersicon esculentum) (3). Comparison of the stress-response curves ofWGA and ABA in wheat seedling roots (Fig. 2) demonstrated that the maximum ABA level (after 2 h) coincided with the commencement of the WGA increase. Moreover, shortly thereafter, the rate of WGA synthesis also reached a maximum (Table I). These data on the kinetics of ABA and WGA induction suggest a causal relationship between stress-induced ABA and WGA accumulation. This hypothesis is further supported by the observation that fluridone, an inhibitor of ABA synthesis, totally repressed stress-induced WGA accumulation in roots. In addition, we observed that administering ABA exogenously in a concentration range from 10-6 M to 10' M, induced a two- to three-fold increase in WGA content of the roots (results are not shown). These results are consistent with previous reports on ABA-stimulated WGA accumulation in different tissues of wheat (1 1, 14, 16). From these results we conclude that (a) water stress induces a transient accumulation of WGA in roots of wheat seedlings, and (b) this accumulation is associated with a stress-induced increase of ABA. LITERATURE CITED 1. Allen AK, Neuberger A, Sharon N (1973) The purification, composition and specificity of wheat germ agglutinin. Biochem
J 131: 155-162 2. Bartels PG, Watson CW (1978) Inhibition of carotenoid synthesis by fluridone and norflurazon. Weed Sci 2: 198-203
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3. Cornish K, Zeevaart JAD (1985) Abscisic acid accumulation by roots of Xanthium strumarium L. and Lycopersicon esculentum Mill. in relation to water stress. Plant Physiol 79: 653-658 4. Fong F, Smith JD, Koehler DE (1983) Early events in maize seed development. Plant Physiol 73: 899-901 5. Gamble PE, Mullet JE (1986) Inhibition of carotenoid accumulation and abscisic acid biosynthesis in fluridone-treated dark-grown barley. Eur J Biochem 160: 117-121 6. Lehmann H, Schutte HR (1984) Abscisic acid metabolism in intact wheat seedlings under normal and stress conditions. J Plant Physiol 117: 201-209 7. Loveys BR (1977) The intracellular location of abscisic acid in stressed and non-stressed leaf tissue. Physiol Plant 40: 6- 10 8. Michel BE, Kaufmann MR (1973) The osmotic potential of polyethylene glycol 6000. Plant Physiol 51: 914-916 9. Mishkind M, Keegstra K, Palevitz BA (1980) Distribution of wheat germ agglutinin in young wheat plants. Plant Physiol 66: 950-955 10. Peumans WJ, Stinissen HM, Carlier AR (1982) Lectin synthesis in developing and germinating wheat and rye embryos. Planta 156: 41-44 11. Quatrano RS, Ballo BL, Williamson JD, Hamblin MT, Mansfield M (1983) ABA controlled expression of embryo-specific genes during wheat grain development. In RB Goldberg, ed, Plant Molecular Biology. Alan R Liss, New York, pp 343-352 12. Raikhel NV, Mishkind ML, Palevitz BA (1984) Characterization of a wheat germ agglutinin like lectin from adult wheat plants. Planta 162: 55-61 13. Raikhel NV, Hughes DW, Galau GA (1986) An enzyme-immunoassay for quantitative analysis of abscisic acid in wheat. In JE Fox, M Jacobs, eds, Molecular Biology of Plant Growth Control. Alan R Liss, New York, pp 197-207 14. Raikhel NV, Palevitz BA, Haigler CH (1986) Abscisic acid control of lectin accumulation in wheat seedlings and callus cultures. Plant Physiol 80: 167-171 15. Rivier L Leonard J-F, Cottier J-P (1983) Rapid effect ofosmotic stress on the content an exodiffusion of abscisic acid in Zea mays roots. Plant Sci Lett 31: 133-137 16. Stinissen HM, Chrispeels MJ, Peumans WJ (1985) Biosynthesis of lectin in roots of germinating and adult cereal plants. Planta 164: 278-286 17. Triplett BA, Quatrano RS (1982) Timing, localization, and control of wheat germ agglutinin synthesis in developing wheat embryos. Dev Biol 91: 491-496 18. Walton DC, Harrison MA, Cote P (1976) The effects of water stress on abscisic-acid levels and metabolism in roots of Phaseolus vulgaris L. and other plants. Planta 131: 141-144 19. Zeevaart JAD, Creelman RA (1988) Metabolism and physiology of abscisic acid. Annu Rev Plant Physiol 39: 439-473
