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Effect of Light Intensity on the Concentrations of Phytohormones in Developing Wheat Grains
Effect of Light Intensity on the Concentrations of Phytohormones in Developing Wheat Grains KONRAD MENGEL, BAERBEL FRIEDRICH and GUENTER KLAus JunEL Institute of Plant Nutrition, Suedanlage 6, D-6300 Giessen Received February 15, 1985 . Accepted April 2, 1985
Summary The effect of light-intensity on the content of phytohormones in developing wheat grains (Triticum aestivum L. vc. Kolibri) was investigated. One day after anthesis plants were grown with a light-intensity of 25 W m -2 (low light treatment) and with 84 W m -2 (high light treatment) in a phytotron. Grain samples taken throughout the grain-filling period and analyzed for phytohormones showed no major influence of light-intensity on the content of cytokinins and free auxins. There was, however, a significant effect of light-intensity on the gibberellin and abscisic acid content; the latter being higher in the low-light treatment, the former being higher in the high-light treatment. Low-light treatment affected grain setting and resulted especially in hampered grain growth and starch synthesis, while the production of grain crude protein was not influenced by light-intensity. From the experimental results it is concluded that light-intensity influences grain initiation by its effect on abscisic acid and gibberellins in the early stage of grain development. In the later stage low light-intensity impedes grain growth and starch production due to the effect of light on supply with sucrose and on abscisic acid and gibberellin metabolism.
Key words: Abscisic acid, auxins, cytokinins, gibberellins, wheat grains.
Introduction Various authors hold the view that processes in developing grains of cereals rather than photosynthesis are of major importance for grain growth and grain production Genner 1980; Michael and Beringer 1980). Stoy (1973) reported that reduced light-intensity, applied shortly after anthesis, considerably decreased the incorporation of photosynthates into grains of cereals. Mengel and Haeder (1976) found that a low-light treatment during the grain filling period especially affected starch formation in wheat grains and to a lesser degree the translocation of photosynthate to the grains. The impeded production of photosynthates under low-light conditions during the grain filling period is compensated to a high degree by the mobilization of non-structural carbohYc:lrates, particularly fructans, already synthesized and stored in the culms before anthesis Gudel and Mengel 1982). In studying the effect of light-intensity on the activity of starch synthesizing enzymes, Mengel and Judel (1981) came to the conclusion that starch synthesis is associated with grain growth and it was suggested that light-intensity may have an impact on the phytohormone level in developing grains
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and thus may control grain growth and starch production. The question whether light-intensity has an impact on the concentration of phytohormones in developing wheat grains was examined in a pot experiment, the results of which are communicated in the following. Materials and Methods Cultivation ofplants
Spring wheat (Triticum aestivum L. cv. Kolibri) was cultivated in Mitscherlich pots on a sandy loam (Luvisol, 7kglpot) mixed with 0.8gN, 0.9gP, and 1.9gK/pot of commercial fertilizer. At tillering, shooting, and ear emergence additional N was supplied, at each stage 0.5 g N / pot in the form of ammonium nitrate. Plants were grown outdoors until anthesis. Then they were transferred into growth chambers, where they were irradiated by mercury-vapor-highpressure-lamps HQi 250 W (Philips, Eindhoven, The Netherlands). Half of the plants (24 pots) stood in a chamber with a light intensity of 25 W m -2 (low light-intensity treatment); the other half in a growth chamber with 84 W m -2 light intensity (high light-intensity treatment). All other conditions were the same for both treatments: day length: 16 h, day temperature: 20°C, night temperature: 14 °C, relative air humidity: 60 % during the day and 80 % at night. Sampling
Throughout the grain filling period 24 samples were collected, on each date the grains of one pot. The 1st sampling date was one day after anthesis aune 6Ih), the last sampling date 57 days after anthesis (August 17lh). Between these days samples were taken at intervals of 1 to 7 days. Ears were cut, immediately immersed in liquid nitrogen in order to stop metabolic activity, and then stored in plastic bags at - 24°C until analysis. For phytohormone analysis as well as for the determination of starch, sucrose, and nitrogen (protein) only ears of the 1'1 order were used and from these only the grains of the medial section. From each ear 20 grains were sampled; with a pair of tweezers the glumes were carefully removed and the karyopses were stored at -24°C. For grain yield and yield component determination all ears were taken into account. Analyses
Dry matter was determined by drying 10 to 30 grains at 105 0c. Total N was analyzed by micro-Kjeldahl. Crude protein content was calculated by multiplying total N with 5.7. Each analysis was carried out with two samples. The starch content of the grains was determined following the hexokinase method of Keppler and Decker (1974). Sucrose was assessed enzymatically according to the technique of Bergmeyer et al. (1974). Each analysis was made in triplicate. The coefficient of variation for the dry matter yield was 4.1, for the crude protein determination 0.8, for the starch determination 3.2, and for the sucrose determination lOA. Isolation ofphytohormone
Grains were homogenized in a Waring Blendor and extracted in triplicate with a five-fold excess of cold 80 % methanol. The combined extracts were purified and the hormones isolated by liquid-liquid chromatography, by TLC and ion exchange chromatography as described by Rademacher and Graebe (1984 a).
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Biotests for phytohormone activity The samples containing phytohormone were investigated for their hormone activity be means of biotests. The reference hormones were received from Sigmar Chemie, Munich.
Cytokinins Preliminary experiments had shown that the primary wheat leaf test according to Kende (1964), (see also Shaw and Srivastava (1964) and the cucumber-cotyledon test yielded reliable results. The two methods were in good agreement. Each sample was analyzed at least 4 times with these two methods so that at least 8 single values for each sample were obtained. The values shown in Fig. 4 are means of the two biotests. Standards were made from kinetin and zeatin solutions in a range of 10- 8 to 10- 5 g. The primary wheat leaf test was carried out with the cultivar «Kolibri», the cucumber cotyledon test with «Chinese Snake».
Gibberellins Gibberellin activity was assessed by the lettuce hypocotyl test (cv. «Arctic King») according to Frankland and Wareing (1960) and with the barley endosperm test (cv. Nudika) according to
Coombe et al. (1967). Preliminary tests had shown that both tests yielded the same results with satisfactory reproducibility. With each biotest technique at least 4 analyses per sample were carried out giving at least 8 single values from which the means (Fig. 5) were calculated. Standards were made from gibberellic acid (GA3) in a range of 10- 10 to 10- 6 g.
Auxins Auxin activity was determined by the wheat coleoptile segment test according to Mitchell and Livingstone (1969). Five determinations per sample were made. Standards contained indoleacetic acid in a range of 10- 10 to 1O- 4 g.ln the samples taken 13, 18,23, and 28 days after anthesis besides free indoleacetic acid (IAA) also the conjugated lAA and the strongly bound IAA were determined. Separation of the three IAA types was carried out as described by Rademacher (W. Rademacher 1978, Thesis Agric. Faculty, University Goettingen, Fed. Rep. of Germany).
A bscisic acid Abscisic acid activity was assessed by two different tests: the wheat leaf transpiration inhibition test according to Rademacher (W. Rademacher 1978, Thesis Agric. Faculty, University Goettingen, Fed. Rep. of Germany) and the barley endosperm test according to Chrispeels and Varner (1966). The cultivars used were «Kolibri» (wheat) and «Nudika» (barley). Both techniques yielded similar values. Each sample was analyzed at least 4 times with each technique so that at least 8 single values per mean were obtained. Standards were made from abscisic acid.
Results
Reduced light intensity resulted in decreased plant growth and grain yield. As shown in Table 1 grain number per ear, single grain weight, spikelet development, and the development of spikelets with three or more grains were affected. Grain growth throughout the grain filling period (Fig. 1) was not affected during the first 13 days after anthesis. From this date on grains having received high light-intensity had a higher growth rate than grains grown with reduced light. The difference in grain development was mainly due to the grain starch production which was much hampered
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Table 1: Effect of light intensity on grain yield and yield components. The data relate to the plants of 1 pot. ------------------------------------------light intensity, W m -2 Grain yield, g Single grain weight, mg No. of ears No. of grains/ear No. of spikelets/ear Spikelets with 3 and more grains in % of total spikelets
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by low light-intensity. As can be seen from Fig. 2 the curves for the starch synthesis resemble those of grain growth (Fig. 1). Interestingly enough light-intensity had no major impact on the N quantity (crude protein) per grain (Fig. 3). Cytokinins in grains were only detectable during the first 2 weeks after anthesis showing a peak 6 days after flowering. Major effects of light-intensity on the content of cytokinins per grain were not observed (Fig. 4). In contrast to cytokinins, gibberellins were clearly influenced by light-intensity (Fig. 5). The treatment with low-light not only resulted in significantly smaller quantities of gibberellins per grain, also the duration of gibberellins during the grain filling period was considerably shorter in the grains with lowlight-intensity.
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Auxin content did not differ much between treatments (Fig. 6) but in the later stage of grain filling, grains having received higher light-intensity had higher auxin contents than those with low-light. The peak in the auxin content occurred earlier in
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Days after flowering Fig. 5: Effect of light intensity on the gibberellin content in grains throughout the grain filling period. ng GA per grain.
the «low light-intensity plants». The effect of light-intensity on auxin was also evident in the content of conjugated auxins which was much higher in the treatment with low-light-intensity (Table 2). The difference in ABA content was particularly obvious during the first 3 weeks after anthesis, ABA being much higher in the grains treated with low-light-intensity (Fig. 7). Sucrose content in grains did not differ very much between treatments
J Plant Physiol. Vol. 120. pp. 255-266 (1985)
Phytohormones
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throughout the grain filling period (Fig. 8). There was a tendency to higher sucrose content in the high-light treatment during the period of maximum grain growth. Discussion Low-light-intensity affected grain yield by a reduced number of grains per ear and by hampered starch synthesis. It is assumed that grain setting was influenced at an early stage of the experimental period which means at flowering or shortly after flowering. According to Michael and Beringer (1980) grain initiation of cereals can be influenced by auxins and by cytokinins. Auxins are supposed to function in a kind of «medical dominance» rather impeding than promoting grain setting whereas cytokinins favour grain initiation. In the earlier stage of grain filling (2 weeks after anthesis) no significant differences in cytokinin levels were found between treatments (Fig. 4). Also the levels of free auxin did not differ between treatments (Fig. 6). The content in conjugated and bound auxins were higher in the low-light treatment
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KONRAD MENGEL, BAERBEL FRIEDRICH and GUENTER KLAus JUDEL
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(Table2) indicating that light-intensity had some influence on auxin metabolism. There were significant differences between treatments for the ABA and gibberellin content. Since at this early stage of grain filling the size of the grain between treatments did not differ (Fig. 1), the content of ABA and gibberellins in the grains dif-
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fered between treatments. ABA is known to be an antagonist of cytokinins (Alvim et al. 1976) and it is feasible that the impeded grain initiation in the low-light treatment was associated with the antagonistic action of ABA. According to Rademacher, gibberellins are involved in processes after fertilization by inducing the synthesis of glycolytic enzymes. Thus the low gibberellin activity in the treatment with low-light-intensity at the early stage of grain development may have had an impact on grain setting. Cytokinin activity reached a peak 6 days after anthesis. This agrees well with findings of Rademacher and Graebe (1984 b). Also the maximum cytokinin content (22 ng cytokinin/grain) found by Rademacher and Graebe (1984 b) by means of gas chromatography is the same as that found by us with biotests. Other authors, however, reported much higher cytokinin content (Wheeler 1972; Herzog and Geisler 1977). About two weeks after anthesis, the cytokinin activity was almost nil. A similar observation was made by Wheeler (1972) and Rademacher and Graebe (1984 b). The maximum of the cytokinin activity coincides with a phase of intensive cell division. At this stage the number of endosperm cells is determined which according to Brocklehurst (1977) may determine the grain filling capacity. An almost linear grain growth was found between the 13 th and 33rd day after anthesis, the growth rate in the reduced-light treatment being much lower than in the high-light-intensity treatment (Fig. 1). During this period also the highest content of gibberellins were found. According to Dela Guardia and Benlloch (1980) gibberellins, like auxins, may activate the plasmalemma-located H+ pump and thus increase the plasticity of cell walls and promote cell elongation. The low gibberellin content found in the treatment with low light-intensity (Fig.5) at the period of highest growth rate is supposed to have led to the poor grain growth found with this treatment. Besides the gibberellins, also the higher ABA content in the grains of the lowlight treatment (Fig. 7) could have contributed to the poor grain growth because of antagonistic effects (Alvim et al. 1976). Throughout the period between 12 and 30 days after anthesis there was a tendency to higher SUcrOse content in the grains of the high-light-intensity treatment. In most cases the difference was significant as can be seen from Fig. 8. During this period also the ABA contents in the grains of this treatment were significantly lower than in the treatment with low light. Thus one may speculate whether ABA impeded the import of sucrose into the storage tissue as suggested by Dorffling (1977). The finding that highest gibberellin content coincides with high rates of grain growth has also been observed by other authors (Mounla and Michael 1973; Radley 1976; Mounla et al. 1980). The assumption that there is a causal relationship between gibberellin content and growth rate is therefore confirmed. The maturation stage lasted, in our experiment, from the 3yd until the 57th day after anthesis. During this period the net import of assimilates into the grains declines, the kernels gradually loose water resulting in a decrease of grain volume. In this late stage ABA was the domi-
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nant phytohormone (Fig. 7). Low quantities of auxin and gibberellin were still present in the «high-light treatment» grains at the beginning of the maturation stage (Figs. S and 6). Whether they still had an impact on grain growth is questioned. ABA is known to induce the maturation stage. This is in good agreement with the finding that the peak in the ABA content coincided with the beginning of the maturation stage (Fig. 1 and Fig. 7) of the plants with high-light application. In the treatment with low light-intensity, however, the ABA peak already occurred 18 days after flowering and it is likely that the process of grain senescence started earlier in this treatment. Although grain growth was much affected by low light-intensity, the N content in the grains (mg N per grain) was not influenced (Fig. 3). But under low-light conditions there is certainly an enhanced breakdown of leaf proteins which results in an increased supply of free amino acids and amides. Consequently protein synthesis in the grains is favoured which causes the higher N content observed in the grains cultivated at low-light-intensity compared to those grown at high-light-intensities (d. Fig. 3). Starch production in grains, on the contrary, was much affected by low light (Fig. 2). It is thus questionable whether the impeded starch synthesis was primarily due to a lack of photosynthates. The fact that the level of free sucrose during the most intense grain filling period (days 10-33) was considerably lower at low light-intensity than at high intensity (Fig. 8) also supports the assumption that there was a deficiency of photosynthates at low light-intensity. It therefore seems likely that the reduced starch synthesis was associated with a limited deficiency of photosynthates and with a poor grain growth of the low light-intensity plants which was again controlled by phytohormones, particularly by gibberellin and ABA. Starch is synthesized and stored in amyloplasts (Preiss 1982). It is possible that also the development of amyloplasts was influenced by phytohormones and thus by light-intensity. How light-intensity may induce synthesis and turnover of phytohormones is still unknown. According to Hewitt and Wareing (1973) and Dorfler and Goring (1978) light effects on phytohormones are likely to occur. Acknowledgements The authors are much obliged to the Deutsche Forschungsgemeinschaft for financial support.
References ALVIM, R., E. W. HEWITI, and P. F. SANDERS: Seasonal variation in the hormone content of willow. I. Changes in abscisic acid content and cytokinin activity in the xylem sap. Plant Physiol. 57, 474-476 (1976). BERGMEYER, H. U., E. BERNT, F. SCHMIDT und H. STORK: D-Glucose. In: Methoden der enzymatischen Analyse (H. U. BERGMEYER, ed.), pp. 1241-1246. Verlag Chemie, Weinheim, 1974. ISBN 3-527255303. BROCKLEHURST, P. A.: Factors controlling grain weight in wheat. Nature 266, 348-349 (1977). CHRISPEELS, M. J. and J. E. VARNER: Inhibition of gibberellic acid induced formation of cx· amylase by abscisin II. Nature 212, 1066-1067 (1966).
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COOMBE, B. G., D. COHEN, and B. G. PALEG: Barley endosperm bioassay for gibberellins. I. Parameters of the response system. Plant Physiol. 42, 105-112 (1967). DELA GUARDIA, M. D. and M. BENLLOCH: Effects of potassium and gibberellic acid on stem growth of whole sunflower plants. Physiol. Plant. 49, 443 -448 (1980). DORFFUNG, K.: Speicherprozesse - die Rolle der Phytohormone. Z. Pflanzenernahr. Bodenkd. 140, 3 - 14 (1977). DORFLER, M. und H. GORING: Der EinfluB verschiedener Lichtqualitat (Blau- und Rodicht) auf den Cytokiningehalt von Kiirbisjungpflanzen. BioI. Rundsch. 16, 186-188 (1978). FRANKLAND, B. and P. F. WAREING: Effects of gibberellic acid on hypocotyl growth of lettuce seedlings. Nature 185, 255-256 (1960). HERZOG, H. und G. GEISLER: Der EinfluB von Cytokininapplikation auf die Assimilateinlagerung und die endogene Cytokininaktivitat der Karyopsen bei 2 Sommerweizensorten. Z. Acker- u. Pflanzenbau 144, 230-242 (1977). HEWlT!", E. W. and P. F. WAREING: Cytokinins in Populus x robusta (Schneid): Light effects on endogenous levels. Planta 114, 119-129 (1973). JENNER, C. F.: The conversion of sucrose to starch in developing fruits. Ber. Deutsch. Bot. Ges. 93,289-298 (1980). JUDEL, G. K. and K. MENGEL: Effect of shading on nonstructural carbohydrates and their turnover in culms and leaves during the grain filling period of spring wheat. Crop. Sci. 22, 958-962 (1982). KENDE, H.: Preservation of chlorophyll in leaf sections by substances obtained from root exudate. Science 145, 1066-1067 (1964). KEPPLER, D. und K. DECKER: Glykogen-Bestimmung mit Amyloglucosidase. In: Methoden der enzymatischen Analyse (H. U. BERGMEYER, ed). pp. 1171-1176. Verlag Chemie, Weinheim, 1974. ISBN 3-527255303. MENGEL, K. and H. E. HAmER: The effect of potassium and light intensity on the grain yield production of spring wheat. 4th International Colloquium on the Control of Plant Nutrition, Vol. II. (A. COTIENIE, ed.), pp. 463 -475, Gent (1976). MENGEL, K. and G. K. JUOEL: Effect of light intensity on the activity of starch synthesizing enzymes and starch synthesis in developing wheat grains. Physiol. Plant. 51, 13-18 (1981). MICHAEL, G. and H. BERINGER: The role of hormones in yield formation. In: Physiological Aspects of Crop Productivity. - 15th Colloquium International Potash Institute, ed., pp. 85-116, Berne (1980). MITCHELL, J. W. and G. A. LIVINGSTONE: Methods of studying plant hormones and growth-regulating substances. Agriculture Handbook No. 336, Agricultural Research Service U.S.D.A. (1969). MoUNLA, M. A. KH. and G. MICHAEL: Gibberellin-like substances in developing barley grain and their relation to dry weight increase. Physiol. Plant. 29,274-276 (1973). MoUNLA, M. A. KH., F. BANGERTH, and V. STOY: Gibberellin-like substances and indole type auxins in developing grains of normal and high lysine genotypes of barley. Physiol. Plant. 48, 568-573 (1980). PREISS, J.: Regulation of the biosynthesis and degradation of starch. Ann. Rev. Plant Physiol. 33,431-454 (1982). RADEMACHER, W. and J. E. GRAEBE: Isolation and analysis by gas-liquid chromatography of auxins, gibberellins, cytokinins, and abscisic acid from a single sample of plant material. Ber. Deutsch. Bot. Ges. 97, 75-85 (1984 a). - - Hormonal changes in developing kernels of two spring wheat varieties differing in storage capacity. Ber. Deutsch. Bot. Ges. 97, 167 -181 (1984 b). RADLY, M.: Effect of variation in ear temperature of gibberellin content of wheat ears. Ann. appl. BioI. 82, 335-340 (1976).
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SHAW, M. and B. J. S. SRIVASTAVA: Purine-like substances from coconut endosperm and their effect on senescence in excised cereal leaves. Plant Physiol. 39,528-532 (1964). STOY, V.: Assimilatbildung und -verteilung als Komponenten fiir Ertragsbildung beim Getreide. Angew. Bot. 47, 17 -26 (1973). WHEELER, A. W.: Changes in growth substance contents during growth of wheat grains. Ann. appl. BioI. 72, 327 -334 (1972).
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Summary The effect of light-intensity on the content of phytohormones in developing wheat grains (Triticum aestivum L. vc. Kolibri) was investigated. One day after anthesis plants were grown with a light-intensity of 25 W m -2 (low light treatment) and with 84 W m -2 (high light treatment) in a phytotron. Grain samples taken throughout the grain-filling period and analyzed for phytohormones showed no major influence of light-intensity on the content of cytokinins and free auxins. There was, however, a significant effect of light-intensity on the gibberellin and abscisic acid content; the latter being higher in the low-light treatment, the former being higher in the high-light treatment. Low-light treatment affected grain setting and resulted especially in hampered grain growth and starch synthesis, while the production of grain crude protein was not influenced by light-intensity. From the experimental results it is concluded that light-intensity influences grain initiation by its effect on abscisic acid and gibberellins in the early stage of grain development. In the later stage low light-intensity impedes grain growth and starch production due to the effect of light on supply with sucrose and on abscisic acid and gibberellin metabolism.
Key words: Abscisic acid, auxins, cytokinins, gibberellins, wheat grains.
Introduction Various authors hold the view that processes in developing grains of cereals rather than photosynthesis are of major importance for grain growth and grain production Genner 1980; Michael and Beringer 1980). Stoy (1973) reported that reduced light-intensity, applied shortly after anthesis, considerably decreased the incorporation of photosynthates into grains of cereals. Mengel and Haeder (1976) found that a low-light treatment during the grain filling period especially affected starch formation in wheat grains and to a lesser degree the translocation of photosynthate to the grains. The impeded production of photosynthates under low-light conditions during the grain filling period is compensated to a high degree by the mobilization of non-structural carbohYc:lrates, particularly fructans, already synthesized and stored in the culms before anthesis Gudel and Mengel 1982). In studying the effect of light-intensity on the activity of starch synthesizing enzymes, Mengel and Judel (1981) came to the conclusion that starch synthesis is associated with grain growth and it was suggested that light-intensity may have an impact on the phytohormone level in developing grains
J Plant Physiol. Vol. 120. pp. 255-266 (1985)
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KONRAD MENGEL, BAERBEL FRIEDRICH and GUENTER KLAus JUDEL
and thus may control grain growth and starch production. The question whether light-intensity has an impact on the concentration of phytohormones in developing wheat grains was examined in a pot experiment, the results of which are communicated in the following. Materials and Methods Cultivation ofplants
Spring wheat (Triticum aestivum L. cv. Kolibri) was cultivated in Mitscherlich pots on a sandy loam (Luvisol, 7kglpot) mixed with 0.8gN, 0.9gP, and 1.9gK/pot of commercial fertilizer. At tillering, shooting, and ear emergence additional N was supplied, at each stage 0.5 g N / pot in the form of ammonium nitrate. Plants were grown outdoors until anthesis. Then they were transferred into growth chambers, where they were irradiated by mercury-vapor-highpressure-lamps HQi 250 W (Philips, Eindhoven, The Netherlands). Half of the plants (24 pots) stood in a chamber with a light intensity of 25 W m -2 (low light-intensity treatment); the other half in a growth chamber with 84 W m -2 light intensity (high light-intensity treatment). All other conditions were the same for both treatments: day length: 16 h, day temperature: 20°C, night temperature: 14 °C, relative air humidity: 60 % during the day and 80 % at night. Sampling
Throughout the grain filling period 24 samples were collected, on each date the grains of one pot. The 1st sampling date was one day after anthesis aune 6Ih), the last sampling date 57 days after anthesis (August 17lh). Between these days samples were taken at intervals of 1 to 7 days. Ears were cut, immediately immersed in liquid nitrogen in order to stop metabolic activity, and then stored in plastic bags at - 24°C until analysis. For phytohormone analysis as well as for the determination of starch, sucrose, and nitrogen (protein) only ears of the 1'1 order were used and from these only the grains of the medial section. From each ear 20 grains were sampled; with a pair of tweezers the glumes were carefully removed and the karyopses were stored at -24°C. For grain yield and yield component determination all ears were taken into account. Analyses
Dry matter was determined by drying 10 to 30 grains at 105 0c. Total N was analyzed by micro-Kjeldahl. Crude protein content was calculated by multiplying total N with 5.7. Each analysis was carried out with two samples. The starch content of the grains was determined following the hexokinase method of Keppler and Decker (1974). Sucrose was assessed enzymatically according to the technique of Bergmeyer et al. (1974). Each analysis was made in triplicate. The coefficient of variation for the dry matter yield was 4.1, for the crude protein determination 0.8, for the starch determination 3.2, and for the sucrose determination lOA. Isolation ofphytohormone
Grains were homogenized in a Waring Blendor and extracted in triplicate with a five-fold excess of cold 80 % methanol. The combined extracts were purified and the hormones isolated by liquid-liquid chromatography, by TLC and ion exchange chromatography as described by Rademacher and Graebe (1984 a).
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Biotests for phytohormone activity The samples containing phytohormone were investigated for their hormone activity be means of biotests. The reference hormones were received from Sigmar Chemie, Munich.
Cytokinins Preliminary experiments had shown that the primary wheat leaf test according to Kende (1964), (see also Shaw and Srivastava (1964) and the cucumber-cotyledon test yielded reliable results. The two methods were in good agreement. Each sample was analyzed at least 4 times with these two methods so that at least 8 single values for each sample were obtained. The values shown in Fig. 4 are means of the two biotests. Standards were made from kinetin and zeatin solutions in a range of 10- 8 to 10- 5 g. The primary wheat leaf test was carried out with the cultivar «Kolibri», the cucumber cotyledon test with «Chinese Snake».
Gibberellins Gibberellin activity was assessed by the lettuce hypocotyl test (cv. «Arctic King») according to Frankland and Wareing (1960) and with the barley endosperm test (cv. Nudika) according to
Coombe et al. (1967). Preliminary tests had shown that both tests yielded the same results with satisfactory reproducibility. With each biotest technique at least 4 analyses per sample were carried out giving at least 8 single values from which the means (Fig. 5) were calculated. Standards were made from gibberellic acid (GA3) in a range of 10- 10 to 10- 6 g.
Auxins Auxin activity was determined by the wheat coleoptile segment test according to Mitchell and Livingstone (1969). Five determinations per sample were made. Standards contained indoleacetic acid in a range of 10- 10 to 1O- 4 g.ln the samples taken 13, 18,23, and 28 days after anthesis besides free indoleacetic acid (IAA) also the conjugated lAA and the strongly bound IAA were determined. Separation of the three IAA types was carried out as described by Rademacher (W. Rademacher 1978, Thesis Agric. Faculty, University Goettingen, Fed. Rep. of Germany).
A bscisic acid Abscisic acid activity was assessed by two different tests: the wheat leaf transpiration inhibition test according to Rademacher (W. Rademacher 1978, Thesis Agric. Faculty, University Goettingen, Fed. Rep. of Germany) and the barley endosperm test according to Chrispeels and Varner (1966). The cultivars used were «Kolibri» (wheat) and «Nudika» (barley). Both techniques yielded similar values. Each sample was analyzed at least 4 times with each technique so that at least 8 single values per mean were obtained. Standards were made from abscisic acid.
Results
Reduced light intensity resulted in decreased plant growth and grain yield. As shown in Table 1 grain number per ear, single grain weight, spikelet development, and the development of spikelets with three or more grains were affected. Grain growth throughout the grain filling period (Fig. 1) was not affected during the first 13 days after anthesis. From this date on grains having received high light-intensity had a higher growth rate than grains grown with reduced light. The difference in grain development was mainly due to the grain starch production which was much hampered
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KONRAD MENGEL, BAERBEL FRIEDRICH and GUENTER KLAus JUDEL
Table 1: Effect of light intensity on grain yield and yield components. The data relate to the plants of 1 pot. ------------------------------------------light intensity, W m -2 Grain yield, g Single grain weight, mg No. of ears No. of grains/ear No. of spikelets/ear Spikelets with 3 and more grains in % of total spikelets
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by low light-intensity. As can be seen from Fig. 2 the curves for the starch synthesis resemble those of grain growth (Fig. 1). Interestingly enough light-intensity had no major impact on the N quantity (crude protein) per grain (Fig. 3). Cytokinins in grains were only detectable during the first 2 weeks after anthesis showing a peak 6 days after flowering. Major effects of light-intensity on the content of cytokinins per grain were not observed (Fig. 4). In contrast to cytokinins, gibberellins were clearly influenced by light-intensity (Fig. 5). The treatment with low-light not only resulted in significantly smaller quantities of gibberellins per grain, also the duration of gibberellins during the grain filling period was considerably shorter in the grains with lowlight-intensity.
f Plant Physiol. Vol. 120. pp.
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Phytohormones
259
'j
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Fig. 3: Effect of light intensity on the content of grain crude protein throughout the grain filling period. mg crude protein N per grain.
Auxin content did not differ much between treatments (Fig. 6) but in the later stage of grain filling, grains having received higher light-intensity had higher auxin contents than those with low-light. The peak in the auxin content occurred earlier in
J. Plant Physiol.
Vol. 120. pp. 255-266 (1985)
260
KONRAD MENGEL, BAERBEL
I
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FRIEDRICH and
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3
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27 30 33 37 41
Days after flowering Fig. 5: Effect of light intensity on the gibberellin content in grains throughout the grain filling period. ng GA per grain.
the «low light-intensity plants». The effect of light-intensity on auxin was also evident in the content of conjugated auxins which was much higher in the treatment with low-light-intensity (Table 2). The difference in ABA content was particularly obvious during the first 3 weeks after anthesis, ABA being much higher in the grains treated with low-light-intensity (Fig. 7). Sucrose content in grains did not differ very much between treatments
J Plant Physiol. Vol. 120. pp. 255-266 (1985)
Phytohormones
130
j".)-84wm-,
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Days after flowering Fig. 6: Effect of light intensity on the auxin content in grains throughout the grain filling period. ng IAA per grain. Table 2: Effect of light intensity on the content of free, conjugated, and strongly bound indoleacetic acid, ng indoleacetic acid per grain. Days after anthesis 13 18 23 28
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free
6 16 21
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throughout the grain filling period (Fig. 8). There was a tendency to higher sucrose content in the high-light treatment during the period of maximum grain growth. Discussion Low-light-intensity affected grain yield by a reduced number of grains per ear and by hampered starch synthesis. It is assumed that grain setting was influenced at an early stage of the experimental period which means at flowering or shortly after flowering. According to Michael and Beringer (1980) grain initiation of cereals can be influenced by auxins and by cytokinins. Auxins are supposed to function in a kind of «medical dominance» rather impeding than promoting grain setting whereas cytokinins favour grain initiation. In the earlier stage of grain filling (2 weeks after anthesis) no significant differences in cytokinin levels were found between treatments (Fig. 4). Also the levels of free auxin did not differ between treatments (Fig. 6). The content in conjugated and bound auxins were higher in the low-light treatment
J. Plant Physiol.
Vol. 120. pp. 255-266 (1985)
262
KONRAD MENGEL, BAERBEL FRIEDRICH and GUENTER KLAus JUDEL
I
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I
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so
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(Table2) indicating that light-intensity had some influence on auxin metabolism. There were significant differences between treatments for the ABA and gibberellin content. Since at this early stage of grain filling the size of the grain between treatments did not differ (Fig. 1), the content of ABA and gibberellins in the grains dif-
J. Plant Physiol. Vol. 120. pp. 255-266 (1985)
Phytohormones
263
fered between treatments. ABA is known to be an antagonist of cytokinins (Alvim et al. 1976) and it is feasible that the impeded grain initiation in the low-light treatment was associated with the antagonistic action of ABA. According to Rademacher, gibberellins are involved in processes after fertilization by inducing the synthesis of glycolytic enzymes. Thus the low gibberellin activity in the treatment with low-light-intensity at the early stage of grain development may have had an impact on grain setting. Cytokinin activity reached a peak 6 days after anthesis. This agrees well with findings of Rademacher and Graebe (1984 b). Also the maximum cytokinin content (22 ng cytokinin/grain) found by Rademacher and Graebe (1984 b) by means of gas chromatography is the same as that found by us with biotests. Other authors, however, reported much higher cytokinin content (Wheeler 1972; Herzog and Geisler 1977). About two weeks after anthesis, the cytokinin activity was almost nil. A similar observation was made by Wheeler (1972) and Rademacher and Graebe (1984 b). The maximum of the cytokinin activity coincides with a phase of intensive cell division. At this stage the number of endosperm cells is determined which according to Brocklehurst (1977) may determine the grain filling capacity. An almost linear grain growth was found between the 13 th and 33rd day after anthesis, the growth rate in the reduced-light treatment being much lower than in the high-light-intensity treatment (Fig. 1). During this period also the highest content of gibberellins were found. According to Dela Guardia and Benlloch (1980) gibberellins, like auxins, may activate the plasmalemma-located H+ pump and thus increase the plasticity of cell walls and promote cell elongation. The low gibberellin content found in the treatment with low light-intensity (Fig.5) at the period of highest growth rate is supposed to have led to the poor grain growth found with this treatment. Besides the gibberellins, also the higher ABA content in the grains of the lowlight treatment (Fig. 7) could have contributed to the poor grain growth because of antagonistic effects (Alvim et al. 1976). Throughout the period between 12 and 30 days after anthesis there was a tendency to higher SUcrOse content in the grains of the high-light-intensity treatment. In most cases the difference was significant as can be seen from Fig. 8. During this period also the ABA contents in the grains of this treatment were significantly lower than in the treatment with low light. Thus one may speculate whether ABA impeded the import of sucrose into the storage tissue as suggested by Dorffling (1977). The finding that highest gibberellin content coincides with high rates of grain growth has also been observed by other authors (Mounla and Michael 1973; Radley 1976; Mounla et al. 1980). The assumption that there is a causal relationship between gibberellin content and growth rate is therefore confirmed. The maturation stage lasted, in our experiment, from the 3yd until the 57th day after anthesis. During this period the net import of assimilates into the grains declines, the kernels gradually loose water resulting in a decrease of grain volume. In this late stage ABA was the domi-
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MENGEL, BAERBEL FRIEDRICH and GUENTER KLAus JUDEL
nant phytohormone (Fig. 7). Low quantities of auxin and gibberellin were still present in the «high-light treatment» grains at the beginning of the maturation stage (Figs. S and 6). Whether they still had an impact on grain growth is questioned. ABA is known to induce the maturation stage. This is in good agreement with the finding that the peak in the ABA content coincided with the beginning of the maturation stage (Fig. 1 and Fig. 7) of the plants with high-light application. In the treatment with low light-intensity, however, the ABA peak already occurred 18 days after flowering and it is likely that the process of grain senescence started earlier in this treatment. Although grain growth was much affected by low light-intensity, the N content in the grains (mg N per grain) was not influenced (Fig. 3). But under low-light conditions there is certainly an enhanced breakdown of leaf proteins which results in an increased supply of free amino acids and amides. Consequently protein synthesis in the grains is favoured which causes the higher N content observed in the grains cultivated at low-light-intensity compared to those grown at high-light-intensities (d. Fig. 3). Starch production in grains, on the contrary, was much affected by low light (Fig. 2). It is thus questionable whether the impeded starch synthesis was primarily due to a lack of photosynthates. The fact that the level of free sucrose during the most intense grain filling period (days 10-33) was considerably lower at low light-intensity than at high intensity (Fig. 8) also supports the assumption that there was a deficiency of photosynthates at low light-intensity. It therefore seems likely that the reduced starch synthesis was associated with a limited deficiency of photosynthates and with a poor grain growth of the low light-intensity plants which was again controlled by phytohormones, particularly by gibberellin and ABA. Starch is synthesized and stored in amyloplasts (Preiss 1982). It is possible that also the development of amyloplasts was influenced by phytohormones and thus by light-intensity. How light-intensity may induce synthesis and turnover of phytohormones is still unknown. According to Hewitt and Wareing (1973) and Dorfler and Goring (1978) light effects on phytohormones are likely to occur. Acknowledgements The authors are much obliged to the Deutsche Forschungsgemeinschaft for financial support.
References ALVIM, R., E. W. HEWITI, and P. F. SANDERS: Seasonal variation in the hormone content of willow. I. Changes in abscisic acid content and cytokinin activity in the xylem sap. Plant Physiol. 57, 474-476 (1976). BERGMEYER, H. U., E. BERNT, F. SCHMIDT und H. STORK: D-Glucose. In: Methoden der enzymatischen Analyse (H. U. BERGMEYER, ed.), pp. 1241-1246. Verlag Chemie, Weinheim, 1974. ISBN 3-527255303. BROCKLEHURST, P. A.: Factors controlling grain weight in wheat. Nature 266, 348-349 (1977). CHRISPEELS, M. J. and J. E. VARNER: Inhibition of gibberellic acid induced formation of cx· amylase by abscisin II. Nature 212, 1066-1067 (1966).
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COOMBE, B. G., D. COHEN, and B. G. PALEG: Barley endosperm bioassay for gibberellins. I. Parameters of the response system. Plant Physiol. 42, 105-112 (1967). DELA GUARDIA, M. D. and M. BENLLOCH: Effects of potassium and gibberellic acid on stem growth of whole sunflower plants. Physiol. Plant. 49, 443 -448 (1980). DORFFUNG, K.: Speicherprozesse - die Rolle der Phytohormone. Z. Pflanzenernahr. Bodenkd. 140, 3 - 14 (1977). DORFLER, M. und H. GORING: Der EinfluB verschiedener Lichtqualitat (Blau- und Rodicht) auf den Cytokiningehalt von Kiirbisjungpflanzen. BioI. Rundsch. 16, 186-188 (1978). FRANKLAND, B. and P. F. WAREING: Effects of gibberellic acid on hypocotyl growth of lettuce seedlings. Nature 185, 255-256 (1960). HERZOG, H. und G. GEISLER: Der EinfluB von Cytokininapplikation auf die Assimilateinlagerung und die endogene Cytokininaktivitat der Karyopsen bei 2 Sommerweizensorten. Z. Acker- u. Pflanzenbau 144, 230-242 (1977). HEWlT!", E. W. and P. F. WAREING: Cytokinins in Populus x robusta (Schneid): Light effects on endogenous levels. Planta 114, 119-129 (1973). JENNER, C. F.: The conversion of sucrose to starch in developing fruits. Ber. Deutsch. Bot. Ges. 93,289-298 (1980). JUDEL, G. K. and K. MENGEL: Effect of shading on nonstructural carbohydrates and their turnover in culms and leaves during the grain filling period of spring wheat. Crop. Sci. 22, 958-962 (1982). KENDE, H.: Preservation of chlorophyll in leaf sections by substances obtained from root exudate. Science 145, 1066-1067 (1964). KEPPLER, D. und K. DECKER: Glykogen-Bestimmung mit Amyloglucosidase. In: Methoden der enzymatischen Analyse (H. U. BERGMEYER, ed). pp. 1171-1176. Verlag Chemie, Weinheim, 1974. ISBN 3-527255303. MENGEL, K. and H. E. HAmER: The effect of potassium and light intensity on the grain yield production of spring wheat. 4th International Colloquium on the Control of Plant Nutrition, Vol. II. (A. COTIENIE, ed.), pp. 463 -475, Gent (1976). MENGEL, K. and G. K. JUOEL: Effect of light intensity on the activity of starch synthesizing enzymes and starch synthesis in developing wheat grains. Physiol. Plant. 51, 13-18 (1981). MICHAEL, G. and H. BERINGER: The role of hormones in yield formation. In: Physiological Aspects of Crop Productivity. - 15th Colloquium International Potash Institute, ed., pp. 85-116, Berne (1980). MITCHELL, J. W. and G. A. LIVINGSTONE: Methods of studying plant hormones and growth-regulating substances. Agriculture Handbook No. 336, Agricultural Research Service U.S.D.A. (1969). MoUNLA, M. A. KH. and G. MICHAEL: Gibberellin-like substances in developing barley grain and their relation to dry weight increase. Physiol. Plant. 29,274-276 (1973). MoUNLA, M. A. KH., F. BANGERTH, and V. STOY: Gibberellin-like substances and indole type auxins in developing grains of normal and high lysine genotypes of barley. Physiol. Plant. 48, 568-573 (1980). PREISS, J.: Regulation of the biosynthesis and degradation of starch. Ann. Rev. Plant Physiol. 33,431-454 (1982). RADEMACHER, W. and J. E. GRAEBE: Isolation and analysis by gas-liquid chromatography of auxins, gibberellins, cytokinins, and abscisic acid from a single sample of plant material. Ber. Deutsch. Bot. Ges. 97, 75-85 (1984 a). - - Hormonal changes in developing kernels of two spring wheat varieties differing in storage capacity. Ber. Deutsch. Bot. Ges. 97, 167 -181 (1984 b). RADLY, M.: Effect of variation in ear temperature of gibberellin content of wheat ears. Ann. appl. BioI. 82, 335-340 (1976).
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SHAW, M. and B. J. S. SRIVASTAVA: Purine-like substances from coconut endosperm and their effect on senescence in excised cereal leaves. Plant Physiol. 39,528-532 (1964). STOY, V.: Assimilatbildung und -verteilung als Komponenten fiir Ertragsbildung beim Getreide. Angew. Bot. 47, 17 -26 (1973). WHEELER, A. W.: Changes in growth substance contents during growth of wheat grains. Ann. appl. BioI. 72, 327 -334 (1972).
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