Planta 137, 25-28 (1977)
P l a n t a 9 by Springer-Verlag 1977
Early Responses to Gibberellic Acid in a Dwarf Maize Mutant (Zea mays L. dl) Dieter Neumann Institut fiir Biochemie der Pflanzen, Akademie der Wissenschaften der DDR, Weinberg 3, DDR-401 Halle (Saale), German Democratic Republic
A.G.S. J/mossy Biological Research Centre, Hungarian Academy of Sciences, Institute of Biophysics, P.O.B. 521, H-6701 Szeged, Hungary
Abstract. Early effects of gibberellic acid (GA3) (1 4 h treatment) on the ion ratios in a dwarf maize mutant (Zea mays L. dl) showing normal growth after hormone treatment, have been investigated by electron microprobe analysis. GA3 exerts a different effect on the ion ratios in plastids, cytoplasm and vacuoles in short term experiments. The C1 content of chloroplasts and cytoplasm increases without a lag phase after GA3 treatment. The K content of plastids increases after a lag phase of 2 h, whereas in the cytoplasm an increase can be observed immediately after GA3 addition. The hormone has only little influence on the Ca content of the cell compartments investigated. Control experiments with water and the physiologically inactive GA3 methylester confirm the specifity of the short-term actions of GA3 on the ion ratios. The primary action of GA3 at the membrane level is discussed. Key words: Gibberellin analysis - Zea.
Ion ratio - Microprobe
Introduction
As in the case with other phytohormones, gibberellic acid (GA3) takes part in numerous reactions which may not express the "primary action" of the hormone. There is no doubt that a particular hormone exerts only one "primary action" and that phenomena observed later could be the consequence of an earlier "primary action". Therefore, investigations of the "primary actions" of a hormone require the study of effects with a short lag phase. The best known system for GA3 action is the barley aleurone layer. In these cells GA3 stimulates the synthesis of membrane components by activation of the corresponding enzymes (Ben-Tal and Varner, 1974; Johnson and Kende, 1971), formation of
endoplasmic reticulum (Evins and Varner, 1971) and polyribosomes (Evins, 1971; Evins and Varner, 1972), and production of amylase (Filner and Varner, 1967). However, these processes occur with lag phases between 2 and 8 h and nothing is known about the "primary action" of the hormone. In Sinapis seeds, GA3 stimulates the synthesis of amino acids and the incorporation of leucine into proteins (Edwards, 1976) with a lag phase of 2 h. These short term effects - stimulation of particular enzymes, amino acid and protein synthesis, formation of membranes and polysomes-are such diverse processes that they cannot be considered as the "primary action" of GA3. A GA3 response with a very short lag phase was observed by Hebard et al. (1976). After 10 min of GA 3 treatment an acidification of the incubation medium takes place, which is very similar to the action of IAA (see review Evans, 1974). This observation together with experiments on ion uptake and translocation (Lfittge et al., 1968) and investigations with artificial membranes (Wood and Paleg, 1974; Wood etal., 1974) indicates that the "primary action" of GA3 takes place at the membrane level. Earlier findings on the action of GA3 on the ion content ratios of particular compartments in mesophyll cells of a dwarf corn mutant support this assumption (Neumann and Jfinossy, 1977). These investigations have been extended to obtain data on the early actions of the hormone on the ion content by experiments analyzed between 0 and 4 h after GA3 treatment.
Material and Methods 1. Plant Material
The dwarf corn mutant (Zea mays L., cv. Phinney dl) was cultivated as described earlier (Neumann and Jfinossy, 1977). After
26
D. N e u m a n n and A.G.S. Jfinossy: Early Responses to Gibberellin
l0 days g r o w t h - t h e plants show 5 to 6 l e a v e s - 1 5 m m discs of the third leaf were used for the experiments. The leaf discs were placed on the surface of water or a 2 x 10 5 M solution of G A 3 or G A 3 methylester. The leaf discs were removed after 1, 2, and 4 h from the solutions and the inner parts (5 x 5 mm) were used for analysis.
GibberelIic Acid Methylester (Fig. 2)
2. Tissue Preparation Tissues were prepared by a slightly modified freeze substitution method (Neumann, 1973; N e u m a n n and J/mossy, 1977). In former experiments A1203-used as a drying agent for the substitution m e d i u m - g a v e a considerable a m o u n t of A1 in all investigated cell compartments. It was replaced by a molecular sieve (Zeocarb). The substitution medium was used without OsO4 because of the overlapping of P and Si peaks by the Os peak.
In the chloroplasts the C1 content shows a very low level and the K and Ca contents decline during the incubation time. In the cytoplasm G A 3 methylester causes a slight increase in C1 content and a decrease in K content whereas the Ca content remains constant except in the sample collected after 1 h incubation. There is a remarkable increase of K and some increase of Ca in the first hour, but the C1, K, and Ca contents of the vacuoles are low after 4 h of incubation.
Water (Fig. 3) 3. Microprobe Analysis The microprobe analyses were performed as described earlier (Neum a n n and Jfinossy, 1977) with a JEOL 100 B and E D A X 707 B energy dispersive X-ray analyzer system in transmission mode of action. For a semiquantitative evaluation of the data thin sections of dentine were used as standards for Ca (Jfinossy and Neumann, 1977) and absolute Ca values were calculated after Hall et al. (1973). The absolute data for the rest of the detected elements were calculated from the weight ratios (Russ, 1975). It should be noted that the results obtained by this method should be regarded as semiquantitative. However, the differences observed were significant enough to allow comparisons o f the ion distribution between the investigated cell compartments.
Results and Discussion
The results of the microprobe analyses for the three prominent elements-C1, K, C a - a r e summarized in Figures 1-3. Each value represents a mean of 6 individual determinations with 6 different cells from 3 plants.
Gibberellic Acid (Fig. 1) The C1 content in the chloroplasts of the dwarf mutant is smaller than the detection limit. G A 3 causes an increase of the C1 content immediately without a lag phase. The K content does not change during the first 2 h, but later shows a great increase, while the Ca content remains constant during the experimental period. In the cytoplasm, G A 3 causes an increase of C1 and Ca contents immediately after the hormone treatment. The Ca content shows only a slight increase during the first 2 h and drops to the initial value after 4 h. The C1 and Ca levels in the vacuoles are not influenced by GA3 during the 4 h incubation. After a lag phase of 1 h we observe a slight increase in the K content.
Water has little effect on the C1, K, and Ca levels of the chloroplasts. In the cytoplasm we observe the same increase of Ca content at 1 h as in experiments with GA3 methylester. The C1 and K levels show a slight decrease in the 1st h of incubation with water. In the vacuoles there is a remarkable increase in the C1 and K contents during the first hour of incubation, while the Ca level is not influenced. These results confirm earlier observations (Neumann and J/mossy, 1977) that changes of the ion content in particular cell compartments may be caused by the action of GA3 on membranes. The short-term effect of the hormone appears more complex than the effect found in earlier long-term experiments. Whereas the C1 content in chloroplasts changed without a measurable lag phase, alteration of the K and Ca levels are observed not earlier than 2 h after hormone treatment. We were unable to observe such differences in the cytoplasm; where all investigated ion contents are influenced immediately after GA3 treatment. Earlier observations on the action of GA3 on the Ca content are confirmed by these experiments. The Ca content of chloroplasts and cytoplasm shows no changes after hormone treatment and that of the vacuoles is only slightly increased. To test the possible specificity of the GA3 action on the ion ratios control experiments were carried out with water and GA3 methylester. The ester is chemically very similar to GAa, but physiologically inactive and has no influence on the growth of the dwarf corn mutant. Water and G A 3 methylester cause basically similar changes: In all cases very low amounts of the investigated ions are present in the cell compartments after 4 h incubation. A remarkably strong increase for K, Ca and C1 in the first hour of the experiment is observed. This effect could be attributed to mechanical damage caused by isolation
D. Neumann and A.G.S. J&nossy: Early Responses to Gibberellin
27
Zeomoys-dworf
+GA$ chloroplasts
/
/ /
a2
//
cytoplasm
/"
/
vacuoles
0.5
Ke .... 9
9
02
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Fig. 1. Ion content in mesophyll cells of Zea mays d~ after GAs- treatment
Zao rno/s-dwar# +6A3- rne~hyles~ar chloropl~TsOs
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i;,xYocuoles
i
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P l a n t a 9 by Springer-Verlag 1977
Early Responses to Gibberellic Acid in a Dwarf Maize Mutant (Zea mays L. dl) Dieter Neumann Institut fiir Biochemie der Pflanzen, Akademie der Wissenschaften der DDR, Weinberg 3, DDR-401 Halle (Saale), German Democratic Republic
A.G.S. J/mossy Biological Research Centre, Hungarian Academy of Sciences, Institute of Biophysics, P.O.B. 521, H-6701 Szeged, Hungary
Abstract. Early effects of gibberellic acid (GA3) (1 4 h treatment) on the ion ratios in a dwarf maize mutant (Zea mays L. dl) showing normal growth after hormone treatment, have been investigated by electron microprobe analysis. GA3 exerts a different effect on the ion ratios in plastids, cytoplasm and vacuoles in short term experiments. The C1 content of chloroplasts and cytoplasm increases without a lag phase after GA3 treatment. The K content of plastids increases after a lag phase of 2 h, whereas in the cytoplasm an increase can be observed immediately after GA3 addition. The hormone has only little influence on the Ca content of the cell compartments investigated. Control experiments with water and the physiologically inactive GA3 methylester confirm the specifity of the short-term actions of GA3 on the ion ratios. The primary action of GA3 at the membrane level is discussed. Key words: Gibberellin analysis - Zea.
Ion ratio - Microprobe
Introduction
As in the case with other phytohormones, gibberellic acid (GA3) takes part in numerous reactions which may not express the "primary action" of the hormone. There is no doubt that a particular hormone exerts only one "primary action" and that phenomena observed later could be the consequence of an earlier "primary action". Therefore, investigations of the "primary actions" of a hormone require the study of effects with a short lag phase. The best known system for GA3 action is the barley aleurone layer. In these cells GA3 stimulates the synthesis of membrane components by activation of the corresponding enzymes (Ben-Tal and Varner, 1974; Johnson and Kende, 1971), formation of
endoplasmic reticulum (Evins and Varner, 1971) and polyribosomes (Evins, 1971; Evins and Varner, 1972), and production of amylase (Filner and Varner, 1967). However, these processes occur with lag phases between 2 and 8 h and nothing is known about the "primary action" of the hormone. In Sinapis seeds, GA3 stimulates the synthesis of amino acids and the incorporation of leucine into proteins (Edwards, 1976) with a lag phase of 2 h. These short term effects - stimulation of particular enzymes, amino acid and protein synthesis, formation of membranes and polysomes-are such diverse processes that they cannot be considered as the "primary action" of GA3. A GA3 response with a very short lag phase was observed by Hebard et al. (1976). After 10 min of GA 3 treatment an acidification of the incubation medium takes place, which is very similar to the action of IAA (see review Evans, 1974). This observation together with experiments on ion uptake and translocation (Lfittge et al., 1968) and investigations with artificial membranes (Wood and Paleg, 1974; Wood etal., 1974) indicates that the "primary action" of GA3 takes place at the membrane level. Earlier findings on the action of GA3 on the ion content ratios of particular compartments in mesophyll cells of a dwarf corn mutant support this assumption (Neumann and Jfinossy, 1977). These investigations have been extended to obtain data on the early actions of the hormone on the ion content by experiments analyzed between 0 and 4 h after GA3 treatment.
Material and Methods 1. Plant Material
The dwarf corn mutant (Zea mays L., cv. Phinney dl) was cultivated as described earlier (Neumann and Jfinossy, 1977). After
26
D. N e u m a n n and A.G.S. Jfinossy: Early Responses to Gibberellin
l0 days g r o w t h - t h e plants show 5 to 6 l e a v e s - 1 5 m m discs of the third leaf were used for the experiments. The leaf discs were placed on the surface of water or a 2 x 10 5 M solution of G A 3 or G A 3 methylester. The leaf discs were removed after 1, 2, and 4 h from the solutions and the inner parts (5 x 5 mm) were used for analysis.
GibberelIic Acid Methylester (Fig. 2)
2. Tissue Preparation Tissues were prepared by a slightly modified freeze substitution method (Neumann, 1973; N e u m a n n and J/mossy, 1977). In former experiments A1203-used as a drying agent for the substitution m e d i u m - g a v e a considerable a m o u n t of A1 in all investigated cell compartments. It was replaced by a molecular sieve (Zeocarb). The substitution medium was used without OsO4 because of the overlapping of P and Si peaks by the Os peak.
In the chloroplasts the C1 content shows a very low level and the K and Ca contents decline during the incubation time. In the cytoplasm G A 3 methylester causes a slight increase in C1 content and a decrease in K content whereas the Ca content remains constant except in the sample collected after 1 h incubation. There is a remarkable increase of K and some increase of Ca in the first hour, but the C1, K, and Ca contents of the vacuoles are low after 4 h of incubation.
Water (Fig. 3) 3. Microprobe Analysis The microprobe analyses were performed as described earlier (Neum a n n and Jfinossy, 1977) with a JEOL 100 B and E D A X 707 B energy dispersive X-ray analyzer system in transmission mode of action. For a semiquantitative evaluation of the data thin sections of dentine were used as standards for Ca (Jfinossy and Neumann, 1977) and absolute Ca values were calculated after Hall et al. (1973). The absolute data for the rest of the detected elements were calculated from the weight ratios (Russ, 1975). It should be noted that the results obtained by this method should be regarded as semiquantitative. However, the differences observed were significant enough to allow comparisons o f the ion distribution between the investigated cell compartments.
Results and Discussion
The results of the microprobe analyses for the three prominent elements-C1, K, C a - a r e summarized in Figures 1-3. Each value represents a mean of 6 individual determinations with 6 different cells from 3 plants.
Gibberellic Acid (Fig. 1) The C1 content in the chloroplasts of the dwarf mutant is smaller than the detection limit. G A 3 causes an increase of the C1 content immediately without a lag phase. The K content does not change during the first 2 h, but later shows a great increase, while the Ca content remains constant during the experimental period. In the cytoplasm, G A 3 causes an increase of C1 and Ca contents immediately after the hormone treatment. The Ca content shows only a slight increase during the first 2 h and drops to the initial value after 4 h. The C1 and Ca levels in the vacuoles are not influenced by GA3 during the 4 h incubation. After a lag phase of 1 h we observe a slight increase in the K content.
Water has little effect on the C1, K, and Ca levels of the chloroplasts. In the cytoplasm we observe the same increase of Ca content at 1 h as in experiments with GA3 methylester. The C1 and K levels show a slight decrease in the 1st h of incubation with water. In the vacuoles there is a remarkable increase in the C1 and K contents during the first hour of incubation, while the Ca level is not influenced. These results confirm earlier observations (Neumann and J/mossy, 1977) that changes of the ion content in particular cell compartments may be caused by the action of GA3 on membranes. The short-term effect of the hormone appears more complex than the effect found in earlier long-term experiments. Whereas the C1 content in chloroplasts changed without a measurable lag phase, alteration of the K and Ca levels are observed not earlier than 2 h after hormone treatment. We were unable to observe such differences in the cytoplasm; where all investigated ion contents are influenced immediately after GA3 treatment. Earlier observations on the action of GA3 on the Ca content are confirmed by these experiments. The Ca content of chloroplasts and cytoplasm shows no changes after hormone treatment and that of the vacuoles is only slightly increased. To test the possible specificity of the GA3 action on the ion ratios control experiments were carried out with water and GA3 methylester. The ester is chemically very similar to GAa, but physiologically inactive and has no influence on the growth of the dwarf corn mutant. Water and G A 3 methylester cause basically similar changes: In all cases very low amounts of the investigated ions are present in the cell compartments after 4 h incubation. A remarkably strong increase for K, Ca and C1 in the first hour of the experiment is observed. This effect could be attributed to mechanical damage caused by isolation
D. Neumann and A.G.S. J&nossy: Early Responses to Gibberellin
27
Zeomoys-dworf
+GA$ chloroplasts
/
/ /
a2
//
cytoplasm
/"
/
vacuoles
0.5
Ke .... 9
9
02
""m 4
:/h
k
.....
e/
"~-~ ....
"ih
1
T- ~ ~
2
~ ~--"
4'/?
Fig. 1. Ion content in mesophyll cells of Zea mays d~ after GAs- treatment
Zao rno/s-dwar# +6A3- rne~hyles~ar chloropl~TsOs
cy~plosm
i;,xYocuoles
i
0.3i
o.3
i i
0.5
\.
i
a2
0.2 - ./
