Planta 9 by Springer-Verlag 1978
Planta 140, 137-142 (1978)
Does Indoleacetic Acid Promote Growth via Cell Wall Acidification? David G. Pope School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, U.K.
Abstract. G r o w t h of Triticum aestivum L. cv. Cappelle Desprez coleoptiles is promoted by 5.7 • 10- 5 M indole acetic acid (IAA) as effectively in p H 3.4 buffer as in water, but I A A is not effective in the presence of buffer at p H 3.0 or 3.2 A combination of 5.7• 1 0 - S M I A A and p H 3.4 buffer promotes growth to a greater extent than p H 3.2 buffer alone, which is optimal for acid-induced growth. I A A employed at 1 0 - T M is still effective at promoting growth in the presence of p H 3.4 buffer, moreover, I A A at 10 7M interacts synergistically with the acidic buffer to promote growth. It is concluded that I A A and acid promote growth via separate mechanisms, and that I A A does not promote cell wall loosening by rendering the cell wall more acid.
Key words: Auxin secretion - Triticum.
Cell elongation
-
Proton
Introduction
Exogenously supplied protons can mimic the effect of I A A on coleoptile growth for a short time (Rayle and Cleland, 1970; Hager et al., 1971). Hager et al. showed that CCCP, which renders membranes more permeable to protons, rapidly inhibits the IAAinduced growth of Arena coleoptiles, while acidinduced growth is unaffected for some ten minutes. These data indicated that I A A acts by causing an increase in cell wall proton concentration, and with other evidence led to the p r o t o n - p u m p hypothesis of IAA-action, according to w h i c h auxin is said to act as an effector of a membrane-bound, anisotropic ATPase, which raises the proton concentration in the cell wall. The resultant lowered p H then promotes Abbreviation : IAA = Indoleacetic acid
cell wall plasticisation by either activating a cell wallloosening enzyme or by breaking labile interpolymeric linkages. Further evidence supporting this hypothesis was presented by Cleland (1973) and Rayle (1973) who showed that peeled Arena coleoptiles, exposed to IAA, acidified the medium in which they floated. The case for IAA-induced acid secretion by dicotyledonous plants is not so well established as that for monocotyledonous ones. Ilan (1973) showed that Helianthus hypocotyls could be stimulated by auxin to secrete protons, but the effect was not discernible for some two or three hours. Marr6 et al. (1973) reported that pea stem segments secrete protons in response to I A A over a four h time period, but Parrish and Davies (1977) found that peeled or slit, green or etiolated pea stem sections secreted protons in both the presence and absence of IAA, and that I A A had no promotive effect on this process, a finding repeated by Vanderhoefet al. (1977) using soybean hypocotyls. However, Jacobs and Ray (1976) using a fine pH electrode inserted into the cell walls of pea stem segments, showed that I A A does stimulate cell wall acidification, and that in both maize coleoptile and pea stem segments acidification preceded the growth response, as it did in Arena coleoptile segments (Cleland, 1976). In spite of the foregoing evidence in support, the role of cell wall acidification in the mechanism of auxin-induced growth has been questioned. Penny et al. (1975) found that I A A enhanced the growth rate of Arena coleoptiles which were simultaneously making their incubation medium more alkaline. Pea stem segments also initially lower the p H of the solution they float in, while exhibiting the normal auxininduced growth response (Parrish and Davies, 1977). Perley et al. (1975) questioned the assumption that I A A and exogenously supplied protons promote growth via the same mechanism, since they found
0032-0935/78/0140/0137/$01.20
138 that the radius of l u p i n hypocotyls decreased d u r i n g a c i d - i n d u c e d growth a n d increased d u r i n g auxini n d u c e d growth. Pope (1977) separated acid-induced growth from I A A - i n d u c e d growth in Arena coleoptiles by a p H 3/pH 7 p r e t r e a t m e n t which m a r k e d l y inhibited acid-induced growth, b u t n o t I A A - i n d u c e d growth. Clearly, the c o n t e n t i o n that I A A p r o m o t e s growth by i n d u c i n g cell wall acidification is n o t wholely accepted. In this paper the relationship between I A A action a n d p r o t o n secretion has been further investigated m a k i n g use of Cleland's (1975) o b s e r v a t i o n that p r o t o n secretion by peeled Arena coleoptile segments is regulated by the external pH. This model predicts that I A A , if acting by p r o m o t i n g cell wall acidification, should have n o effect o n growth in the presence o f a high external c o n c e n t r a t i o n of protons. This prediction is tested here, by d e t e r m i n i n g the effect of I A A o n the growth of wheat coleoptile segments incub a t e d in acidic buffer.
Materials and Methods
Growth and Preparation of Plant Material Grains of Triticum aestivum L. vat. Cappelle Desprez were soaked in water for approximately 6h, they were then washed for 5 rain in 3-fold diluted sodium hypochlorite solution. The grains were grown on a double layer of wet absorbent paper, in seed trays covered with aluminium foil which were kept in an incubator at 25~ C. After approximately 100 h incubation, those coleoptiles between 30 to 35 mm long were harvested, and decapitated by removal of the 5 mm apical portion. The upper ten mm of the remainder was de-leafed and stored in ice-cold water. When a sufficient number of coleoptile segments had been collected they were transferred to distilled water at room temperature, and stored for at least a further 30rain in the dark. From this point the coleoptile sections were kept in darkness ; any manipulations necessary were performed in green light. Replicated groups of ten segments were floated on ten ml of the appropriate test solution in the dark at 25~ C. IAA, when present, was at a concentration of either 5.7• 10 S M (10 gg/ml) or 10-7 M; buffers used were pH 3.0, 3.2, 3.4, 3.8 10 mM citrate-sodium citrate and pH 6, 1 mM potassium phosphate. Each experiment was repeated several times and mean growth + S.D. was calculated for each treatment. The significance of differences between treatments in each experiment was determined by applying the t test.
Measurement of Growth Growth was measured by a photographic method. Groups of ten coleoptiles were placed on a, glass plate, and using a photographic enlarger fitted with a green filter, were projected • 9 on to photographic paper. An initial shadowgraph was taken immediately before the addition of the coleoptile segments to their respective solutions and further ones were made at the intervals indicated for each experiment. The lengths of both sides of the coleoptile images were estimated accurately by measuring the traced image on 1 mm squared graph paper. The amount of growth in each
D.G. Pope: IAA Action via Cell Wall Acidification? treatment was determined by subtracting the total side lengths of the initial shadowgraph from the subsequent totals: the difference was expressed as mm growth per coleoptile segment.
Results
Acid Induced Growth." Optimum p H Acidic buffers stimulate coleoptile e l o n g a t i o n m a r k edly (Table 1). The o p t i m a l p H appears to be 3.2, but the growth i n d u c e d by this t r e a t m e n t is n o t significantly different from that i n d u c e d by p H 3.0 or p H 3.4. This result is similar to that f o u n d for Arena coleoptiles, where the m a x i m a l extension response is achieved at a b o u t p H 3.0 (Rayle a n d Cleland, 1972). Table I. Effect of acidic pH's on wheat coleoptile extension growth. Duplicate groups of ten coleoptiles were incubated for 50 rain at 25~ in i0 mM citrate buffer at the pH's indicated. The control groups were incubated in 1 mM potassium phosphate buffer (pH 6). Each figure represents the mean +S.D. of 6replicates fiom 3 separate experiments Treatment pH
Growth per segment (mm)
3.0 3.2 3.4 3.8 6.0
0.39• 0.47• 0.36• 0.27• 0.08•
i0
Duration of Coleoptile Extension at p H ' s 3.0, 3.2 and 3.4 Acidic solutions p r o m o t e coleoptile extension, b u t they also d a m a g e the tissues causing a loss of turgor. This complicates the d e t e r m i n a t i o n of the o p t i m a l p H for a c i d - i n d u c e d growth, because if the solutions being tested d a m a g e the tissues at different rates, then the p H o p t i m u m is d e p e n d e n t on the d u r a t i o n of the experiment. The validity of the results shown in T a b l e 1 was checked by d e t e r m i n i n g the d u r a t i o n of the coleoptile growth in solutions buffered at p H ' s 3.0, 3.2 a n d 3.4. The result of this e x p e r i m e n t is p o r t r a y e d in Figure 1. The coleoptiles r e m a i n turgid at all p H ' s tested for 80 min, which exceeds the d u r a t i o n of the experiment designed to determine the o p t i m a l pH. After 80 rain coleoptiles i n c u b a t e d at p H 3 lose turgor, b u t those i n c u b a t e d at pHs 3.2 a n d 3.4 extend with similar kinetics for 160 min.
IAA-induced Growth in Acidic Buffer I A A p r o m o t e s coleoptile extension for 24 h or more, b u t a c i d - i n d u c e d growth is of relatively short d u r a t i o n (Fig. 1). T h u s if I A A p r o m o t e s cell wall acidification, some m e c h a n i s m must be present to prevent the pro-
D.G. Pope: IAA Action via Cell Wall Acidification?
139
pH3g
1.0
g
Table 3. Effect of pH 3.4 citrate buffer on growth induced by 10- v M IAA. Duplicate groups of coleoptile segments were preincubated for 16 min at R.T. in the solutions indicated. Segment length was measured at the beginning and end of a subsequent 50 min incubation at 25 ~ C. The _+IAA (10 -~ M) treatments were carried out in either the presence of 10 m M citrate buffer (pH 3.4) or 1 m M potassium phosphate buffer (pH 6). Each figure is the mean + S.D. of 16 replicates from 8 separate experiments. IAAinduced growth at each p H employed is the mean + S.D. of 8 figures
o~ 0.5
60
T20
180
240
Time in rain
Fig. l. Time course of wheat coleoptile segment growth in pH 3.0, 3.2 and 3.4 citrate buffer. Duplicate groups of segments were incubated at 2 5 ~ in either pH 3.0 (o) pH 3.2 (e) or pH 3.4 (R) 10 m M citrate buffer. Segment length was recorded at 40 min intervals
ton concentration form rising too high (Cleland, 1975). Therefore, IAA-induced growth should be effectively neutralized by pH 3.4 citrate buffer, which must decrease cell wall pH to the level normally induced by IAA, since it is as effective at promoting growth (Table 2). Table 2. Effect of p H 3.4 citrate buffer on IAA-induced growth. Duplicate groups of coleoptile segments were preincubated for 16 min at R.T. in the solutions indicated. Segment length was measured at the beginning and end of a subsequent 50 min incubation at 25 ~ C. The • (5.7x10 -5 M) treatments were carried out in either 10 m M citrate buffer (pH 3.4) or 1 m M potassium phosphate buffer (pH 6). Each figure is the mean • S.D. of twelve replicates from 6 separate experiments. IAA-induced growth at each pH employed is the m e a n • S.D. of 6 figures Treatment
Growthpersegment (mm)
IAA-inducedgrowth persegment(mm)
IAA/3.4 3.4 IAA/6 6
0.64+0.09 0.44+0.10 0.46• 0.25•
0.2•
Treatment
Growth per segment (ram)
IAA-induced growth per segment (mm)
IAA/3.4 3.4 IAA/6 6
0.51• 0.38• 0.22• 0.16•
0i3• 0.07•
Table 4. Growth effect of I A A (5.7 x 10- 5 M) in p H 3.4 citrate buffer compared with that of pH 3.2 citrate buffer. Duplicate groups of coleoptile segments were preincubated in the solutions indicated for 1 6 m i n at R.T. Segment length was measured at the beginning and end of a subsequent 50 min incubation at 25 ~ C. Each figure is the mean + S . D . of 12 replicates from 6 separate experiments Treatment
Growth per segment (mm)
3.2 3.4 IAA/3.2 IAA/3.4
0.46• 0.44+0.10 0.48• a 0.65+0.14
a
Mean _+S.D. of 6 replicates e~ co
_
Planta 140, 137-142 (1978)
Does Indoleacetic Acid Promote Growth via Cell Wall Acidification? David G. Pope School of Biological Sciences, University of Sussex, Falmer, Brighton BN1 9QG, U.K.
Abstract. G r o w t h of Triticum aestivum L. cv. Cappelle Desprez coleoptiles is promoted by 5.7 • 10- 5 M indole acetic acid (IAA) as effectively in p H 3.4 buffer as in water, but I A A is not effective in the presence of buffer at p H 3.0 or 3.2 A combination of 5.7• 1 0 - S M I A A and p H 3.4 buffer promotes growth to a greater extent than p H 3.2 buffer alone, which is optimal for acid-induced growth. I A A employed at 1 0 - T M is still effective at promoting growth in the presence of p H 3.4 buffer, moreover, I A A at 10 7M interacts synergistically with the acidic buffer to promote growth. It is concluded that I A A and acid promote growth via separate mechanisms, and that I A A does not promote cell wall loosening by rendering the cell wall more acid.
Key words: Auxin secretion - Triticum.
Cell elongation
-
Proton
Introduction
Exogenously supplied protons can mimic the effect of I A A on coleoptile growth for a short time (Rayle and Cleland, 1970; Hager et al., 1971). Hager et al. showed that CCCP, which renders membranes more permeable to protons, rapidly inhibits the IAAinduced growth of Arena coleoptiles, while acidinduced growth is unaffected for some ten minutes. These data indicated that I A A acts by causing an increase in cell wall proton concentration, and with other evidence led to the p r o t o n - p u m p hypothesis of IAA-action, according to w h i c h auxin is said to act as an effector of a membrane-bound, anisotropic ATPase, which raises the proton concentration in the cell wall. The resultant lowered p H then promotes Abbreviation : IAA = Indoleacetic acid
cell wall plasticisation by either activating a cell wallloosening enzyme or by breaking labile interpolymeric linkages. Further evidence supporting this hypothesis was presented by Cleland (1973) and Rayle (1973) who showed that peeled Arena coleoptiles, exposed to IAA, acidified the medium in which they floated. The case for IAA-induced acid secretion by dicotyledonous plants is not so well established as that for monocotyledonous ones. Ilan (1973) showed that Helianthus hypocotyls could be stimulated by auxin to secrete protons, but the effect was not discernible for some two or three hours. Marr6 et al. (1973) reported that pea stem segments secrete protons in response to I A A over a four h time period, but Parrish and Davies (1977) found that peeled or slit, green or etiolated pea stem sections secreted protons in both the presence and absence of IAA, and that I A A had no promotive effect on this process, a finding repeated by Vanderhoefet al. (1977) using soybean hypocotyls. However, Jacobs and Ray (1976) using a fine pH electrode inserted into the cell walls of pea stem segments, showed that I A A does stimulate cell wall acidification, and that in both maize coleoptile and pea stem segments acidification preceded the growth response, as it did in Arena coleoptile segments (Cleland, 1976). In spite of the foregoing evidence in support, the role of cell wall acidification in the mechanism of auxin-induced growth has been questioned. Penny et al. (1975) found that I A A enhanced the growth rate of Arena coleoptiles which were simultaneously making their incubation medium more alkaline. Pea stem segments also initially lower the p H of the solution they float in, while exhibiting the normal auxininduced growth response (Parrish and Davies, 1977). Perley et al. (1975) questioned the assumption that I A A and exogenously supplied protons promote growth via the same mechanism, since they found
0032-0935/78/0140/0137/$01.20
138 that the radius of l u p i n hypocotyls decreased d u r i n g a c i d - i n d u c e d growth a n d increased d u r i n g auxini n d u c e d growth. Pope (1977) separated acid-induced growth from I A A - i n d u c e d growth in Arena coleoptiles by a p H 3/pH 7 p r e t r e a t m e n t which m a r k e d l y inhibited acid-induced growth, b u t n o t I A A - i n d u c e d growth. Clearly, the c o n t e n t i o n that I A A p r o m o t e s growth by i n d u c i n g cell wall acidification is n o t wholely accepted. In this paper the relationship between I A A action a n d p r o t o n secretion has been further investigated m a k i n g use of Cleland's (1975) o b s e r v a t i o n that p r o t o n secretion by peeled Arena coleoptile segments is regulated by the external pH. This model predicts that I A A , if acting by p r o m o t i n g cell wall acidification, should have n o effect o n growth in the presence o f a high external c o n c e n t r a t i o n of protons. This prediction is tested here, by d e t e r m i n i n g the effect of I A A o n the growth of wheat coleoptile segments incub a t e d in acidic buffer.
Materials and Methods
Growth and Preparation of Plant Material Grains of Triticum aestivum L. vat. Cappelle Desprez were soaked in water for approximately 6h, they were then washed for 5 rain in 3-fold diluted sodium hypochlorite solution. The grains were grown on a double layer of wet absorbent paper, in seed trays covered with aluminium foil which were kept in an incubator at 25~ C. After approximately 100 h incubation, those coleoptiles between 30 to 35 mm long were harvested, and decapitated by removal of the 5 mm apical portion. The upper ten mm of the remainder was de-leafed and stored in ice-cold water. When a sufficient number of coleoptile segments had been collected they were transferred to distilled water at room temperature, and stored for at least a further 30rain in the dark. From this point the coleoptile sections were kept in darkness ; any manipulations necessary were performed in green light. Replicated groups of ten segments were floated on ten ml of the appropriate test solution in the dark at 25~ C. IAA, when present, was at a concentration of either 5.7• 10 S M (10 gg/ml) or 10-7 M; buffers used were pH 3.0, 3.2, 3.4, 3.8 10 mM citrate-sodium citrate and pH 6, 1 mM potassium phosphate. Each experiment was repeated several times and mean growth + S.D. was calculated for each treatment. The significance of differences between treatments in each experiment was determined by applying the t test.
Measurement of Growth Growth was measured by a photographic method. Groups of ten coleoptiles were placed on a, glass plate, and using a photographic enlarger fitted with a green filter, were projected • 9 on to photographic paper. An initial shadowgraph was taken immediately before the addition of the coleoptile segments to their respective solutions and further ones were made at the intervals indicated for each experiment. The lengths of both sides of the coleoptile images were estimated accurately by measuring the traced image on 1 mm squared graph paper. The amount of growth in each
D.G. Pope: IAA Action via Cell Wall Acidification? treatment was determined by subtracting the total side lengths of the initial shadowgraph from the subsequent totals: the difference was expressed as mm growth per coleoptile segment.
Results
Acid Induced Growth." Optimum p H Acidic buffers stimulate coleoptile e l o n g a t i o n m a r k edly (Table 1). The o p t i m a l p H appears to be 3.2, but the growth i n d u c e d by this t r e a t m e n t is n o t significantly different from that i n d u c e d by p H 3.0 or p H 3.4. This result is similar to that f o u n d for Arena coleoptiles, where the m a x i m a l extension response is achieved at a b o u t p H 3.0 (Rayle a n d Cleland, 1972). Table I. Effect of acidic pH's on wheat coleoptile extension growth. Duplicate groups of ten coleoptiles were incubated for 50 rain at 25~ in i0 mM citrate buffer at the pH's indicated. The control groups were incubated in 1 mM potassium phosphate buffer (pH 6). Each figure represents the mean +S.D. of 6replicates fiom 3 separate experiments Treatment pH
Growth per segment (mm)
3.0 3.2 3.4 3.8 6.0
0.39• 0.47• 0.36• 0.27• 0.08•
i0
Duration of Coleoptile Extension at p H ' s 3.0, 3.2 and 3.4 Acidic solutions p r o m o t e coleoptile extension, b u t they also d a m a g e the tissues causing a loss of turgor. This complicates the d e t e r m i n a t i o n of the o p t i m a l p H for a c i d - i n d u c e d growth, because if the solutions being tested d a m a g e the tissues at different rates, then the p H o p t i m u m is d e p e n d e n t on the d u r a t i o n of the experiment. The validity of the results shown in T a b l e 1 was checked by d e t e r m i n i n g the d u r a t i o n of the coleoptile growth in solutions buffered at p H ' s 3.0, 3.2 a n d 3.4. The result of this e x p e r i m e n t is p o r t r a y e d in Figure 1. The coleoptiles r e m a i n turgid at all p H ' s tested for 80 min, which exceeds the d u r a t i o n of the experiment designed to determine the o p t i m a l pH. After 80 rain coleoptiles i n c u b a t e d at p H 3 lose turgor, b u t those i n c u b a t e d at pHs 3.2 a n d 3.4 extend with similar kinetics for 160 min.
IAA-induced Growth in Acidic Buffer I A A p r o m o t e s coleoptile extension for 24 h or more, b u t a c i d - i n d u c e d growth is of relatively short d u r a t i o n (Fig. 1). T h u s if I A A p r o m o t e s cell wall acidification, some m e c h a n i s m must be present to prevent the pro-
D.G. Pope: IAA Action via Cell Wall Acidification?
139
pH3g
1.0
g
Table 3. Effect of pH 3.4 citrate buffer on growth induced by 10- v M IAA. Duplicate groups of coleoptile segments were preincubated for 16 min at R.T. in the solutions indicated. Segment length was measured at the beginning and end of a subsequent 50 min incubation at 25 ~ C. The _+IAA (10 -~ M) treatments were carried out in either the presence of 10 m M citrate buffer (pH 3.4) or 1 m M potassium phosphate buffer (pH 6). Each figure is the mean + S.D. of 16 replicates from 8 separate experiments. IAAinduced growth at each p H employed is the mean + S.D. of 8 figures
o~ 0.5
60
T20
180
240
Time in rain
Fig. l. Time course of wheat coleoptile segment growth in pH 3.0, 3.2 and 3.4 citrate buffer. Duplicate groups of segments were incubated at 2 5 ~ in either pH 3.0 (o) pH 3.2 (e) or pH 3.4 (R) 10 m M citrate buffer. Segment length was recorded at 40 min intervals
ton concentration form rising too high (Cleland, 1975). Therefore, IAA-induced growth should be effectively neutralized by pH 3.4 citrate buffer, which must decrease cell wall pH to the level normally induced by IAA, since it is as effective at promoting growth (Table 2). Table 2. Effect of p H 3.4 citrate buffer on IAA-induced growth. Duplicate groups of coleoptile segments were preincubated for 16 min at R.T. in the solutions indicated. Segment length was measured at the beginning and end of a subsequent 50 min incubation at 25 ~ C. The • (5.7x10 -5 M) treatments were carried out in either 10 m M citrate buffer (pH 3.4) or 1 m M potassium phosphate buffer (pH 6). Each figure is the mean • S.D. of twelve replicates from 6 separate experiments. IAA-induced growth at each pH employed is the m e a n • S.D. of 6 figures Treatment
Growthpersegment (mm)
IAA-inducedgrowth persegment(mm)
IAA/3.4 3.4 IAA/6 6
0.64+0.09 0.44+0.10 0.46• 0.25•
0.2•
Treatment
Growth per segment (ram)
IAA-induced growth per segment (mm)
IAA/3.4 3.4 IAA/6 6
0.51• 0.38• 0.22• 0.16•
0i3• 0.07•
Table 4. Growth effect of I A A (5.7 x 10- 5 M) in p H 3.4 citrate buffer compared with that of pH 3.2 citrate buffer. Duplicate groups of coleoptile segments were preincubated in the solutions indicated for 1 6 m i n at R.T. Segment length was measured at the beginning and end of a subsequent 50 min incubation at 25 ~ C. Each figure is the mean + S . D . of 12 replicates from 6 separate experiments Treatment
Growth per segment (mm)
3.2 3.4 IAA/3.2 IAA/3.4
0.46• 0.44+0.10 0.48• a 0.65+0.14
a
Mean _+S.D. of 6 replicates e~ co
_
