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A difference between auxin-induced and hydrogen ion-induced growth
Plant Science Letters, 4 (1975) 133--136 133 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
A D I F F E R E N C E B E T W E E N AUXIN-INDUCED AND HYDROGEN ION-
INDUCED GROWTH
J.E. PERLEY*, DAVID PENNY** and PAULINE PENNY
Department of Botany and Zoology, Massey University, Paimerston North (New Zealand)
(Received October 21st, 1974)
SUMMARY Displacement transducers were used to measure simultaneously short-term changes in the radius and length of growing lupin hypocotyl segments. Auxin, low pH, and CO2 saturated water all stimulate the elongation rate but the radius decreases in low pH and increases in auxin and CO2. From this and other evidence it is concluded that H ÷ ions cannot be the "second messenger" for auxin. INTRODUCTION There has recently been considerable interest in the hypothesis that auxins increase growth rate in plant segments by lowering the pH of the cell wall. Cleland [1] has suggested that "hydrogen ions act as a second messenger in auxin-induced elongation and can be the wall-loosening factor" and Rayle [2] has suggested that "H + ion secretion is responsible, at least in part, for the initiation of auxin-induced cell wall loosening and extension growth". These conclusions are based on the observations that low pH will stimulate the elongation of growing tissues [3], will cause cell wall loosening of isolated cell walls [4], and that eventually auxin will cause a lowering in pH of the medium surrounding elongating tissues [1, 2, 5, 6]. It has also been suggested [7] that CO: stimulates growth by lowering the pH of the cell wall. The concept of auxin acting via hydrogen ions has been supported by other workers [8, 9] but some evidence against it has been presented [10--12]. Experiments in this laboratory which were designed to test the recently published growth equation [13] have shown that there are major differences between auxin-induced and acid-induced growth. In these experiments we have used displacement transducers [12, 14] to measure simultaneously both *Permanent address: Department of Biology, The College of Wooster, Wooster, Ohio 44691 (U.S.A.). **To whom reprint requests should be addressed.
134 changes in length and diameter of light-grown lupin hypocotyls in response to such experimental treatments as auxin, low pH, CO2 saturated solutions, and mannitol solutions. Short-term measurements of diameter have not been possible in the past but the use of displacement transducers has allowed a 20-fold increase in resolution over the other main automatic system for measuring plant growth [5] and in addition is used on a single segment rather than on a file of 10 or more segments. METHODS The plant material and growing conditions have been described previously [16]. A hypocotyl segment was held horizontally in the same chamber [12] that was used for simultaneously measuring elongation rate and extracellular pH and the method of measuring length was identical to that used in that paper. The diameter measurements were also made with a Philips displacement transducer [14] but with the core attached over a pulley to a counterbalancing weight resting on the lupin segments. Estimates of volume were made fromthe length and diameter measurements. RESULTS AND DISCUSSION While a full description of both the technique and results will be published elsewhere, the results presented here (Figs. 1--3) show that the diameter initially decreases in low pH solutions but increases in auxin and CO2 saturated solutions. Fig. 1 presents results for the relative rate of increase in length (dl/1.dt) A
B
c
6.0
3.790
5.0
3474
4.0
3.~58'
6.2
3.0' 2.842,
3.8
20-2.52
11.0
-.C ...... '"':::•".i::: .....
I
8.6
A i 4
i:.:!::'::-.....--
10"221 2'0
40
-
-1.0
Time in minutes
Fig. 1. Relative elongation rate for lupin h y p o c o t y l segments. 25-ram segments were changed at the downward pointing arrow from 1 mM phosphate buffer pH 6.5 to the experimental treatments which were: A, 30 uM indolyl-3-acetic acid in I mM phosphate buffer pH 6.5; B, 10 mM citrate-phosphate buffer, pH 4.0; C, CO 2 saturated in 1 mM phosphate buffer. Segments were taken from 4-day-old lupin seedlings grown in continuous light as described previously [6]. Units are m i n - ' • 10 -4 .
135 A 1.4310
C .802270
B
.975250
['"'--.l
"B
°.°"
c
1.4296
..,..,. ..........
.974616
°.o
•801764
.°
1.4282
.973982
1.4268
.973348
:. •
.801258 ""
"" A
-....
. ."
.°
1.4254
.972714
• .....-
. . . .. ... . .
...°.........
1.4240
.97208
....800752
.
" "N° . . . . . .
.800246
"-..o.•..
....
'
2'0 Time
"'
4'o
.799740
in m i n u t e s
•Fig. 2. Radius of the same lupin hypocotyl segments before and after the experimental treatments shown in Fig. 1. A is the radius of the segment for which the elongation rate was shown in Fig. 1, and similarly for B and C. Units are in ram.
for auxin, low pH, and CO2 saturated buffer and these results are in agreement with other published work where changes in length alone have been measured. The results shown in Fig. 2 represent the first published short-term measurements of change in radius of growing segments. With auxin and CO2 the results show that the pattern of changes in radius is similar to the changes in the elongation rate. However, when segments are placed in low pH, there is a consistent decrease in the radius which continues for about 30 rain before a new level is reached. There is considerable diversity in the radius results after this time and the radius may start increasing again. This result indicates that the mechanism of growth induced by hydrogen ions is different from that in auxin or CO2 saturated solutions and, therefore, it is unlikely that hydrogen ions could be the second messenger for auxin action on growth. B
A 164
75.6
163
75.4
,
•- •
. .o .
51.2 B
162
C ..~:: 51.5
...""
••
"°"
75.2
.°.."
50.9
A .'-° . ° • °
161
75.0
°° C.'°°
160
74.8
159
74.6
.
50.6
. . °°°° . . - . . o . - °"
50.3 20 40 T i m e in m i n u t e s
50.0
Fig. 3. Volume of hypocotyl segments shown in Figa I and 2, estimated from the length (derived from Fig. 1) and the radius (Fig. 2). Units are in mm 3.
136 T h e decrease in radius in low pH is sufficient t o result in an initial decrease in volume even t h o u g h t he length is still increasing (see Fig. 3). T h e decrease in radius does eventually stop and it is at this time t h a t the volume begins to increase again because the length is still increasing. These results emphasize t h e i m p o rt an ce o f being able to measure t he changes of the radius at short intervals and also to obtain estimates of the volume change. In evaluating the h ypot hes i s t hat h y d r o g e n ions mediate the auxin response on elongation it is i m p o r t a n t t o stress t h a t there is as y e t no evidence t hat the pH o f the wall decrease be f or e growth increases. On the contrary, we have previously shown [12] t hat pH microelectrodes could n o t d e t e c t an auxininduced pH d r o p within t w o different tissues before elongation was stimulated. T h e lack o f a d etecta bl e pH drop, t he effect o f lactone inhibitors stimulating growth [17] rather than reducing it as predicted [18], t oget her with the present results on radius change (Fig. 2) seriously challenge the possibility that h y d r o g e n ions are the " s e c o n d messenger" for either auxin or CO2 on growth. F u r t h e r m o r e , models of the growing plant cell wall [9] will need t o be modified to a c c o m m o d a t e differential effects on t he length and on t he radius o f growing plants. ACKNOWLEDGMENTS J.E.P. acknowledges with thanks t he research leave awarded by the College o f Wooster, Wooster, Ohio, U.S,A. The w o r k has been support ed in part by t he Research Grant~ C o m m i t t e e of t he N.Z. University Grants Committee. REFERENCES
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
R. Cleland, Proc. Natl. Acad. Sci. (U.S.), 70 (1973) 3092. D.L. Rayle, Planta, 114 (1973) 63. M.L. Evans, BioScience, 23 (1973) 711. D.L. Rayle, P.M. Haughton and R. Cleland, Proc. Natl. Acad. Sci. (U.S.), 67 (1970) 1814. E. MarrY, P. Lado, F. Rasi Caldogno and R. Colombo, Plant Sci. Letters, 1 (1973) 179. E. MarrY, P. Lado, F. Rasi Caldogno and R. Colombo, Plant Sci. Letters, 1 (1973) 185. D.L. Rayle and R. Cleland, Plant Physiol., 46 (1970) 250. A. Hager, H. Manzel and A. Krauss, Planta, 100 (1971) 47. K. Keegstra, K.W. Talmadge, W.D. Bauer and P. Albersheh~n, Plant Physiol., 51 (1973) 188. I. Ilan, Physiol. Plant., 28 (1973) 146. G.M. Barkley and A.C. Leopold, Plant Physiol., 52 (1973) 76. P. Penny, J. Dunlop, J.E. Perley and D. Penny, Plant Sci. Letters, 4 (1975) 35. P.M. Ray,, P.B. Green and R. Cleland, Nature, 239 (1972) 163. D. Penny, P. Penny and D.C. Marshall, Can. J. Bot., 52 (1974) 959. M.L. Evans and P.M. Ray, J. Gen. Physiol., 53 (1969) 1. P. Penny, N.Z.J. Bot., 7 (1969) 290. J.E. Perley and D. Penny, N.Z.J. Bot., 12 (1974) 503. K.D. Johnson, D. Daniels, M.J. Dowler and D.L. Rayle, Plant Physiol., 53 (1974) 224.
A D I F F E R E N C E B E T W E E N AUXIN-INDUCED AND HYDROGEN ION-
INDUCED GROWTH
J.E. PERLEY*, DAVID PENNY** and PAULINE PENNY
Department of Botany and Zoology, Massey University, Paimerston North (New Zealand)
(Received October 21st, 1974)
SUMMARY Displacement transducers were used to measure simultaneously short-term changes in the radius and length of growing lupin hypocotyl segments. Auxin, low pH, and CO2 saturated water all stimulate the elongation rate but the radius decreases in low pH and increases in auxin and CO2. From this and other evidence it is concluded that H ÷ ions cannot be the "second messenger" for auxin. INTRODUCTION There has recently been considerable interest in the hypothesis that auxins increase growth rate in plant segments by lowering the pH of the cell wall. Cleland [1] has suggested that "hydrogen ions act as a second messenger in auxin-induced elongation and can be the wall-loosening factor" and Rayle [2] has suggested that "H + ion secretion is responsible, at least in part, for the initiation of auxin-induced cell wall loosening and extension growth". These conclusions are based on the observations that low pH will stimulate the elongation of growing tissues [3], will cause cell wall loosening of isolated cell walls [4], and that eventually auxin will cause a lowering in pH of the medium surrounding elongating tissues [1, 2, 5, 6]. It has also been suggested [7] that CO: stimulates growth by lowering the pH of the cell wall. The concept of auxin acting via hydrogen ions has been supported by other workers [8, 9] but some evidence against it has been presented [10--12]. Experiments in this laboratory which were designed to test the recently published growth equation [13] have shown that there are major differences between auxin-induced and acid-induced growth. In these experiments we have used displacement transducers [12, 14] to measure simultaneously both *Permanent address: Department of Biology, The College of Wooster, Wooster, Ohio 44691 (U.S.A.). **To whom reprint requests should be addressed.
134 changes in length and diameter of light-grown lupin hypocotyls in response to such experimental treatments as auxin, low pH, CO2 saturated solutions, and mannitol solutions. Short-term measurements of diameter have not been possible in the past but the use of displacement transducers has allowed a 20-fold increase in resolution over the other main automatic system for measuring plant growth [5] and in addition is used on a single segment rather than on a file of 10 or more segments. METHODS The plant material and growing conditions have been described previously [16]. A hypocotyl segment was held horizontally in the same chamber [12] that was used for simultaneously measuring elongation rate and extracellular pH and the method of measuring length was identical to that used in that paper. The diameter measurements were also made with a Philips displacement transducer [14] but with the core attached over a pulley to a counterbalancing weight resting on the lupin segments. Estimates of volume were made fromthe length and diameter measurements. RESULTS AND DISCUSSION While a full description of both the technique and results will be published elsewhere, the results presented here (Figs. 1--3) show that the diameter initially decreases in low pH solutions but increases in auxin and CO2 saturated solutions. Fig. 1 presents results for the relative rate of increase in length (dl/1.dt) A
B
c
6.0
3.790
5.0
3474
4.0
3.~58'
6.2
3.0' 2.842,
3.8
20-2.52
11.0
-.C ...... '"':::•".i::: .....
I
8.6
A i 4
i:.:!::'::-.....--
10"221 2'0
40
-
-1.0
Time in minutes
Fig. 1. Relative elongation rate for lupin h y p o c o t y l segments. 25-ram segments were changed at the downward pointing arrow from 1 mM phosphate buffer pH 6.5 to the experimental treatments which were: A, 30 uM indolyl-3-acetic acid in I mM phosphate buffer pH 6.5; B, 10 mM citrate-phosphate buffer, pH 4.0; C, CO 2 saturated in 1 mM phosphate buffer. Segments were taken from 4-day-old lupin seedlings grown in continuous light as described previously [6]. Units are m i n - ' • 10 -4 .
135 A 1.4310
C .802270
B
.975250
['"'--.l
"B
°.°"
c
1.4296
..,..,. ..........
.974616
°.o
•801764
.°
1.4282
.973982
1.4268
.973348
:. •
.801258 ""
"" A
-....
. ."
.°
1.4254
.972714
• .....-
. . . .. ... . .
...°.........
1.4240
.97208
....800752
.
" "N° . . . . . .
.800246
"-..o.•..
....
'
2'0 Time
"'
4'o
.799740
in m i n u t e s
•Fig. 2. Radius of the same lupin hypocotyl segments before and after the experimental treatments shown in Fig. 1. A is the radius of the segment for which the elongation rate was shown in Fig. 1, and similarly for B and C. Units are in ram.
for auxin, low pH, and CO2 saturated buffer and these results are in agreement with other published work where changes in length alone have been measured. The results shown in Fig. 2 represent the first published short-term measurements of change in radius of growing segments. With auxin and CO2 the results show that the pattern of changes in radius is similar to the changes in the elongation rate. However, when segments are placed in low pH, there is a consistent decrease in the radius which continues for about 30 rain before a new level is reached. There is considerable diversity in the radius results after this time and the radius may start increasing again. This result indicates that the mechanism of growth induced by hydrogen ions is different from that in auxin or CO2 saturated solutions and, therefore, it is unlikely that hydrogen ions could be the second messenger for auxin action on growth. B
A 164
75.6
163
75.4
,
•- •
. .o .
51.2 B
162
C ..~:: 51.5
...""
••
"°"
75.2
.°.."
50.9
A .'-° . ° • °
161
75.0
°° C.'°°
160
74.8
159
74.6
.
50.6
. . °°°° . . - . . o . - °"
50.3 20 40 T i m e in m i n u t e s
50.0
Fig. 3. Volume of hypocotyl segments shown in Figa I and 2, estimated from the length (derived from Fig. 1) and the radius (Fig. 2). Units are in mm 3.
136 T h e decrease in radius in low pH is sufficient t o result in an initial decrease in volume even t h o u g h t he length is still increasing (see Fig. 3). T h e decrease in radius does eventually stop and it is at this time t h a t the volume begins to increase again because the length is still increasing. These results emphasize t h e i m p o rt an ce o f being able to measure t he changes of the radius at short intervals and also to obtain estimates of the volume change. In evaluating the h ypot hes i s t hat h y d r o g e n ions mediate the auxin response on elongation it is i m p o r t a n t t o stress t h a t there is as y e t no evidence t hat the pH o f the wall decrease be f or e growth increases. On the contrary, we have previously shown [12] t hat pH microelectrodes could n o t d e t e c t an auxininduced pH d r o p within t w o different tissues before elongation was stimulated. T h e lack o f a d etecta bl e pH drop, t he effect o f lactone inhibitors stimulating growth [17] rather than reducing it as predicted [18], t oget her with the present results on radius change (Fig. 2) seriously challenge the possibility that h y d r o g e n ions are the " s e c o n d messenger" for either auxin or CO2 on growth. F u r t h e r m o r e , models of the growing plant cell wall [9] will need t o be modified to a c c o m m o d a t e differential effects on t he length and on t he radius o f growing plants. ACKNOWLEDGMENTS J.E.P. acknowledges with thanks t he research leave awarded by the College o f Wooster, Wooster, Ohio, U.S,A. The w o r k has been support ed in part by t he Research Grant~ C o m m i t t e e of t he N.Z. University Grants Committee. REFERENCES
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
R. Cleland, Proc. Natl. Acad. Sci. (U.S.), 70 (1973) 3092. D.L. Rayle, Planta, 114 (1973) 63. M.L. Evans, BioScience, 23 (1973) 711. D.L. Rayle, P.M. Haughton and R. Cleland, Proc. Natl. Acad. Sci. (U.S.), 67 (1970) 1814. E. MarrY, P. Lado, F. Rasi Caldogno and R. Colombo, Plant Sci. Letters, 1 (1973) 179. E. MarrY, P. Lado, F. Rasi Caldogno and R. Colombo, Plant Sci. Letters, 1 (1973) 185. D.L. Rayle and R. Cleland, Plant Physiol., 46 (1970) 250. A. Hager, H. Manzel and A. Krauss, Planta, 100 (1971) 47. K. Keegstra, K.W. Talmadge, W.D. Bauer and P. Albersheh~n, Plant Physiol., 51 (1973) 188. I. Ilan, Physiol. Plant., 28 (1973) 146. G.M. Barkley and A.C. Leopold, Plant Physiol., 52 (1973) 76. P. Penny, J. Dunlop, J.E. Perley and D. Penny, Plant Sci. Letters, 4 (1975) 35. P.M. Ray,, P.B. Green and R. Cleland, Nature, 239 (1972) 163. D. Penny, P. Penny and D.C. Marshall, Can. J. Bot., 52 (1974) 959. M.L. Evans and P.M. Ray, J. Gen. Physiol., 53 (1969) 1. P. Penny, N.Z.J. Bot., 7 (1969) 290. J.E. Perley and D. Penny, N.Z.J. Bot., 12 (1974) 503. K.D. Johnson, D. Daniels, M.J. Dowler and D.L. Rayle, Plant Physiol., 53 (1974) 224.