In a previous paper (1) a relation was shown to exist between the respiration of the plant cell and its elongation under the influence of the plant growth hormone. A more extensive investigation of this relation was therefore undertaken with the hope that elongation would exhibit a close correlation with some relatively accessible property of the respiration, for example with the magnitude or the respiratory quotient of the latter. It may be said at once, however, that this was not the case, and that the work reported in the present paper, while revealing several points of interest and defining more clearly the dependence of elongation upon respiration, has not resulted in any explanation of the way in which respiration is essential to growth

Bonner, James

English

PubMed

Caltech Authors - Main

THE GROWTH AND RESPIRATION OF THE AVENA 
COLEOPTILE 
BY JAMES BONNER 
(From the William G. Kerckhoff Laboratories of the Biological Sciences, California 
Institute of Technology, Pasadena) 
(Accepted for publication, November 25, 1935) 
INTRODUCTION 
In a previous paper (1) a relation was shown to exist between the 
respiration of the plant cell and its elongation under the influence of 
the plant growth hormone. A more extensive investigation of this 
relation was therefore undertaken with the hope that elongation would 
exhibit a close correlation with some relatively accessible property of 
the respiration, for example with the magnitude or the respiratory 
quotient of the latter. It may be said at once, however, that this was 
not the case, and that the work reported in the present paper, while 
revealing several points of interest and defining more clearly the 
dependence of elongation upon respiration, has not resulted in any 
explanation of the way in which respiration is essential to growth. 
II 
Materials and Methods 
Arena coleoptiles from seedlings of the pure line "Sieges Haler" were used 
throughout. They were grown under the usual controlled conditions (2). The 
measurements of respiration were made by the method of Warburg with the 
apparatus of Dr. Robert Emerson to whom the author is greatly indebted. Coni- 
cal vessels possessing a central alkali well and side wells for reagents to be added 
during the course of an experiment were used. Twenty coleoptile sections (1) 
suspended in the desired solution were placed in each vessel. The measure- 
ments of elongation were carried out in the manner described in the previous 
paper (1). 
1 
The Journal of General Physiology
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GROWTH AND RESP IRAT ION OF AVENA COLEOPT ILE  
III. 
Steps of the Growth Process Dependent upon Respiration 
It was earlier shown that coleoptile sections do not elongate in 
growth substance solution when placed in an atmosphere of oxygen- 
free nitrogen. When such sections were subsequently placed in growth 
substance solution in air they elongated rapidly, showing that they 
had not been harmed. If they were, however, placed in water without 
growth substance they did not elongate, showing that they had not 
taken up and stored growth substance; that is, that the transport of 
the hormone into the sections does not take place in the absence of 
aerobic metabolism. In order to show that the action of growth 
TABLE I 
Inhibition o/the Action of Growth Substance Already in the Plant 
Agar block (2 hrs.) Subsequent solution Subsequent elongation 
Water agar 
Water agar 
1200 plant units 
1200 plant units 
1200 plant units 
1200 plant units 
Distilled water 
10 u./cc. 
Distilled water 
KCN, 3 X 10-SN 
KCN, I X 10-sN 
Phenylurethane, 0.05 per cent 
per cent in 8 hrs. 
3.4 
27.6 
12.8 
2.1 
3.7 
4.9 
substance already in in the section is also inhibited under these condi- 
tions, the following type of experiment was performed. Two lots of 
coleoptile sections were prepared. Upon the apical ends of one lot 
were placed agar blocks containing no growth substance. Upon the 
apical ends of the other set were placed agar blocks containing 1200 
plant units of the hormone. The sections were left at 25°C in a satu- 
rated atmosphere for 2 hours. At the end of this time some of those 
which had had no growth substance were placed in water, the re- 
mainder in a solution containing 10 units of growth substance percubic 
centimeter. The sections which had had 1200 plant unit blocks were 
distributed among the following solutions, KCN, 3 × 10-3~¢; KCN, 
1 × 10 -3 N; phenylurethane, 0.05 per cent; water. Table I gives the 
results of a typical experiment. There is no question but that growth 
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J/~M~S BONNER 3 
substance passed into the sections which had had 1200 plant unit blocks, 
since these sections elongated more in water than did those which had 
had plain agar and were also placed in water. This elongation 
brought about by growth substance already in the plant is, however, 
dearly inhibited by both KCN and phenylurethane. We must 
conclude, therefore, that both growth substance transport and growth 
substance action are dependent upon aerobic metabolism. 
IV  
Effect of Growth Substance upon the Magnitude of Respiration 
In the earlier paper (1) it was shown that crude growth substance 
preparations cause an increase in the rate of respiration of coleoptile 
TABLE I I  
Effect of Crystalline Auxine B on Coleoptile Respiration* 
Solution Qo2 before___addition Qo I after addition 
0.0 units, per cc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  0.70 0.60 
10 units, per cc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  I 0.70 0.60 
* In this and in all subsequent tables, Qo2 denotes mm) of oxygen consumed 
per section per hour, measured atstandard conditions oftemperature and pressure. 
sections. It was suggested that although this increase might prove 
to be due to the hormone itself, it might on the other hand be due to 
associated impurities. The latter supposition can now be shown to be 
the correct one since, as has also been found by KSgl (3) the respira- 
tory stimulant is absent from crystalline auxine and can be removed 
from crude hetero-auxlne (4) preparations by further purification. 
Table I I  shows that crystalline auxine B (kindly supplied by Professor 
F. KSgl, Utrecht) does not affect coleoptile respiration. Table I I I  
shows that while a crude hetero-auxine preparation (from cultures of 
Rhizopus uinus (4)) having an activity of 500,000 plant units per 
milligram causes a considerable increase in respiration, a purification 
of this extract o 1,500,000 plant units per milligram removes most 
of the stimulating power. 
The respiratory effect of the more impure hormone preparation is
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GROWTH AND RESPIRATION OF AVENA COLEOPT ILE  
probably due to a specific stimulating substance rather than to a 
nutritive action of the associated impurities for the following reasons; 
(a) it has been found that fructose, which should also have a nutritive 
action, has but little effect upon coleoptile respiration under the condi- 
tions used and (b) it has been shown (1) that the stimulating substance 
is destroyed by oxidation with H202. 
That growth substance does not influence the magnitude of the 
respiration may be demonstrated in another manner. As has been 
described elsewhere (2) the activity of growth substance in promoting 
cell elongation depends upon the undissociated growth substance 
present. However, the pH of the cell contents i such that only 4 to 5 
per cent of the hormone present i  he plant is in the active form. If 
the cell acidity is increased by placing the coleoptile section in an acid 
TABLE IH  
Effect of Crude Hetero-Auxine Preparations upon Respiration 
Solution of 
10 units per co. (2500 units, or 500,000 plant 
units, per rag.) . . . . . . . . . . . . . . . . . . . . . . . . .  
10 units per co. (7500 units, or 1,500,000 plant 
units, per rag.) . . . . . . . . . . . . . . . . . . . . . . . . .  
Qo~ before 
addition 
1.56 
1.53 
Qos after addition 
I hour 2 hours 
1.92 1.95 
1.63 1.43 
buffer solution, more of the cell's own growth substance becomes 
active and its rate of elongation is increased even without he addition 
of more growth substance. If, on the other hand, the section is 
placed in a neutral or basic buffer solution, the acidity of the cell con- 
tents becomes less than normal, growth substance is dissociated, and 
the rate of elongation of the section is decreased. A comparison of the 
respiratory rates in buffer solutions of pH 4.1 and pH 7.2 was therefore 
made. Since the buffer of pH 7 retains a portion of the CO2 given off 
by the respiring tissue it was necessary to add an excess of acid at the 
end of each experiment, in order to drive off all of the accumulated 
CO2 into the gas space of the vessel, where it could be absorbed by the 
KOH of the alkali well. Table IV shows that there is no difference in
the respiratory rates at the two pH values although the rates of 
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ja~n~.s BOZ~-I~ER 5 
elongation are greatly different. It is clear, then, that growth sub- 
stance has no effect upon the magnitude of coleoptile respiration, a 
conclusion also reached by K6gl (3). 
Since changes in the growth rate of the coleoptile induced by growth 
substance are not paralleled by changes in the intensity of respiration 
TABLE IV 
Effect of pH upon tke Elongation and tke Respiration of Coleoptile Sections 
Elongation in 2 hrs., per cent . . . . . . . . . . . . . . . . . . . . .  9.4PI-I 4.1 2.7 pH 7.2 
Qo~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  1.53 4- 0.05 1.49 4- 0.05 
1o0 
] ' I h 
I I i 
I 
t 
i 
I 
I I I 
2 4 6 
P Io~ a~t¢~ cut t ing  
B 
! 
FIG. 1. Changes in the growth rate and in the respiration rate of coleoptile 
sections with time. Curve A, respiration rate; Curve B, growth rate. 
it might be expected that changes in the growth rate induced by other 
causes hould also be without effect. Fig. 1 compares graphically the 
changes with time of (a) the growth rate, and (b) the rate of respira- 
tion, of sections immersed in growth substance solution. The growth 
rate even in the presence of excess growth substance decreases steadily 
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GROWTH AND RESPIRATION OF AVENA COLEOPT ILE  
until after 8 hours it practically reaches 0. At this time then, sub- 
stances or conditions other than the growth hormone are limiting 
elongation. The rate of respiration, on the other hand, sinks only 
about 20 per cent in this time. Similarly, the distribution of growth 
rate and respiratory rate over the length of the coleoptile do not 
parallel one another markedly, as is shown in Fig. 2. Even when the 
C °q-q 
O~. 
~¢.,o 
0 
~ ' B  
/ \ c  / \\\ 
- -  I I I" 
X'Irn. 10 20 30 
Di~±ance ~r~m apex 
FIG. 2. Distribution of growth rate and of respiration rate in the coleoptile. 
Curve A, rate of respiration per milligram; Curve B, rate of respiration per 
milligram protein; Curve C, growth rate (after Went (9)). 
respiration per milligram protein is used as the basis of comparison, 
the respiration of the slowly growing base of the coleoptile is 80 per 
cent of that in the zone of maximum growth rate. Although there is 
in these two cases no strict parallelism between elongation and respira- 
tion, still it is to be noted that a slight decrease in respiratory rate is 
accompanied by a large decrease in growth rate. 
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JAMES BONNER 7 
v 
Does Growth Substance Change the Nature of the Respiration? 
A search for qualitative differences in the nature of the respiration 
in the presence and in the absence of growth substance was next made. 
The respiratory quotient of sections under different conditions was 
determined by the method of Warburg. This method consists in the 
measurement of the gas which may be driven out of the tissue by acid 
at the beginning of the experiment ("preformed CO~"), the net gas 
exchange, the gas driven off by acid at the end of the experiment, and 
the oxygen consumption during the experiment. The determination 
TABLE V 
Effect of Growtk Substance upon the Respiratory Quotient 
Total gas (h uptake l.Q. 
Solution exchange ram. 8 mm.3 
pH4.1 
pH 4.1 + growth substance 
pH 7.2 
pH 7.2 + growth substance 
1 per cent fructose 
1 per cent fructose + growth substance 
+51.3 
+51.2 
--41.1 
--43.5 
--9.1 
--8.1 
175.0 
175.0 
243.0 
243.0 
228.0 
228.0 
1.29 
1.29 
0.83 
0.82 
0.96 
0.96 
of the respimt0ry quotients revealed a peculiar fact which has, how- 
ever,--so far as can be seen at present,--no relation to the action of 
growth substance, namely, that the respiratory quotient is markedly 
higher for sections uspended in an acidic buffer than for sections 
suspended in a neutral buffer. In buffer of pH 4.1 the quotients are 
very constantly greater than one, in buffer of pH 7.2 they are more 
irregular but are markedly below one. It must be emphasized that 
the fact that during the main body of the experiment most of the CO2 
is driven off by buffer of pH 4.1 and a portion of it retained by buffer 
of pH 7.2 does not affect directly the determination f the respiratory 
quotient since in both cases all of the CO, is ultimately driven off with 
excess acid. Moreover, this difference in respiratory quotient be- 
tween acid and neutral solutions has no direct relation to growth, 
since, as is shown in Table V, in unbuffered fructose solution the 
quotient is the same irrespective of the elongation, and is practically 
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GROWTH AND RESP IRAT ION OF AVENA COLEOPT ILE  
one, as would be expected. The sample experiment of Table V shows 
that in buffer of pH 4.1 the R. Q. is greater than one, in buffer of pH 7.2 
less than one, and in unbuffered solution approximately one, and that 
in all three of these cases growth substance is without effect. In spite 
of the unexplained ifference between acidic and neutral solutions it 
seems afe to believe that growth substance itself has no influence upon 
the respiratory quotient. 
"Per,  
c~t  
100 
o 
..4 
.o 
.oA 
J ' B  
I I I I 
2 4 6 8 
HCN concent ra t ion  mola l  x 10 4 
FIG. 3. Inhibition of growth and of respiration by HCN. Curve A, inhibition 
of respiration; Curve B, inhibition of growth. 
Strugger (5) has suggested that fermentation favors elongation. 
The high quotient at pH 4 might indeed be interpreted as a kind of 
fermentation. However, this high quotient is not essential to growth. 
Moreover, if fermentation were conducive to cell elongation one might 
expect that anaerobiosis should stimulate growth, or that I-ICN in 
suitable amounts should, by inhibition of the "Pasteur reaction," 
increase longation. An increase of growth rate by either anaerobic 
conditions or HCN has never been observed in the course of these 
experiments. 
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JAMES BONNER 9 
v I  
Effect of HCN on Growth and on Respiration 
Up to the present point the independence, both quantitative and 
qualitative, of cell elongation and respiration has been stressed. In 
Part I I I  and in the earlier paper (1) it has, however, been pointed out 
that this independence is by no means complete, since, for example, 
HCN concentrations of about the same order are needed to inhibit 
the two processes. A more quantitative study of this relation has now 
been made and the results are presented in Fig. 3, in which per cent 
inhibition is plotted against HCN concentration. The two curves 
parallel one another very closely. A similar parallel inhibition of 
growth and of respiration was demonstrated arlier for phenylure- 
thane (1). 
v i i  
DISCUSSION 
The experimental results which have been presented show that: (a) 
during the growth of the cell, either in the normally growing coleoptile 
or in the excised coleoptile section, the rate of elongation decreases 
much more rapidly than does the rate of respiration; (b) HCN and 
phenylurethane decrease elongation and respiration to he same xtent. 
Low temperatures also slow down both processes, although respira- 
tion is somewhat more affected than is elongation (6). Neutral 
buffers, however, decrease elongation without exerting any effect upon 
respiration. 
From (a) and (b) it is necessary to conclude that decreases in respira- 
tion decrease longation but not the reverse; i.e., no considerable por- 
tion of the respiration is the result of elongation processes. This 
dependence of growth upon a large respiration may be viewed in two 
ways; (1) the entire respiration is a "formal prerequisite" of growth 
(Pfeffer (7)); (2) one or more of the processes of growth is itself a 
respiratory process but one of small oxygen uptake. Calculations of 
the amount of mechanical work done during cell elongation have 
shown that only a small portion of the energy liberated uring respira- 
tion can be used in this way, and many workers have shown that when 
the respiratory quotient is near one, the energy of respiration all 
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I0 GROWTH AND RESPIRATION OF AVENA COLEOPTILE 
appears ultimately asheat (Algera (8)). (1) can give therefore no clear 
idea of why a reduction of respiration by HCN or phenylurethane 
should decrease longation to even approximately the same extent. 
This brings up the question as to the way in which respiration might 
be a formal prerequisite of growth. It  is dear that it is necessary only 
if it performs functions which are necessary for growth. The principal 
function which it performs is the production of heat, which it is difficult 
to visualize as useful for elongation. It  seems more likely therefore 
that only a portion of the respiration is needed in the actual growth 
processes, and (1) would become identical with (2). 
Experimentally also the distinction between (1) and (2) is difficult 
if not impossible. (1) Growth is decreased when respiration is 
decreased because th whole respiration is essential. (2) Growth is 
decreased when respiration is decreased because the two portions of 
the respiration have similar properties. Upon (1) elongation can 
cease and respiration continue because the latter is a "prerequisite" 
of the former. Upon (2) elongation can cease and respiration con- 
tinue because of the inhibition of other portions of the growth process. 
Concept (1) therefore cannot be either theoretically or experi- 
mentally strictly differentiated from concept (2). It would seem 
desirable then to avoid the ambiguity of meaning in statement (1), 
and to express the necessity of respiration for growth in the statement 
that a process of respiratory nature is one (or more) of the component 
processes of elongation. 
VIII 
SUM.MARY 
1. Transport of the plant growth hormone into the Arena coleoptile 
as well as the action of the hormone on cell elongation in the coleoptile 
are shown to depend upon aerobic metabolism. 
2. Crystalline auxine, in contrast with impure preparations, affects 
neither the magnitude nor the respiratory quotient of coleoptile 
respiration. 
3. Increasing age of the coleoptile cell decreases its rate of elongation 
much more than its rate of respiration. HCN or phenylurethane on
the other hand decrease the two processes to the same extent, in spite 
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JAMES BONNER l i  
of the fact that only a small portion of the energy liberated by respira- 
tion can be used in the mechanical process of growth. 
4. From 2 and 3 it is concluded that processes ofa respiratory nature 
but of relatively small magnitude form one or more integral steps in 
the chain of reactions by which the plant growth hormone brings about 
cell elongation. 
LITERATURE 
1. Bonner, J., J . Gen. Physiol., 1933, 17, 63. 
2. Bonner, J., Protoplasma, 1934, 9.1, 406. 
3. Kt~gl, F., Bet. chem. Ges., 1935, 68, 16. 
4. Thimann, K. V., J. Biol. Chem., 1935, 109, 279. 
5. Strugger, S., .Ber. bot. Ges., 1933, 51, 193. 
6. Bonner, J., and Thimann, K. V., J. Gen. Physiol., 1935, 18, 649. 
7. Pfeffer, W., in Ewart, J., Plant physiology, Oxford, Clarendon Press, English 
edition, 1900. 
8. Algera, L., Rec. tray. bot. nAerl., 1932, 29~ 47. 
9. Went, F., Rec. tray. bot. n~erl., 1928, 9.8, 1. 
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The growth and respiration of the Avena coleoptile

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The growth and respiration of the Avena coleoptile

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