P l a n t a n d Soil X, no. 3

M a r c h 1959

SOME FACTORS INFLUENCING CATION UPTAKE BY EXCISED ROOTS OF PERENNIAL RYE GRASS

(LOLIUM PERENNE) by R O D E R I C K P. MURPHY * Soil Laboratory, Johnstown Castle, Wexford, Ireland

INTRODUCTION

A number of mechanisms have been postulated to explain the uptake by plant roots of cations against apparent concentration gradients 1 3. One of the most widely accepted of these involves the following reaction: H R -~ K + ~ K R ~- H + where H R is a cation carrier produced by a metabolic reaction in some region of the plant cell. In connection with some work being done in this laboratory on the ability of certain types of plant roots to absorb nutrients from both culture media and soil, it was decided to examine this reaction. It was assumed that being a reversible reaction, it obeys the law of Mass Action i.e., it can be driven either to the right or to the left by varying the concentrations of the hydrogen ion in the culture medium. As this work was ultimately to be related to the relatively complex solutions found in soil, the uptake experiments were conducted in a solution similar in nature to that in which the plant roots were grown. .METHODS T h e m a t e r i a l used was excised roots of p e r e n n i a l r y e g r a s s ( W e s t e r n w o l t h s vat.). The p l a n t s were c u l t u r e d on P y r e x glass dishes (26cm × 18cm X 5cm) across t h e t o p of w h i c h a grid m a d e of P y r e x rods was laid. Over t h i s a n d d i p p i n g into t h e t r a y s a t each end, was a s h e e t oI b u t t e r m u s l i n on w h i c h a b o u t 50 g of seeds were placed. The t r a y s were filled w i t h c u l t u r e m e d i u m to w i t h i n 0.5 c m of t h e t o p a n d t h e whole p l a c e d in t h e i n c u b a t o r a t 26 ° C. * Present address: Dept. of Pathology, St. Finbarr's Hospitals, Cork, Ireland.

-- 242 --

CATION UPTAKE BY EXCISED GRASS ROOTS

243

The plants were allowed to grow for 5 days. At the end of this period the roots had grown to about 6 cm in length. On the day of the experiment these were excised as close to the seeds as possible, washed in distilled water, blotted between sheets of filter paper to remove excess moisture and weighed in 2-g lots. The proportion of 2 g excised roots to 40 ml absorption solution was maintained in all experiments done. The absorption solution was prepared so t h a t it contained the following molar concentrations: -Ions Ca++

Mg++ K+ NH4

+

NO3

-

P04

~--

MillimoIes per litre 2 1

2.5 (approx.) 2 8 2

The calcium, magnesium and ammonium ions are added as their nitrates and the phosphate is added as orthophosphoric acid. This gives a solution of approx, p H 3.2. The p H is raised to the required level with K O H and sufficient KC1 added to bring the potassium concentration to 2.5 millimoles per litre. This solution diluted fifty times was used for growing the roots. Variations in the absorption solution will be described in another section of this paper. As the roots were excised, it was thought that during the course of the potassium absorption period, they might become deficient in the carbohydrate necessary to produce the energy required for active absorption of potassium. To offset this possible limiting factor, enough sucrose was added to the absorption solution to give a concentration of 2% of this compound. The absorption of potassium was measured by suspending 2 g of excised roots in 40 ml solution in a 150-ml Erlenmeyer flask in a water bath at 26°C and aerating -with oxygen. A bank of 10 such flasks was set up and one removed every 30 minutes and the concentration of potassium in the solution was measured flamephotometrically. The decrease in concentration gave the amount absorbed by the roots. To prevent loss by evaporation due to the aeration, the oxygen was passed through several bottles of distilled water before being bubbled through the solutions. To ensure uniformity of aeration in each flask, they were also connected at the outlet side to gas bubblers and the rate at which the gas bubbled through each was carefully regulated so that all flasks were aerated at the same rate. RESULTS T h e r e s u l t s r e c o r d e d h e r e s h o w t h e e f f e c t of p H f r o m 3.5 t o 7.0 on t h e r a t e of a b s o r p t i o n of p o t a s s i u m . As d e s c r i b e d a b o v e , t h e p H w a s a d j u s t e d b y t h e a d d i t i o n of a l k a l i b e f o r e s t a r t i n g e a c h e x p e r i ment.

I t is s e e n (Fig. 1) t h a t t h e r a t e of a b s o r p t i o n rises as t h e

244

RODERICK P. MURPHY

initial p H of the absorption solution is raised. The rate of uptake tends to decrease slightly with time. This was explained b y the fact that in the absorption solution used, the pH invariably dropped to about 3.9 (Fig. 2) over the course of the experiment i.e., in about 5 hours. It was seen that between pH 6.0 and 7.0 there is very little difference in the rate of uptake of potassium. It will also be seen that at the lowest pH there is an initial secretion of potassium (recorded as a negative absorption) which is reversed when the pH 30 -K uptakeln mM/kg

25

pH 7.O PH 6.O

_~ ~ ~ o j j

20

a~~-

~

.

pHs . c

15

~0

. . . .

0

~"

I I~OJ--

¢ /

I

I

I

2.0

3.0

4.0

~,me ,n hour~ S.O

-5

_10

Fig. 1. Graph showing increase in the rate of potassium absorption as initial pH of culture solution is raised from 3.5 to 7.0. of the absorption solution rises above 3.8 as is shown in Fig. 2. Unrecorded experiments have shown that below pH 3.0 the roots !ose potassium to the external solution and that they ultimately lose the power to absorb potassium at higher pH values. This is in agreement with the work of various other authors 1 ~ Examination of these results shows that the rate of absorption during the first 30 minutes is only slightly influenced b y changes in p H between 4.0 and 7.0. This is interpreted as meaning that the initial apparent absorption is mostly due to passive diffusion of

CATION

BY EXCISED GRASS ROOTS

UPTAKE

245

potassium into to the free space in the roots. That it is not completely due to this is shown by the temporary loss of potassium at and beiow pH 3.5 and to the slight decrease in absorption rate in the first 30 minutes at pH 5.0. pN V.O

6.0

5.0'

4.0

time in hours 3.0 - -

1

o

2

I 4

~ 6

R 8

___1 IC

Fig. 2. Rate of fall of pH during l~2+-uptake varying the initial pH in slightly buffered solution containing sucrose.

As described earlier, the absorption solution used in the above experiments contained 2 per cent sucrose as a source of carbohydrate. In view of the fact that there was such an unusual decrease in the pH in the above experiments, it was decided to repeat some of these experiments in the absence of sucrose. The potassium uptake was examined at pH 6.0 and pH 5.0, and it was seen {Fig. 3) that the presence of sucrose had no marked depressant effect on the rate of potassium absorption over an extended period. It was also seen that in the absence of sucrose, the pH decreased by only one unit over the time of the experiment. Because of this sucrose effect, the uptake of sucrose was measured.

246

R O D E R I C K P. M U R P H Y

This was done colorimetrically, using the anthrone reagent 4. This was done by suspending 20 g of excised roots in 80 ml of absorption solution containing varying quantities of sucrose and removing 1 ml at hourly intervals for analysis. This was diluted 25 times with distilled water and the sucrose content estimated. The results Kuptake mMoles/ kg roots 4O

35

30

25

20

15

tO

5

/.

time in hours I I

O Fig. 3.

I 2

I 3

I 4

I 5

I 6

I 7

E f f e c t of v a r y i n g c o n c e n t r a t i o n of s u c r o s e o n K + - u p t a k e b y e x c i s e d roots. Initial pH •

=

1.0 mM

sucrose

(~) =

2.0 mM

sucrose

-- 0.0 mM

sucrose

@

of culture

solution (~

=

=

6.0

3.0 mM

sucrose

/~ -- 4.0 mM

sucrose

given in Fig. 4. show that the sucrose is absorbed by the plant roots and that the rate of absorption is governed by the concentration of this compound below a sucrose concentration of 3.0 millimoles per litre, but that above this figure the absorption does not increase as the sucrose content is raised above this level. It is evident from these data that some factor imposes a limit on the rate

HERBICIDAL EFFICIENCY OF 2,4-DINITRO-0-CRESOL

247

L o w e r i n g t h e surface t e n s i o n of t h e s p r a y solution or p r e t r e a t i n g p l a n t s of P . saXivum w i t h t r i c h l o r o a c e t i c acid d i m i n i s h e s t h e c o n t a c t angle m a d e b e t w e e n tile d r o p s a n d t h e leaf surface a n d this r e d u c t i o n in t h e angle is a s s o c i a t e d w i t h a h i g h e r r e t e n t i o n ratio. T h e r e l a t i o n b e t w e e n t h e a m o u n t of s p r a y r e t a i n e d a n d t h e c o n c e n t r a t i o n of 2,4-dinitro-o-cresol (DNC) r e q u i r e d to kill half t h e p l a n t s of F. esculentum, P. sativum a n d B. n a p u s was s t u d i e d . F o r e a c h species, i r r e s p e c t i v e of s p r a y o u t p u t or m e a n d r o p l e t size t h e equi-effective dose of D N C r e m a i n e d c o n s t a n t i.e. t h e equi-effective c o n c e n t r a t i o n was c o n s t a n t l y a n d i n v e r s e l y prop o r t i o n a l t o t h e level of r e t e n t i o n p e r u n i t surface, S t a t i s t i c a l analysis of a series of e x p e r i m e n t s u n d e r t a k e n a t different seasons of t h e y e a r on B. ~ a p u s d e m o n s t r a t e d t h a t t h e m a g n i t u d e of t h e equi-effective dose of DNC was p r i m a r i l y d e p e n d e n t on t h e t o t a l solar r a d i a t i o n received d u r i n g t h e first few h o u r s a f t e r t h e s p r a y application. ACKNOWLEDGMENTS I a m g r e a t l y i n d e b t e d to P r o f e s s o r G. E. B l a c k m a n , F . R . S . of t h e D e p a r t m e n t of Agriculture, U n i v e r s i t y of O x f o r d n o t o n l y for his assistance in d r a f t i n g this p a p e r b u t also for his help in t h e i n t e r p r e t a t i o n of some of t h e d a t a . I w o u l d also like to t h a n k Dr. R. If. P f e i f f e r of C h e s t e r f o r d P a r k R e s e a r c h S t a t i o n for his i n t e r e s t a n d e n c o u r a g e m e n t . Received June 29, 1959 REFERENCES 1 Adam, N. K., The Physics and Chemistry of Surfaces. Oxford Univ. Press (1942). 2 B l a c k m a n , G. E., Studies in the principles of phytotoxicity. I. The assessment of relative toxicity. J. Exptl. Botany 3, 1-27 (1952). 3 B l a c k m a n , G. E., Bruce, R. S. and Holly, K., Studies in the principles of phytotoxicity. V. Interrelationships between specific differences in spray retention and selective toxicity. J. ExptI. Botany 9, 175-205 (1958). 4 B l a c k m a n , G. E., Holly, K. and R o b e r t s , H. A., The comparative toxicity of phytocidal substances. Symposia Soe. Exptl. Biol. 3, 283-317 (1949). 5 B l a c k m a n , G. E. and R o b e r t s o n - C u n i n g h a m e , R. C., Interrelationships between light intensity and the physiological effects of 2,4-diehloro-phenoxyacetic acid on the growth of Helic~ntlms annuus. J. Exptl. Botany 6, 177-211 (1955). 6 Bliss, C. I., The method of probits. Science 70, 38-39 (1934). 7 Brullskill, R. T., Physical factors affecting the retention of spray droplets on leaf surfaces. Proc. Brit. Weed Control Conf. 3rd Conf., 593-603 (1956). 8 Dewey, O. R., G r e g o r y , P. and P f e i f f e r , R. K., Factors affecting the susceptibility of peas to dinitroherbicides. Proc. Brit. Weed Control Conf. 3rd Conf., 313-326 (1956). 9 E g g i n k , H. J. and R i e p m a Kzn, P., De voor onkruidbestrijdingsmiddelen gevoelige stadia van graangewassen. Verslag Centraal Inst. Landbouwk. Onderzoek 1950, 121-128 (1951). I0 E i c h b o r n , J. L. von, Zur Frage des gegenseitigen Haftens riiumlich nicht mischbarer Stoffe. Problelne der Randwinkel insbesondere yon Fltissigkeiten an Festobertl/ichen. Kolloid Z. 107, 107-113 (1944).

248

RODERICK

P. MURPHY

H+i0n secretion /

kg excised roots 24

20

16 •~°

o/J

°

•

12

o / ~

o/_

o / °

4 I///l~/" I/// 'A

I//'/A oil V

I

I

I

, I

t

I

2

3

4

5

t~m¢ ,n hours t I

6

7

Fig. 5. H+-ion secretion into culture solution by excised roots in varying concentrations of sucrose in the culture solution. A = 1.0 m M s u c r o s e O = 2.0 m M s u c r o s e

(]) = 3 . 0 m M s u c r o s e • = 4.0 mM sucrose

161-" mM sucrose absorbed/ k 9 excised roots I4

.../ /.

,° r 6

I/

O+

Fig. 6.

1

¢i " 4

H+secretion in mM/k 9 excised roots I I MI I t 8 12 16 20 24

R e l a t i o n s h i p of H + - s e c r e t i o n t o s u c r o s e u p t a k e .

CATION U P T A K E BY E X C I S E D GRASS ROOTS

249

CONCLUSIONS

It has been shown that the hydrogen-ion concentration in the external medium affects potassium absorption in the same way as would be expected from the equation K + + H R -~ K R + H +

i.e. the potassium uptake is diminished in increasing concentrations of hydrogen ion. It has been shown that at p H 3.5 the reaction is driven in the reverse direction liberating potassium and that at slightly higher pH values, the potassium uptake is as would be expected, relatively slow. This is in agreement with similar work done on barley roots by F a w z y et al. 1. It has also been shown that in the presence of sucrose there is a large secretion of hydrogen ions, but that the sucrose concentration does not affect the potassium uptake, except to decrease the uptake rate slightly as the external p H falls. It is therefore assumed' that the potassium uptake is not directly related to the carbohydrate metabolism. The uptake of sucrose has been investigated and it was seen that the rate of uptake increases with a limited increase in the sucrose concentration showing that sucrose does not diffuse passively into the cells but is absorbed b y some metabolic reaction which is saturated at a definite sucrose concentration in the absorption solution. ACKNOWLEDGEMENTS I wish to thank my assistant, Mr. S. M o i i o y, who did most of the analytical work involved in the above experiments. I would also like to thank Dr. T. Walsh of the Department of Agriculture for his advice in compiling this paper. Received J u n e 16, 1958 REFERENCES 1 F a w z y ~ H., O v e r s t r e e t , R., and J a c o b s o n , L., The influence of hydrogen ion concentration on cation absorption by barley roots. Plant Physiol. 29, 234-237 (1954). 2 H a n d , R. G., and S u t c l i f f e , J. F., Effect of p H on salt uptake by plants. Nature (London) 18@, 238 (1957). 3 J a c o b s o n , L., O v e r s t r e e t , R., H a n g , H. M., and H a n d l e y , R., A s t u d y of potassium absorption by barley roots. Plant Physiol. 25, 639-647 (t950). 4 M u r p h y , R. P., A method for the extraction of plant samples and the determination of total soluble carbohydrates. J. Sci. Food Agr. (In press). 5 N i e l s o n , T. R. and O v e r s t r e e t , R., A s t u d y of the role of the hydrogen ion in the m e c h a n i s m of potassium absorption by excised barley roots. Plant Physiol. 80, 303-309 (1955).