Planta (Berl.) 111, 1--12 (1973) 9 by Springer-Verla,g 1973

The Influence of Dinitrophenol on Phloem Transport along the Stolon of Saxifraga sarmentosa F . A . Qureshi * and D. C. Spanner Botany Department, Bedford College, London, NW1 4NS, U.K. Received November ll, 1972

Summary. Dinitrophenol in concentrations of 5 • 10-31~ applied to the centre 30 em of 60-70 cm stolons of Saxl/raga produces a strong and reversible inhibition of the phloem transport of laTCsor 14C-assimilates. There is every reason to believe that this effect is loealised in the sieve tubes; callose formation does not occur. This evidence is very difficult to reconcile with the M/inch hypothesis; it seems on the contrary to demand a theory of active pumping. Introduction The work reported in this communication is the third in a series dealing with the effect of metabolic inhibitors on phloem transport in the stolon of Saxifraga. Both anoxia and cyanide have been found to exert a strong and reversible effect on the sieve tubes (Qureshi and Spanner, 1973 a, c). Careful earlier work with these and other inhibitors (see Willenbrink, 1968) had already reached similar conclusions, although it had been challenged; but in the case of 2,4 dinitrophenol (DNP) the evidence rested on a less solid foundation, Willenbrink reporting that he "could obtain no satisfactory result" with it, and Harel and l~einhold (1966) in an interesting paper concluding that any "inhibitory effect of D N P ... was probably due to an effect on uptake and/or secretion into the sieve tubes, not to an effect on the conducting cells themselves". Indeed, in their work D X P appeared to actually Tromote downward transport; and the absence of a definite inhibitory effect on the sieve tubes appeared to them " t o exclude the close participation of ATP-dependent processes" at this locus. This would be a most important conclusion if it were inescapable; however, I-Iarel and Reinhold's work leaves open other possibilities, and it was therefore very desirable to re-investigate the action of D N P in circumstances enabling a clearer indication of its behaviour and locus of action * This work formed part of that submitted for the degree of Ph.D. of London University. 1

Planta (Berl.), Bd. 111

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F . A . Qureshi and D. C. Spanner:

to be o b t a i n e d . T h e p r e s e n t p a p e r r e p o r t s some p r e l i m i n a r y w o r k to this end.

Materials and Methods These have been adequately described in previous papers (Qureshi and Spanner, 1971, 1973a, b, e). In the earlier stages of the work DNP was applied to portions of the stolon only 2 cm long. Results were slight and erratic; and as in the case of cyanide treatment it was decided to expose longer lengths to the inhibitor. Eventually 30 cm was fixed on, and plants were therefore selected which had particularly long stolons (60 to 70 cm). The inhibitor was made up in 0.05 M phosphate buffer of pH 7.2, and applied to the stolon by running the latter along a groove milled in a perspex block. The tracers were made up in similar buffer and applied in a small reservoir (Qureshi and Spanner, 1971, Fig. 2), while the 14C0~ was given to the subtending leaf in a small chamber of flexible transparent plastic. On termination of the experiment the stolon was cut up as indicated on the horizontal axes of the diagrams; the la~Cs was assayed under an automatic endwindow counter and the 14C in an automatic liquid scintillation counter (for details see Qureshi and Spanner, 1973a, b). Callose was examined by plunging the stolon quickly into acetic alcohol (Eschrich and Currier, 1964) at --20~ and leaving it at this temperature for 24 h before processing for electron microscopy. Autoradiographs were prepared in the conventional manner after heat- or freeze drying. Plants were in continuous light during the experimental period, either daylight or (at night), low pressure mercury illumination of 3 750 lux. They were mamtained otherwise under ordinary greenhouse conditions, with a temperature ranging from 15~ to 25~ depending on the season. The daughter plants were enclosed in small black polythene bags internally dampened to reduce both transpiration and photosynthesis.

Results

Movement o/137C8 I n t h e earlier w o r k w i t h ~37Cs regression lines were f i t t e d b y c o m p u t e r t o t h e v e r y s t r a i g h t semi-logarithmic plots. I t was t h e failure to find a n y r e l a t i o n s h i p b e t w e e n t h e slope of these lines a n d t h e c o n c e n t r a t i o n of a p p l i e d D N P t h a t led t o a lengthening of t h e zone of a p p l i c a t i o n t o 30 em. Fig. 1 records t h e e x p e r i m e n t a l results when la7Cs was a p p l i e d to t h e stolons b e t w e e n t h e p a r e n t p l a n t a n d t h e zone of t r e a t m e n t . I n h i b i t o r t r e a t m e n t was s t a r t e d 4 h prior to t r a c e r application, a n d t h e d u r a t i o n of t h e e x p e r i m e n t , including this 4 h, was 22 h. T h e stolons were t h e n s e g m e n t e d a n d assayed. I t is e v i d e n t t h a t t h e r e is a small effect a t 10 -4 M concentration. I t increases progressively u p to 5 • 10 -3 M. A t this level no t r a c e r a t all passes t h r o u g h t h e zone of t r e a t m e n t . I n t h e earlier w o r k (Qureshi a n d Spanner, 1971) i t h a d been f o u n d t h a t along w i t h t h e long d i s t a n c e t r a n s p o r t t h e r e is always a s y m m e t r i c a l s h o r t - d i s t a n c e s p r e a d i n t e r p r e t e d as t a k i n g place p r o b a b l y in t h e apoplast. I t seemed desirable t o see w h e t h e r this m o v e m e n t was affected b y

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Fig. 1. Unidirectional movement of 187Cs down stolon of Saxi]raga under the influence of DiNvPapplied continuously to a 30 cm zone. DNP applied 4 h prior to la~Cs; total duration of experiment 22 h. Curves for lower concentrations have been displaced horizontally for clarity

D N P . Accordingly, excised pieces of stolon 40 cm long, with vaseline smeared over the cut ends, were soaked in 5 x 10-3 M D N P for 4 h. T h e y were then supported so t h a t a short length at the centre was out of the solution; and to this region 13:Cs was applied in the usual way. After a further 18 h the stolons were assayed. Fig. 2 shows the results. I t is evident t h a t the inhibitor has n o t influenced the movement. This seems to confirm t h a t the spread occurs as suggested in the apoplast.

Transport o/Naturally-assimilated 14C The slope of the semi-logarithmic lines p r o b a b l y reflects the balance between lateral leakage of tracer and longitudinal transport. I t is k n o w n t h a t D N P can affect leakage of ions from parenehyma. However, were the change in slope due to increased leakage this would be revealed b y a localised raising of the general level of tracer at e n t r y to the zone of treatment. This clearly did n o t occur; hence we m u s t conclude t h a t it is principally the longitudinal velocity t h a t is influenced b y the inhibitor. 1"

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Where natural assimilates are concerned the leakage in the Saxi/raga stolon is believed to be slight (ibid, 1973b); hence it was of obvious interest to repeat the pattern of the previous experiment using 14C02. Figs. 3a and b report the results of a series in which laCO~ was administered to the subtending leaf 4 h after treatment with D N P began. After a further 4 h the stolons were segmented and assayed. I t can be seen that there was a small effect with 10-4 ~ DNP. I t increased progressively and was extremely pronounced at 10-2 M. At this concentration there was occasional evidence of damage to the external tissues. Accordingly treatment was limited subsequently to 5 • 10-a M. I n order to test how rapidly the poison acts an experiment was run in which the treatment was begun simultaneously with the administration of 14C02. Four hours later the stolons were harvested. I t is clear from Fig. 4 that a somewhat belated inhibition has occurred (of. Fig. 3 b). However, in view of the general rapidity with which natural assimilate is formed and loaded into the sieve tubes it is still impressive.

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The influence of dinitrophenol on phloem transport along the stolon of Saxifraga sarmentosa.

Dinitrophenol in concentrations of 5×10(-3) M applied to the centre 30 cm of 60-70 cm stolons of Saxifraga produces a strong and reversible inhibition...
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