Planta

Planta 146, 603 605 (1979)

9 by Springer-Verlag 1979

The Effect of Temperature on the Velocity of Exogenous Auxin Transport in Intact Chilling-Sensitive and Chilling-Resistant Plants D.A. Morris Department of Biology, Building 44, The University, Southampton, SO9 5NH, U.K.

Abstract. The velocity of exogenous indol-3yl-acetic acid ([1-a4C]IAA) transport f r o m the apical buds o f intact pea, sunflower and c o t t o n plants was determined f r o m 0.5 ~ C to 47 ~ C. The m i n i m u m temperature at which transport occurred varied f r o m 2 ~ (pea and sunflower) to 7 ~ (cotton). A b o v e these temperatures the velocity of transport increased steadily to m a x i m a near 4 4 ~ in all three species. F u r t h e r increase in temperature resulted in a complete cessation o f transport, suggesting a sudden high-temperature b r e a k d o w n o f the auxin transport system. Temperature coefficients (Qto) for transport velocity calculated f r o m Arrhenius plots were low (1.36 to 1.41 between 15 ~ C and 30 ~ C). Arrhenius plots for the chilling-sensitive c o t t o n and sunflower plants exhibited abrupt discontinuities at 14.6 ~ C and 8.7 ~ C respectively. A n Arrhenius plot for the chilling-resistant pea exhibited no such discontinuity over the whole temperature range at which transport occurred. Key words: Auxin transport M e m b r a n e viscosity.

Chilling sensitivity -

Introduction

Studies o f the transport o f 14C- or 3H-labelled indol3yl-acetic acid (IAA) applied to the shoot apex of the intact plant have shown that the resulting longdistance basipetal transport possesses m a n y of the well-known characteristics of the slow polar transport o f auxin observed in excised tissue segments. The transport is slow (10-15 m m h 1), polar, specific to I A A and related growth-active synthetic auxins, sensitive to specific auxin transport inhibitors and, in dicotyledons, p r o b a b l y occurs in the vascular c a m b i u m and differentiating vascular elements (Goldsmith, 1977; Morris, 1977; Morris and Thomas, 1978; and literature cited by these authors). In this system auxin Abbreviation." IAA = indoi-3yl-acetic acid

applied to the shoot apex is transported t h r o u g h the whole length o f the shoot and accumulates in developing lateral r o o t primordia (Rowntree and Morris, 1979). The velocity o f this transport is independent of cell length in the intact stem and o f the activity of ' s i n k s ' for auxin in the r o o t (Eliezer, 1978). Relatively little is k n o w n a b o u t the effects on auxin transport in the intact plant o f external environmental factors. D a t a on the effects o f temperature on transport, in particular, might be expected to provide information of value in assessing the validity of suggested mechanisms o f polar auxin transport (see Goldsmith, 1977). This paper reports a study of the effect o f temperature on the velocity of exogenous auxin m o v e m e n t in intact plants o f three species deliberately chosen for their widely different temperature tolerances and chilling sensitivities. Materials and Methods Plant Material

The plants chosen for the study were: P•um sativum L. cv Meteor (a chilling-resistant dwarf pea); Gossypium hirsutum L. cv Albar A(61)21 (a chilling-sensitive inbred line of American Upland cotton); and Helianthus annuus L. (sunflower a locally-grown, chilling-sensitive stock). Pea seedlings were germinated in John Innes No. 1 compost in a growth cabinet (21~ C; photoperiod 16 h at 19 klx from 'daylight' fluorescent lamps). Cotton and sunflower seedlings were raised in compost in a warm greenhouse (minimum temperature ca. 20~ C) with supplementary illumination from mercury vapour lamps (photoperiod 16h). Uniform plants were selected for transport experiments 14 d after sowing. They were transferred to unlit incubators at the required temperatures (from 0.5~ to 47~ C) and allowed to equilibrate overnight (ca. 16 h) before the application of [1-14C]IAA. Application and Estimation

of [14C]IAA

[1-14C]IAA (specific activity 1.92 GBq mM 1) was obtained from the Radiochemical Centre, Amersham in solution in benzene/acetone. The solvents were removed at room temperature under a gentle stream of nitrogen and the residue was redissolved in an appropriate volume of distilled water containing 0.01% Tween 20. The radiochemical purity of the compound was checked before use by ascending paper chromatography. The compound was applied to the apex of the intact shoots as a 5 gl droplet containing

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D.A. Morris: Effect of Temperature on Auxin Transport

detected at 0.5~ a transport velocity approaching 5 mm h - ~ was observed in plants at 2 ~ C. Changes in the estimated transport velocity with increasing temperature are shown for each species in Fig. 1. An unexpected feature of the results was the absence of a clear temperature optimum for velocity. In all three species the velocity increased with temperature above the minimum until a temperature was reached in the region of 44 47 ~ C at which the transport system suddenly ceased to function at all. This upper limit was similar in all three species and coincided with the temperature at which symptoms of high temperature injury (wilting) were visible in some of the experimental plants by the end of the equilibration period. Arrhenius plots of the experimental data were constructed (Fig. 2). In the chilling-resistant pea the plot yielded a single straight line over the temperature range 2 ~ C to 39 ~ C (Fig. 2c). In the chilling-sensitive cotton and sunflower plants the Arrhenius plots exhibited abrupt discontinuities at temperatures of 14.6 ~ C and 8.7 ~ C resPectively (Fig. 2a and b). Below these temperatures the temperature-dependence of transport velocity increased markedly and calculated 'activation energies' (Ea) increased 4-fold in cotton and 6-fold in sunflower (Fig. 2a and b). Temperature coefficients (Q~0) computed from the Arrhenius plots were low, and in the range 15~ to 30 ~ C were 1.36, 1.41 and 1.39 in cotton, sunflower and pea respectively. Previous experiments with intact pea seedlings transporting IAA in light have yielded

ca 2.9 kBq 14C (0.27 ~tg IAA). Droplets were applied to the pea between the stipules of leaf 6, which were still enclosed within the stipules of the partially-unfolded leaf 5. In cotton the dropiet was applied to the upper surface of leaf 2. This leaf was still partially folded and 5 7 m m long; the first internode was about 5 m m long at the time of labelling. In sunflower the droplet was applied to the upper surface of the second pair of foliage leaves (each ca. 4 m m long), and in the plants used the first internode was approximately 20 m m long. The plants were alIowed to transport in darkness for an accurately measured period between 3 and 4 h. At harvest the labelled apex was removed and the stem was quickly cut into successive 5 m m segments which were extracted overnight at 2 ~ C in darkness in polyethylene counting vials containing 1.0 ml ethanol and 10.0 mI dioxan-based scintillation fluid. The ~ C in each segment was then determined by liquid scintillation counting. Semi-logarithmic profiles of radioactivity were plotted for each plant individually and curves were fitted to the profile fronts. The velocity of m o v e m e n t of the front through the stem was estimated by extrapolating the fitted curve to background radioactivity on the distance axis.

Results and Discussion

The species differed in the minimum temperature at which transport could occur. In cotton no transport was detected below 7 ~ C and the cotyledons of plants subjected to temperatures of 5~ C or lower had wilted by the end of the 16-hour equilibration period and bore water-soaked lesions indicative of membrane leakage. Sunflower plants remained turgid at all low temperatures except 0.5 ~ C and transport was detected at 2 ~ C. No signs of low temperature injury were observed in the pea seedlings at any of the temperatures tested, and although no transport was

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Fig. 2a-c. Arrhenius plots for estimated transport velocity in the stem of intact cotton, sunflower and dwarf pea of [1-t4C]IAA applied to the apical bud. Each point is the mean of four individual determinations. Ea = activation energy in kJ molsimilarly low Q l o values for velocity, r a n g i n g f r o m 1.17 to 1.44 over the range 5 ~ to 3 5 ~ (Eliezer, 1978), a n d low Q10 values for velocity have consistently been r e p o r t e d f o r e x p e r i m e n t s involving p o l a r t r a n s p o r t in cut tissue segments (e.g. H e r t e l a n d Leopold, 1963; K a l d e w e y , 1965). These values are far lower t h a n w o u l d be e x p e c t e d for a t r a n s p o r t process limited by m e t a b o l i s m , a n d t a k e n t o g e t h e r with the lack o f a clear t e m p e r a t u r e o p t i m u m for t r a n s p o r t velocity a n d the relatively high t e m p e r a t u r e s at which m a x i m u m velocities were found, suggest t h a t the o b s e r v e d velocity with which the tracer f r o n t moves t h r o u g h the stem is d e t e r m i n e d largely by physical features o f the t r a n s p o r t system. The A r r h e n i u s plots for auxin t r a n s p o r t velocity are similar to those o b t a i n e d b y o t h e r w o r k e r s for the rates o f p h y s i o l o g i c a l a n d b i o c h e m i c a l processes m e d i a t e d by m e m b r a n e - b o u n d enzyme systems, a n d those for the chilling-sensitive c o t t o n a n d sunflower exhibit the s a m e c h a r a c t e r i s t i c d i s c o n t i n u i t y close to the critical t e m p e r a t u r e for chilling injury (Lyons, 1973). E l e c t r o n spin r e s o n a n c e m e a s u r e m e n t s have shown t h a t at this critical t e m p e r a t u r e the cell m e m b r a n e lipids u n d e r g o a p h a s e c h a n g e f r o m a fluid state to a m o r e o r d e r e d crystalline f o r m with a corres p o n d i n g l y h i g h e r viscosity (e.g., see R a i s o n , 1974). A l t h o u g h the m e c h a n i s m o f p o l a r auxin t r a n s p o r t in the intact p l a n t r e m a i n s to be elucidated, evidence f r o m w o r k on cut tissue segments a n d P a r t h e n o c i s s u s c r o w n gall tissue has p o i n t e d to the p a r t i c i p a t i o n o f m e m b r a n e - a s s o c i a t e d p r o t e i n carriers in the t r a n s p o r t (for reviews see G o l d s m i t h , 1977; L a m b , 1978). The m o b i l i t y o f such carriers across o r in the plane o f the m e m b r a n e s w o u l d be highly sensitive to changes

in m e m b r a n e viscosity. C h a n g e s in m e m b r a n e viscosity m i g h t therefore p r o v i d e a n e x p l a n a t i o n for the o b s e r v e d response o f a u x i n t r a n s p o r t velocity to temp e r a t u r e change a n d w o u l d a c c o u n t for the essentially physical n a t u r e o f this response. I am indebted to Mrs. R.P. Bell for assistance with the experiments.

References Eliezer, J.: Some factors influencing exogenous auxin transport in intact plants, pp. 114 Ph.D. thesis, University of Southampton, 1978 Goldsmith, M.H.M.: The polar transport of auxin. Annu. Rev. Plant Physiol. 28, 439M78 (1977) Hertel, R., Leopold, A.C. : Versuche zur Analyse des Auxintransports in der Koleoptile von Zea mays L. Planta 59, 535 562 (1963) Kaldewey, H. : Wuchstofftransport, Temperatur und Pflanzenalter. Ber. Dtsch. Bot. Ges. 78, 128-143 (1965) Lamb, C.J.: Hormone binding it1 plants. Nature (London) 274, 312 314 (1978) Lyons, J.M.: Chilling injury in plants. Annu. Rev, Plant Physiol. 24, 445466 (1973) Morris, D.A. : Transport of exogenous auxin in two-branched pea seedlings (Pisum sativum L. ). Planta 136, 91 96 (1977) Morris, D.A., Thomas, A.G.: A microautoradiographic study of auxin transport in the stem of intact pea seedlings (Pisum sat# vurn L.). J. Exp. Bot. 29, 147-157 (1978) Raison, J.K. : A biochemical explanation of low temperature stress in tropical and sub-tropical plants. In : Mechanism of regulation of plant growth, pp. 487 497, Bieleski, R.L., Ferguson, A.R., and Cresswell, M.M., eds. Wellington: Roy. Soc. New Zealand 1974 Rowntree, R.A., Morris, D.A.: Accumulation of t4C from exogenous labelled auxin in lateral root primordia of intact pea seedlings (Pisum sativum L.). Ptanta 144, 463466 (t979) Received 12 April; accepted 12 July 1979