Planta (Berl.) 82, 123--144 (1968)

Auxin Movement in Corn Coleoptiles* I~ASKNER HERTEL a n d RAnD FLORr MSU/AEC Plant Research Laboratory, Michigan State University, East Lansing Received April 24, 1968

Summary. Movement of radioactive auxins was analysed in corn coleoptile sections. The results support the idea that processes involved in the transport of indoleacetie acid (IAA) are specific for growth-promoting auxins. Inhibition of IAA transport by triiodobenzoic acid is caused by a reversible block of the exit; the auxin held back remains in the transport pool. The observed increase in immobilization may be a secondary effect caused by the increased concentration of free IAA in the tissue. Auxin molecules are most likely transported by a non-covalent mechanism. IAA and naphthaleneacetic acid (NAA) move through the cell and exit as free molecules. A search for a transient auxin complex, chaseable as required for any transport carrier intermediate, yielded negative results. No 1so was lost from NAA labeled with 180 in the carboxyl group during transport of the auxin through coleoptfle tissue. After application of IAA to auxin-depleted tissue, the transport rate undergoes oscillations with a period length of ca. 25 min. The movement of the auxin 2.4-diehlorophenoxyacetic acid which is usually sluggish, increased several times if some IAA was added. Auxin, thus, stimulates its own transport. A model is discussed in which auxin-binding to the plasma membrane and reversible changes of membrane conformation may provide a basis for active secretion and for the observed cooperativity. A. Introduction The m o v e m e n t of a u x i n t h r o u g h riving p l a n t tissue (latest review b y GOLDSMITH, 1968) is n o t a simple diffusion process, b u t includes u p t a k e , i m m o b i l i z a t i o n , d e s t r u c t i o n a n d active t r a n s p o r t . T h e basic m e t h o d s a n d p h e n o m e n a of a u x i n t r a n s p o r t were established in coleoptiles b y VAn DEl~ WEIJ (1932). A u x i n t r a n s p o r t is fast (ca. 1 cm/hour), p o l a r (preferentially from t i p to base) a n d a c t i v e ; i t can proceed a g a i n s t a c o n c e n t r a t i o n g r a d i e n t a n d i t requires m e t a b o l i c e n e r g y (GoLDSmITH, 1966, 1967). The a u x i n is t r a n s p o r t e d t h r o u g h p a r e n e h y m a tissue; p l a s m a t i c c o n t i n u i t y is n o t required, a n d some evidence suggests t h a t t r a n s p o r t processes are r e p e a t e d from cell to cell, t h e a c t i v e a n d specific s t e p being a secretion o u t of each cell (I-IE~TEL a n d LEOPOLD, 1963; LEOPOLD a n d HALL, 1966). * LEO B~AUN]m zum 70. Geburtstag gewidmet.

124

R. HE~TEr~and R. F L O R Y

:

The e x p e r i m e n t s reported i n this paper were performed to analyse certain aspects of the p o s t u l a t e d secretion m e c h a n i s m , e.g. the t y p e of chemical b o n d involved.

B. Materials and Methods

a) Chemicals. 3-Indoleacetic acid (IAA), 1-naphthaleneacetic acid (NAA), 1-naphthaleneacetie acid methylester (NAA-methylester), 2.4-diehlorophenoxyacetic acid (2.4-D), were obtained from Nutritional Bioehemicals Corp., Cleveland, Ohio; 2,3,5-triiodobenzoic acid (TIBA) was purchased from J. T. Baker Chem. Co., Phillipsburg, N.J. Buffers were prepared according to Go~o~I (1955). b) Enzyme. fl-Gineosidase (emulsin) from Worthington :Biochem. Co., Freehold, N.J. was used to characterize an auxin conjugation product. The enzyme was dissolved in H20 (1 mg/ml) ; for each assay, 0.5 ml of enzyme solution were mixed with 0.5 ml of substrate and 0.3 ml of Na-acetate buffer (0.1 M, pH 5). The mixture was incubated at 37~ and the reaction stopped by boiling, by adjusting to pH 3 and extracting with ether or by simply transferring 0.2 ml to a chromatogram. c) Thin-Layer Chromatography. Compounds to be tested were spotted on Eastman Chromatogram sheets (K 301 Rs) and the chromatograms were developed in an Eastman Chromatogram developing apparatus (both from Distillation Products Industries, Rochester N.Y.). Solvents: A 1 (chloroform: ethyl acetate: formic acid = 5: 4:1) ; A 2 (chloroform: ethyl acetate: formic acid = 2.5: 6.5:1) ; B (ethyl acetate: isopropanol: 25 % ammonia = 5: 4:1); N I (isopropanot: water = 4:1) ; N 2 (ethyl acetate:isopropanohwater = 5.5:4:0.5):

d) Radioactive Compounds. IAA-I-laC (specific activity 10.4me/raM) was obtained from New England Nuclear, Boston, Mass., NAA-I-I'C (specific activity 16 me/raM) from Tracerlab, Waltham, Mass., 2.4-D-1J4C (12 me/raM) from Nuclear Chicago. When IAA-1-1~C(33 mc/mM) from Nuclear Chicago Corp. and IAA-5-ZH (1000 me/raM) from Schwarz Bioreseareh, Inc., Orangeburg, N.Y. was used, it will be specified. The radiochemieal purity of 2.4-D-14C was > 99%. NAA-I~C and IAA-14C (freshly diluted into acetonitrile) were rechecked by chromatography for some experiments (e.g. Fig. 5) and found to be > 98% pure (see also Results). The impurities of the IAA-att ( > 10%) did not interfere with the conclusions. e) Measurement of Radioactivity. Agar blocks and ethanol extracts (see below) were transferred to plastic vials containing 10 ml of BP~Y's solution (4 g POP and 0,2 g POPOP, both from Packard Instr, Co., Downers Grove, Ill.; 60 g naphthalene, 100 ml me,hanoi; diluted to 1000 ml wi~h p-dioxane) and courted in a Iiquid scintillation counter (Beckman CPM-100) at room temperature. Since no sig-nificant differential quenching for ~C was observed, the counts were corrected only for background. Chromatograms were assayed for radioactivity on a Packard Chromatogram scanner Model 7201 (thin-layer set-up), or (especially in ease of SH) cut into various zones and placed in B~AY's solution for counting. I) Preparation of NAA-180, and 1sO-Analysis. NAA-methylester (0.01 g of oily liqued) was placed in a glass capillary; 0.02 ml of H~sO (60 to 80 % 1sO; from Yeda, Rehovot, Israel), 0.005 ml of 10 N NaOH and some broken glass were added. The capillary was sealed, shaken (to mix the two phases) and incubated for 40 min in boiling water. The crude product was taken up in a large excess of cold I-I20, adjusted to pH 3 and extracted once into ethyl ether. The product was then extracted

Auxin Movement in Corn Coleoptiles

125

into buffer (pH 7.4) ; it was acidified and reextracted with ethyl ether from which the compound was crystallized into ice-cold water, filtered and dried in vacuo, and subsequently dissolved and stored in acetonitrile at --20 ~ The yield was ~ 50%. The product was identified as NAA from the form of the crystals, from the fluorescence spectrum (HE,TEL, unpublished data), from chromatographic behaviour (in 4 solvents cochromatographing with NAA-14C) and from its characteristic transport behavior (see below, Fig. 7). The product was found to contain 25% of its oxygen as 1so, in agreement with the original H20 isotope ratio and a hydrolysis mechanism where ca. half of the 160 of the ester exchanged with oxygen 1sO from water (see :BENDER, 1951). Samples (ca. 5 mg each) to be tested for the lsO/t60 radio were redried, combusted and analysed as CO S in a mass spectrometer by Dr. A. S. OSTASHEVE~at Analytica Corp., Westbury, L.I., N.Y. g) Coleoptiles. Corn seeds (Zea mays L., hybrid W F 9 • 38, lot 4243 from :Bear Hybrid Corn Co., Decatur, Ill.) were soaked overnight in running tap water and planted in plastic boxes (covered, with air inlet) on 4 layers of paper towels. Water was added to ca. 1 mm above the paper. The seedlings grew in a darkroom (24 ~ 80 to 90 % humidity, 2 hours of red light each night). Coleoptiles were used when 2 to 3 cm long, 80 to 90 hours after soaking. Oats (Arena sativa L., var. VICTORY), husked, soaked in vacuo for 2 hours, and wheat (Triticum vulgare VILL., var. H]~N~Y), soaked overnight, were grown as described above for corn and used after 3 days. h) Transport Tests (see VAN DER WEIJ, 1932; GOLDSMITHand THL~ANN, 1962; HE,TEL and LEOPOLD, 1963). All work with living tissue was performed in the described darkroom under dim green light. Agar (:Bacto-Agar from Difco; 1.5 % ) was poured in steel molds on microscopic slides (ice cooled from underneath) and cut flat (blocks:23 mm diameter, 1 mm high). The receptor blocks were made of agar only (plus inhibitors where indicated); radioactive compounds or other auxins were added to the molten agar which was used for the donor blocks. Coleopti]es were always freed from the leaves inside, and sections were cut with two razor blades mounted at a fixed distance. The sections were then placed on a receptor. In the corners of the receptor glass slide four plasticine columns were attached somewhat higher than sections plus blocks. The slide with the donor was pressed down on the plastieine until all sections made good contact with the donor surface. Donor, tissue and receptor were disassembled after a given time; the agar was transferred to the counting vial, and if desired the sections were extracted twice with 0.75 ml ethanol for several hours, and the extract was assayed for radioactivity. (There was no difference between hot or cold ethanol extraction.)

C. R e s u l t s

1. Transpor~ o / I A A , N A A and 2,4-D (a) N A A is s i m i l a r a n d n e a r l y as e f f e c t i v e as I A A in its g r o w t h a c t i v i t i e s (e.g., JOENSSO~, 1961) a n d is p r e f e r a b l e in s o m e t e s t s d u e t o its s t a b i l i t y (e. g., ZENK, 1962). I t s m o v e m e n t in tissue is w e l l d o c u m e n t e d t o be p o l a r a n d a c t i v e (LEOPOLD a n d LAM, 1961 ; VEEN, 1967) a n d t h e r e is e v i d e n c e f o r a s e c r e t i o n m e c h a n i s m s i m i l a r t o t h a t s u g g e s t e d f o r I A A ( H E , T E L a n d LEOPOLD, 1963). I n t h e s t u d y r e p o r t e d here, i t w a s conf i r m e d t h a t I A A a n d N A A m o v e m e n t i n coleoptiles was p o l a r a n d i n h i b i t e d b y T I B A as is t h e I A A t r a n s p o r t . T h e r e l a t i o n b e t w e e n m o v e -

B. H]~BTELand R. FI, ORY:

126

m e n t a n d donor c o n c e n t r a t i o n was c o m p a r a b l e for N A A a n d I A A , s a t u r a t i o n occurring a t ca. 10-sM in t h e donor. N A A a t 5 • 10-sM inhib i t e d I A A t r a n s p o r t a n d vice versa, again suggesting t h a t the t w o c o m p o u n d s m o v e in t h e same system. (a)

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Fig. 1 a and b. Comparison of movement o4 IAA and NAA. Corn coleoptile sections (10 mm long; 30 per block) were placed on receptors, and radioactive auxin at 5 • 10-8 hi was applied in a donor at the apical end for 7.5 rain. The sections were quickly transferred to new receptors at the time indicated by the end of each column in the figure. The used receptors were assayed for radioactivity, as was the ethanol extract of the sections at the end of the experiment. In (a) the epm in the receptors, delivered per 7.5 rain, are related to cpm in donor and represent by the height of the dashed columns. In (b) EXPORTED = sum of cpm in receptors • 104/cpm in donor; HELD IN TISSUE = epm in final extract • 104/cpm indonor; TOTAL UPTAKE = EXPORTED -~ HELD IN TISSUE. T h e v e l o c i t y of m o v e m e n t of a pulse of N A A was d e t e r m i n e d a n d comp a r e d w i t h t h a t of I A A . T h e t e c h n i q u e (VAN ])~B W~IJ, 1932; H~BTEL a n d LEOPOLD~ 1963) consisted of a p p l y i n g labeled a u x i n t o t h e apical end for a short t i m e only; t h e a p p e a r a n c e of label w i t h t i m e was t h e n followed b y changing t h e basal receptors a t brief intervals. Fig. 1 shows t h a t N A A m o v e d slower t h a n I A A : t h e I A A p e a k was delivered f r o m t h e base of t h e 1 c m sections after c~. 45 m i n (velocity ~ 1.3 cm/hr~ ; w i t h IqAA t h e m a x i m u m a p p e a r e d after ca. 70 min (velocity = 0.9 em/hr.).

Auxin Movement in Corn Coleoptiles

127

T h e N A A pulse was b r o a d c o m p a r e d w i t h t h a t of I A A . T o t a l u p t a k e was g r e a t e r for N A A , m o r e was r e t a i n e d in t h e tissue, a n d r e l a t i v e l y less was exported. (b) 2.4-D, a n o t h e r auxin, shows a q u a l i t a t i v e l y similar, a l t h o u g h less efficient m o v e m e n t when c o m p a r e d w i t h I A A . T h e criteria of a u x i n t r a n s p o r t - - p o l a r i t y , i n h i b i t i o n b y certain specific substances - - h a v e

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I.LJ I- 9 9 % ) and 2,4-D ( > 95 %) reappeared in the receptor blocks as free substances as shown b y direct chromatography with or without previous ethanol extraction. This finding confirms the literature reports previously mentioned. The radioactivity in the tissue was examined after exposing coleoptile sections (mostly 2 m m long) to 14C-auxin for 5, 10 or 20 min, either in the transport setup (with 5 • l0 -5 M auxin in the donor block) or in a buffer (Na-citrate-phosphate, 0.01 M, p H 5; plus 10-s M or 5 • 10-6 M auxin). I n the latter method 0.2, 0.4 or 1 g of tissue were shaken in 2 ml of solution. Before extraction the sections were quickly filtered and rinsed with cold buffer. The chase period with unlabeled auxin, or buffer, or agar was as long or slightly longer than the expos~trc. Boiling and cold ethanol, methanol and buffers in large excess to tissue mass were used for extraction. I n some cases the tissue was extracted in a glass grinder. The residue after ethanol extraction was never found to contain significant activity. The bulk of the activity in the tissue (always > 98 %) was chromatographically defined as "free" auxin. This technique would not differentiate the immobilized auxin (WizqT]~g, 1967). Among the other labeled compounds no auxin complex was found to be chaseable as would be required ~or a transport intermediate. Ca. 0.1% of the total activity of the extract would have been detected on the chromatograms in regions away from the major peak, especially in reruns after discarding the fraction containing the bulk of free auxin. b) I n agreement with results of ZmCK (1961) and VEEZr (1967) small amounts ( < 0 . 5 % of total after 5 rain) of NAA-fi-glucose were found (Fig. 5, at Rf 0.3). This conjugate was identified b y the following criteria : (1) I n the solvents used the Rf values of the radioactive compound formed in the tissue correspond with the reported values for the glucose conjugate (e.g. ZE~K, 1964); (2) when the suspected product was eluted from the chromatogram and incubated with fl-glucosidase as described above, free 14C-NAA was produced (ca. 50% in 15 rain).

132

R. H E R T E L a n d R . F L O R Y :

C o m p a r i n g Fig. 5, left versus right, no t u r n o v e r of t h e NAA-14Cfl-glucose can be seen; on t h e c o n t r a r y , t h e c o m p o u n d c o n t i n u e d to a c c u m u l a t e d u r i n g t h e chase. S i m i l a r d a t a were o b t a i n e d w i t h coleoptiles of w h e a t a n d sweet corn, a n d also when I A A was used i n s t e a d of N A A . The results agree w i t h conclusions of ZENK (1964). 5000

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Fig. 5. •AA-tr and its products in tissue extracts after pulse and chase.Coleoptile sections (twice 0.2 g; each ca. 1.5 rata long) were incubated in buffer for 30 rain. Then NAAI~C (5 • 10-~ M) was added. After 5 rain the sections were quickly filtered and rinsed on cheesecloth. One saraple was extracted with hot 95 % ethanol, while the other was transferred to buffer containing 5 • 10-6 ]~ unlabeled NAA. After another 5 rain, these sections, too, were filtered, rinsed and extracted. The extracts were reduced in volume under a cold fan, applied to thin-layer chromatograms and developed in solvent A 1 (upper scans), then the regions from start to Rf 0.6 of the chroraatogTaras were cut out, eluted with ethanol and rechromatographed in A 1 (lower scans). Rf 0.8 = free NAA; Rf 0.3 ~ fl-glucose-NAA T h e f r a c t i o n of r a d i o a c t i v i t y i d e n t i f i e d as NAA-14C-fl-glucose was e l u t e d from c h r o m a t o g r a m s . W h e n r e a p p l i e d f r o m t h e outside t o coleoptiles t h i s c o n j u g a t e was shown t o be split v e r y efficiently (e.g. Fig. 6). T h e s a m e conjugate, however, w h e n o b s e r v e d being f o r m e d a n d r e m a i n ing inside t h e tissue, p r e s u m a b l y in t h e cells, was n o t split a n d no significant t u r n o v e r could be seen (Fig. 5). This difference in b r e a k d o w n of NAA-fl-glucose, d e p e n d i n g on its origin or t h e r o u t e of i n t r o d u c t i o n , i n d i c a t e s c o m p a r t m e n t a t i o n in t h e tissue (see M A c L E N N ~ et al., 1963). Fig. 6 shows t h e l i b e r a t i o n of N A A f r o m t h e conjugate. T h e f a c t t h a t free a u x i n a p p e a r s even in t h e d o n o r (left) suggests t h a t a significant a m o u n t of free a u x i n l i b e r a t e d f r o m t h e c o n j u g a t e in t h e tissue m o v e s

Auxin Movement in Corn Coleoptiles

133

b a c k into t h e donor. N o s p l i t t i n g a c t i v i t y could be o b s e r v e d in t h e d o n o r when r e m o v e d from t h e tissue. D u r i n g t h e s h o r t t i m e t h e sections were e x p o s e d to laC-auxin no f u r t h e r p r o d u c t s could be f o u n d in t h e extracts. W i t h longer i n c u b a t i o n (e.g. 20 min) some r a d i o a c t i v i t y was n o t i c e d in p e r h a p s two spots

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Fig, 6. Formation of I~AA from applied fl-glucose-NAA. Coleoptile sections (1 g) were incubated in 10-5 M NAA-14C for 45 min, otherwise under the same conditions as in Fig. 5. The ethanol extract was separated in solvent A 1. The region of the chromatogram around RI 0.3 was cut out and eluted. The material was then rechromatographed and re-eluted in the same way. The radioactive compound (at ca. 5 • 10-~ M) was applied in donor blocks to each 40 sections. Donor (0 rain) did not contain significant free NAA (Rf 0.8). In the tissue (20 rain) the glucoseconjugate taken up (Rf 0.3) was presumably split efficiently liberating free NAA, which appeared in the receptors where no conjugate could be found (receptor, 60 rain). In the donors the ratio conjugate vs. free IAA decreased with time (see e.g. donor 60 rain) b e t w e e n R f 0.4 a n d 0.6 (solvent A1), a m o u n t i n g to ca. 10% of t h a t of t h e /%glucose conjugate. These compounds, too, d i d n o t show t u r n o v e r when chased. c) F o r a n y covalent interactions, t h e m o s t likely a t t a c h m e n t site of t h e a u x i n molecule is t h e e a r b o x y l group where t h e h e a v y isotope 1sO can easily be i n t r o d u c e d , Because of t h e s t a b i l i t y of N A A , this a u x i n was used a n d l a b e l e d w i t h 1sO. (No significant 1sO loss was o b s e r v e d a f t e r a m o n t h of storage in acetonitrile a n d d u r i n g t h e 2 t o 3 hours n e e d e d for t h e e x p e r i m e n t ; c o m p a r e w i t h MAsoIq, 1957.) The fate of t h e isO-label in t h e c a r b o x y l group of N A A was followed after t r a n s p o r t t h r o u g h eoleoptile sections. Methods a n d results from t w o

134

R. HERTEL ~nd R. FLORY:

s e p a r a t e e x p e r i m e n t s a r e s h o w u i n F i g . 7. N o 180 was l o s t f r o m t h e c a r b o x y l g r o u p of N A A d u r i n g m o v e m e n t t h r o u g h t h e tissue. T h i s f i n d i n g e l i m i n a t e s c e r t a i n c h e m i c a l m e c h a n i s m s as possible t r a n s p o r t steps.

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expt.

#

NAA NAA-leO DONOR MATERIAL RECEPTORMATERIAL:

I

2 I

2 I

2 I

2

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26 % 25 %

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25 % 27 %

Fig. 7. Fate of NAA-X80 during transport. NAA was labeled with 180 and incorporated into donors at "physiological" concentrations (10 -5 M in Expt. 1; 1.5 • 10 -5 M in Expt. 2). Some NAA-14C (10 -7 M) was added to monitor movement and re-isolation. Transport through corn coleoptile sections (2 mm long, 30/block; 1200 in each expt.) was allowed for 120 min in Expt. 1 and for 90 rain in Expt. 2. All receptors and a number of donors (4 in Expt. 1; all 40 in Expt. 2) were quickly extracted and NAA was purified without appreciable losses as judged by the NAA-14C, In Expt. 1, a 10% sample of the partitioned extract of donor material contained 124 cpm NAA-~4C, 10% of the receptor extract contained 145 epm; in Expt. 2, the radioactivity was found to be 398 cpm (5% of donor material) and 136 cpm (5% of receptors). The material left was then diluted with a large excess of standard NAA (12C, 1GO). (The dilution factor given in the figure is based on the cpm of NAA-I~C and the NAA-180 concentrations in unused donors.) Then, NAA was twice reerystallized, dried and analysed by Analytiea Corp. ; the relative instrument error did not exceed =t=0.002% 1sO-excess

Auxin Movement in Corn Coleoptiles

135

4. Oscillations o] Transport Rate The time course of auxin arrival in receptors has been represented by a straight line (e.g. VA~ n~B WwIJ, 1932; GOLDSMITI{ and TnzMANN, 1962; H]~RTEL and LEOPOLD, 1963). This suggests that some time after the arrival of the "front % the export rate is constant. On the other hand, I

i

l

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