Planta (Berl.) 73, 281--295 (1967)

ON T H E R E L A T I O N B E T W E E N A U X I N T R A N S P O R T AND A U X I N METABOLISM I N E X P L A N T S OF COLEUS H. Vv,E~ Plant Physiological Research Centre, Wageningen, The Netherlands Received September 17, 1966

Summary. Transport and metabolism of naphthylacetie acid, labelled with 14C or with 3H, were studied by means of the liquid scintillation counting technique in combination with thin layer chromatography. The amounts of radioactivity reaching the receiver blocks as well as the loss from donor blocks greatly depended on the donor concentration. The relative amounts in receiver blocks increased with decreasing auxin concentrations in donor blocks. This phenomenon may be ascribed to the low immobilization capacity of the tissue at very low auxin concentrations. The relative amounts of radioactivity lost from donor blocks increased with decreasing auxin concentrations in donor blocks. Different characteristics of auxin transport can be explained by assuming a movement in symplast or in apoplast. During transport in the symplast the auxin is immobilized. Auxin immobilization governs many characteristics of auxin transport and could have a regulating effect on the free auxin content in plant tissues. Introduction The integrity of the comphcated structure of higher organisms depends to a great extent on regulations which co-ordinate the different parts of the whole. I n plants these regulations are of hormonal nature. Since production centre and action centre are often located at different places in the plant body, a transport of hormones takes place. I n a number of correlative processes like apical dominance and abscission, hormone transport might be of great physiological importance. The ultimate goal of our investigations is to find a relation between auxin transport and various physiological phenomena like petiole abscission, geotropic- and epinastie curvatures and root initiation. Coleus explants are excellently suited to study these relationships. I n previous publications ( G o g T ~ and V~v,~, 1966; V~E~, 1966) some characteristics were given of the transport of naphthylaeetic acid in Coleus explants. Naphthylacetie acid (NAA) was used instead of indoleacetic acid (IAA), because it has been found by many authors that NAA had a stronger abscission retarding effect. I t was stated earlier (Go~Tv,~ and VEEr, 1966; Vv,]~, 1966) that if the auxin was applied at high concentrations (100 to 400 ~M) almost all the auxin remained in the tissue. Only 1.4% of the radioactivity lost from the donor blocks reached the receiver blocks. All activity found in the

282

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receivers at the basal e n d appeared to be still i n the form of n a p h t h y l acetic acid. C h r o m a t o g r a p h e d tissue extracts d e m o n s t r a t e d a n i n t e n s e metabolic t u r n o v e r of the auxin. The t i m e - d e p e n d e n c y of the p r o d u c t i o n of several c o m p o u n d s was studied. I n the p r e s e n t p a p e r i n f o r m a t i o n will be given o n t r a n s p o r t a n d m e t a b o l i s m of a u x i n a t low d o n o r concentrations. I n the discussion a t t e n t i o n will be paid to a general scheme of a u x i n t r a n s p o r t i n stem sections. Material a n d Methods The material used has been described in detail before (V]~v.l% 1966). The explants consisted of a node with 5 mm pieces of stem above and below and two petiole stumps of 5 mm each (see Fig. 1). The explants were placed horizontal in petri dishes on small foam-plastic cushions. The dishes remained in the 2/

('afler GozTzz,zs~) g]

,4~sc/s.,/on zozes Fig. 1. Schemeof an explantused in the experiments

dark at 20~ Each petri dish contained 5 explants, for one record 20 explants were used. ~-Naphthylaeetie aeid-l-14C, earboxyl labelled (NAA-X4C),as well as tritiated NAA (NAA-aH), generally labelled, were applied to position I I of an explant in a donor block of agar gel, see Fig. 1. The transport of 1~Cwas measured by counting the radioactivity appearing in the receiver blocks of p]ain agar gel present at position I I I (see Fig 1). Both NAAJ4C and NAA-aH were obtained from the Radioehemieal Centre, Amersham, England. The x4C-labelled compound had a speeific activity of 8.27 mc/mmole (44.5 ~e/mg) ; the tritiated NAA had a specific activity of 733 mc/mmole (3.93 me/rag). The purity of these compounds was checked by thin-layer chromatography. This technique was Mso used re1' identifying different compounds in the tissue extracts. The method for 14C-labelled material has already been described (Vv.E~, 1966). Different samples were spotted on the ehromatograms of silieagel G and developed with isopropanol/ammonia 25 per cent/water (8 : 1 : 1). The chromategrams were covered with a "Melinex" polyester film to avoid chemical reactions on the film plate. A Kodak medical X-ray no screen film was then placed on the ehromatogram ("sandwich"); the film was exposed for at least one week. Places on the ehromatogram which corresponded to black spots on the developed film were marked and the silicagel of each area (including areas which had given no blackening on the film) was carefully scraped off the glass and transferred to

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a counting vial. I n the case of thin-layer chromatography of tritiated compounds, the silicagel G was mixed with an equal amount of finely ground anthracene (Li~Tnz and WASrR, 1965). The energy of the tritium particles is extremely low, therefore a high self-absorption loss of radiation in the thin layer will take place. By mixing the silicagel with anthracene, the tritium radiation can be made visible by the fluorescence induced in the scintillator. According to L i ) T ~ and WASrR it is essential that a low temperature is maintained throughout the exposure time. Therefore the thin-layer chromatogram was covered with a film and placed in a deep-freeze between layers of dry ice. I n general an exposure time of one week was sufficient to obtain a "fluorogram". After developing the film the chromatogram was treated in the same way as is described for the I40 samples. As described earlier (V~E~, 1966), radioactive assay was done by the liquid scintillation counting technique. Each counting vial contained: 2 m l water, 1 ml ethylalcohol and 10 ml scintillation liquid. The scintillation liquid contained per litre: 800 ml dioxan, 160 ml ethyleneglycolmonoethylether, 48 g naphthalene, 9.6 g 2.5-diphenyloxazole and 0.48 g 1.4-bis-2-(5-phenyloxazolyl)benzene. The radioactivity in each vial was determined in a Packard liquid scintillation spectrometer. Samples were counted for at least 10 rain at 0~ The counting efficiency in these experiments was 51% for an aqueous standard of 14C in the 100--1000 window at a high voltage setting of 1040 V. With an aqueous tritium standard a counting efficiency of 7 % in the 50--1000 window at a high voltage setting of 1100 V. was obtained. The background in our experiments usually was 40 45 counts per minute. Under these conditions 1 cpm 14C corresponded to approximately 1.5 • 10-1~ mmole ~qAA-14C (-~ 0.28 • 10-4 ~g NAA), while 1 cpm aI-I corresponded to approximately 0.97 • 10-1~mmole NAA-3H ( = 0.18 • 10-5 ~g ~AA). To measure the degree of quenching the counts were recorded in two separate channels. The ratio of these two channels provided a rapid measure of correlation with counting effieiencies determined with the aid of an internal standard. A disadvantage of this method is that the precision of the channel ratio decreased with samples having a very low activity. Therefore quenched samples having a low activity were counted applying an internal standard according to WAnG and W~LIS (1965). The radioactivity in donor as well as in receiver blocks was measured by transferring these blocks into counting vials with 2 ml water. After shaking for a given period ethylalcohol and the scintillation liquid were added to the vials. The total amount of aeetonitrilc-soluble 14C and aH was recovered by extracting the tissue for several hours with hot acetonitrile. The final 12 ml acetonitrile was evaporated to precisely 5 ml. From this 5 ml a sample of 1 In] was pipetted into a counting vial. To this 1 ml sample, 2 ml water and 10 ml of the scintillation liquid were added. The sample was then counted in the spectrometer. The remaining 4 mi was evaporated to dryness and the residue was taken up in 0.5 ml acetonitrile and spotted onto thin-layer chromatograms. Results T h e s t u d y of a u x i n t r a n s p o r t i n p l a n t s e c t i o n s is g r e a t l y f a c i l i t a t e d b y u s i n g c o m p o u n d s l a b e l l e d w i t h r a d i o a c t i v e isotopes. I n t r a n s p o r t e x p e r i m e n t s u s e h a s b e e n m a d e m a i n l y of 1dO-labelled a u x i n s . E v e n w i t h t h e h i g h e s t possible specific a c t i v i t y a n d t h e m o s t s e n s i t i v e detection technique it remained difficult to carry out experiments with p h y s i o l o g i c a l (i.e., low) d o n o r c o n c e n t r a t i o n s . A c o n s i d e r a b l e i n c r e a s e in

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H. VEEN:

the specific activity can only be obtained b y using tritium-labelled compounds. However, particularly with tritiated compounds a radiation-induced self-decomposition m a y be expected. Therefore the establishment of the purity of the compounds has to be the initial step in any radiotracer experiment (WA~o and W~LLIS, 1965). The purity was checked b y means of thin-layer chromatography. NAA-14C appeared to be stable and showed n o radioehemical impurities. NAA-aH, however, showed a radiochemical artefact (1.5% of the total amount of the radioactivity chromatographed). Besides this contamination, the formation of a " t a i l " in front of the NAA-aH spot was observed. This " t a i l " formation is indicative of an exchange of tritium atoms with hydrogen of the solution during storage or with the solvent system during chromatography (see Fig. 4). Yet 90 per cent of the t e r m amount of radioactivity chromatographed was recovered as pure naphthylaeetie acid.

The E//ect el Di//erent Donor Concentrations on A u x i n Transport NAA-laC and NAA-SH were added at different concentrations to the donor blocks. After a transport period of 3 and 7 hours, respectively, the donor blocks and receiver blocks were removed and counted. The results are given in the same w a y as those of PIL]~T (PLainT, 1965). The initial counts in the donor blocks are represented as Do; D t and R t represent the radioactivity after a given time t in the donor and the receiver blocks, respectively. The difference between D O and D t is called the net loss from the donor (A D). This value is expressed as a percentage of the initial concentration (1). The radioactivity found in receiver blocks (Rt) is expressed as a percentage of the amount lost from donor (2). (1)

Do--Dt Do 9100

(2)

Rt Do--D--~" 100

The data of these experiments are given in Table 1. F r o m the results shown in Table I it m a y be concluded t h a t the amounts of radioactivity reaching the receiver blocks as well as the amounts lost from the donor blocks greatly depended on the auxin concentration in donors. The absolute amounts of radioactivity in receiver blocks increased with increasing donor concentrations, but the relative amounts (as a percentage of A D), decreased. The absolute loss from the donor blocks increased with increasing donor concentrations, but the relative loss (as a percentage of the initial concentration) decreased. So the uptake of the auxin in the tissue cannot be considered a simple diffusion process. Since in t h a t case the toss from donor blocks would be expected to be proportional to the initial concentration.

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Table 1. The relation between di/]erent concentrations o/lVAA in the donor blocks and the transport o/14C. Transport time 3 and 7 hours. Experiment carried out with NAA14C and ]VAA-aH. The data are given in counts per minute Concentration in donor blocks, ~z/r 0.1

0.7

0.9

7

8

90

100

aI~

I~C

aH

x*C

all

14C

aH

Transport time 3 hours Do 260 144 D3 156 101 AD 104 43 % olD 0 40 30 R3 44 4* % of AD 42 9*

2,095 1,587 508 24 151 30

1,391 1,075 316 23 31 10

18,219 14,025 4,194 23 388 9

19,286 15,463 3,823 20 69 2

225,571 201,426 24,237 ll 777 3

Transport time 7 hours DO 260 144 D7 117 84 AD 143 60 % of D o 55 42 R7 76 19 % of zJ D 53 32

2,095 1,230 865 41 194 22

1,391 773 618 44 106 17

18,219 12,311 5,908 32 2,543 43

19,286 13,533 5,753 30 306 5

225,571 162,397 63,174 28 4,276 7

Tracer

* n. sign. Percentage Recovery /rom Transport Experiments. LVCKWILL a n d LLOYD-JoNEs (1962) showed t h a t ultraviolet r a d i a t i o n h a d a distinct effect on the d e g r a d a t i o n of the N A A molecule, a n d resulted i n the loss of the carboxyl group. As the experiments presented here were carried out i n a d a r k i n c u b a t o r , such a loss of the 14C isotope was n o t to be expected. Nevertheless, i n a n u m b e r of experiments the recovery of the 14C was determined. To measure the percentage of the recovery of laC i n the t r a n s p o r t experiments the a m o u n t s of r a d i o a c t i v i t y in donor a n d receiver blocks as well as i n tissue extracts were counted. All d a t a on recovery of laC r a d i o a c t i v i t y v a r y between 90 a n d 105 per cent, with a m e a n value of 97 per cent. Therefore it was concluded t h a t all the r a d i o a c t i v i t y i n the tissue was acetonitrfle soluble a n d was i n fact extracted b y this compound. F u r t h e r m o r e no d e c a r b o x y l a t i o n occurs d u r i n g the first 5 hours, as stated before (VEEN, 1966). These recovery experiments are at variance with the results described i n a previous paper (VIXEN, 1966). This can be explained b y a different t r e a t m e n t of the acetonitrile extract. I n former experiments this e x t r a c t was evaporated to dryness a n d the residue was t a k e n u p i n 0.5 ml of acetonitrile. I t was f o u n d t h e n t h a t some of the r a d i o a c t i v i t y was deposited on the glass wall of the vial a n d did n o t redissolve in the acetonitrile. So a lower percentage of recovery (about 85 per cent) was found. 19

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286

H. VE~.~:

The E//ect o] Di//erent Donor Concentrations on Auxin Metabolism Earlier (VIsioN, 1966) the intense metabolic turnover of the auxin was studied. The time-dependency of the production of several compounds and the ratio between the compounds in extracts of different parts of the explant were investigated. The differences found were ascribed to different auxin levels in the tissue. I t was suggested t h a t the formation of the auxin complexes was greatly dependent on the concentration of the substrate (NAA) which probably acted as an inductor in the formation of adaptive enzymes. Three types of complexes

Fig. 2. P h o t o g r a p h of a K o d a k no screen X - r a y film, which h a d covered a thin-layer c h r o m a t o g r a m for a b o u t two weeks. I t shows the metabolic t u r n o v e r of N A A a t three different donor concentrations a n d after two t r a n s p o r t periods. Chrom. A Donor cone. 1 ~zlVIT r a n s p o r t period: 3 hours ]3 D o n o r cone. 1 ~M T r a n s p o r t period: 7 hours D D o n o r conc. 10 ~IE[ T r a n s p o r t period: 3 hours E D o n o r conc. 10 ~M T r a n s p o r t period: 7 hours F D o n o r conc. 100 ~ [ T r a n s p o r t period: 3 hours G D o n o r conc. 100 g.l~ T r a n s p o r t period: 7 hours C: a control r u n of the initial c o m p o u n d NAA-14C

have been reported in the literature, viz. a) conjugation with aspartic acid, b) esterification with glucose and e) hydroxylation which m a y be followed b y a glucoside formation ( K ~ X ~ T , 1961 and Z ~ K , 1962). To study the dependency of the production of the various compounds on the donor concentration the following experiment was earned out. After 3 and 7 hours, respectively, the transport experiments were finished and the metabolic turnover was studied. Fig. 2 shows the spots on the film obtained from tissue extracts made after these two transport periods and with three different donor concentrations. After removal of the film the radioactivity of different spots on the thin layer was quantitatively measured in the spectrometer. The

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287

Table 2. The relative amounts o] various products o / N A A metabolism at three di//erent donor concentrations and alter two different transport periods. NAA-14C was used as tracer compound D onor conc.

Time in hours

A

1 ~M 10 ~M 100 ~M

3 3 3

0 0 0

1 ~M

7

0

l0 ~M 100 ~M

7 7

0 0.7

B

C

0 3.2 8.1

0 3.9 9.3 1.6

11.5 15.7

24.6 23.2

D

E

F

0 1.8 3.0

98.6 86.0 75.0

1.4 3.8 3.8

2.3

88.1

5.0 4.2

50.0 49.5

7.8 8.1 4.0

activity present at different Rf-values is expressed as a percentage of the total chromatographed amount (Table 2). A previous publication (VEEN, 1966) gives a survey of the literature on different I~AA metabolites. In this publication it was suggested that the identities of the compounds arc as follows: Compound A ~-unknown, Rf-value: 0.07; B ~--fl-D-glucoside of 8-hydroxynaphthylacctic acid, l~f-value 0.15; C = naphthylacetie aspartic acid, Rf-value : 0.22 ; D = 8-hydroxynaphthylacetic acid, Rf-value : 0.36; E--~ naphthylacetic acid, l~f-valuc 0.64; F = naphthylacetylfi-D-glucose, Rf-value: 0.87. The data shown in Table 2 show that the amounts of various mctabolites increased with the donor concentration as well as with the transport period (see also Fig. 2). At the low donor concentration of 1 ~M, only c o m p o u n d F ( p r o b a b l y NAA-glucose) could be d e t e c t e d after 7 hours. A f t e r 24 hours t h e m e t a b o l i c conversion a t this low donor c o n c e n t r a t i o n is v e r y clear (Fig. 3). I t has been a l r e a d y described t h a t t h e f o r m a t i o n of c o m p o u n d F precedes t h e f o r m a t i o n of other complexes (V~E~, 1966). W i t h NAA-14C t h e a c c u r a c y of t h e m e t h o d becomes d o u b t f u l a t d o n o r c o n c e n t r a t i o n s below 1 ~5~ (about 200 c p m per a g a r block). W i t h t r i t i a t e d N A A , which h a d a m u c h higher specific a c t i v i t y , a concent r a t i o n as low as 0.1 ~M in t h e donor block was sufficient to give detectable a m o u n t s of r a d i o a c t i v i t y in t h e receiver blocks (see Table 1). W i t h t h e t r i t i a t e d c o m p o u n d t h e r e l a t i v e presence of t h e different complexes was m e a s u r e d in t h e s a m e w a y as described earlier for 14C. A c o m p a r i s o n of t h e p r o p e r t i e s of NAA-14C (carboxyl labelled) a n d NAA-~I-I (generally labelled) is e x p e c t e d to give a d d i t i o n a l i n f o r m a t i o n concerning t h e m e t a b o l i s m . Fig. 4 shows a film w i t h which a 3H chrom a t o g r a m was covered. The results o b t a i n e d from these e x p e r i m e n t s are in v e r y good a g r e e m e n t with those of t h e ~4C experiments. Therefore it m a y be concluded t h a t no other molecular s t r u c t u r e t h a n r a d i o a c t i v e N A A is i n v o l v e d in t h e f o r m a t i o n of the complexes. 19"

288

~-~. V E E N :

100

after

24

hours

._~ E L

2 c ou

75

T

I--

o

I

I

.5

1.0

Rf

Fig. 3. D i s t r i b u t i o n of r a d i o a c t i v i t y in a thin-layer e h r o m a t o g r a m of tissue e x t r a c t m a d e f r o m sections used for a t r a n s p o r t e x p e r i m e n t w i t h a donor concentration of 1 ~zM NAA-ZdC. T r a n s p o r t period: 24 hours

Fig. 4. P h o t o g r a p h of a K o d a k no screen X - r a y film, which h a d covered a n anthracene/silica gel 1/1 thin-layer c h r o m a t o g r a m for 6 days. I t shows the metabolic t u r n o v e r of ~ A A a t two different donor concentrations a n d after two t r a n s p o r t periods. Chrom. ~ Donor cone. 10 ~M T r a n s p o r t period: 3 hours I Donor cone. 10 ~]V[ T r a n s p o r t period: 7 hours K Donor cone. 100 ~M T r a n s p o r t period: 3 hours L D o n o r cone. 100 ~M T r a n s p o r t period: 7 hours J : a control r u n of the initial c o m p o u n d ~AA-SI-I

The lowest NAA-3H concentration tested was 0.1 ~M. E v e n in that case there is evidence of the formation of the compounds B and C after 7 hours (see Fig. 5).

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289

Earlier it was found (GORTEI~and V ~ , 1966) t h a t all radioactivity in the receiver blocks appeared to be in the form of NAA. I t was therefore apparent t h a t the metabolic products were not transported. The data presented here strongly suggest t h a t the immobilization capacity of the tissue is determined b y the auxin concentration in the tissue. An increased immobilization activity will decrease the amounts of free 57AA in the transport system. This causes a relative decrease of the transported amounts. I n the present paper it was shown t h a t

175 E

after

7 hours

~150

o u 125

T

100-

75-

oo

_--J

0

i

/

.5

1.o

Rf ~ig. 5. Distribution of r a d i o a c t i v i t y in a thin-layer c h r o m a t o g r a m of tissue e x t r a c t m a d e f r o m sections used for a t r a n s p o r t e x p e r i m e n t w i t h a donor concentration of 0.1 tzlV[ NAA-aH. T r a n s p o r t period: 7 hours

at low donor concentrations a relatively great deal of the activity was transported into receiver blocks and only a small part remained in the tissue. This phenomenon m a y now be ascribed to the low immobilization capacity in this particular case. The immobilization of the applied auxin strongly affected the auxin transport and could have a regulating effect on the free auxin content in plant tissue. I n the discussion more attention will be paid to the relationship between auxin transport and auxin metabolism.

Discussion Transport through tissue sections m a y take place in three different ways: 1. through the apoplast; 2. through the symplast; 3. through both. 19b

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290

H. V~E~:

The apoplast is the system of interconnected cell walls and intercellular spaces and is equivalent to the "free space". X y l e m vessels present in the sections are considered to be a p a r t of the apoplast, too. The symplast is the continuous living phase of the plant; it consists of cytoplasm connected from cell to cell b y plasmodesmata and includes phloem cells. The transport from donor to receiver block can be divided into three phases: uptake, transport, and secretion. The dependency of the uptake process on the auxin concentration in the donor block is indicative of the non-physical nature of this process.

I

syrnplast

f ~ ~--

~

apoplast epical side

=

7A

~-

~ basal side

Fig. 6. A diagram of the model for auxin transport in tissue sections

I t is suggested t h a t during the uptake some metabolic " s i t e s " are involved, the extent of which might form a limiting factor at high donor concentrations. F r o m our studies on the decrease with time of the radioactivity of the donor blocks (GogT~g and V ~ , 1966) an increased net loss from donor blocks was observed during the first few hours. Then, after about 3 - - 4 hours, the loss from donor blocks became less rapid. These data agree very well with T m ~ A ~ q ' s hypothesis t h a t the auxin enters the "free space" (apoplast) b y diffusion and then moves from the apoplast laterally into the symplast; see Fig. 6 (1) and (2). This latter process is an active uptake dependent on metabolism (THIMANN, 1964). The actual transport takes place within the symplast (3) and its mechanism is unknown. Protoplasmic streaming, bioelectrie potentials or a membrane transport m a y all be involved in this nonpolar transport. The transport from cell to cell m a y be supposed to take place in two different ways. One, in which the material moves into the cytoplasm of other cells without even entering the apoplast system, namely through the plasmodesmata (7]3) (see AgIsz et al., 1966). Another possibility is t h a t the activity is secreted from the symplast into the apoplast (6), and then diffuses through the apoplast to the next cell (7A) where it is t a k e n up again (2). At the moment it is impossible to weigh these two possibilities against one another. During the m o v e m e n t of auxin in the symplast (3) it is subjected to immobilization. I t was suggested before (V~,~,~, 1966) t h a t at in-

Auxin Transport and Auxin Metabolism in Coleus

291

creasing auxin concentration in the tissue a threshold was passed for enzyme induction. The present experiments do not show any evidence of such a threshold. Even at the lowest NAA concentration tested (0.1 ~M) the formation of complexes is evident. Yet the amount of immobilization is dependent on the substrate concentration, the transport period as well as the age of the tissue (unpublished results). A factor like gibberellie acid could perhaps decrease the amount of immobilization (FAzcG et al., 1960) while other factors m a y stimulate the reaction (5 and 4, resp. in l~ig. 6). The nature of the different immobilization products in the so-called auxin-pool was elaborately discussed in a previous paper ( V ~ , 1966). Finally there must be a secretion from the cells into the apoplast and from the apoplast a free diffusion into the receiver block (8). The relative amount of radioactivity which was secreted into receiver blocks decreased strongly with increasing amounts of A D. This points to some limiting factor in this secretion process. Arguments for such a factor are given b y H ~ T E L and L~OPOLD (1963). As the relative decrease of secretion is much stronger than the decrease of the uptake at increasing donor concentrations, it is assumed that, if the transport as a whole is saturated (ScoTT and JAC0BS, 1963), the secretion will be the limiting factor. One of the most characteristic properties of the transport of auxin is its polarity. Very recently GOLDSMITH (1966) stated t h a t an unequal distribution of radioactivity in basal and in apical receivers could be achieved if auxin moved acropetally only b y diffusion and could be recycled into the basal source again b y transport. I n our scheme this means an acropetal diffusion (7 A) in the apoplast and an active uptake in the symplast (2). I n her experiments GOLDSMITHcould not find any evidence of an aeropctal movement other than a diffusion. As we could not find a distinct acropetal transport ( G o ~ T ~ and V~z~, 1966) it is supposed t h a t in our experiments with donor blocks at the basal end recycling takes away all the auxin from the apoplast into the symplast. I n fact, the immobilization in the tissue near donor blocks is the same for acropetal or basipetal movement (unpublished results). I t is very well known t h a t older Coleus tissue shows a pronounced aeropetal movement (L~orOLD and Gc~gzcs~u 1953; NAQW and GogDo~, 1965). This is probably caused b y a decreased metabolic uptake or secretion of the anxin. This causes a decrease of the recycling and therefore an increase in the free auxin in the apoplast which can diffuse in acropetal direction. As GOLDSlVHTHstated, it is "impossible to distinguish between the uptake or exit from the ceils because inhibiting any one of a sequential series of steps required for transport from donor to receiver is very likely to decrease the net flux of auxin through prior steps". Therefore experiments with anti-auxins, like triiodobenzoic

292

H. VEEr:

acid (TIBA), do not give us a definite answer to the question whether secretion or uptake is inhibited. I n general there seems to be no differences in behaviour between Arena coleoptfles and Coleus sections, so we fully agree with GOLDSMITH'S concept of auxin transport (GOLDSMITH, 1966a and b). Yet the immobilization of I A A in Avena sections is divergent from the immobilization of NAA in Coleus (WI~TEU and TmMA~N, 1966). Still the phenomenon of polarity remains obscure. HEUTEL and LEOPOLD (1962 and 1963) discussed a model in which structures necessary for the secretion are distributed in a polar manner within the cell

at the plasma membrane. Movable particles -- like starch grains -would enhance the activity of the secretive structures. HEUTEL and LEOPOLD also suggested a carrier theory in which the auxin-glucose complex crosses the membrane. According to ZE~K (1964), IAA-glucose can be synthesized from IAA and uridinediphosphoglucose (UDPGIu). Pl~IDHAM (1965) assumes that in general nucleoside diphosphate sugars are involved in the biosynthesis of phenolic glycosides. It is therefore likely that also IqAA is esterified in accordance with the following reaction: NAA

-~ UDPGIu-+NAAGlu

~- UDP

Probably, UDPGIu is synthesized from glucose-l-phosphate (gluc-l-ph) and uridinetriphosphate (UTP). UTP is synthesized from adenosinetriphosphate and UDP: ATP Jr UDP-+UTP -~ ADP gluc-l-ph Jr UTP-+UDPGIu ~- PP The synthesis of the auxin-glucose complex is located at the inner side of the plasmalemma and involves glucose-l-phosphate. This" Cori ester" cannot be transported, because its permeability is extremely low (KuEcH, 1954). In this hypothesis transport of auxin depends on the presence of gincose-l-phosphate at the inner side of the outer membrane. This Cori ester can be found in great amounts in the amyloplasts surrounding the starch granule (BADENHUIZEN, 1959). /ks soon as the starch granules have been displaced downwards, there willbe a close contact between the basal membrane structure and the Cori ester present in the amyloplast. In the basal membrane the enzymatic potentialities are found to synthesize the auxin-glucose complex. The view of auxin transport presented here is based on the concepts of vAz~ ])ER WEIJ (1932), HEUTEL and LEOPOLD (1963), PILET (1953) and THIIVIAZ~N (1964). Apart from its role as a carrier, l~AA-glucose may be considered acting as a detoxieation product. The presence of this compound in plant

Auxin Transport and Auxin Metabolism in Coleus

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extracts was proved m a n y times b y KLXMBT and ZE~K (KLXm~T, 1961, 1964; ZE~K, 1962, 1964), in amounts which were m u c h higher t h a n could be expected on a basis of the carrier function. If in experiments with intact plants a clear auxin transport is observed, this m u s t be ascribed to the ability of the auxin to be transported in the phloem (symplast) or xylem (apoplast) (LITTLE and BLACKMAN, 1963). This non-specific auxin t r a n s p o r t is determined b y the "source-sink" relations in the plant. According to CRAFTS (1966) herbicide transport in plants is inherently tied to these two p a t h w a y s : phloem and xylem. I n sections a transport " f r o m source to s i n k " will n o t take place. Phloem as well as xylem vessels do not p l a y a role in the transport of auxins in sections (HERTEL and LEOPOLD, 1963; JACOBS, 1965). The observed polarity in these sections m u s t be ascribed to a specific p r o p e r t y of the p a r e n c h y m a t i c tissue. I t is still questionable whether transport of endogenous auxin "in v i v o " takes place in the vascular bundles or exclusively in parenchymatic tissue. I n recent studies evidence of a cytokinin and a gibberellin movem e n t in xylem have been found (cytokinins : LOEFFLE~ and v a n 0VE~BE]~K, 1964 ; gibberellins : McCo~B, 1964). As stated b y 1V[cCI~EADu (1966), the t y p e of experiment, presented here, is artificial in m a n y respects. One has to be v e r y careful in applying results obtained in experiments with sections to the auxin transport in the whole plant. A closer analysis is therefore needed of the auxin transport in the whole plant as compared with t h a t in tissue sections. The author wishes to express his gratitude to Dr. D. DE ZEEUWfor his hospitality at the Institute for Atomic Sciences in Agriculture at Wageningen. Thanks are also due to Dr. C. J. GO~TE~ for her interest and for supplying him with material, and to Miss M. I)E BRVXNfor her technical assistance. The help of Dr. H. K o ~ s in the preparation of the manuscript is acknowledged with thanks. Literature

ARISZ, W.H., and E.P. WI]~RSE~A: Symplasmatic long distance transport in Vallisneria plants investigated by means of autoradiograms. Proc. kon. ned. Akad. Wet. C 69, 223--241 (1966). BADE~VIZEN, 1~.P. : Chemistry and biology of the starch granule. ProtoplasmMogia 2 B 1--74 (1959). C~AFTS, A. S. : Relation between food and herbicide transport. In: Isotopes in weed research, p. 3--7. Proc. IAEA Syrup. Vienna 1966. FANG, S. C., J. B. BOU~K~.,V. L. STEVV,NS, and J. S. BUTTS:Influences of gibberellic acid on metabolism of indoleacetic acid, acetate, and glucose in roots of higher plants. Plant Physiol. 35, 251--255 (1960).

294

H. Vxx~:

GOLDSMITH, M. H. M. : Suspension of polarity by total inhibition of the basipetal transport. Plant Physiol. 41, 15--27 (1966). - - M a i n t e n a n c e of polarity of auxin movement by basipetal transport. Plant Physiol. 41, 749--754 (1966). GOI~TER,C]Y~. J. : Studies on abscission in explants of Coleus. Physiol. Plantarum (Copenh.) 17, 331--345 (1964). - - , and H. VEEN: Auxin transport in explants of Coleus. Plant Physiol. 41, 83--86 (1966). HERTEL, 1~., and A. C. L:EOPOLD: Auxintransport und Schwerkraft. Naturwissenschaften 49, 377--378 (1962). - - - - Versueh zur Analyse des Auxintransports in der Koleoptile yon Zea mays L. Planta (]3erl.) 59, 535--562 (1963). J~COBS, W. P. : Polar transport of IAA-I~C and 2,4-DlaC through pith and vascular tissue of Coleus 5 internodes. Plant Physiol. 40, Suppl. p. xxxiii (1965). KLs H. D. : Stoffwechselprodukte der Naphthyl-l-essigsaure und 2,4-Dichlorphenoxyessigsaure und der Vergleich mit jenen der Indol-3-essigs~ure und ]3enzoesaure. Planta (Berl.) 57, 339--353 (1961). - - D i e Identifizierung eines Stoffwechselproduktes der Naphthyl-l-essigsaure, alas mSglicherweise ]3ezieh~mg zur Waehstun~6rderung besitzt. PIanta (]3erl.) 57, 391--401 (1961). - - 2-Itydroxyindole-3-acetic acid and similar compounds in seeds and other plant parts. I n : R6gu]ateurs naturels de la Croissance v6g4tale: Fifth Internat. Conf. on Plant Growth Substances, p. 235---240. Paris: C.N.R.S. 1964. KR~c~, E. : ~ b e r die Phosphorylasen in hSheren Pflanzen. ]3eitr. Biol. Pflanzen 30, 379--405 (1954). L~OPOLD, A. C., and F. S. G~ER~SnY: Auxin polarity in the Coleus plant. ]3ot. Gaz. 115, 147--154 (1953). LXTTT,E,E. C. S., and G. E. ]3LACKM-4:~:Comparative studies of transport inPhaseolus vulgaris. New Phytologist 62, 173--197 (1963). LOEFrLER, J. E., and J. vA~ OVERBE:EK: K i n i n activity in coconut milk. R6gulateurs naturels de la Croissance v6g~tale: Fifth Internat. Conf. on Plant Growth Substances, p. 77--82. P~ris: C.N.R.S. 1964. L v c K w ~ , L.C., and C.P. LLOYD-Jo~s: The absorption, transtocation and metabolism of 1-naphthaleneacetic acid applied to apple leaves. J. Horticult. Sci. 37, 190--206 (1962). LiiT~i, U., and P. G. WAs]~: Low-temperature fluorography induced by tritiumlabelled compounds on thin-layer chromatograms. Nature (Lond.) 205, 1190-1191 (1965). )][CCREADY, C. C. : Translocation of growth regulators. Ann. Rev. Plant Physiol. 17, 283--294 (1966). McCoMB, A. J. : The stability and movement of gibberellic acid in pea seedlings. Ann. ]3ot. 28, 669--687 (1964). NAQVI, S. 1~r and S.A. GORDOn: Auxin transport in flowering and vegetative shoots of Coleus blumei ]3E~CTH. Plant Physiol. 40, 116--118 (1965). P~nT, P. E. : Essais d'interpr~tation du g~otropisme des racines du Lens culinaris Medikns. Bull. Soc. vaud. Sci. nat. 6~, 409--~21 (1953). - - Polar transport of radioactivity from ~4C-l~belled-fi-indolyt~cetic acid in stems of Lens eulinaris. Physiol. Plantarum (Copenh.) 18, 687--702 (1965). P~IDHA~, J. ]3. : Low molecular weight phenols in higher plants. Ann. Rev. Plant Physiol. 16, 13--36 (1965). SCOTT, T. K., and W. P. JAoo]~s: Auxin in Coleus stems; limitation of transport at higher concentrations. Science 1;~9, 589--590 (1963).

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TnZ~A~N, K. V. : Studies on the movement of auxin in tissues and its modification by gravity and light. In: R6gulateurs naturels de la Croissance v6g6tale: Fifth Internat. Conf. on Plant Growth Substances, p. 575--585. Paris: C.N.R.S. 1964. VEEZq, It.: Transport, immobilization and localization of naphthylacetic acid -1-1aC in Coleus explants. Acta bot. neerl. 15, 4 1 9 4 3 3 (1966). WANG, C.H., and D. L. WILLIS: Radiotracer methodology in biological science. Englewood Cliffs, New Yersey: Prentice-Hall, Inc. 1965. WEIJ, H. G., VAN DER: Der Meehanismus des Wuchsstofftransportes. Rec. Tray. bot. necrl. 29, 379--496 (1932). WI~TEg, A., and K. V. T~TMANN: Bound indoleacetie acid in Avena coleoptiles. Plant Physiol. 41, 335--342 (1966). ZENK, M . H . : Aufnahme und Stoffwechsel yon ~-Naphthyl-essigs~ure dutch Erbsenepicotyle. Planta (BEE.) 58, 75--94 (1962). - - Isolation, biosynthesis and function of indoleacetic acid conjugates. In: R6gulateurs naturels de la Croissanee v6g6talc: Fifth Intemat. Conf. on Plant Growth Substances, p. 241--249. Paris: C.N.R.S. 1964. Dr. H. VEEN Plant Physiological Research Centre 47 Bornsesteeg Wageningen, The Netherlands