Planta (Berl.) 127, 201--206 (1975) 9 by Springer-Verlag 1975
The Induction of Transport Channels by Auxin* Tsvi Sachs Department of Botany, The Hebrew University, Jerusalem, Israel Received 15 Juli; accepted 24 July 1975
Summary. This
work deals with measurements of the transport of radio-activity from
[3H] indole acetic acid during early stages of auxin-induced vascular differentiation. Transverse wounds were made in stems of Phaseolus vulgaris L. and the radioactivity which was transported around these wounds was measured. Treatments with unlabelled auxin above the wounds increased transport within 16 hours of the time they were first applied while the maturation of vascular elements in response to these treatments started only on the 3rd day. It is concluded that increased transport is an early stage of vascular differentiation, and this phenomenon can account for the organized differentiation of vascular elements in vertical rows and defined bundles.
Introduction Auxin causes vascular differentiation in a variety of systems (Snow, 1935; Camus, 1949; Jaeobs, 1952; Wangermann, 1967; Sachs, 1969). I t has been suggested that an early stage in auxin-induced vascular differentiation is an increase in polar auxin transport through the differentiating cells (Sachs, 1969, 1974, 1975). This hypothesis was based on the control of contact formation between strands; its possible importance is that a direct relation between transport and differentiation could account for the vertical arrangement of vascular elements of the same type (see Discussion) and could, therefore, be an important mechanism in the cellular interactions which are responsible for organized differentiation. Auxin transport can be studied directly, especially with methods using radioactive IAA (Goldsmith, 1969). The hypothesis that increased auxin transport is an early stage of vascular differentiation should therefore be tested. Published results show that auxin can be transported much more rapidly through intact plants than through sections in which the phloem is presumably non-functionM (Eschrich, 1968; Morris and Kadir, 1972; Morris etal., 1973; Goldsmith etal., 1974). Rapid transport is found only when auxin is applied to relatively mature leaves, and this might be due to a dependance of this transport on the movement of carbohydrates (Morris and Kadir, 1972 ; Morris et al., 1973). There is no available evidence, however, concerning the stage of differentiation at which auxin transport becomes relatively rapid, and it is therefore not possible to conclude that increased transport occurs early enough to determine the pattern of differentiation. I t is also known that the application of auxin increases auxin transport (Leopold and Lain, 1962 ; Hcrtel and Flory, 1968; Rayle et al., 1969; Osborne and Mullins, 1969). This result is exactly that which would be expected if increased transport were an early stage in auxin-induced differentiation. The available work, however, * Abbreviation: IAA = indole acetic acid. 1 Planta (BerL)
202
T. Sachs
can also be i n t e r p r e t e d to m e a n t h a t a u x i n t r e a t m e n t s only m a i n t a i n the a c t i v i t y of t r a n s p o r t channels which are present at the time the e x p e r i m e n t is started, either for a long t e r m (Leopold a n d Lain, 1962) Or a short t e r m (Hertel a n d Flory, 1968), rather t h a n causing the differentiation of new channels. The e x p e r i m e n t presented below, therefore, is a direct a t t e m p t to test whether a u x i n t r e a t m e n t can cause the d e v e l o p m e n t of increased t r a n s p o r t capacity before it causes the differentiation of a n y m a t u r e vascular elements.
Materials and Methods
Phaseolus vulgaris L. plants were grown from seeds supplied by W. Atlee Burpee Co. The seeds were soaked over-night and then planted in vermiculite in a growth chamber at a constant temperature of 25~ and 16 h of light. Well water with no added nutrients was used throughout. The plants were used when the first leaves were in their final stage of expansion, about one week after the seeds were planted. Sections composed of parts of the hypocotyl, the epicotyl and one cotyledon were cut as shown in Fig. 1. The sections were placed in closed petri dishes with moist filter paper, sealed with parafilm. These petri dishes were kept in the same growth chamber in which the plants were grown. The sections were wounded to cause transverse transport (Fig. 1) and were treated above this wound with lanolin or lanolin with unlabelled IAA. Half an hour before the application of radioactive material the lanolin (or lanolin with IAA) was removed by a slanted cut (Fig. 1). Control wounds and the preparation of sections from intact plants were performed at this time. The surface of the cuts where the agar with radioactive IAA was to be applied was kept moist with well water for half an hour, to prevent water tensions from affecting the transport of radioactive materials. [5-3H] IAA (28.103 Ci/mmole; Schwarz-Mann, Orangeburg, N.Y., U.S.A.) was applied at a concentration of 3-10-7 M in 3 X 2 X 1 mm blocks of 1.5% agar. The agar was left on the plants for only 12 rain. Transport in the plants was allowed for 3 h (includingthe initial 12 rain). The plants were then cut along the cotyledonary node and the cotyledon was discarded. The epicotyl and hypocotyl were placed separately in Aquasol scintillation fluid and kept cold for a few days before being counted for 10 min. Cpm was used directly in Table 1, since all the treatments were the same in terms of the quantity of tissue and its pigment content and the conclusions are based only on a comparison of the different treatments.
Results and Discussion The work reported here was performed on stem sections of beans with one cotyledon which supplied the n u t r i e n t s necessary for d e v e l o p m e n t (Fig. 1). Sections left i n petri dishes for 3 days regenerated new roots at their basal ends a n d the buds i n the axil of the cotyledon grew, so t h a t the sections could be readily converted to n e w plants. To d e m o n s t r a t e the formation of new t r a n s p o r t channels it was necessary to find conditions i n which t r a n s p o r t rate or capacity is increased b y a u x i n t r e a t m e n t s b e y o n d t h a t f o u n d i n intact, u n t r e a t e d controls. Maximal t r a n s p o r t rate is k n o w n to be m u c h slower i n sections t h a n i n i n t a c t p l a n t s (Goldsmith st al., 1974) a n d it therefore appeared unlikely t h a t this requirem e n t could be fulfilled concerning t r a n s p o r t along the vertical axis of the plant, The e x p e r i m e n t a l system was therefore designed to test t r a n s p o r t in a transverse direction, in which the n o r m a l channels were n o t expected to be developed in i n t a c t plants. The diversion of t r a n s p o r t towards this transverse axis was achieved b y a deep w o u n d (Fig. 1), a n d the t r e a t m e n t s a n d m e a s u r e m e n t s were m a d e i n such a way so t h a t all factors other t h a n the t r a n s p o r t above this w o u n d could
Induction of Transport Channels by Auxin
203
Fig. 1. Schematic representation of experimental material cut from bean plants. The radioactivity in the hypoeotyl provided a measure of the transverse transport above the wound. The stems above the cotyledon were 1.5-2.5 m m in diameter, the depth of the wound was 2/a of this diameter. The arrow marks the region to which lanolin or 0.02% IAA in lanolin was applied. After 16 h these lanolin treatments were removed b y a cut along the dotted line. This cut did not quite reach the transverse wound, so t h a t a narrow region of euticte prevented the movement of water on the surface of the plant. The agar with the radioactive IAA was placed on this fresh cut, which was just deep enough to reach the vascular tissues
Table 1. Influence of a u x i n p r e t r e a t m e n t on transport around a wound Treatment
Percent radioactivity transported (hypocotyl/epicotyl. 100)
Average epm, epicotyl
Average cpm, hypocotyl
1. Cut from intact plants; wounded late
2.7-L 0.1
4689
125
2. Lanolin treated; wounded late
3.2-L 0.2
5451
17'7
3. Lanolin treated; wounded early
2 . 6 • 0.1
4378
113
4. Auxin treated; wounded late
2.8 4- 0.3
4498
126
5. Auxin treated; wounded early
4.9 • 0.4
4:254
207
:For experimental system, see :Fig. 1. The transverse wound was performed either late, 0.5 h before the application of the radioactivity, or 16 h earlier, a t the time the material was cut from the plants. Auxin t r e a t m e n t consisted of 0.02% IAA in lanolin and the radioactive auxin applied to all plants was 3 • 10.7 M [aH]IAA in agar. There were 8 plants per t r e a t m e n t and the percent t r a n s p o r t is given with the standard error of the mean.
b e e x p e c t e d t o b e e q u a l . T h e i n d u c t i o n of n e w t r a n s p o r t c h a n n e l s a b o v e t h e w o u n d w a s a c h i e v e d b y a n a p p l i c a t i o n of 0.02 % I A A i n l a n o l i n , a n d t h i s p r e p a r a tion was removed only a short time before it was replaced by radioactive IAA in a g a r f o r t r a n s p o r t m e a s u r e m e n t s (Fig. 1). T a b l e 1 s h o w s t h e a m o u n t of r a d i o a c t i v i t y c o m i n g f r o m a 3" l 0 -7 M [3H] I A A which was transported transversely above the wound and down to the hypoeotyl.
204
T. Sachs
The differences in the radioactivity which reached the hypocotyl reflect differences in rate or capacity of transport above the wound. I n intact plants transverse transport may be expected to take place through parenchyma and through phloem anastomoses (Aloni and Sachs, 1973), and a measure of this transport in freshly cut sections is given by the first treatment. Treatments 2 and 3 show that the transverse transport did not change greatly when the sections were prepared 16 h before the measurements were taken, regardless of whether the wounds were made when the sections were cut or just before the radioactive IAA was applied. Auxin treatment increased transport significantly and this effect of auxin was not due to the mere presence of auxin but required the presence of the wound as well (compare treatments 4 and 5). I t may be concluded, therefore, that auxin changes the cells above the wound within 16 h so that a signi/icant increase in the transport o/radioactivity could be measured. Similar experiments were performed in which the sections were left in the petri dishes for either shorter periods (8 h) or longer periods (20-30 h and 48 h). I n the short experiments no significant difference was found between the different treatments. I n the longer periods the effect of auxin seen in Table 1 was found repeatedly. The variation between experiments, however, was too great to establish a clear relation between the length of the period of treatment and the effect of auxin on transport. The results did indicate that an increase of the effect after 16 h, if it is present at all, is not very great. At periods of 24 h or longer, however, an increase of transport of radioactive material was found above wounds which were treated with lanolin with no IAA. This increase, which was also found repeatedly, was not as great as that found after auxin treatments. Within 3 days after the wounds were made it was possible to observe mature vessels and sieve tubes, which differentiated parallel to the upper surface of the wound. When auxin was not supplied above the wounds this differentiation was very limited and at most 3 transverse vessels were found above the wound on each side of the stem. The radioactivity which was measured in the hypocotyls was not chemically characterized. Considering the low concentration and short period of transport it appears highly likely that at least most of this radioactivity is of auxin molecules, as shown by the work of Goldsmith et al. (1974) on the transport of the same auxin. The main point which is important for the conclusions reached below, furthermore, is that a transport capacity of the plant was demonstrated and not the exact nature of the transported material. The central conclusion from the experiment presented above is that auxin induces an increase of a directional transport long before the differentiation of new vascular elements is completed. Using fluorescent dyes, Eschrich (1953) was also able to demonstrate a channelling of transport around a wound after phloem differentiation was seen to start but was definitely not completed. Eschrich did not demonstrate that the regeneration he observed around wounds was induced by auxin, but the work of Jacobs (1952) and LaMotte and Jacobs (1963) clearly indicates that the vascular regeneration was limited by auxin coming from the leaves above the wound. I t may therefore be concluded that increased transport capacity is an early stage of vascular differentiation. I t is known that a major differentiation stimulus coming from maturing leaves is auxin (Snow,
Induction of Transport Channels by Auxin
205
1935; Camus, 1949; Jacobs, 1952; W a n g e r m a n n , 1967) a n d t h e work p r e s e n t e d here d e m o n s t r a t e s t h a t t h e t r a n s p o r t of either a u x i n or its p r o d u c t s is increased during e a r l y stages of v a s c u l a r differentiation. I t follows t h a t when a cell s t a r t s to differentiate it d r a i n s stimuli for differentiation a w a y from a d j a c e n t cells a n d supplies t h e n in r e l a t i v e l y large q u a n t i t i e s to t h e cells below. This means t h a t t h e first cells to s t a r t differentiating i n h i b i t t h e l a t e r a l neighbors from differentiating in t h e same w a y a n d induce t h e cells i m m e d i a t e l y below t h e m t o become v a s c u l a r elements. T h e coupling b e t w e e n increased t r a n s p o r t c a p a b i l i t y a n d d i f f e r e n t i a t i o n is therefore a m e c h a n i s m for t h e control of t h e relations b e t w e e n differentiating cells, causing v a s c u l a r elements to be a r r a n g e d in v e r t i c a l rows a n d defined bundles (Sachs, 1969, 1975). A u x i n n o t only causes t h e f o r m a t i o n of new t r a n s p o r t channels, as was shown above, b u t is also necessary for t h e m a i n t e n a n c e of existing channels (Leopold a n d Lain, 1962). A s t r e a m of t r a n s p o r t e d a u x i n is therefore required n o t o n l y during t h e entire period of v a s c u l a r differentiation (Sachs, 1974) b u t also for t h e m a i n t e n a n c e of t h e i n t e g r i t y of t h e p h l o e m p a r t of t h e v a s c u l a r system. This t r a n s p o r t of differentiation stimuli t h r o u g h m a t u r e tissues regulates c a m b i a l a c t i v i t y a n d v a s c u l a r differentiation in dicotyledons so t h a t it d e p e n d s n o t only oft t h e p r o d u c t i o n of stimuli b y leaves b u t also on t h e c a p a c i t y of t h e available f u n c t i o n a l p h l o e m ( B e n a y o u n et al., 1975). I wish to thank Prof. I. M. Sussex, in whose laboratory at Yale University most of this work was performed, for his help and advice. Thanks are due to Prof. M. H. M. Goldsmith for essential advice, stimulating discussions, help with the manuscript and the supply of the radioactive IAA. I also thank Prof. L. I~einhold and Dr. E. Wangermann for advice concerning the manuscript. References Aloni, t~., Sachs, T.: The three-dimensional structure of primary phloem systems. Planta (Berl.) 113, 345-353 (1973) Benayoun, J., Aloni, R., Sachs, T. : l~egeneration around wounds and the control of vascular differentiation. Ann. Bot. 39, 447-454 (1975) Camus, G.: Reeherches sur le r61e des bourgeons dans los ph6nom~nes de morphogen~se. Rev. Cytol. Biol. V~g. 11, 1-199 (1949) Eschrich, W. : Beitri~ge zur Kenntnis der Wundsiebr6hrenentwicklung bei Impatiens holsti. Planta (Berl.) 43, 37-74 (1953) Eschrich, W. : Translokation radioaktiver markierter Indolyl-3-essigss in Siebr5hren yon Vicia/aba. Planta (Berl.) 78, 144 157 (1968) Goldsmith, M. H. M. : Transport of plant growth regulators. In: Physiology of plant growth and development, (M. B. Wilkins, ed.), p. 127-162. London: McGraw Hill 1969 Goldsmith, M. H. M., Cataldo, D. A., Karn, J., Brenneman, T., Trip, P. : The rapid non-polar transport of auxin in the phloem of intact Coleus plants. Planta (Berl.) 116, 301-317 (1974) tIertel, I~., Flory, 1~. : Auxin movement in corn eoleoptites. Planta (Berl.) 82, 123-144 (1968) Jacobs, W. P. : The role of auxin in the differentiation of xylem around a wound. Amer. J. Bot. 39, 301-309 (1952) LaMotte, C. E., Jacobs, W. P. : A role of auxin in phloem regeneration in Coleus internodes. Develop. Biol. 8, 80-98 (1963) Leopold, A. C., Lain, S. L.: The auxin transport gradient. Physiol. Plantarum (Copenh.) 15, 631-638 (1962) Morris, D. A., Kadir, G. O. : Pathways of auxin transport in the intact pea seedling (Pisum sativum L.) Planta (Berl.) 107, 171-182 (1972)
206
T. Sachs
Norris, D. A., Kadir, G. O., Barry, A. J.: Auxin transport in intact pea seedlings (Pisum sativum L.): The inhibition of transport by 2,3,5-triiodobenzoic acid. Planta (Berl.) l l 0 , 173-182 (1973) Osborne, D., Mullins, M. G. : Auxin, ethylene and kinetin in a carrier-protein model system for the polar transport of auxins in petiole segments of Phaseolus vulgaris. New Phytol. 68, 977-991 (1969) Rayle, D. L., Ouitrakul, 1~., Hertel, 1~. : Effects of auxins on the auxin transport system in coleoptiles. Planta (Berl.) 87, 49-53 (1969) Sachs, T. : Polarity and the induction of organized vascular tissues. Ann. Bot. 88, 263-275 (1969) Sachs, T. : The induction of vessel differentiation by auxin. In: Plant growth substances 1973. Proc. 8th Int. Conf. Plant Growth Substances, p. 900-906. Tokyo: Hirokawa 1974 Sachs, T. : The control of differentiation of vascular networks. Ann. Bot. 89, 197-204 (1975) Snow, R. : Activation of cambial growth by pure hormones. New Phytol. 84, 347-360 (1935) Wangermann, E. : The effect of the leaf on differentiation of primary xylem in the inteI~ode of Coleus blumei Benth. New Phytol. 66, 747-754 (1967)
The Induction of Transport Channels by Auxin* Tsvi Sachs Department of Botany, The Hebrew University, Jerusalem, Israel Received 15 Juli; accepted 24 July 1975
Summary. This
work deals with measurements of the transport of radio-activity from
[3H] indole acetic acid during early stages of auxin-induced vascular differentiation. Transverse wounds were made in stems of Phaseolus vulgaris L. and the radioactivity which was transported around these wounds was measured. Treatments with unlabelled auxin above the wounds increased transport within 16 hours of the time they were first applied while the maturation of vascular elements in response to these treatments started only on the 3rd day. It is concluded that increased transport is an early stage of vascular differentiation, and this phenomenon can account for the organized differentiation of vascular elements in vertical rows and defined bundles.
Introduction Auxin causes vascular differentiation in a variety of systems (Snow, 1935; Camus, 1949; Jaeobs, 1952; Wangermann, 1967; Sachs, 1969). I t has been suggested that an early stage in auxin-induced vascular differentiation is an increase in polar auxin transport through the differentiating cells (Sachs, 1969, 1974, 1975). This hypothesis was based on the control of contact formation between strands; its possible importance is that a direct relation between transport and differentiation could account for the vertical arrangement of vascular elements of the same type (see Discussion) and could, therefore, be an important mechanism in the cellular interactions which are responsible for organized differentiation. Auxin transport can be studied directly, especially with methods using radioactive IAA (Goldsmith, 1969). The hypothesis that increased auxin transport is an early stage of vascular differentiation should therefore be tested. Published results show that auxin can be transported much more rapidly through intact plants than through sections in which the phloem is presumably non-functionM (Eschrich, 1968; Morris and Kadir, 1972; Morris etal., 1973; Goldsmith etal., 1974). Rapid transport is found only when auxin is applied to relatively mature leaves, and this might be due to a dependance of this transport on the movement of carbohydrates (Morris and Kadir, 1972 ; Morris et al., 1973). There is no available evidence, however, concerning the stage of differentiation at which auxin transport becomes relatively rapid, and it is therefore not possible to conclude that increased transport occurs early enough to determine the pattern of differentiation. I t is also known that the application of auxin increases auxin transport (Leopold and Lain, 1962 ; Hcrtel and Flory, 1968; Rayle et al., 1969; Osborne and Mullins, 1969). This result is exactly that which would be expected if increased transport were an early stage in auxin-induced differentiation. The available work, however, * Abbreviation: IAA = indole acetic acid. 1 Planta (BerL)
202
T. Sachs
can also be i n t e r p r e t e d to m e a n t h a t a u x i n t r e a t m e n t s only m a i n t a i n the a c t i v i t y of t r a n s p o r t channels which are present at the time the e x p e r i m e n t is started, either for a long t e r m (Leopold a n d Lain, 1962) Or a short t e r m (Hertel a n d Flory, 1968), rather t h a n causing the differentiation of new channels. The e x p e r i m e n t presented below, therefore, is a direct a t t e m p t to test whether a u x i n t r e a t m e n t can cause the d e v e l o p m e n t of increased t r a n s p o r t capacity before it causes the differentiation of a n y m a t u r e vascular elements.
Materials and Methods
Phaseolus vulgaris L. plants were grown from seeds supplied by W. Atlee Burpee Co. The seeds were soaked over-night and then planted in vermiculite in a growth chamber at a constant temperature of 25~ and 16 h of light. Well water with no added nutrients was used throughout. The plants were used when the first leaves were in their final stage of expansion, about one week after the seeds were planted. Sections composed of parts of the hypocotyl, the epicotyl and one cotyledon were cut as shown in Fig. 1. The sections were placed in closed petri dishes with moist filter paper, sealed with parafilm. These petri dishes were kept in the same growth chamber in which the plants were grown. The sections were wounded to cause transverse transport (Fig. 1) and were treated above this wound with lanolin or lanolin with unlabelled IAA. Half an hour before the application of radioactive material the lanolin (or lanolin with IAA) was removed by a slanted cut (Fig. 1). Control wounds and the preparation of sections from intact plants were performed at this time. The surface of the cuts where the agar with radioactive IAA was to be applied was kept moist with well water for half an hour, to prevent water tensions from affecting the transport of radioactive materials. [5-3H] IAA (28.103 Ci/mmole; Schwarz-Mann, Orangeburg, N.Y., U.S.A.) was applied at a concentration of 3-10-7 M in 3 X 2 X 1 mm blocks of 1.5% agar. The agar was left on the plants for only 12 rain. Transport in the plants was allowed for 3 h (includingthe initial 12 rain). The plants were then cut along the cotyledonary node and the cotyledon was discarded. The epicotyl and hypocotyl were placed separately in Aquasol scintillation fluid and kept cold for a few days before being counted for 10 min. Cpm was used directly in Table 1, since all the treatments were the same in terms of the quantity of tissue and its pigment content and the conclusions are based only on a comparison of the different treatments.
Results and Discussion The work reported here was performed on stem sections of beans with one cotyledon which supplied the n u t r i e n t s necessary for d e v e l o p m e n t (Fig. 1). Sections left i n petri dishes for 3 days regenerated new roots at their basal ends a n d the buds i n the axil of the cotyledon grew, so t h a t the sections could be readily converted to n e w plants. To d e m o n s t r a t e the formation of new t r a n s p o r t channels it was necessary to find conditions i n which t r a n s p o r t rate or capacity is increased b y a u x i n t r e a t m e n t s b e y o n d t h a t f o u n d i n intact, u n t r e a t e d controls. Maximal t r a n s p o r t rate is k n o w n to be m u c h slower i n sections t h a n i n i n t a c t p l a n t s (Goldsmith st al., 1974) a n d it therefore appeared unlikely t h a t this requirem e n t could be fulfilled concerning t r a n s p o r t along the vertical axis of the plant, The e x p e r i m e n t a l system was therefore designed to test t r a n s p o r t in a transverse direction, in which the n o r m a l channels were n o t expected to be developed in i n t a c t plants. The diversion of t r a n s p o r t towards this transverse axis was achieved b y a deep w o u n d (Fig. 1), a n d the t r e a t m e n t s a n d m e a s u r e m e n t s were m a d e i n such a way so t h a t all factors other t h a n the t r a n s p o r t above this w o u n d could
Induction of Transport Channels by Auxin
203
Fig. 1. Schematic representation of experimental material cut from bean plants. The radioactivity in the hypoeotyl provided a measure of the transverse transport above the wound. The stems above the cotyledon were 1.5-2.5 m m in diameter, the depth of the wound was 2/a of this diameter. The arrow marks the region to which lanolin or 0.02% IAA in lanolin was applied. After 16 h these lanolin treatments were removed b y a cut along the dotted line. This cut did not quite reach the transverse wound, so t h a t a narrow region of euticte prevented the movement of water on the surface of the plant. The agar with the radioactive IAA was placed on this fresh cut, which was just deep enough to reach the vascular tissues
Table 1. Influence of a u x i n p r e t r e a t m e n t on transport around a wound Treatment
Percent radioactivity transported (hypocotyl/epicotyl. 100)
Average epm, epicotyl
Average cpm, hypocotyl
1. Cut from intact plants; wounded late
2.7-L 0.1
4689
125
2. Lanolin treated; wounded late
3.2-L 0.2
5451
17'7
3. Lanolin treated; wounded early
2 . 6 • 0.1
4378
113
4. Auxin treated; wounded late
2.8 4- 0.3
4498
126
5. Auxin treated; wounded early
4.9 • 0.4
4:254
207
:For experimental system, see :Fig. 1. The transverse wound was performed either late, 0.5 h before the application of the radioactivity, or 16 h earlier, a t the time the material was cut from the plants. Auxin t r e a t m e n t consisted of 0.02% IAA in lanolin and the radioactive auxin applied to all plants was 3 • 10.7 M [aH]IAA in agar. There were 8 plants per t r e a t m e n t and the percent t r a n s p o r t is given with the standard error of the mean.
b e e x p e c t e d t o b e e q u a l . T h e i n d u c t i o n of n e w t r a n s p o r t c h a n n e l s a b o v e t h e w o u n d w a s a c h i e v e d b y a n a p p l i c a t i o n of 0.02 % I A A i n l a n o l i n , a n d t h i s p r e p a r a tion was removed only a short time before it was replaced by radioactive IAA in a g a r f o r t r a n s p o r t m e a s u r e m e n t s (Fig. 1). T a b l e 1 s h o w s t h e a m o u n t of r a d i o a c t i v i t y c o m i n g f r o m a 3" l 0 -7 M [3H] I A A which was transported transversely above the wound and down to the hypoeotyl.
204
T. Sachs
The differences in the radioactivity which reached the hypocotyl reflect differences in rate or capacity of transport above the wound. I n intact plants transverse transport may be expected to take place through parenchyma and through phloem anastomoses (Aloni and Sachs, 1973), and a measure of this transport in freshly cut sections is given by the first treatment. Treatments 2 and 3 show that the transverse transport did not change greatly when the sections were prepared 16 h before the measurements were taken, regardless of whether the wounds were made when the sections were cut or just before the radioactive IAA was applied. Auxin treatment increased transport significantly and this effect of auxin was not due to the mere presence of auxin but required the presence of the wound as well (compare treatments 4 and 5). I t may be concluded, therefore, that auxin changes the cells above the wound within 16 h so that a signi/icant increase in the transport o/radioactivity could be measured. Similar experiments were performed in which the sections were left in the petri dishes for either shorter periods (8 h) or longer periods (20-30 h and 48 h). I n the short experiments no significant difference was found between the different treatments. I n the longer periods the effect of auxin seen in Table 1 was found repeatedly. The variation between experiments, however, was too great to establish a clear relation between the length of the period of treatment and the effect of auxin on transport. The results did indicate that an increase of the effect after 16 h, if it is present at all, is not very great. At periods of 24 h or longer, however, an increase of transport of radioactive material was found above wounds which were treated with lanolin with no IAA. This increase, which was also found repeatedly, was not as great as that found after auxin treatments. Within 3 days after the wounds were made it was possible to observe mature vessels and sieve tubes, which differentiated parallel to the upper surface of the wound. When auxin was not supplied above the wounds this differentiation was very limited and at most 3 transverse vessels were found above the wound on each side of the stem. The radioactivity which was measured in the hypocotyls was not chemically characterized. Considering the low concentration and short period of transport it appears highly likely that at least most of this radioactivity is of auxin molecules, as shown by the work of Goldsmith et al. (1974) on the transport of the same auxin. The main point which is important for the conclusions reached below, furthermore, is that a transport capacity of the plant was demonstrated and not the exact nature of the transported material. The central conclusion from the experiment presented above is that auxin induces an increase of a directional transport long before the differentiation of new vascular elements is completed. Using fluorescent dyes, Eschrich (1953) was also able to demonstrate a channelling of transport around a wound after phloem differentiation was seen to start but was definitely not completed. Eschrich did not demonstrate that the regeneration he observed around wounds was induced by auxin, but the work of Jacobs (1952) and LaMotte and Jacobs (1963) clearly indicates that the vascular regeneration was limited by auxin coming from the leaves above the wound. I t may therefore be concluded that increased transport capacity is an early stage of vascular differentiation. I t is known that a major differentiation stimulus coming from maturing leaves is auxin (Snow,
Induction of Transport Channels by Auxin
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1935; Camus, 1949; Jacobs, 1952; W a n g e r m a n n , 1967) a n d t h e work p r e s e n t e d here d e m o n s t r a t e s t h a t t h e t r a n s p o r t of either a u x i n or its p r o d u c t s is increased during e a r l y stages of v a s c u l a r differentiation. I t follows t h a t when a cell s t a r t s to differentiate it d r a i n s stimuli for differentiation a w a y from a d j a c e n t cells a n d supplies t h e n in r e l a t i v e l y large q u a n t i t i e s to t h e cells below. This means t h a t t h e first cells to s t a r t differentiating i n h i b i t t h e l a t e r a l neighbors from differentiating in t h e same w a y a n d induce t h e cells i m m e d i a t e l y below t h e m t o become v a s c u l a r elements. T h e coupling b e t w e e n increased t r a n s p o r t c a p a b i l i t y a n d d i f f e r e n t i a t i o n is therefore a m e c h a n i s m for t h e control of t h e relations b e t w e e n differentiating cells, causing v a s c u l a r elements to be a r r a n g e d in v e r t i c a l rows a n d defined bundles (Sachs, 1969, 1975). A u x i n n o t only causes t h e f o r m a t i o n of new t r a n s p o r t channels, as was shown above, b u t is also necessary for t h e m a i n t e n a n c e of existing channels (Leopold a n d Lain, 1962). A s t r e a m of t r a n s p o r t e d a u x i n is therefore required n o t o n l y during t h e entire period of v a s c u l a r differentiation (Sachs, 1974) b u t also for t h e m a i n t e n a n c e of t h e i n t e g r i t y of t h e p h l o e m p a r t of t h e v a s c u l a r system. This t r a n s p o r t of differentiation stimuli t h r o u g h m a t u r e tissues regulates c a m b i a l a c t i v i t y a n d v a s c u l a r differentiation in dicotyledons so t h a t it d e p e n d s n o t only oft t h e p r o d u c t i o n of stimuli b y leaves b u t also on t h e c a p a c i t y of t h e available f u n c t i o n a l p h l o e m ( B e n a y o u n et al., 1975). I wish to thank Prof. I. M. Sussex, in whose laboratory at Yale University most of this work was performed, for his help and advice. Thanks are due to Prof. M. H. M. Goldsmith for essential advice, stimulating discussions, help with the manuscript and the supply of the radioactive IAA. I also thank Prof. L. I~einhold and Dr. E. Wangermann for advice concerning the manuscript. References Aloni, t~., Sachs, T.: The three-dimensional structure of primary phloem systems. Planta (Berl.) 113, 345-353 (1973) Benayoun, J., Aloni, R., Sachs, T. : l~egeneration around wounds and the control of vascular differentiation. Ann. Bot. 39, 447-454 (1975) Camus, G.: Reeherches sur le r61e des bourgeons dans los ph6nom~nes de morphogen~se. Rev. Cytol. Biol. V~g. 11, 1-199 (1949) Eschrich, W. : Beitri~ge zur Kenntnis der Wundsiebr6hrenentwicklung bei Impatiens holsti. Planta (Berl.) 43, 37-74 (1953) Eschrich, W. : Translokation radioaktiver markierter Indolyl-3-essigss in Siebr5hren yon Vicia/aba. Planta (Berl.) 78, 144 157 (1968) Goldsmith, M. H. M. : Transport of plant growth regulators. In: Physiology of plant growth and development, (M. B. Wilkins, ed.), p. 127-162. London: McGraw Hill 1969 Goldsmith, M. H. M., Cataldo, D. A., Karn, J., Brenneman, T., Trip, P. : The rapid non-polar transport of auxin in the phloem of intact Coleus plants. Planta (Berl.) 116, 301-317 (1974) tIertel, I~., Flory, 1~. : Auxin movement in corn eoleoptites. Planta (Berl.) 82, 123-144 (1968) Jacobs, W. P. : The role of auxin in the differentiation of xylem around a wound. Amer. J. Bot. 39, 301-309 (1952) LaMotte, C. E., Jacobs, W. P. : A role of auxin in phloem regeneration in Coleus internodes. Develop. Biol. 8, 80-98 (1963) Leopold, A. C., Lain, S. L.: The auxin transport gradient. Physiol. Plantarum (Copenh.) 15, 631-638 (1962) Morris, D. A., Kadir, G. O. : Pathways of auxin transport in the intact pea seedling (Pisum sativum L.) Planta (Berl.) 107, 171-182 (1972)
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Norris, D. A., Kadir, G. O., Barry, A. J.: Auxin transport in intact pea seedlings (Pisum sativum L.): The inhibition of transport by 2,3,5-triiodobenzoic acid. Planta (Berl.) l l 0 , 173-182 (1973) Osborne, D., Mullins, M. G. : Auxin, ethylene and kinetin in a carrier-protein model system for the polar transport of auxins in petiole segments of Phaseolus vulgaris. New Phytol. 68, 977-991 (1969) Rayle, D. L., Ouitrakul, 1~., Hertel, 1~. : Effects of auxins on the auxin transport system in coleoptiles. Planta (Berl.) 87, 49-53 (1969) Sachs, T. : Polarity and the induction of organized vascular tissues. Ann. Bot. 88, 263-275 (1969) Sachs, T. : The induction of vessel differentiation by auxin. In: Plant growth substances 1973. Proc. 8th Int. Conf. Plant Growth Substances, p. 900-906. Tokyo: Hirokawa 1974 Sachs, T. : The control of differentiation of vascular networks. Ann. Bot. 89, 197-204 (1975) Snow, R. : Activation of cambial growth by pure hormones. New Phytol. 84, 347-360 (1935) Wangermann, E. : The effect of the leaf on differentiation of primary xylem in the inteI~ode of Coleus blumei Benth. New Phytol. 66, 747-754 (1967)
