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Importance of the Tip on the (5-3H)-Indol-3yl-acetic Acid Transport in Maize Root
Institute of Plant Biology and Physiology of the University, Lausanne, Switzerland
Importance of the Tip on the (5-3H)-Indol-3yl-acetic Acid Transport in Maize Root JEAN-JACQUES PERNET and PAUL-EMILE PILET With 2 figures Received February 15, 1979 . Accepted March 7, 1979
Summary Basipetal and acropetal movement of radioactivity from (5- 3H)-IAA (10-7 M) was followed for 1 and 3 hours in intact or decapitated 10 mm apical maize root segments. Beside a weak acropetal transport - significant radioactivity could only be detected in apical receivers on decapitated segments - a typical basipetal movement was also obtained. A clear acropetal polarity was only observed in root tissues near the receiver blocks, the presence of the root tip significantly reducing such polarity. Chromatographic analyses of root extracts showed a great increase in IAA proportion in function of the distance counted from the tip.
Key words: Auxin gradient, auxin polarity, fAA chromatography, fAA-oxidase, fAA transport, labelled auxin, maize, root tip.
Introduction It has been clearly demonstrated that the IAA movement in the root was preferentially acropetal (PILET, 1964; SCOTT and WILKINs,1968; PILET, 1975), occurring predominantly in the central cylinder (BOWEN et aI., 1972; BRIDGES et aI., 1973; GREENWOOD et aI., 1973). When IAA is given to the shoot of dicotyledonous plants, it may move into the root (DIGBY and W ANGERMANN, 1965; MORRIS et aI., 1969; DAVIES and MITCHELL, 1972; ELIASSON, 1972; KALDEWEY and KRAUS, 1972; BOURBOULOUX and BONNEMAIN, 1974). In maize, the application of IAA to the cut mesocotyl surface or to the caryopsis led to the appearance of radioactivity in the root (TEPPER and BOSSARD, 1969; BATRA et aI., 1977). When IAA was applied on the tip of the coleoptile, it accumulated inside the root cap (MARTIN et aI., 1978) and when it was given on the root cap, it entered the root tip (cap and apex) but with a basipetal transport very limited (PERNET and PILET, 1976). However it has to be noticed that the existence of a not negligible basipetal movement has been Abbreviations: IAA = Indol-3yl-acetic Acid.
z. Pflanzenphysiol. Bd. 94. s. 273-279. 1979.
274
JEAN-JACQUES PERNET and PAUL-EMILE PILET
demonstrated in apical root segments (PILET, 1964; DAVIES and MITCHELL, 1972; SHAW and WILKINS, 1974; MITCHELL and DAVIES, 1975; MIGINIAC et aI., 1978; TSUMURI and OHWAKI, 1978). On the other hand, mass spectrometry analyses (GC/MS) clearly indicate that IAA was present in the maize roots (ELLIOTT and GREENWOOD, 1974) and its higher concentration was found in the cap cells (RIVIER and PILET, 1974; PILET, 1977). The aim of the present work was to study the role of the tip on the basipetal and acropetal movement of tritium from (5- 3H)-IAA using intact and decapitated apical maize root segments. Material and Methods Conditions for preparing the maize root segments have been previously discussed (PILET, 1977). Selected caryopses of Zea mays L. cv. Kelvedon 33 were germinated in darkness (22°C) and the primary roots allowed to elongate vertically on moist paper towels. When they reached 15 ± 3 mm, apical segments 10 ± 0.2 mm in length were mounted in plastic frames with their apical and basal cut ends applied on agar blocks. For the decapitated segments, 0.5 mm fragments were cut from the tip of intact roots. The cylindrical blocks (diameter 2.5 mm; thickness 1.8 mm) were prepared with purified agar Difco (1.5 Ofo), the donor containing (5-3H)-IAA (21 CilmM, CEA, Gif-sur-Yvette, France) at 10-7 M (PERNET and PILET, 1976). Root segments were then kept in closed boxes (25°C ± 0.1). At the end of the experiment, the agar blocks were collected in small plastic tubes (two blocks per vial) containing 2.8 ml of scintillation solution (PILET and PERNET, 1969). The segments were cut in 5 fragments and kept in plastic tubes (two fragments per vial) containing 0.2 ml of Soluen-350 (Packard Instrument Compagny, Downers Grove, Illinois, USA). After 24 h at 55°C, 2.6 ml of scintillation mixture was added. After correction for background and efficiency, the radioactivity was calculated as desintegrations per minute (DPM). In order to take small fluctuations of initial donor radioactivity (Do) into account results were expressed as 0/000 of Do (relative radioactivity) and, for the fragments, calculated as relative radioactivity per mm or per mg fresh weight. To determine the IAA content of blocks and segments, the following technique was used: 50 fragments or blocks were put into liquid nitrogen and freeze dried. After grinding, the powder was extracted 3 times with 4 ml of methanol (3 h -18°C). After drying under vacuum, 100 ,ttl of BSTFA (Pierce Chemical Compagny, Rockford, Illinois, USA) were added to ensure silylation of extracted substances. The labelled compounds were then separated on a Packard GC mod. 427 (10 Ofo SE-30, injector 250°C, oven 180 °C for 2 minutes then 5 DC/min, last 10 minutes at 250°C), and their radioactivity simultaneously counted on a Packard gas prop. counter mod. 894. The radioactivity of each peak was expressed as % total radioactivity found in the chromatogram.
Results and Discussion The acropetal and basipetal movement of radioactivity from 3H-IAA was followed both in intact and decapitated apical root segments (Table 1). As can be seen for the intact segments, no significant radioactivity (at P = 0.05) was obtained in both apical and basal receiver blocks. Considering the radioactivity of the fragment situated near the receiver blocks, a significant acropetal polarity can be observed. Similar results were obtained by SCOTT and WILKINS (1968). Such a Z. P/lanzenphysiol. Bd. 94. S. 273-279.1979.
Importance of the root tip
275
Table 1: Relative radioactivity (± standard error of mean) per mg fresh weight in intact and decapitated maize (cv. Kelvedon 33) root segments and in receiver blocks, after 3 hours of basal or apical 3H-IAA (at 10-7 M) application; acropetal (AT) and basipetal (BT) transport. Distance from donor blocks (in mm)
Intact root segments
Decapitated root segments
AT
BT
AT
BT
0- 2 2- 4 4- 6 6- 8 8-10*) Receiver blocks:
816.50 ± 50.56 304.81 ± 13.49 158.00±19.79 50.58 ± 4.53 62.99± 6.50 NS
1669.08 ± 116.10 190.11± 13.43 85.35± 4.73 9.76± 1.76 0.94± 0.10 NS
1056.65 ± 44.93 275.94 ± 17.76 118.49± 3.74 40.19± 2.32 17.92± 1.59 33.01 ± 2.08
963.63 ± 80.75 264.38 ± 14.37 113.21 ± 8.77 12.68 1.49 1.55 ± 0.12 NS
±
'f) This depends on the final length of the apical root segments. NS: no significant radioactivity above background (P = 0.05).
polarity was also reported for the Lens roots when only testing the radioactivity in the total root segment (PILET, 1964). For the decapitated segments, a significant radioactivity was observed only in the apical receiver blocks. Radioactivity accumulation in the apical fragments was reduced and basipetal movement enhanced, thus reducing the observed polarity in the tissues. Consequently the presence of the cap has to be related to a stronger polarity of the IAA transport. If the radioactivity of the tissues were expressed not only per mg fresh weight but also per Ofo total absorbed radioactivity (in order to take the difference in uptake between acropetal and basipetal transport into account), a clear acropetal polarity could only be observed in the 4 mm near the receiver blocks (both for intact or decapitated segments), as previously reported by WILKINS et al., 1972. It is necessary to notice that, for a 3 h experiment, the decapitation did not change significantly the growth rate of the segments. This was already observed by PILET, 1972. Basipetal transport of radioactivity in the intact (Fig. 1 A) and the decapitated (Fig. 1 B) root segments was followed for 1 and 3 hours. The present data allow several comments. 1. A typical basipetal movement of the radioactivity from tritiated IAA can be obtained, and this for both intact and decapitated root segments, as, after 1 h, a significant radioactivity was detected at 10 mm from the donor blocks. 2. This indicates a transport velocity which is, at least, of 10 mm per hour. 3. Only a very limited proportion of radioactivity was implicated in such transport, as 90 Ofo of the radioactivity in the segments was located in the apical part - essentially inside the root cap (PERNET and PILET, 1976). 4. It is clear that, when removing the tip, the basipetal movement was enhanced: in decapitated segments only 75 % - then 66 0/0 - was detected, after 1 and 3 h, in the first 2 mm. It was of interest to analyse how the measured radioactivity was transported in the root segments. Only results for a 3 h experiment of basipetal transport using intact
z. PJlanzenphysiol. Bd. 94. s. 273-279. 1979.
276
JEAN-JACQUES PERNET
and
PAUL-EMILE PILET
segments were reported here. Radioactivity was analysed for both donor and receiver blocks and fragments. Considering the great difference in the radioactivity content between the fragments (see Table 1), extracts of fragments 2-4 and 4-6 mm (medium) were pooled. Unfortunately, specific radioactivity of fragments 6-8 and
10
10
I
3
®
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Q
2
....z
w 10
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w 0::
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2
4
6
8
DISTANCE IN
2
4
6
10
0
mm
FROM DONOR BLOCK
8
10
Fig. 1: Distribution of the radioactivity in the intact (A) and the decapitated (B) apical segments of maize (cv. Kelvedon 33) roots after 1 and 3 hours of apical application (basipetal movement of 3H) of (S-3H)-IAA. The points and the vertical lines represent the mean value ± standard error of mean respectively.
z. P/lanzenphysiol. Bd. 94. s. 273-279. 1979.
Importance of the root tip
277
IAA
@ 200 CPM
Ul UJ
..... :::> z
~
a::
UJ
Q.
Ul .....
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0
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2
4
6
10 12 8 TIME IN MINUTES
14
Fig. 2: Radiochromatograms of the extracts prepared from donor blocks (D), apical (A: the first 2 mm counted from the tip) and medium (M: 4 mm located from 2 mm counted from the tip, i. e. 2-6 mm) fragments after 3 hours of apical application of (S-3H)-IAA on the intact apical root segments of maize (cv. Kelvedon 33). Dotted lines indicating the background.
z. PJlanzenphysiol. Bd. 94. S. 273-279. 1979.
278
JEAN-JACQUES PERNET and PAUL-EMILE PILET
8-10 mm, even pooled, was too weak to be detected. The same situation was observed for the receiver blocks extract. Radiochromatograms of extracts of donor blocks (D), apical (A) and medium (M) segments are presented in Fig. 2. A quantitative evaluation of labelled compounds was made, assuming that the total surface above background (dotted line) was 100010. In donor blocks, 75 010 of radioactivity was identified as IAA. Three other unidentified compounds [1 (1.4 010); 2 (7.7010); 3 (15.9010)] were also observed. These compounds are probably related to a contamination of donor blocks by mucilage and cap cells, control of initial donor blocks showing a radiochemical purity of at least 98010. In apical fragments (0-2 mm), the IAA level was only 29 010, most of the radioactivity was found in compound 3 (52010), and 2 (17010). In medium fragments (2-6 mm), a great increase in IAA proportion (68010) as well as an important decrease in compound 3 level (10 %) can be observed, the compound 2 (19010) remaining relatively constant (19010). Therefore, it seems that the compounds which were formed from IAA, both by biodestruction and conjugation, were scarcely basipetally transported. The longitudinal IAA-oxidase gradient (LEE a,nd PILET, 1977) cannot be responsible for the raise in IAA proportion in relation to the distance from the tip: in terms of IAA destroyed per fragment, the IAA-oxidase did not change enough to explain this large biodestruction in the 2 first mm of the apical segments as compared with the 4 following mm. This could be interpretated by the fact that IAA moved preferentially in the stele - where IAA degradation was found to be weak (GREENWOOD et a!., 1973; GRISON and PI LET, 1978) - rather than in the cortex, the cells of which containing much more active IAA-oxidase (BRIDGES et aI., 1973).
References BATRA, M. W., K. L. EDWARDS, and T. K. SCOTT: Auxin transport in roots: its characteristics and relationship to growth. In: J. G. TORREY and D. T. CLARKSON (Eds.): The Development and Function of Roots, pp. 299-325. Academic Press, London, 1975. BOURBOULOUX, A. and J. L. BONNEMAIN: Transport, distribution et metabolisme de l'auxine dans la racine de Vicia faba L. apres application de (HC) AlA ou de (3H) AlA sur Ie bourgeon. Planta 119, 169-182 (1974). BOWEN, M. R., M. B. WILKINS, A. R. CANE, and I. MCCORQUODALE: Auxin transport in roots VIII. The distribution of radioactivity in the tissues of Zea root segments. Planta 105, 273-292 (1972).
BRIDGES, I. G., J. R. HILLMAN, and M. B. WILKINS: Identification and localization of auxin in primary roots of Zea mays by mass spectrometry. Planta 115, 189-192 (1973). DAVIES, P. J. and E. K. MITCHELL: Transport of indoleacetic acid in intact roots of Phaseolus coccineus. Plant a 105, 139-154 (1972). DIGBY, J. and E. WANGERMANN: A note on the effect of the shoot and root apex on secondary thickening in pea radicles. New Phytol. 64, 168-170 (1965). ELIASSON, L.: Translocation of shoot-applied indolylacetic acid into the roots of Populus tremula. Physiol. Plant. 27, 412-416 (1972). Z. Pflanzenphysiol. Bd. 94. S. 273-279. 1979.
Importance of the root tip
279
ELLIOTT, M. C. and M. S. GREENWOOD: Indol-3yl-acetic acid in roots of Zea mays. Phytochern. 13,239-241 (1974). GREENWOOD, M. S., J. R. HILLMAN, S. SHAW, and M. B. WILKINS: Localization and identification of auxin in roots of Zea mays. Plant a 109, 369-374 (1973). GRISON, R. and P. E. PILET: Analyse critique des peroxydases de la racine de mais. Plant Sci. Letters 13, 213-218 (1978). KALDEWEY, H. and L. KRAUS: Translocation and immobilisation of radiocarbon in the hypocotyl and the primary root of Gossypium hirsutum L. after application of IAA-2-14 C to intact light-grown seedlings. In: H. KALDEWEY and Y. VARDAR (Eds.): Hormonal Regulation in Plant Growth and Development, pp. 137-153. Verlag CheMie, Weinheim, 1972. LEE, T. T. and P. E. PILET: IndoLe-3-acetic acid oxidase and peroxidase in maize roots. Plant Sci. Letters 9, 147-151 (1977). MARTIN, H. V., M. C. ELLIOTT, E. WANGERMANN, and P. E. PILET: Auxin gradient along the root of the maize seedling. Plant a 141, 179-181 (1978). MIGINIAC, E., L. SOSSOUNTZOV, and N. DUGUE: Modalites du transport et du metabolisme de l'AIA 14C ou AlA 3H en relation avec la morphogenese des bourgeons axillaires chez Ie Scophularia arguta. Physiol. Plant. 44, 335-344 (1978). MITCHELL, E. K. and P. J. DAVIES: Evidence for three different systems of movement of indoleacetic acid in intact roots of Phaseolus coccineus. Physiol. Plant 33, 290-294 (1975). MORRIS, D. A., R. E. BRIANT, and P. G. THOMSON: The transport and metabolism of 14C_ labeled indoleacetic acid in intact pea seedlings. Planta 89,178-197 (1969). PILET, P. E.: Auxin transport in roots. Nature 204, 561-562 (1964). - Root cap and root growth. Planta 106, 169-171 (1972). - Abscisic acid as a root growth inhibitor: physiological analyses. Planta 122, 299-302 (1975). - Growth inhibitors in growing and geostimulated maize roots. In: P. E. PILET (Ed.): Plant Growth Regulation, pp. 115-128. Springer-Verlag, Berlin - Heidelberg - New York,1977. PILET, P. E. and J. J. PERNET: Polarite de transport in vitro de l'auxine radioactive (tige de Lens). Bull. Soc. Bot. Suisse 80,5-16 (1970). - - Indoleacetic acid movement in the root cap. Planta 128, 183-184 (1976). RIVIER, L. and P. E. PILET: Indolyl-3-acetic acid in cap and apex of maize roots: identification and quantification by mass fragmentography. Plant a 120, 107-112 (1974). SCOTT, T. K. and M. B. WILKINS: Auxin transport in roots II. Polar flux of IAA in Zea roots. Planta 83, 323-334 (1968). SHAW, S. and M. B. WILKINS: Auxin transport in roots X. Relative movement of radioactivity from IAA in the stele and cortex of Zea root segments. J. Exp. Bot. 25, 199-207 (1974). TEPPER, H. and D. BROSSARD: Voie de transport de l'acide indolylacetique chez Ie Zea et chez Ie Coleus. C. R. Acad. Sci. (Paris) 269, 567-569 (1969). TSURUMI, S. and Y. OHWAKI: Transport of 14C-Iabeled indol.eacetic acid in Vicia root segments. Plant Cell Physiol. 19, 1195-1206 (1978). WILKINS, M. B., A. R. CANE, and 1. MCCORQUODALE: Auxin transport in roots VII. Uptake and movement of radioactivity from IAA-14 C by Zea roots. Planta 105, 93-115 (1972). Prof. Dr. P. E. PILET, Institute of Plant Biology and Physiology of the University, 6, Pi. de la Riponne, Ch-1005 Lausanne, Switzerland.
Z. Pflanzenphysiol. Bd. 94. S. 273-279. 1979.
Importance of the Tip on the (5-3H)-Indol-3yl-acetic Acid Transport in Maize Root JEAN-JACQUES PERNET and PAUL-EMILE PILET With 2 figures Received February 15, 1979 . Accepted March 7, 1979
Summary Basipetal and acropetal movement of radioactivity from (5- 3H)-IAA (10-7 M) was followed for 1 and 3 hours in intact or decapitated 10 mm apical maize root segments. Beside a weak acropetal transport - significant radioactivity could only be detected in apical receivers on decapitated segments - a typical basipetal movement was also obtained. A clear acropetal polarity was only observed in root tissues near the receiver blocks, the presence of the root tip significantly reducing such polarity. Chromatographic analyses of root extracts showed a great increase in IAA proportion in function of the distance counted from the tip.
Key words: Auxin gradient, auxin polarity, fAA chromatography, fAA-oxidase, fAA transport, labelled auxin, maize, root tip.
Introduction It has been clearly demonstrated that the IAA movement in the root was preferentially acropetal (PILET, 1964; SCOTT and WILKINs,1968; PILET, 1975), occurring predominantly in the central cylinder (BOWEN et aI., 1972; BRIDGES et aI., 1973; GREENWOOD et aI., 1973). When IAA is given to the shoot of dicotyledonous plants, it may move into the root (DIGBY and W ANGERMANN, 1965; MORRIS et aI., 1969; DAVIES and MITCHELL, 1972; ELIASSON, 1972; KALDEWEY and KRAUS, 1972; BOURBOULOUX and BONNEMAIN, 1974). In maize, the application of IAA to the cut mesocotyl surface or to the caryopsis led to the appearance of radioactivity in the root (TEPPER and BOSSARD, 1969; BATRA et aI., 1977). When IAA was applied on the tip of the coleoptile, it accumulated inside the root cap (MARTIN et aI., 1978) and when it was given on the root cap, it entered the root tip (cap and apex) but with a basipetal transport very limited (PERNET and PILET, 1976). However it has to be noticed that the existence of a not negligible basipetal movement has been Abbreviations: IAA = Indol-3yl-acetic Acid.
z. Pflanzenphysiol. Bd. 94. s. 273-279. 1979.
274
JEAN-JACQUES PERNET and PAUL-EMILE PILET
demonstrated in apical root segments (PILET, 1964; DAVIES and MITCHELL, 1972; SHAW and WILKINS, 1974; MITCHELL and DAVIES, 1975; MIGINIAC et aI., 1978; TSUMURI and OHWAKI, 1978). On the other hand, mass spectrometry analyses (GC/MS) clearly indicate that IAA was present in the maize roots (ELLIOTT and GREENWOOD, 1974) and its higher concentration was found in the cap cells (RIVIER and PILET, 1974; PILET, 1977). The aim of the present work was to study the role of the tip on the basipetal and acropetal movement of tritium from (5- 3H)-IAA using intact and decapitated apical maize root segments. Material and Methods Conditions for preparing the maize root segments have been previously discussed (PILET, 1977). Selected caryopses of Zea mays L. cv. Kelvedon 33 were germinated in darkness (22°C) and the primary roots allowed to elongate vertically on moist paper towels. When they reached 15 ± 3 mm, apical segments 10 ± 0.2 mm in length were mounted in plastic frames with their apical and basal cut ends applied on agar blocks. For the decapitated segments, 0.5 mm fragments were cut from the tip of intact roots. The cylindrical blocks (diameter 2.5 mm; thickness 1.8 mm) were prepared with purified agar Difco (1.5 Ofo), the donor containing (5-3H)-IAA (21 CilmM, CEA, Gif-sur-Yvette, France) at 10-7 M (PERNET and PILET, 1976). Root segments were then kept in closed boxes (25°C ± 0.1). At the end of the experiment, the agar blocks were collected in small plastic tubes (two blocks per vial) containing 2.8 ml of scintillation solution (PILET and PERNET, 1969). The segments were cut in 5 fragments and kept in plastic tubes (two fragments per vial) containing 0.2 ml of Soluen-350 (Packard Instrument Compagny, Downers Grove, Illinois, USA). After 24 h at 55°C, 2.6 ml of scintillation mixture was added. After correction for background and efficiency, the radioactivity was calculated as desintegrations per minute (DPM). In order to take small fluctuations of initial donor radioactivity (Do) into account results were expressed as 0/000 of Do (relative radioactivity) and, for the fragments, calculated as relative radioactivity per mm or per mg fresh weight. To determine the IAA content of blocks and segments, the following technique was used: 50 fragments or blocks were put into liquid nitrogen and freeze dried. After grinding, the powder was extracted 3 times with 4 ml of methanol (3 h -18°C). After drying under vacuum, 100 ,ttl of BSTFA (Pierce Chemical Compagny, Rockford, Illinois, USA) were added to ensure silylation of extracted substances. The labelled compounds were then separated on a Packard GC mod. 427 (10 Ofo SE-30, injector 250°C, oven 180 °C for 2 minutes then 5 DC/min, last 10 minutes at 250°C), and their radioactivity simultaneously counted on a Packard gas prop. counter mod. 894. The radioactivity of each peak was expressed as % total radioactivity found in the chromatogram.
Results and Discussion The acropetal and basipetal movement of radioactivity from 3H-IAA was followed both in intact and decapitated apical root segments (Table 1). As can be seen for the intact segments, no significant radioactivity (at P = 0.05) was obtained in both apical and basal receiver blocks. Considering the radioactivity of the fragment situated near the receiver blocks, a significant acropetal polarity can be observed. Similar results were obtained by SCOTT and WILKINS (1968). Such a Z. P/lanzenphysiol. Bd. 94. S. 273-279.1979.
Importance of the root tip
275
Table 1: Relative radioactivity (± standard error of mean) per mg fresh weight in intact and decapitated maize (cv. Kelvedon 33) root segments and in receiver blocks, after 3 hours of basal or apical 3H-IAA (at 10-7 M) application; acropetal (AT) and basipetal (BT) transport. Distance from donor blocks (in mm)
Intact root segments
Decapitated root segments
AT
BT
AT
BT
0- 2 2- 4 4- 6 6- 8 8-10*) Receiver blocks:
816.50 ± 50.56 304.81 ± 13.49 158.00±19.79 50.58 ± 4.53 62.99± 6.50 NS
1669.08 ± 116.10 190.11± 13.43 85.35± 4.73 9.76± 1.76 0.94± 0.10 NS
1056.65 ± 44.93 275.94 ± 17.76 118.49± 3.74 40.19± 2.32 17.92± 1.59 33.01 ± 2.08
963.63 ± 80.75 264.38 ± 14.37 113.21 ± 8.77 12.68 1.49 1.55 ± 0.12 NS
±
'f) This depends on the final length of the apical root segments. NS: no significant radioactivity above background (P = 0.05).
polarity was also reported for the Lens roots when only testing the radioactivity in the total root segment (PILET, 1964). For the decapitated segments, a significant radioactivity was observed only in the apical receiver blocks. Radioactivity accumulation in the apical fragments was reduced and basipetal movement enhanced, thus reducing the observed polarity in the tissues. Consequently the presence of the cap has to be related to a stronger polarity of the IAA transport. If the radioactivity of the tissues were expressed not only per mg fresh weight but also per Ofo total absorbed radioactivity (in order to take the difference in uptake between acropetal and basipetal transport into account), a clear acropetal polarity could only be observed in the 4 mm near the receiver blocks (both for intact or decapitated segments), as previously reported by WILKINS et al., 1972. It is necessary to notice that, for a 3 h experiment, the decapitation did not change significantly the growth rate of the segments. This was already observed by PILET, 1972. Basipetal transport of radioactivity in the intact (Fig. 1 A) and the decapitated (Fig. 1 B) root segments was followed for 1 and 3 hours. The present data allow several comments. 1. A typical basipetal movement of the radioactivity from tritiated IAA can be obtained, and this for both intact and decapitated root segments, as, after 1 h, a significant radioactivity was detected at 10 mm from the donor blocks. 2. This indicates a transport velocity which is, at least, of 10 mm per hour. 3. Only a very limited proportion of radioactivity was implicated in such transport, as 90 Ofo of the radioactivity in the segments was located in the apical part - essentially inside the root cap (PERNET and PILET, 1976). 4. It is clear that, when removing the tip, the basipetal movement was enhanced: in decapitated segments only 75 % - then 66 0/0 - was detected, after 1 and 3 h, in the first 2 mm. It was of interest to analyse how the measured radioactivity was transported in the root segments. Only results for a 3 h experiment of basipetal transport using intact
z. PJlanzenphysiol. Bd. 94. s. 273-279. 1979.
276
JEAN-JACQUES PERNET
and
PAUL-EMILE PILET
segments were reported here. Radioactivity was analysed for both donor and receiver blocks and fragments. Considering the great difference in the radioactivity content between the fragments (see Table 1), extracts of fragments 2-4 and 4-6 mm (medium) were pooled. Unfortunately, specific radioactivity of fragments 6-8 and
10
10
I
3
®
@
Q
2
....z
w 10
:::2: C)
w VI
E E
0:: W
Q..
....>-
:> ~
u
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0
C
«0::
w >
¢
~
«...J
w 0::
0
2
4
6
8
DISTANCE IN
2
4
6
10
0
mm
FROM DONOR BLOCK
8
10
Fig. 1: Distribution of the radioactivity in the intact (A) and the decapitated (B) apical segments of maize (cv. Kelvedon 33) roots after 1 and 3 hours of apical application (basipetal movement of 3H) of (S-3H)-IAA. The points and the vertical lines represent the mean value ± standard error of mean respectively.
z. P/lanzenphysiol. Bd. 94. s. 273-279. 1979.
Importance of the root tip
277
IAA
@ 200 CPM
Ul UJ
..... :::> z
~
a::
UJ
Q.
Ul .....
Z
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0
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2
4
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10 12 8 TIME IN MINUTES
14
Fig. 2: Radiochromatograms of the extracts prepared from donor blocks (D), apical (A: the first 2 mm counted from the tip) and medium (M: 4 mm located from 2 mm counted from the tip, i. e. 2-6 mm) fragments after 3 hours of apical application of (S-3H)-IAA on the intact apical root segments of maize (cv. Kelvedon 33). Dotted lines indicating the background.
z. PJlanzenphysiol. Bd. 94. S. 273-279. 1979.
278
JEAN-JACQUES PERNET and PAUL-EMILE PILET
8-10 mm, even pooled, was too weak to be detected. The same situation was observed for the receiver blocks extract. Radiochromatograms of extracts of donor blocks (D), apical (A) and medium (M) segments are presented in Fig. 2. A quantitative evaluation of labelled compounds was made, assuming that the total surface above background (dotted line) was 100010. In donor blocks, 75 010 of radioactivity was identified as IAA. Three other unidentified compounds [1 (1.4 010); 2 (7.7010); 3 (15.9010)] were also observed. These compounds are probably related to a contamination of donor blocks by mucilage and cap cells, control of initial donor blocks showing a radiochemical purity of at least 98010. In apical fragments (0-2 mm), the IAA level was only 29 010, most of the radioactivity was found in compound 3 (52010), and 2 (17010). In medium fragments (2-6 mm), a great increase in IAA proportion (68010) as well as an important decrease in compound 3 level (10 %) can be observed, the compound 2 (19010) remaining relatively constant (19010). Therefore, it seems that the compounds which were formed from IAA, both by biodestruction and conjugation, were scarcely basipetally transported. The longitudinal IAA-oxidase gradient (LEE a,nd PILET, 1977) cannot be responsible for the raise in IAA proportion in relation to the distance from the tip: in terms of IAA destroyed per fragment, the IAA-oxidase did not change enough to explain this large biodestruction in the 2 first mm of the apical segments as compared with the 4 following mm. This could be interpretated by the fact that IAA moved preferentially in the stele - where IAA degradation was found to be weak (GREENWOOD et a!., 1973; GRISON and PI LET, 1978) - rather than in the cortex, the cells of which containing much more active IAA-oxidase (BRIDGES et aI., 1973).
References BATRA, M. W., K. L. EDWARDS, and T. K. SCOTT: Auxin transport in roots: its characteristics and relationship to growth. In: J. G. TORREY and D. T. CLARKSON (Eds.): The Development and Function of Roots, pp. 299-325. Academic Press, London, 1975. BOURBOULOUX, A. and J. L. BONNEMAIN: Transport, distribution et metabolisme de l'auxine dans la racine de Vicia faba L. apres application de (HC) AlA ou de (3H) AlA sur Ie bourgeon. Planta 119, 169-182 (1974). BOWEN, M. R., M. B. WILKINS, A. R. CANE, and I. MCCORQUODALE: Auxin transport in roots VIII. The distribution of radioactivity in the tissues of Zea root segments. Planta 105, 273-292 (1972).
BRIDGES, I. G., J. R. HILLMAN, and M. B. WILKINS: Identification and localization of auxin in primary roots of Zea mays by mass spectrometry. Planta 115, 189-192 (1973). DAVIES, P. J. and E. K. MITCHELL: Transport of indoleacetic acid in intact roots of Phaseolus coccineus. Plant a 105, 139-154 (1972). DIGBY, J. and E. WANGERMANN: A note on the effect of the shoot and root apex on secondary thickening in pea radicles. New Phytol. 64, 168-170 (1965). ELIASSON, L.: Translocation of shoot-applied indolylacetic acid into the roots of Populus tremula. Physiol. Plant. 27, 412-416 (1972). Z. Pflanzenphysiol. Bd. 94. S. 273-279. 1979.
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ELLIOTT, M. C. and M. S. GREENWOOD: Indol-3yl-acetic acid in roots of Zea mays. Phytochern. 13,239-241 (1974). GREENWOOD, M. S., J. R. HILLMAN, S. SHAW, and M. B. WILKINS: Localization and identification of auxin in roots of Zea mays. Plant a 109, 369-374 (1973). GRISON, R. and P. E. PILET: Analyse critique des peroxydases de la racine de mais. Plant Sci. Letters 13, 213-218 (1978). KALDEWEY, H. and L. KRAUS: Translocation and immobilisation of radiocarbon in the hypocotyl and the primary root of Gossypium hirsutum L. after application of IAA-2-14 C to intact light-grown seedlings. In: H. KALDEWEY and Y. VARDAR (Eds.): Hormonal Regulation in Plant Growth and Development, pp. 137-153. Verlag CheMie, Weinheim, 1972. LEE, T. T. and P. E. PILET: IndoLe-3-acetic acid oxidase and peroxidase in maize roots. Plant Sci. Letters 9, 147-151 (1977). MARTIN, H. V., M. C. ELLIOTT, E. WANGERMANN, and P. E. PILET: Auxin gradient along the root of the maize seedling. Plant a 141, 179-181 (1978). MIGINIAC, E., L. SOSSOUNTZOV, and N. DUGUE: Modalites du transport et du metabolisme de l'AIA 14C ou AlA 3H en relation avec la morphogenese des bourgeons axillaires chez Ie Scophularia arguta. Physiol. Plant. 44, 335-344 (1978). MITCHELL, E. K. and P. J. DAVIES: Evidence for three different systems of movement of indoleacetic acid in intact roots of Phaseolus coccineus. Physiol. Plant 33, 290-294 (1975). MORRIS, D. A., R. E. BRIANT, and P. G. THOMSON: The transport and metabolism of 14C_ labeled indoleacetic acid in intact pea seedlings. Planta 89,178-197 (1969). PILET, P. E.: Auxin transport in roots. Nature 204, 561-562 (1964). - Root cap and root growth. Planta 106, 169-171 (1972). - Abscisic acid as a root growth inhibitor: physiological analyses. Planta 122, 299-302 (1975). - Growth inhibitors in growing and geostimulated maize roots. In: P. E. PILET (Ed.): Plant Growth Regulation, pp. 115-128. Springer-Verlag, Berlin - Heidelberg - New York,1977. PILET, P. E. and J. J. PERNET: Polarite de transport in vitro de l'auxine radioactive (tige de Lens). Bull. Soc. Bot. Suisse 80,5-16 (1970). - - Indoleacetic acid movement in the root cap. Planta 128, 183-184 (1976). RIVIER, L. and P. E. PILET: Indolyl-3-acetic acid in cap and apex of maize roots: identification and quantification by mass fragmentography. Plant a 120, 107-112 (1974). SCOTT, T. K. and M. B. WILKINS: Auxin transport in roots II. Polar flux of IAA in Zea roots. Planta 83, 323-334 (1968). SHAW, S. and M. B. WILKINS: Auxin transport in roots X. Relative movement of radioactivity from IAA in the stele and cortex of Zea root segments. J. Exp. Bot. 25, 199-207 (1974). TEPPER, H. and D. BROSSARD: Voie de transport de l'acide indolylacetique chez Ie Zea et chez Ie Coleus. C. R. Acad. Sci. (Paris) 269, 567-569 (1969). TSURUMI, S. and Y. OHWAKI: Transport of 14C-Iabeled indol.eacetic acid in Vicia root segments. Plant Cell Physiol. 19, 1195-1206 (1978). WILKINS, M. B., A. R. CANE, and 1. MCCORQUODALE: Auxin transport in roots VII. Uptake and movement of radioactivity from IAA-14 C by Zea roots. Planta 105, 93-115 (1972). Prof. Dr. P. E. PILET, Institute of Plant Biology and Physiology of the University, 6, Pi. de la Riponne, Ch-1005 Lausanne, Switzerland.
Z. Pflanzenphysiol. Bd. 94. S. 273-279. 1979.