3H-GA1 Transport in Growing Maize Root

Institute of Plant Biology and Physiology of the University, Lausanne, Switzerland

3H-GA t Transport in Growing Maize Root

J. J. PERNET and P. E. PILET With 7 figures Received July 5,1980 . Accepted July 14,1980

Summary Movement of (3,4- 3 H) GAl (10- 7 M) was investigated in maize (cv. ORLA 264) primary roots (intact roots attached to the caryopses, apical root segments) decapitated or not. In segments, a weak basipetal transport - enhanced in decapitated segments - was observed. A clear acropetal polarity was only noticed in rOOt tissues near the receivers. Movement of radioactivity and export to receivers were reduced after 3 hours, the rate of uptake from donors remaining unchanged. A much higher basipetal transport was observed in decapitated roots. Chromatographic analyses of root extracts showed the presence of an unidentified translocatable labelled compound. Patterns of basipetal transport seemed to be very different if the effects in root segments were compared with those in the whole roots.

Key words: Gibberellin transport, labelled Gibberellin, maize, root, root cap.

Introduction Gibberellins (GA) can be formed in both young leaves of apical buds and root apices of seedlings (LANG, 1970). Exchanges of GA or precursors might occur between these two sites of biosynthesis (CROZIER and REID, 1971). In shoots, transport of GA has been reported to be either non polar (WILKINS and NASH, 1974; PHILLIPS and HARTUNG, 1974), basipetally polar (MOST and SCOTT, 1971) or acropetally polar (NASH and CROZIER, 1975). Endogenous GA may accumulate in roots (JONES and PHILLIPS, 1966; SKENE, 1967; CROZIER and REID, 1971), but in root segments the transport seems to be preferentially basipetal (JACOBS and PRUETT, 1973; HARTUNG and PHILLIPS, 1974). The evidence of an upward movement of GA in geotropically stimulated roots (WEBSTER and WILKINS, 1974) might indicate a possible role of GA in the growth and geotropic responses of roots (EL ANTABLY, 1974); EL ANTABLY and LARSEN, 1974). Such GA action may well be regulated by both indol-3yl-acetic acid (IAA) and abscisic acid (ABA) which also undergo polar translocation in roots. While it has been shown that GA may cause an increase in both the uptake and movement (velocity) of IAA in plant (lentil) tissues (PILET, 1965), it seems clear now that IAA movement in roots is preferentially acropetal Abbreviations: ABA

=

Abscisic Acid; GA

=

Gibberellin; IAA

=

Indol-3yl-acetic Acid.

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26

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and P. E. PILET

1964; SHAW and WILKINS, 1974; PILET, 1975). It accumulates in the root tip (RIVIER and PILET, 1974; PI LET, 1977; PILET et al., 1979). In contrast, ABA moves basipetally, from the cap cells, in maize roots (PILET, 1975). But it has been supposed - from data obtained by some physiological analyses (PILET and NOCERAPRZYBECKA, 1978; PILET, 1979) and by some GC/MS determinations (RIVIER et al., 1977) - that ABA could be produced to a large extent in the lower part of the cap of roots maintained in a horizontal position (PILET, 1976). And this ABA could accumulate in the lower half of the extending zone of geostimulated roots (PILET, 1979). (PI LET,

The aim of the present work was to study the movement of (3,4-:lH)-GAl apical root segments, decapitated or not, and in decapitated maize roots.

111

Materials and Methods The conditions for preparing maize root s·egments have been previously presented (PI LET, 1977). Selected caryopses of Zea mays cv. Orla 264 were germinated in darkness (20 DC) 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 between two agar blocks (darkness, 25 ± 0.2 DC), the donor-containing (3,4- 3H(N))GAl at 10-7 M. For the decapitated segments, the tips (cap + apex; 0.5 mm) from intact roots were removed and were then prepared as previously described. For the decapitated roots, 0.5 mm apical fragments were cut from intact roots and a donor block applied on the cut section. At the end of the assays, the segments were cut into 5 fragments and the decapitated roots into 6 fragments (2 mm), the last being considered as receiver. They were collected in counting vials containing 1 ml of soluene 350 (3 fragments per vial). After 24 h (50 DC) 15 ml of scintillation solution were added (PILET and PERNET, 1970). Control assays were also made for the blocks. After correction for background and efficiency, the radioactivity was calculated as disintegrations per minute (DPM). In order to take small fluctuations of initial donor radioactivity (Do) into account, results were expressed as 10-2 % of Do (relative radioactivity) and, for the fragments, calculated as relative radioactivity per mm. After a 6-hour transport experiment, decapitated roots and root segments (50-70) were cut into fragments as previously described and frozen in liquid nitrogen. After grinding, the cold powder was extracted overnight (-18 DC) with 0.5 ml 80 % methanol. After centrifugation, the residues were extracted twice with 0.3 ml 80 % methanol (-18 DC; 6 h). The pooled supernatant was evaporated to dryness under nitrogen and diluted with 50,u1 80 % methanol. The donor and receiver blocks were freeze dried and extracted in the same way. As observed in control experiments, less than 0.5 % of fragment radioactivity remained in the final residues. Chromatographic analyses of extracts was made by TLC, using hand-spread inactivated silica gel H plates (thickness 250 ft; 20 X 10 cm; 75 g Kiesel gel H (Merck) in 75 ml water). The methanolic extracts were streaked onto a plate with two spots of 3H-GA l stock solution and developed for 15 cm in benzene-butanol-l-acetic acid solvent (70/25/5; KAGAWA et aI., 1963). Each chromatogram was divided into 30 strips (0.5 X 2 cm), each being scraped and the silica powder transferred in counting vials containing 15 ml of scintillation solution. After correction for background, results were expressed as % of total chromatogram radioactivity (the low radioactivity remaining on the initial spOt was not taken into account). Z. Pflanzenphysiol. Rd. 101. S. 25-35. 1981.

3H-GA t transport in maize root

27

Results and Discussion In the first series of experiments, basipetal movement of radioactivity from 3H_ GAt will be analysed in relation to time (0 to 7 h) in both intact (Fig. 1) and decapitated (Fig. 2) apical root segments. A weak basipetal transport is obtained in intact segments (Fig. 1 A). As can be seen, after a significant increase during the first hour, accumulation of the radioactivity in the receiver blocks slows down considerably and changes linearly as a function of time (P = 0.05). It will be noticed that total segment radioactivity increases in a linear manner during the experiment (P = 0.01), showing no such modification of the slope. Gradients of radioactivity in these segments (Fig. 1 B) clearly indicate that most of the radioactivity is located in the apical 4 mm (more than 98 % of total segment, unchanged for all times), as previously described by WEBSTER and WILKINS (1974). Only 0.2 Ofo arc detected in the basal 2 mm. In decapitated segments, basipetal movement of radioactivity to receivers is enhanced, especially in the first 3 hours, as given in Fig. 2 A. As before, total segment radioactivity increases linearly with time (P = 0.01). Gradients of radioactivity

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Fig. 1: Basipetal transport of radioactivity from 3H-GA t (at 10- 7 M) in intact root segments. A) Relative radioactivity in receiver blocks (R) and in total segments (5) as a function of time (in h). - B) Relative radioactivity per mm fragments as a function of time (in 11) and distance from donor (in mm).

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Fig.2: Basipetal transport of radioactivity from 3H-GA 1 (at 10-7 M) in decapitated root segments. A) Relative radioactivity in receiver blocks (R) and in total segments (5) as a function of time (in h). - B) Relative radioactivity per mm fragments as a function of time (in h) and distance from donor (in mm).

along these segments (Fig. 2 B) indicate that accumulation in the apical fragment is reduced and the radioactivity level in the basal fragment enhanced, as compared with data obtained in intact segments. Under such conditions, 98 % of total segment radioactivity is still located in the first apical 4 mm. Removing the tip (cap + apex) did not change the uptake in the root segments (as tested by covariance analysis), but in contrast it clearly enhanced the basipetal displacement of radioactivity. The velocity cannot be calculated by the intercept method, but a significant activity is observed in the basal fragment (9-10.5 mm from donor) after 1 h. This velocity can be estimated as being at least 9 mm/h. In the second set of assays, acropetal movement of radioactivity from 3H-GA 1 is analysed in relation to time (0 to 7 h) using decapitated root segments (Fig. 3). Radioactivity detected in apical receiver blocks is presented in Fig. 3 A. After a large increase for the first 3 hours, no significant changes are observed for the next 4 hours. Gradients of radioactivity along segments (Fig. 3 B) clearly indicate that accumulation in the apical fragment continues after 3 hours, though at a reduced rate. It has to be noticed that total segment radioactivity increases linearly

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3H-GA t transport in maize root

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Fig.3: Acropetal transport of radioactivity from 3H-GA t (at 10-7 M) in decapitated root segments. A) Relative radioactivity in receiver blocks (R) and in total segments (5) as a function of time (in h). - B) Relative radioactivity per mm fragments as a function of time (in h) and distance from donor (in mm).

with time within the duration of the experiment. It seems that in apical root segments, especially in decapitated ones, movement of radioactivity and export to receivers is reduced after 3 hours, the rate of uptake from donor remaining unchanged. Considering the radioactivity collected in receiver blocks after 3 hours on decapitated segments (see Fig. 2 A and 3 A), there seems to be a net acropetal polarity (AT/BT = 3.2); but if corrected for uptake area it is reduced to 1.6 which is not significantly different from 1. Nevertheless, it is clear (see Fig. 2 Band 3 B) that a stronger acropetal polarity may be calculated along the segment (19 for the fragment near the receiver block). But if we consider the total segment radioactivity, no significant acropetal polarity (1.6) can be shown. HARTUNG and PHILLIPS (1974), using 6 mm root segments of Phaseolus coccineus taken at 2 mm from the tip, calculated a basipetal polarity of 9.2 in receiver blocks and of 0.9 for the total segment. It has to be borne in mind that 3H-GA t concentration was higher (2.10- 6 M) and the transport time fixed to 18 h. In the last set of experiments, basipetal movement of radioactivity from 3H-GA t is analysed in relation to time (0 to 7 hours) using decapitated roots. In these assays, Z. Pflanzenphysiol. Bd. 101. S. 25-35.1981.

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Fig. 4: Basipetal transport of radioactivity from 3H-GA t (at 10-7 M) in decapitated roots. A) Relative radioactivity in receiver fragments (R) and in total segments (5) as a function of time (in h). - B) Relative radioactivity per mm fragments as a function of time (in h) and distance from donor (in mm). 2.3 mm root fragments, taken 11.3 mm from donor, are considered as receivers. Time-course of radioactivity in receivers is given in Fig. 4 A, from which it can be seen that no decrease in accumulation rate is observed after 3 hours, radioactivity increasing exponentially (P = 0.01) with time. Gradients of radioactivity along the root (Fig. 4 B) clearly indicate that, after a large increase between the first and the third hours, radioactivity steadily rises for the last four hours. When comparing these data with those obtained for segments (see Fig. 2 B), it can be concluded that higher levels of radioactivity are detected everywhere along the tissues, the difference being larger in the basal fragments. This could very well explain the enhanced radioactivity found in receivers. Patterns of basipetal transport of radioactivity are clearly different in decapitated root segments as compared with decapitated roots.

It was of interest to know if radioactivity movement was representative of GAt transport. Chromatographic analyses of decapitated segments after 6 hours' apical application of 3H-GA t are presented in Fig. 5. Beside GAl (47 % of total chromatogram radioactivity) another major unidentified labelled compound X (35 0/0) was observed. These proportions were significantly unchanged along the segment. The Z. P/lanzenphysiol. Bd. 101. S. 25-35. 1981.

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Fig. 5: Chromatographic analyses of decapitated root segments after 6 h apical application of 3H-GA, (at 10-7 M). The segments were subdivided into 5 fragments (A ... E) dessignated by their distances from the donor blocks. A (0-2.1 mm); B (2.1-4.4 mm); C (4.4-6.7 mm); D + E (6.7-11.1 mm). Radioactivity was expressed as the % of the total radioactivity of the chromatogram. Horizontal bars indicate R f value of 3H-GA , in the solvent system used.

receiver's radioactivity was too weak to be chromatographed (see Fig. 3). After 6 hours of acropetal transport, decapitated segments and receiver blocks were analysed. Results presented in Fig. 6 clearly showed that the proportion of GA, decreased linearly with distance from the donor (68 Ofo in the basal fragment, 37 Ofo in the apical one), the opposite process being observed for the unidentified compound X (200/0, 42 %). In receiver blocks, both GA, (38 %) and X (29 %) are present. It seemed that X was acropetally transported and GA, to X conversion took place all along the root segment. From a comparison of Fig. 5 with Fig. 6 it can be concluded that the transformation of GA, to X is much larger in the apical fragment than in the basal one. This difference could be explained by: 1) the higher Z. P/lanzenphysiol. Ed. 101. S. 25-35. 1981.

32

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Fig. 6: Chromatographic analyses of decapitated root segments and receiver blocks (R) after 6 h basal application of 3H-GA l (at 10-7 M). The segments were subdivided into 5 fragments (A ... E) designated by their distances from the donor blocks. A (0-2.1 mm); B (2.1-4.4 mm); C 4.4-6.7 mm); D (6.7-9.1 mm); E (9.1-11.4 mm). Radioactivity was expressed as the % of the total radioactivity of the chromatogram. Horizontal bars indicate R' value of 3H-GA l in the solvent system used.

level of GAl in the apical fragment, 2) the differences in histological structures, 3) the differences in the level of some endogenous growth regulators which might interact with GAl transport. Chromatographic analyses of decapitated roots after 6 hours of apical applications of 3H-GA l are presented in Fig. 7. In view of the low level of radioactivity in the basal and «receiver» fragments, these were pooled before chromatography. The proportion of GAl decreased linearly with distance from the donor (68 % in the apical fragment, 46 0 10 in the «basal + receiver» one), the opposite trend being Z. P/lanzenphysiol. Bd. 101. S. 25-35.1981.

3H-GA I transport in maize root

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Fig. 7: Chromatographic analyses of decapitated roots after 6 h apical application of 3H_ GAl (at 10-7 M). The roots were subdivided into 6 fragments (A ... R) - the last being considered as «receiver» - designated by their distances from the donor blocks. A (0-2.1 mm); B (2.1-4.4 mm); C (4.4-6.7 mm); D (6.7-9.1 mm); E + R (9.1-13.7 mm). Radioactivity was expressed as the DID of the total radioactivity of the chromatogram. Horizontal bars indicate R f value of 3H-GA I in the solvent system used. observed for the compound X (20 vs 340/0). From a comparison of Fig. 5 with Fig. 7, it can be observed that in the apical fragment the concentration of the X compound is much greater (35 % as opposed to 200/0) in the segment than in the root. This seems to indicate a larger metabolism in the segment. Such an observation is confirmed by the higher GAl level all along the root as compared with the segment. The patterns of basipetal transport seem to be very different if the effects in root segments are compared with those in whole roots. It is noteworthy that control chromatography of donor blocks, before or after basal or apical applications, showed only one radioactive spot (GAl). There is no exogenous modification of GAl like that observed for IAA (PERNET and PILET, 1979). The chemical nature of Z. P/lanzenphysiol. Bd. 101. S. 25-35. 1981.

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J. J.

PERNET and P. E. PILET

the X compound is not yet determined, but this compound producted from GAt could be GAs. The conversion of 3H-GA t to 3H-GAs has been observed in several plant materials: Pisum sativum (PATTERSON and RAPPAPORT, 1974), Phaseolus coccineus (REEVE et al., 1975), Zea mays (DAVIES and RAPPAPORT, 1975). In roots, other labelled metabolites from GAl have been detected (DAVIES and RAPPAPORT, 1975). Basipetal 3H-GA t transport experiments in closed boxes showed the presence of labelled volatile compounds (possibly 3HHO), which represent 1 Ofo of the initial donor radioactivity. This seems to indicate that unlabelled metabolites of GAt are present, as previously obsereved by NASH and CROZIER (1975). It is thus clear that, after application of 3H-GA l on roots or root segments, other radioactive compounds are present in the tissues (HARTUNG and PHILLIPS, 1974), some of them being acropetally and basipetally translocated. The radioactivity measured does not necessarily agree with the GAt gradient. In the present experiments, the shape and slope of the radioactivity gradients are not fundamentally different from those obtained for 3H-GA t . References CROZIER, A. and D. M. REID: Do roots synthesize gibberellins? Canad. J. Bot. 49, 967-975 (1971). DAVIES, L. J. and L. RAPPAPORT: Metabolism of tritiated gibberellins in d-5 dwarf maize. I. In excised tissues and in intact dwarf and normal plants. Plant Physio!. 55, 620-625 (1975). EL ANTABLY, H. M.: Redistribution of endogenous indoleacetic acid, abscisic acid and gibberellins in geotropically stimulated Ribes nigrum roots. Z. Pflanzenphysio!. 75, 15-24 (1975). EL ANTABLY, H. M. and P. LARSEN: Distribution of gibberellin and abscisic acid in geotropically stimulated Vicia faba roots. Physio!. Plant. 32, 322-329 (1974). - - Redistribution of endogenous gibberellins in geotropically stimulated roots. Nature 250, 76-77 (1974). HARTUNG, W. and I. D. J. PHILLIPS: Basipetally polarized transport of eH) gibberellin A, and (HC) gibberellin A3, and acropetal polarity of (HC) indole-3-acetic acid transport in stellar tissues of Phaseolus coccineus roots. Planta 118, 311-322 (1974). JACOBS, W. P. an P. E. PRUETT: The time-course of polar movement of gibberellins through Zea roots. Amer. J. Bot. 60, 896-900 (1973). JONES, R. L. and 1. D. J. PHILLIPS: Organs of gibberellin synthesis in light-grown sunflower plants. Plant Physio!. 41,1381-1386 (1966). KAGAWA, T., T. FUKINBARA, and Y. SUMIKI: Thin-layer chromatography of gibberellins. Agr. Bio!. Chern. 27, 598-599 (1963). LANG, A.: Gibberellins: Structure and metabolism. Ann. Rev. Plant Physio!. 21, 537-570 (1970). MOST, B. H. and T. K. SCOTT: Transport of growth regulators in sugar cane. Plant Physio!. 47, supp!. 41 (1971). NASH, L. J. and A. CROZIER: Translocation and metabolism of (3H) gibberellins by lightgrown Phaseolus coccineus seedlings. Planta 127,221-231 (1975). PATTERSON, R. J. and L. RAPPAPORT: The conversion of gibberellin At to gibberellin AR by a cell-free enzyme system. Planta 119, 183-191 (1974).

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3H-GA t transport in maize root

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PERNET, J. J. and P. E. PILET: Importance of the tip on the (5- 3H)-indol-3yl-acetic acid transport in maize root. Z. Pflanzenphysiol. 94, 273-279 (1979). PHILLIPS, 1. D. J. and W. HARTUNG: Basipetal and acropetal transport of (3,4-3H) gibberellin At in short and long segments of Phaseolus coccineus second internode. Planta 116, 109-121 (1974). PILET, P. E.: Auxin transport in roots. Nature 204, 561-562 (1964). - Action of gibberellic acid on auxin transport. Nature 208, 1344-1345 (1965). - Abscisic acid as a root growth inhibitor: Physiological analyses. Planta 122, 299-302 (1975). - Effects of gravity on the growth inhibitors of geostimulated roots of Zea mays L. Planta 131,91-93 (1976). - 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). - The role of the cap in the geotropism of roots exposed to light. Z. Pflanzenphysiol. 89,411-426 (1978). - Kinetics of the light-induced georeactivity of maize roots. Planta 145, 403-404 (1974). PILET, P. E., M. C. ELLIOTT, and M. M. MOLONEY: Endogenous and exogenous auxin III the control of root growth. Planta 146, 405-408 (1979). PILET, P. E. and D. NOCERA-PRZYBECKA: Abscisic acid effect on the DNA microgradients of decapped maize roots. Plant and Cell Physiol. 19, 1475-1481 (1978). PI LET, 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). REEVE, D. R., A. CROZIER, R. C. DURLEY, D. M. REID, and P. R. PHARIS: Metabolism of 3H-gibberellin At and 3H-gibberellin A4 by Phaseolus coccineus seedlings. Plant Physiol. 55,42-44 (1975). RIVIER, L., H. MIL ON, and P. E. PILET: Gas chromatography-mass spectrometric determinations of abscisic acid levels in the cap and the apex of maize roots. Planta 134, 23-27 (1977). RIVIER, L. and P. E. PILET: Indolyl-3-acetic acid in cap and apex of maize roots: Identification and quantification by mass fragmentography. Planta 120, 107-112 (1974). 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). SKENE, K. G. M.: Gibberellin-like substances in root exudate of Vitis vini/era. Planta 74, 250-262 (1967). WEBSTER, J. H. and M. B. WILKINS: Lateral movement of radioactivity from (14C) gibberellic acid (GA3) in roots and coleoptiles of Zea mays seedlings during geotropic stimulation. Planta 121, 303-308 (1974). WILKINS, M. B. and L. J. NASH: Movement of radioactivity from (3H) GA3 in geotropically stimulated coleoptiles of Zea mays. Planta 115, 245-251 (1974). Prof. Dr. P. E. PILET, Institut de Biologie et de Physiologie vegetales, Universite de Lausanne, Place de la Riponne 6, CH-1005 Lausanne.

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