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Role of Endogenous Auxins and Gibberellins in Growth-correlative Effects of Root Metabolism and in the Hypocotyl Regeneration of Flax Seedlings
iMZ±&M&
Hio('hem. Physiol. Pflanzen 11'4, 691-695 (1979)
Role of Endogenous Auxins and Gibberellins in Growth-correlative Effeets of Root Metabolism and in the Hypocotyl Regeneration of Flax Seedlings J. SEBANEK, HOA~G MINH TAN and S. KLi60VA Department of Bot
Sununary The p
Introduction
Flax seedlings represent a popular model of experimental plant morphology. This is above all due to a marked correlative-promoting effect of cotyledons both on growth of cytyladonar buds (KmIAREK 1930) and on bending of hypocotyl stumps (DOSTAL 1968; RAHMAN et aI. 1977) on the one hand and due to a marked promoting effect of root on growth of cotylcdonar buds (DOSTAL 1955). Finally, flax seedlings rcpresent 11 rcmarkable model of experimental plant morphology also due to a marked regeneration of adYClltitiollS buds on hypocotyl stumps (DOSTAL 1967). This paper contributes to studics on the ph~-tohormonal naturc of growth correlations in flax seedlings in relation to endogenous auxins and gibberelins partly with regard to correlative functions of root and partly with regard to the regeneration of adventitious buds on hypocotyl stumps. Material and Methods Experiments were carried out with flax seedlings (Lillum usilalissill1ulI1) cv. Vent grown in vegetation pots containing garden soil under the continuous illumination of 3,000 Ix. To study correlative effeets of roots upon the level of endogenous auxins and gibberellins in cotyledons, ll-day-old seedlings were divided into 5 series: Series I was used for analyses of cotyledons obtained from intact plants, Series II to V for analyses of cotyledons obtained from plants with excised roots (together with the 30-mm-Iong basal segment of hypocotyl); in Series II, the root and hypocotyl base were absent for 12 h, in Series III for 24 h, in Series IV for 48 h and in Series V vor 72 h. Plants deprived of roots and hypocotyl bases were cultivated in vessels containing water in which the hypocotyl stumps were submerged.
u.
a_i"
692
J. SEBANEK, H. 1\I. TAN and S KLicoVA
To study the phytohormonal nature of regenemtion of adventitious buds on the hypocotyl stums, ll-day-old seedlings were divided into four series: Series 1 served for analyses of 20-mm-Iong apical part of the hypocotyl immediately after th~ decapitation of plants cut immediately below cotyledons, Series 2, 3, and 4 for analyses carried out 12, 24, and 48 h after decapitation, resp. In each esperimental series, endogenous auxins and gibberellins were estimated in 2 grams of material. Fresh matter was homogenized and the homogenates of individual samples extracted with 30 ml of methylalcohol for 24 h at 6°e. The extracts obtained were decanted and the homogenate re-ex-tracted with methylalcohol for a further period of 24 h. The pooled extmcts were then filtered and evapomted to dryness in a stream of could air. The residues were dissolved in 1 ml of ethylacetate and subjected to chromatographic separation. In the case of gibberellins this separation was performed
220
1&0 A
140
ao
120
110
90
1
2
.Fig. 1. Chromatographic separation of endogenous auxins in cotyledons of flax seedlings A - intact plants; B - seedlings deprived of root and basal part of hypocotyl for 12 h; e - for 24 h; D - for 48 h; E - for 72 h. Y-axis, elongation of coleoptile segments against control (100) in per cent; x-axis, RF values. Fig. 2. Chromatographic separation of endogeuous gibberellins in cotyledons of flax seedlings A - intact plants; B- seedlings deprived of root and basal part ofhypocotyl for 12 h; e - for 24 h; D - for 48 h; E - for 72 h. Y-axis, elongation of hypocotyls of lettuce seedlings ag,tinst control {100) in per cent; x-,txis, RF values.
693
Endogenous Auxins and Gibberellins of Flax Seedlings
on thin layers of silicagel G using chloroform: ethylacetate: acetic acid (60: 40: 5) as a solvent system (SEMBDNER et a!. 1962). The extracts of chromatograms corresponding to the individual RF were tested using lettuce seedlings (Lactuca sativa, cv. Knil maje) and evaluated as described earlier (KOPECKY et al. 1975). In the case of endogenous auxins the chromatographic separation took place on thin layers of silicagel G using isopropylalcohol: ammonium hydroxide: water (80: 5: 5) as a solvent system (LABLER and SCHWARZ 1965). The extracts corresponding to the individual Rfs were tested using coleoptile segments of wheat (Triticum aestivum, cv, Kharkovskaya 159) and the results were evaluated as described in our earlier paper (KOPECKY et a!. 1975). All analyses were repeated twice with analogical results.
130
A
110 110
95 130
".~
110
95 130
'''~ =
c
110
,
u==
95
130
110
'"'~
_
~
~ =c;:::rU 0
95
4 3 Fig. 3. Ohromatographic separation of endogenous auzins in the apicaZ part of hypocotyZs of (la:c seed-
Zings A - intact plants; B- seedlings decapitated immediately below cotyledons after 12 h; 0 after 24 h; D - after 48 h. Y-axis, elongation of wheat coleoptile segments against controls (100) in per cent; x-axis, RF values. Fig. 4. Ohromatographic separation of endogenous gibberelUns in the apical part of hypocotyZs of (la:c seedlings A - intact plants; B - seedlings decapitated immediately below cotyledons after 12 h; 0 after 24 h; D - after 48 h. Y-axis, elongation of lettuce seedling hypocotyls against control (100) in per cent; x-axis, RF values. 44&
•
r
694
J. SEBANEK, H. M. TAN and S. KLfcoVA
Results
The chromatographic estimation of endogenous auxins and gibberellins in intact plants and those deprived roots and basal parts of hypocotyls for 12 to 72 h is presented in Figs. 1 and 2, resp. It is obvious that the excision of roots and hypocotyl bases resulted in an increase in the level of endogenous auxins in cotyledons. This increase was directly proportional to the time interval after the excision of roots and hypocotyl bases. On the other hand, a reverse relationship was observed in the case of endogenous gibberellins: the excision of roots and hypocotyl bases resulted in a decrease in the level of endogenous gibberellins directly proportional to the time of absence of roots and hypocotyl bases. In Figs. 3 and 4, the chromatographic estimation of endogenous auxins and endogenous gibberellins, resp., is presented immediately after the decapitation of hypocotyls below cotyledons and/or after 12 to 48 h. It is obvious that the level of endogenous auxins decreased and that of endogenous gibberellins increased proportionally to the time interval after decapitation. This suggests that the preparation of the formation of adventitous buds on hypocotyl stumps was associated with a decrease in the level of endogenous auxins on the one hand and an increase of endogenous gibberellins on the other. Discussion
If the epicotyl of ilax seedlings is excised immediately above cotyledons together with one of these organs, the axillary bud of the remaining cotyledon grows always more intensively; this suggests a promoting effect 01 epigeic cotyledons of flax seedlings upon the growth of cotyledonar buds (KOMAREK 1930). However, this promoting effect of flax cotyledons can be observed only on plants with intact roots; if the root of decapitated and one-cotyledon-deprived seedlings is excised together with the hypocotyl base (so that only a 20-mm-long hypocotyl remains on such plants) a more intensive growth of axillary bud of the excised cotyledon can be observed (DOSTAL 1955). However, the excision of roots does not result in the induction of such a growth-inhibiting effect of cotyledon. This suggests that also the hypocotyl base has its own root metabolism because its exicision together with the root results in a reversal of the growth-promo tin effect of cotyledon into a growth-inhibiting one (DOSTAL 1968; TAN et a1. 1978). The promoting effect of cotyledons is, therfore, associated with the phytohormonal metabolism of roots and hypocotyl bases. Its nature is indicated by results of this study because the level of endogenous gibberellins decreases while that of endogenous auxins increases after the excision of root and hypocotyl bases. According to this observation the root metabolism contributes to the promoting effect of epigeic cotyledons in such a way that it decreases its endogenous level of auxin and increases that of gibberellin. On the other hand, in plants with excised roots and hypocotyl bases, the inhibitng effect of epigeic cotyledon is manifested by an increase in the level of endogenous auxin and a decrease in that of endogenous gibberellin in it. The decreased level of endogenous auxin in cotyledons after the excision of root and hypocotyl base and the resulting
Endogenous Auxin, ,md Gibberellins of Flax Seedlings
695
elimination of root metabolism is obviousl~' associated with a well-known ability of roots to inactiyate auxin (PILET 1964); the simultaneous increase in the level of gibberellins in cot:'ledons is, on the other hand, associated with the root's cnpacity to, synthetize endogenous gibberellins (CARR et a1. 1964; SEBANEK 1965). According to our results, the regeneration of adventitious buds on hypocotyl stumps is preceded b:' nn increase in the level of endogenous gibberellins and a decrease in endogenous nuxins in this stump. This agrees with the fact that the regeneration of adventitious buds on the hypocotyl stump is either markedly reduced or completely suppressed by the application of 0.03 and 0.06% IAA paste on this stump. On the other hnnd, the application of 0.06 to 0.5% gibberellin pnste promoted the regenration of adventitious buds on the hypocotyl stump by 50 to 100% (TAN et a1. 1978). References CARR, D. J., READ, D. }I., and SKENE, K. G.M.: The Supply of Gibberellins from the Root to the Shoot. Planta 63, 382-392 (1964). DOSTAL, R.: On the Correlative Morphogenesis Exemplified by Flax/Linum usilatissimum. Prace Brnenske Zakladny CSAV 27, 193-267 (1955). - On Integration in Plants. Harvard Univ. Press, Cambridge 1967. - Correlative Curvatures of the Linu111 Hypocotyl and Growth Regulators. Beitr. BioI. Pflanzen 4';, 357-370 (1968). I\:mIAREK, V.: Zur experimentellen Beeinflussung der Korelationstiitigkeit von epigiiischen Keimbliittem. Flont 124, 300-314 (1930). KOPECKY, K., SEBA:UK, J., and BLAhov A, J.: Time Course ofthe Chang~s in the Level of Endogen.ous Growth Regulators during the Stratification of the Seed of the" Panenske Ceske" Apple. BioI. Plant. 17,81-87 (1975). L.\BLER, L., an.d SCHWARZ, V.: Thin Layer Chromatography. NCSA V Praha 1965. PILET, P. E.: Auxin Transport in Roots Len.s culinaris. Nature 204, 561-562, (1964). RUDIAN, M. 1\1., SEBANEK, J., an.d HRADILiK, J.: Cotylar Dominance in Pea and Flax Seedlin.gs with Regard to Growth Regulators. Biochem. Physiol. Pflanzen 171, 165-169 (1977). SEMBDNER, G., GROSS, R, and SCHREIBER, K.: Die Diin.n.schichtchromatographie von Gibberellinen. Experientia 18, 584-585 (1962). SOBAXEK, J.: Die Interaktion endogener Gibberelline in. der Korrelation zwischen Wurzel und'Epikotyl bei Pisum-Keimlingen. Flora 1a6 A, 303-311 (1965). TXN, H. 1\1., SEB.~NEK, J., and KLicoVA, S.: Contribution to the Experimental Morphogenesis on Flax seedlings/Linum usilalissimum. Acta Univ. Agr. Bmo, A (in press) (1978).
Received April 17, 1979. Authors' address: Prof. Dr. JIRi SEBANEK, Dr. HOANG Mnm TAN and lng. SARKA KLicovA, Vniversity of Agriculture, Zemedl>lska 1, 66265 Bmo, CSSR.
45
Biochem. Physiol. Pflanzen, Bd. 174
Hio('hem. Physiol. Pflanzen 11'4, 691-695 (1979)
Role of Endogenous Auxins and Gibberellins in Growth-correlative Effeets of Root Metabolism and in the Hypocotyl Regeneration of Flax Seedlings J. SEBANEK, HOA~G MINH TAN and S. KLi60VA Department of Bot
Sununary The p
Introduction
Flax seedlings represent a popular model of experimental plant morphology. This is above all due to a marked correlative-promoting effect of cotyledons both on growth of cytyladonar buds (KmIAREK 1930) and on bending of hypocotyl stumps (DOSTAL 1968; RAHMAN et aI. 1977) on the one hand and due to a marked promoting effect of root on growth of cotylcdonar buds (DOSTAL 1955). Finally, flax seedlings rcpresent 11 rcmarkable model of experimental plant morphology also due to a marked regeneration of adYClltitiollS buds on hypocotyl stumps (DOSTAL 1967). This paper contributes to studics on the ph~-tohormonal naturc of growth correlations in flax seedlings in relation to endogenous auxins and gibberelins partly with regard to correlative functions of root and partly with regard to the regeneration of adventitious buds on hypocotyl stumps. Material and Methods Experiments were carried out with flax seedlings (Lillum usilalissill1ulI1) cv. Vent grown in vegetation pots containing garden soil under the continuous illumination of 3,000 Ix. To study correlative effeets of roots upon the level of endogenous auxins and gibberellins in cotyledons, ll-day-old seedlings were divided into 5 series: Series I was used for analyses of cotyledons obtained from intact plants, Series II to V for analyses of cotyledons obtained from plants with excised roots (together with the 30-mm-Iong basal segment of hypocotyl); in Series II, the root and hypocotyl base were absent for 12 h, in Series III for 24 h, in Series IV for 48 h and in Series V vor 72 h. Plants deprived of roots and hypocotyl bases were cultivated in vessels containing water in which the hypocotyl stumps were submerged.
u.
a_i"
692
J. SEBANEK, H. 1\I. TAN and S KLicoVA
To study the phytohormonal nature of regenemtion of adventitious buds on the hypocotyl stums, ll-day-old seedlings were divided into four series: Series 1 served for analyses of 20-mm-Iong apical part of the hypocotyl immediately after th~ decapitation of plants cut immediately below cotyledons, Series 2, 3, and 4 for analyses carried out 12, 24, and 48 h after decapitation, resp. In each esperimental series, endogenous auxins and gibberellins were estimated in 2 grams of material. Fresh matter was homogenized and the homogenates of individual samples extracted with 30 ml of methylalcohol for 24 h at 6°e. The extracts obtained were decanted and the homogenate re-ex-tracted with methylalcohol for a further period of 24 h. The pooled extmcts were then filtered and evapomted to dryness in a stream of could air. The residues were dissolved in 1 ml of ethylacetate and subjected to chromatographic separation. In the case of gibberellins this separation was performed
220
1&0 A
140
ao
120
110
90
1
2
.Fig. 1. Chromatographic separation of endogenous auxins in cotyledons of flax seedlings A - intact plants; B - seedlings deprived of root and basal part of hypocotyl for 12 h; e - for 24 h; D - for 48 h; E - for 72 h. Y-axis, elongation of coleoptile segments against control (100) in per cent; x-axis, RF values. Fig. 2. Chromatographic separation of endogeuous gibberellins in cotyledons of flax seedlings A - intact plants; B- seedlings deprived of root and basal part ofhypocotyl for 12 h; e - for 24 h; D - for 48 h; E - for 72 h. Y-axis, elongation of hypocotyls of lettuce seedlings ag,tinst control {100) in per cent; x-,txis, RF values.
693
Endogenous Auxins and Gibberellins of Flax Seedlings
on thin layers of silicagel G using chloroform: ethylacetate: acetic acid (60: 40: 5) as a solvent system (SEMBDNER et a!. 1962). The extracts of chromatograms corresponding to the individual RF were tested using lettuce seedlings (Lactuca sativa, cv. Knil maje) and evaluated as described earlier (KOPECKY et al. 1975). In the case of endogenous auxins the chromatographic separation took place on thin layers of silicagel G using isopropylalcohol: ammonium hydroxide: water (80: 5: 5) as a solvent system (LABLER and SCHWARZ 1965). The extracts corresponding to the individual Rfs were tested using coleoptile segments of wheat (Triticum aestivum, cv, Kharkovskaya 159) and the results were evaluated as described in our earlier paper (KOPECKY et a!. 1975). All analyses were repeated twice with analogical results.
130
A
110 110
95 130
".~
110
95 130
'''~ =
c
110
,
u==
95
130
110
'"'~
_
~
~ =c;:::rU 0
95
4 3 Fig. 3. Ohromatographic separation of endogenous auzins in the apicaZ part of hypocotyZs of (la:c seed-
Zings A - intact plants; B- seedlings decapitated immediately below cotyledons after 12 h; 0 after 24 h; D - after 48 h. Y-axis, elongation of wheat coleoptile segments against controls (100) in per cent; x-axis, RF values. Fig. 4. Ohromatographic separation of endogenous gibberelUns in the apical part of hypocotyZs of (la:c seedlings A - intact plants; B - seedlings decapitated immediately below cotyledons after 12 h; 0 after 24 h; D - after 48 h. Y-axis, elongation of lettuce seedling hypocotyls against control (100) in per cent; x-axis, RF values. 44&
•
r
694
J. SEBANEK, H. M. TAN and S. KLfcoVA
Results
The chromatographic estimation of endogenous auxins and gibberellins in intact plants and those deprived roots and basal parts of hypocotyls for 12 to 72 h is presented in Figs. 1 and 2, resp. It is obvious that the excision of roots and hypocotyl bases resulted in an increase in the level of endogenous auxins in cotyledons. This increase was directly proportional to the time interval after the excision of roots and hypocotyl bases. On the other hand, a reverse relationship was observed in the case of endogenous gibberellins: the excision of roots and hypocotyl bases resulted in a decrease in the level of endogenous gibberellins directly proportional to the time of absence of roots and hypocotyl bases. In Figs. 3 and 4, the chromatographic estimation of endogenous auxins and endogenous gibberellins, resp., is presented immediately after the decapitation of hypocotyls below cotyledons and/or after 12 to 48 h. It is obvious that the level of endogenous auxins decreased and that of endogenous gibberellins increased proportionally to the time interval after decapitation. This suggests that the preparation of the formation of adventitous buds on hypocotyl stumps was associated with a decrease in the level of endogenous auxins on the one hand and an increase of endogenous gibberellins on the other. Discussion
If the epicotyl of ilax seedlings is excised immediately above cotyledons together with one of these organs, the axillary bud of the remaining cotyledon grows always more intensively; this suggests a promoting effect 01 epigeic cotyledons of flax seedlings upon the growth of cotyledonar buds (KOMAREK 1930). However, this promoting effect of flax cotyledons can be observed only on plants with intact roots; if the root of decapitated and one-cotyledon-deprived seedlings is excised together with the hypocotyl base (so that only a 20-mm-long hypocotyl remains on such plants) a more intensive growth of axillary bud of the excised cotyledon can be observed (DOSTAL 1955). However, the excision of roots does not result in the induction of such a growth-inhibiting effect of cotyledon. This suggests that also the hypocotyl base has its own root metabolism because its exicision together with the root results in a reversal of the growth-promo tin effect of cotyledon into a growth-inhibiting one (DOSTAL 1968; TAN et a1. 1978). The promoting effect of cotyledons is, therfore, associated with the phytohormonal metabolism of roots and hypocotyl bases. Its nature is indicated by results of this study because the level of endogenous gibberellins decreases while that of endogenous auxins increases after the excision of root and hypocotyl bases. According to this observation the root metabolism contributes to the promoting effect of epigeic cotyledons in such a way that it decreases its endogenous level of auxin and increases that of gibberellin. On the other hand, in plants with excised roots and hypocotyl bases, the inhibitng effect of epigeic cotyledon is manifested by an increase in the level of endogenous auxin and a decrease in that of endogenous gibberellin in it. The decreased level of endogenous auxin in cotyledons after the excision of root and hypocotyl base and the resulting
Endogenous Auxin, ,md Gibberellins of Flax Seedlings
695
elimination of root metabolism is obviousl~' associated with a well-known ability of roots to inactiyate auxin (PILET 1964); the simultaneous increase in the level of gibberellins in cot:'ledons is, on the other hand, associated with the root's cnpacity to, synthetize endogenous gibberellins (CARR et a1. 1964; SEBANEK 1965). According to our results, the regeneration of adventitious buds on hypocotyl stumps is preceded b:' nn increase in the level of endogenous gibberellins and a decrease in endogenous nuxins in this stump. This agrees with the fact that the regeneration of adventitious buds on the hypocotyl stump is either markedly reduced or completely suppressed by the application of 0.03 and 0.06% IAA paste on this stump. On the other hnnd, the application of 0.06 to 0.5% gibberellin pnste promoted the regenration of adventitious buds on the hypocotyl stump by 50 to 100% (TAN et a1. 1978). References CARR, D. J., READ, D. }I., and SKENE, K. G.M.: The Supply of Gibberellins from the Root to the Shoot. Planta 63, 382-392 (1964). DOSTAL, R.: On the Correlative Morphogenesis Exemplified by Flax/Linum usilatissimum. Prace Brnenske Zakladny CSAV 27, 193-267 (1955). - On Integration in Plants. Harvard Univ. Press, Cambridge 1967. - Correlative Curvatures of the Linu111 Hypocotyl and Growth Regulators. Beitr. BioI. Pflanzen 4';, 357-370 (1968). I\:mIAREK, V.: Zur experimentellen Beeinflussung der Korelationstiitigkeit von epigiiischen Keimbliittem. Flont 124, 300-314 (1930). KOPECKY, K., SEBA:UK, J., and BLAhov A, J.: Time Course ofthe Chang~s in the Level of Endogen.ous Growth Regulators during the Stratification of the Seed of the" Panenske Ceske" Apple. BioI. Plant. 17,81-87 (1975). L.\BLER, L., an.d SCHWARZ, V.: Thin Layer Chromatography. NCSA V Praha 1965. PILET, P. E.: Auxin Transport in Roots Len.s culinaris. Nature 204, 561-562, (1964). RUDIAN, M. 1\1., SEBANEK, J., an.d HRADILiK, J.: Cotylar Dominance in Pea and Flax Seedlin.gs with Regard to Growth Regulators. Biochem. Physiol. Pflanzen 171, 165-169 (1977). SEMBDNER, G., GROSS, R, and SCHREIBER, K.: Die Diin.n.schichtchromatographie von Gibberellinen. Experientia 18, 584-585 (1962). SOBAXEK, J.: Die Interaktion endogener Gibberelline in. der Korrelation zwischen Wurzel und'Epikotyl bei Pisum-Keimlingen. Flora 1a6 A, 303-311 (1965). TXN, H. 1\1., SEB.~NEK, J., and KLicoVA, S.: Contribution to the Experimental Morphogenesis on Flax seedlings/Linum usilalissimum. Acta Univ. Agr. Bmo, A (in press) (1978).
Received April 17, 1979. Authors' address: Prof. Dr. JIRi SEBANEK, Dr. HOANG Mnm TAN and lng. SARKA KLicovA, Vniversity of Agriculture, Zemedl>lska 1, 66265 Bmo, CSSR.
45
Biochem. Physiol. Pflanzen, Bd. 174