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No effect of fusicoccin on phytochrome controlled nitrate reductase activity
319
Plant Science Letters. 18 (1980) o Elsevier/North-Holland
319-323 Scientific Publiiens
Ltd.
NO EFFECT OF FUSICOCCIN ON PHYTOMROME CONTROLLED NITRATE REDUCTASE ACTIVITY
J.S. KNYPL
Plant Growth Substances Labomtory, Lodz (Poland)
University of Lodz, ul. Banacha 12/16, 90-237
(Received October 22nd, 1979) (Revision received March lst, 1980) (Accepted March lst, 1980)
SUMMARY
Nitrate reductase activity (NRA; EC 1.6.6.1) was detected by an in vivo assay in light-sensitive lettuce (Lactuca sutiua L. var. Grand Rapids) seeds which had been imbibed in 0.06 M KNOJ in darkness. Illumination with white light (10 min) more than doubled the enzyme activity; 5-min far red illumination following white light impulse reduced NRA to the level showed by the dark-imbibed seeds. Fusicoccin (FC) did not affect the phytochrome controlled NRA, although it substituted light requirement for the seed germination. Phytochrome possibly facilitates the assembly of nitrate reductase components on cell membranes. FC did not affect NRA in cucumber (Cucumis satiuus L.) cotyledons induced with KN03, while it produced a remarkable activity in the absence of external nitrate. The latter effect was possibly dependent on acidification of the medium caused by FC-stimulated proton extrusion from the cotyledons.
INTRODUCTION
Nitrate reductase activity is controlled by phytochrome in some plants [l---5]. The stimulatory effect of red light on NRA in light sensitive seeds of lettuce markedly diminished after piercing the seed coats, with a concomitant many-fold increment of the enzyme activity. This suggested that: (i) penetration of nitrate to intact seeds was restricted; and (ii) the Addreee correepondence
to: Dr. J.S. Knypl, Plant Growth Substances Laboratory, of Lodz, ul. Banacha 12/16,90-237 Lodz, Poland. Abbreuiatione: CAP, chloramphenicol; FC, fusicoccin; FR, far red light; NR, nitrate reductase; NRA, NR activity. University
320
rate of NO; diffusion to the embryo was possibly one of the factors limiting the final NRA [ 61. If this assumption was correct, then fusicoccin, a diterpene glucoside produced by Fusicoccum amygdali Del. [ 71, should enhance NRA in germinating seeds. FC strongly promotes the energydependent proton extrusion and uptake of monovalent cations and anions
[a*
This study presents the evidence that FC had no effect on the light enhanced NRA in both lettuce seeds and cucumber cotyledons. MATERIALS
AND METHODS
NR induction in lettuce seeds Seeds of Lactuca satiua L. var. Grand Rapids were purchased from Ferry Morse Seed Co. (Orchard Park, N.Y.) and stored under silica gel at 6 -8°C. Triple distilled water and KN03 solutions used for NR induction and germination were supplemented with 10e3 M CAP as a bacteriostatic. Batches of 100 seeds were placed in lo-cm Petri dishes containing two discs of prewashed Whatman No. 1 filter paper and 5 ml of 0.05 M KNOJ supplemented with FC. After 1 h in darkness the seeds were exposed to white light for 10 min and/or to FR for 5 min [9], and returned to darkness. Duplicate sets were germinated in permanent illumination (4.6 W m-‘; fluorescent tubes ‘Flora LF’) or in darkness at 25°C. NR activity was determined after 12 h imbibition; none of the seeds had germinated during this time. Since preliminary experiments revealed that 10 min white light produced similar effects as red (660 nm) light impulse, the former illumination was applied throughout the study. Determination of NRA in imbibed seeds NR activity in non-germinated lettuce seeds could not be detected in vitro. Satisfactory conditions for the in vivo NRA assay were established in preliminary experiments. The concentration of n-propanol in an incubation mixture was found to be a highly important factor for reproducibility of the results. If n-propanol was added to a final concentration of 4% (v/v), the release of NO; from imbibed, non-germinated seeds was proportional to time for at least 5 h after an initial 60-min lag period. Anaerobic atmosphere did not improve the results. Two hundred seeds which had been imbibed for 12 h were washed with distilled water, blotted dry on a paper towel, and transferred to 15-ml screwcapped vials containing 5.5-ml aliquots of the incubation mixture: 0.1 M K’-phosphate buffer (pH 7.5), 0.1 M KN03, 4% n-propanol, and two drops of 8 mM CAP. The seeds were twice vacuum infiltrated and left at 30°C in darkness with occasional stirring. After 2 and 4 h 0.5-ml aliquots of the incubation medium were withdrawn for calorimetric NO; determination [lo]. Results of the assay were highly reproducible if the same lot of seeds was used.
321
Induction of NRA in cucumber cotyledons
Cotyledons which had been separated from 5day-old etiolated Cucumis sutiuus L. var. ‘Delicatess’ seedlings were induced with 0.01 M KNOJ for 20 h in light or in darkness at 25”C, and then used for in vivo NRA assays as described [ 111. Experiments were run in triplicate and repeated at least twice. RESULTS
Fusicoccin in a range of concentrations from 10m6to 5 X lo-’ M did not affect NRA in the seeds imbibed in darkness, despite the fact that germination was increased to 100% in comparison with 1. -2 and 15% germination in 0.05 M KNO, and distilled water, respectively. However, NRA in seeds imbibed in permanent illumination in the presence of 5 X 10e6 M FC was enhanced by around 30% in comparison with control seeds, induced with only KN03. Some NRA (35 nmol NO; h-‘/1000 seeds) could be detected also,in seeds imbibed in distilled water in light; the seeds seemingly contained a limited pool of endogenous nitrate. Seeds that were continuously illuminated showed more than twice the activity of those imbibed in darkness (Table I). The same effect was produced by a lo-min white light impulse (or R) followed by darkness. FR illumination after white light reduced the enzyme activity to the level of activity in the seeds continuously maintained in darkness. FC had no effect TABLE I NO EFFECT OF FC ON THE PHYTOCHROME LETTUCE SEEDS
CONTROLLED
NR ACTIVITY
IN
NRA was determined after 12 h imbibition in 0.05 M KNO, (0) or 0.05 M KNO, and 5 x 10m6 M FC (FC). Germinated seeds were counted after 30 h. L, R and FR, white,. red and far red light; D, darkness; -+ change of illumination. Illumination regime after 60 min in darkness
L D lOminL-+D lOminL-+5minFR+D lOminL+ 5minFR-+ + 10minLd D lOminR+D
NRA, nmol NO; h^‘/ 1000 seeds
% Germination
Oa
FC
Ob
FC
155 70 150 70
189 70 160 72
73 1 64 1
100 80 100 80
140 153
158 166
60 68
100 95
a NRA in seeda imbibed for 12 h in distilled water was 5 and 36 nmol NO;/1000 seeds in D and L, respectively. b 15 and 85% seeds germinated during 30 h in distilled water in D and L, respectively.
322 TABLE II EFFECT OF FUSICOCCIN
ON NR ACTIVITY
IN CUCUMBER
COTYLEDONS
Cotyledons were incubated in solutions of FC (non-induced cotyledons) or FC supplemented with 0.01 M KNO, without CAB for 20 h, then NRA determined in vivo. Superscript letters indicate statistically significant differences among treatments, P = 0.01. Concentration of FC (M)
NRA, nmol NO; h-‘/cotyledon Non-induced
-_ 0 1o-6 5 x10-6 1o‘5 10W5; assay mixture without KNO,
cotyledons
KNO,-induced
cotyledons
Darkness
Light
Darkness
Light
0.P 3.lb 3.lb 6.2c 0
0.7a 3.lb 3.lb 8.9 0
6.2’ 6.1c 6.2c 6.2c 4.0b
13.6e 14.2e 14.4e 14.1e 6.2c
on this phytochrome controlled enhancement of enzyme activity, although it induced 80% germination in comparison with 1% germinated seeds in the proper control series. FC did also not affect NRA in excised cucumber cotyledons, when it was applied together with 0.01 M KN03 either in the dark or in light. In contrast, the cotyledons which had been grown in FC solutions without KN03 released remarkable quantities of NO; upon subsequent incubation in the KNO,-supplemented medium for the in vivo NRA assay. FC at the highest concentration tested (10m5 M) induced NR activity equal to that in the KN03-induced cotyledons in darkness, and only by a one third lower than in the nitrate-supplied greening cotyledons. No nitrite was released by the FC-treated cotyledons when the medium for NRA assay was deprived of KNOJ (Table II). This is an indication that the cotyledons contained no measurable pool of endogenous nitrate. Growth of the cotyledons was markedly stimulated by K’ [ll], and FC did not enhance the effect of potassium. FC slightly increased the fresh weight of cotyledons grown in the absence of KNOJ (data not illustrated). DISCUSSION
Although FC mimics the symptoms of action of red light as concerns germination of lettuce seeds [ 121, it has virtually no effect on induction of NR activity in the activation phase of seed germination. This phytotoxin tends to enhance NRA only in the seeds permanently illuminated, the magnitude of the effect being comparable to that produced by kinetin or ethylene [ 61; this effect may be due to FC-stimulated uptake and flow of NO; to an active nitrate pool. It should be stressed, however, that NR activity is detectable in the seeds induced with KNO, in darkness; white
323
light or R impulse only doubles the enzyme activity (Table I). This means that phytochrome does not control NRA at the genome level. It seems that phytochrome affects either the assembly of the nitrate reductase components on a,heterogenous cell constituent such as a membrane or a fraction of endoplasmic reticulum, or nucleotidation of the NR complex [13,14]. None of these two hypothetical sites is sensitive to FC. The phytotoxin can indirectly affect the‘enzyme activity in conditions when the rate of NO; influx to the cell may be a’limiting factor as in the case of seeds and cotyledons maintained in permanent illumination, i.e. in conditions of a rapid activation of many concurrent metabolic pathways. NRA in cucumber cotyledons may be induced by many factors in the absence of external NO;, including alcohols, CAP [6], low pH and organic acids [ 151. Since FC stimulates H’ extrusion from the cells [8] and increases intracellular level of malate [ 16,171, the nitrate reductase activity detected in the non-induced cotyledons may be regarded as a result of increased intracellular malate level [cf. 181. ACKNOWLEDGEMENTS
The author would like to express his gratitude to Professor Alessandro Ballio (Institute of Biological Chemistry, University of Rome, Italy) for the generous gift of FC. REFERENCES 1 R.W. Jones and R.W. Sheard, Plant Phyeiol., 55 (1975) 954. 2 R.W. Jones and R.W. Sheard, in E.J. Hewitt and C.V. Cutting (Eds.),Nitrogen Assimilation of Plants, Academic Press, London, New York and San Fran&co, 1979, p. 521. 3 S.H. Duke and S.O. Duke, Plant Cell Physiol., 19 (1978) 481. 4 H. SPrakawa and P. Yamamoto, Plant Physiol., 63 (1979) 1098. 5 G.C. Whitelam, C.B. Johnson and H. Smith, Photochem. Photobiol., 30 (1979) 589. 6 J.S. Knypl, in E.J. Hewitt and C.V. Cutting (Eda.), Nitrogen Amimilation of Plants, Academic Press, London, New York and San Francisco, 1979, p. 541. 7 A. Ballio, M. Brufani, C.G. Casinovi, S. Cerrini, W. Feldi, R. Pellicciari, B. Santurbano and A. Vaciago, Ezperientia, 24 (1968) 631. 8 E. Ma&, Annu. Rev. Plant Physiol., 30 (1979) 273. 9 T. Tanada, Planta (Berlin), 134 (1977) 57. 10 R.H. Hageman and D.P. Huckleeby, Methods Enzymol., 23 (1971) 491. 11 J.S. Knypl, Z. Pflanzenphysiol., 70 (1973) 1. 12 P. Lado, F. Rasi-Caldogno and R. Colombo, Physiol. Plant., 31 (1974) 149. 13 E.J. Hewitt, D.P. Hucklesby, A.F. Mann, B.A. Notton and G.J. Rucklidge, in E.J. Hewitt and C.V. Cutting (E&e.), Nitrogen Assimilation of Plants, Academic Press, London, New York and San Francisco, 1979, p. 266. 14 R.G. Butz and W.A. Jackson, Phytochemistry, 16 (1977) 409. 15 J.S. Knypl and A.R. Ferguson, Z. Pflanzenphysiol., 74 (1976) 434. 16 E. MarrB, in P.E. Pilet (Ed.), Plant Growth Regulation, Springer Verlag, Berlin and Heidelberg 1977, p. 54. 17 R. Stout and R.E. Cleland, Planta (Berlin), 139 (1978) 43. 18 C.K.M. Rathnam, Plant Physiol., 62 (1978) 220.
Plant Science Letters. 18 (1980) o Elsevier/North-Holland
319-323 Scientific Publiiens
Ltd.
NO EFFECT OF FUSICOCCIN ON PHYTOMROME CONTROLLED NITRATE REDUCTASE ACTIVITY
J.S. KNYPL
Plant Growth Substances Labomtory, Lodz (Poland)
University of Lodz, ul. Banacha 12/16, 90-237
(Received October 22nd, 1979) (Revision received March lst, 1980) (Accepted March lst, 1980)
SUMMARY
Nitrate reductase activity (NRA; EC 1.6.6.1) was detected by an in vivo assay in light-sensitive lettuce (Lactuca sutiua L. var. Grand Rapids) seeds which had been imbibed in 0.06 M KNOJ in darkness. Illumination with white light (10 min) more than doubled the enzyme activity; 5-min far red illumination following white light impulse reduced NRA to the level showed by the dark-imbibed seeds. Fusicoccin (FC) did not affect the phytochrome controlled NRA, although it substituted light requirement for the seed germination. Phytochrome possibly facilitates the assembly of nitrate reductase components on cell membranes. FC did not affect NRA in cucumber (Cucumis satiuus L.) cotyledons induced with KN03, while it produced a remarkable activity in the absence of external nitrate. The latter effect was possibly dependent on acidification of the medium caused by FC-stimulated proton extrusion from the cotyledons.
INTRODUCTION
Nitrate reductase activity is controlled by phytochrome in some plants [l---5]. The stimulatory effect of red light on NRA in light sensitive seeds of lettuce markedly diminished after piercing the seed coats, with a concomitant many-fold increment of the enzyme activity. This suggested that: (i) penetration of nitrate to intact seeds was restricted; and (ii) the Addreee correepondence
to: Dr. J.S. Knypl, Plant Growth Substances Laboratory, of Lodz, ul. Banacha 12/16,90-237 Lodz, Poland. Abbreuiatione: CAP, chloramphenicol; FC, fusicoccin; FR, far red light; NR, nitrate reductase; NRA, NR activity. University
320
rate of NO; diffusion to the embryo was possibly one of the factors limiting the final NRA [ 61. If this assumption was correct, then fusicoccin, a diterpene glucoside produced by Fusicoccum amygdali Del. [ 71, should enhance NRA in germinating seeds. FC strongly promotes the energydependent proton extrusion and uptake of monovalent cations and anions
[a*
This study presents the evidence that FC had no effect on the light enhanced NRA in both lettuce seeds and cucumber cotyledons. MATERIALS
AND METHODS
NR induction in lettuce seeds Seeds of Lactuca satiua L. var. Grand Rapids were purchased from Ferry Morse Seed Co. (Orchard Park, N.Y.) and stored under silica gel at 6 -8°C. Triple distilled water and KN03 solutions used for NR induction and germination were supplemented with 10e3 M CAP as a bacteriostatic. Batches of 100 seeds were placed in lo-cm Petri dishes containing two discs of prewashed Whatman No. 1 filter paper and 5 ml of 0.05 M KNOJ supplemented with FC. After 1 h in darkness the seeds were exposed to white light for 10 min and/or to FR for 5 min [9], and returned to darkness. Duplicate sets were germinated in permanent illumination (4.6 W m-‘; fluorescent tubes ‘Flora LF’) or in darkness at 25°C. NR activity was determined after 12 h imbibition; none of the seeds had germinated during this time. Since preliminary experiments revealed that 10 min white light produced similar effects as red (660 nm) light impulse, the former illumination was applied throughout the study. Determination of NRA in imbibed seeds NR activity in non-germinated lettuce seeds could not be detected in vitro. Satisfactory conditions for the in vivo NRA assay were established in preliminary experiments. The concentration of n-propanol in an incubation mixture was found to be a highly important factor for reproducibility of the results. If n-propanol was added to a final concentration of 4% (v/v), the release of NO; from imbibed, non-germinated seeds was proportional to time for at least 5 h after an initial 60-min lag period. Anaerobic atmosphere did not improve the results. Two hundred seeds which had been imbibed for 12 h were washed with distilled water, blotted dry on a paper towel, and transferred to 15-ml screwcapped vials containing 5.5-ml aliquots of the incubation mixture: 0.1 M K’-phosphate buffer (pH 7.5), 0.1 M KN03, 4% n-propanol, and two drops of 8 mM CAP. The seeds were twice vacuum infiltrated and left at 30°C in darkness with occasional stirring. After 2 and 4 h 0.5-ml aliquots of the incubation medium were withdrawn for calorimetric NO; determination [lo]. Results of the assay were highly reproducible if the same lot of seeds was used.
321
Induction of NRA in cucumber cotyledons
Cotyledons which had been separated from 5day-old etiolated Cucumis sutiuus L. var. ‘Delicatess’ seedlings were induced with 0.01 M KNOJ for 20 h in light or in darkness at 25”C, and then used for in vivo NRA assays as described [ 111. Experiments were run in triplicate and repeated at least twice. RESULTS
Fusicoccin in a range of concentrations from 10m6to 5 X lo-’ M did not affect NRA in the seeds imbibed in darkness, despite the fact that germination was increased to 100% in comparison with 1. -2 and 15% germination in 0.05 M KNO, and distilled water, respectively. However, NRA in seeds imbibed in permanent illumination in the presence of 5 X 10e6 M FC was enhanced by around 30% in comparison with control seeds, induced with only KN03. Some NRA (35 nmol NO; h-‘/1000 seeds) could be detected also,in seeds imbibed in distilled water in light; the seeds seemingly contained a limited pool of endogenous nitrate. Seeds that were continuously illuminated showed more than twice the activity of those imbibed in darkness (Table I). The same effect was produced by a lo-min white light impulse (or R) followed by darkness. FR illumination after white light reduced the enzyme activity to the level of activity in the seeds continuously maintained in darkness. FC had no effect TABLE I NO EFFECT OF FC ON THE PHYTOCHROME LETTUCE SEEDS
CONTROLLED
NR ACTIVITY
IN
NRA was determined after 12 h imbibition in 0.05 M KNO, (0) or 0.05 M KNO, and 5 x 10m6 M FC (FC). Germinated seeds were counted after 30 h. L, R and FR, white,. red and far red light; D, darkness; -+ change of illumination. Illumination regime after 60 min in darkness
L D lOminL-+D lOminL-+5minFR+D lOminL+ 5minFR-+ + 10minLd D lOminR+D
NRA, nmol NO; h^‘/ 1000 seeds
% Germination
Oa
FC
Ob
FC
155 70 150 70
189 70 160 72
73 1 64 1
100 80 100 80
140 153
158 166
60 68
100 95
a NRA in seeda imbibed for 12 h in distilled water was 5 and 36 nmol NO;/1000 seeds in D and L, respectively. b 15 and 85% seeds germinated during 30 h in distilled water in D and L, respectively.
322 TABLE II EFFECT OF FUSICOCCIN
ON NR ACTIVITY
IN CUCUMBER
COTYLEDONS
Cotyledons were incubated in solutions of FC (non-induced cotyledons) or FC supplemented with 0.01 M KNO, without CAB for 20 h, then NRA determined in vivo. Superscript letters indicate statistically significant differences among treatments, P = 0.01. Concentration of FC (M)
NRA, nmol NO; h-‘/cotyledon Non-induced
-_ 0 1o-6 5 x10-6 1o‘5 10W5; assay mixture without KNO,
cotyledons
KNO,-induced
cotyledons
Darkness
Light
Darkness
Light
0.P 3.lb 3.lb 6.2c 0
0.7a 3.lb 3.lb 8.9 0
6.2’ 6.1c 6.2c 6.2c 4.0b
13.6e 14.2e 14.4e 14.1e 6.2c
on this phytochrome controlled enhancement of enzyme activity, although it induced 80% germination in comparison with 1% germinated seeds in the proper control series. FC did also not affect NRA in excised cucumber cotyledons, when it was applied together with 0.01 M KN03 either in the dark or in light. In contrast, the cotyledons which had been grown in FC solutions without KN03 released remarkable quantities of NO; upon subsequent incubation in the KNO,-supplemented medium for the in vivo NRA assay. FC at the highest concentration tested (10m5 M) induced NR activity equal to that in the KN03-induced cotyledons in darkness, and only by a one third lower than in the nitrate-supplied greening cotyledons. No nitrite was released by the FC-treated cotyledons when the medium for NRA assay was deprived of KNOJ (Table II). This is an indication that the cotyledons contained no measurable pool of endogenous nitrate. Growth of the cotyledons was markedly stimulated by K’ [ll], and FC did not enhance the effect of potassium. FC slightly increased the fresh weight of cotyledons grown in the absence of KNOJ (data not illustrated). DISCUSSION
Although FC mimics the symptoms of action of red light as concerns germination of lettuce seeds [ 121, it has virtually no effect on induction of NR activity in the activation phase of seed germination. This phytotoxin tends to enhance NRA only in the seeds permanently illuminated, the magnitude of the effect being comparable to that produced by kinetin or ethylene [ 61; this effect may be due to FC-stimulated uptake and flow of NO; to an active nitrate pool. It should be stressed, however, that NR activity is detectable in the seeds induced with KNO, in darkness; white
323
light or R impulse only doubles the enzyme activity (Table I). This means that phytochrome does not control NRA at the genome level. It seems that phytochrome affects either the assembly of the nitrate reductase components on a,heterogenous cell constituent such as a membrane or a fraction of endoplasmic reticulum, or nucleotidation of the NR complex [13,14]. None of these two hypothetical sites is sensitive to FC. The phytotoxin can indirectly affect the‘enzyme activity in conditions when the rate of NO; influx to the cell may be a’limiting factor as in the case of seeds and cotyledons maintained in permanent illumination, i.e. in conditions of a rapid activation of many concurrent metabolic pathways. NRA in cucumber cotyledons may be induced by many factors in the absence of external NO;, including alcohols, CAP [6], low pH and organic acids [ 151. Since FC stimulates H’ extrusion from the cells [8] and increases intracellular level of malate [ 16,171, the nitrate reductase activity detected in the non-induced cotyledons may be regarded as a result of increased intracellular malate level [cf. 181. ACKNOWLEDGEMENTS
The author would like to express his gratitude to Professor Alessandro Ballio (Institute of Biological Chemistry, University of Rome, Italy) for the generous gift of FC. REFERENCES 1 R.W. Jones and R.W. Sheard, Plant Phyeiol., 55 (1975) 954. 2 R.W. Jones and R.W. Sheard, in E.J. Hewitt and C.V. Cutting (Eds.),Nitrogen Assimilation of Plants, Academic Press, London, New York and San Fran&co, 1979, p. 521. 3 S.H. Duke and S.O. Duke, Plant Cell Physiol., 19 (1978) 481. 4 H. SPrakawa and P. Yamamoto, Plant Physiol., 63 (1979) 1098. 5 G.C. Whitelam, C.B. Johnson and H. Smith, Photochem. Photobiol., 30 (1979) 589. 6 J.S. Knypl, in E.J. Hewitt and C.V. Cutting (Eda.), Nitrogen Amimilation of Plants, Academic Press, London, New York and San Francisco, 1979, p. 541. 7 A. Ballio, M. Brufani, C.G. Casinovi, S. Cerrini, W. Feldi, R. Pellicciari, B. Santurbano and A. Vaciago, Ezperientia, 24 (1968) 631. 8 E. Ma&, Annu. Rev. Plant Physiol., 30 (1979) 273. 9 T. Tanada, Planta (Berlin), 134 (1977) 57. 10 R.H. Hageman and D.P. Huckleeby, Methods Enzymol., 23 (1971) 491. 11 J.S. Knypl, Z. Pflanzenphysiol., 70 (1973) 1. 12 P. Lado, F. Rasi-Caldogno and R. Colombo, Physiol. Plant., 31 (1974) 149. 13 E.J. Hewitt, D.P. Hucklesby, A.F. Mann, B.A. Notton and G.J. Rucklidge, in E.J. Hewitt and C.V. Cutting (E&e.), Nitrogen Assimilation of Plants, Academic Press, London, New York and San Francisco, 1979, p. 266. 14 R.G. Butz and W.A. Jackson, Phytochemistry, 16 (1977) 409. 15 J.S. Knypl and A.R. Ferguson, Z. Pflanzenphysiol., 74 (1976) 434. 16 E. MarrB, in P.E. Pilet (Ed.), Plant Growth Regulation, Springer Verlag, Berlin and Heidelberg 1977, p. 54. 17 R. Stout and R.E. Cleland, Planta (Berlin), 139 (1978) 43. 18 C.K.M. Rathnam, Plant Physiol., 62 (1978) 220.