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The Physiological Role of Indole Acetic Acid in the Moss Funaria Hygrometrica Hedw.
J.PlantPhysiol. Vol. 135.pp. 522-525 (1989)
The Physiological Role of Indole Acetic Acid in the Moss Funaria Hygrometrica Hedw. 1. Quantification of Indole-3-Acetic Acid in Tissue and Protoplasts by Enzyme Immunoassay and Gas Chromatography-Mass Spectrometry RAINER ATZORN!, ULRIKE GEIER!, 1
2
and GORAN SANDBERG2
Botanisches Institut der Universitat, 1m Neuenheimer Feld 360, 6900 Heidelberg, Federal Republic of Germany Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 90183 Umea, Sweden
Received July 15, 1989 . Accepted August 14, 1989
Summary A procedure for extraction, purification and quantification by reversed-phase high-performance liquid chromatography (HPLC) - enzyme immunoassay (EIA) was developed which allowed the specific and reliable determination of indole-3-acetic acid (IAA) contents in tissue and protoplasts of protonema of the moss Funaria hygrometrica Hedw. The results were validated by gas chromatography-mass spectrometry (GC-MS).
Key words: Indole-3-acetic acid, Funaria hygrometrica, protoplasts, enzyme immunoassay, gas chromatography-mass spectrometry. Abbreviations: BHT = Butyl hydroxy toluene; EIA = Enzyme immunoassay; GC-MS = Gas chromatography-mass spectrometry; HPLC = High performance liquid chromatography; IAA = indole-3-acetic acid; IAAme = Methyl ester of indole-3-acetic acid; PVP = Polyvinylpyrrolidone.
Introduction Immunoassays for the quantitative analysis of plant growth regulators are now widely applied (Weiler 1986, Crozier et a1. 1985). However, especially in the case of IAA determination, this method is strongly dependent on the kind of plant material and its grade of purification (Pengelly and Meins 1979, Crozier et a1. 1985). Cohen et a1. (1986) demonstrated that determination of IAA by enzyme immunoassay in various tissues led to overestimations when HPLC-purification was omitted, but in most of the analyses, the combined use of HPLC and EIA showed good agreement with results obtained by GC-MS analysis_ Therefore, it was necessary to validate IAA amounts quantified by EIA in moss protonema prior to routine application. This © 1989 by Gustav Fischer Verlag, Stuttgart
can be achieved by the use of physicochemical methods like GC-MS. In this paper, we report a convenient method using HPLC-EIA to determine the endogenous IAA in tissue and protoplasts of Funaria hygrometrica_
Materials and Methods Plant material Source and growth 0/ moss protonema. Protonema cultures of Funaria hygrometrica Hedw. were raised on cellophane sheets laid on Knop's medium with 2 % Difco-Bacto agar and maintained in a growth chamber (2W m -2,20 h light per day, 20°C) for 12 days, as described by Lehnert and Bopp 1983.
Physiology of auxins in Funaria
Isolation of protoplasts. The procedure is described in detail by Bopp and Geier (1988): Briefly, 15 g moss protonema was plasmolysed in 0.6M mannitol, pH 5.6, at 20°C for 1 h. Maceration (5% Cellulase TC, 5% Pectinase P5, 0.6M mannitol pH 5.6) occurred over night at 20°C. Protoplasts were isolated by centrifugation at < 500 g for 8 min. The pellet was resuspended in an isoosmotic medium, and the yield of intact protoplasts was determined in the usual way. Quantitative analysis of indole-3-acetic acid by HPLC and enzyme Immunoassay Extraction and purification. Up to 2 g moss protonema was frozen with liquid nitrogen and freeze-dried. This material (5 - 10% of the initial fresh weight) was extracted twice with 70 % methanol containing antioxidant butyl hydroxy toluene (BHT) at 4°C on an overhead shaker in darkness for 3 h. After centrifugation, 14C_lAA was added as internal standard to the combined supernatants, which were then stirred for 10 min with insoluble polyvinylpyrrolidone (PVP), filtered and passed through a CIS Sep-pak cartridge (Waters). The extracts were concentrated in vacuo, acidified with 0.1 M HCl and partitioned three times against an equal volume of diethyl ether. The combined organic phase was methylated with etherial diazomethane, dried under a nitrogen stream and redissolved in a small volume of methanol. The extracts were stored at - 20°C. Endogenous IAA of isolated protoplasts from 15 g moss protonema was extracted and purified in the same way. High-performance liquid chromatography. Solvents were delivered at a flow rate of 1 ml min - I by a Varian liquid chromatograph. Reversed-phase HPLC utilized a 10 ~m ODS Eurochrome (Knauer) column (250 mm long, 5 mm i.d.) eluted with a 20-min gradient of 20-100% methanol in 0.1 % glacial acetic acid. The retention property of 14C_IAAme was determined by liquid scintillation counting of aliquots of the collected 0.5 ml-fractions. Those fractions containing radioactivity and co-chromatographing with IAAme were combined and reduced to dryness in vacuo, then dissolved in 1 ml of trisbuffered saline (TBS), pH 7.8, and stored at - 20°C prior to analysis by enzyme immunoassay. Enzyme immunoassay. Raising of antibodies and synthesis of IAA-alkaline phosphatase was performed according to Weiler et al. (1981), as well as the immunoassay procedure. Percentages of crossreactivity of the antiserum with natural and synthetic auxins were in the following range: indole-3-acetone, 32.4 %; indole-3-acetaldehyde, 11 %; indole-3-acetamide, 9.7 %; indole-3-butyric acid, 9 %; indole-3-acetonitrile, 3.5 %: indole-3-pyruvic acid, 2 %; indole-3-propionic acid, 2.3 %; I-naphthyl acetic acid, 5.8 %; 5-hydroxyindole-3acetic acid, 0.2 %; 2,4-dichlorphenoxy-acetic acid, tryptamine and Ltryptophan did not cross-react with the antibodies. The assay had a linear detection range of 0.2 - 20 pmol IAAme. Data were calculated via logit-log trans-formation (Rodbard 1974).
523
model· 320 liquid chromatograph. A 250 x 5 mm i.d. 5 ~m ODS Nucleosil column eluted over 25 min with a gradient of 15-75% methanol in 1 % acetic acid was used. Column effluent was monitored with a Shimadzu fluorescence detector (excitation 280 nm, emission 350 nm). Gas chromatography-mass spectrometry. GC-MS analysis of the trimethylsilyl (TMS) derivatives was performed with an HP 5890 gas chromatograph linked via a direct capillary inlet to an HP 5970B mass selective detector equipped with an HP 9000 computer system. Samples were introduced in the splitless mode (2 min splitless time) at 225°C onto a 25 m x 0.31 mm i.d. cross linked methyl silicone capillary column with a 0.52/-1m film thickness. The column temperature was initially held at 60°C for 3 min, then programmed by 30 °C min - I to 130°C followed by a 7°C min - I gradient to 235°C. The interface temperature was maintained at 250 0C. IAAme-fractions were silylated at 70°C for 15 min, reduced to dryness and dissolved in 20 ~l n-hexane prior to GC-MS; 2 - 3 ~l was injected in the splitless mode, and relative intensities at m/z 202/208 (base peaks), 261/267 (M+) for TMS-IAAme were recorded. The M+ ratio was used to check peak homogenity, while the base peakratio was used to quantify the amount of endogenous substance by reference to a standard curve obtained by analysing increasing amounts of TMS-IAAme in the presence of a fixed amount of C3C 6)TMS-IAAme.
Results The sensitivity and specificity of the developed enzyme immunoassay for IAA is in a similar range as known for other IAA-antisera (e.g. Weiler et al. 1981). All data were corrected with internal standards for losses during purification. Comparing the amounts of IAA determined by EIA at different stages of purification (Table 1), it is evident that crude "0
c
40
::J
0
Q.I
E
« «
3
-0
E
Cl.
Quantitative analysis by combined gas chromatography-mass spectrometry (GC-MS) Purification. Freeze-dried moss protonema was extracted for 2 h at 4°C with 10 ml methanol containing 0.02 % DDC, 50.7 ng 13 ( C 6)lAA. After filtration, the methanolic extract was reduced to dryness in vacuo. The sample was dissolved in 5 ml 0.1 M phosphate buffer, pH 8.0, slurried with 0.3 g PVP, filtered through a Waters reversed-phase Sep pak, diluted with an equal volume of H 20 and partitioned three times against an equal volume of diethyl ether. The pH was then adjusted to 2.7 with 1 M HCl and the sample was partitioned three times against an equal volume of diethyl ether. The latter organic phases were combined, reduced to dryness and methylated prior to further purification by reversed-phase HPLC: Solvents were delivered at a flow rate of 1 ml min - I by a Beckman
o~-----+------+-----~-
10
20 30 pmol IAAme added
Fig. 1: Internal standardization of extract aliquots by adding increasing amounts of IAAme. A slope different from 1.0 indicates the presence of nonspecific inhibitors in the extract. 0 semi-purified protonema extract; slope = 1.323 .• HPLC-purified protonema extract; slope = 1.030.... semi-purified protoplast extract; slope = 0.996.
524
RAINER ATZORN, ULRIKE GEIER, and GORAN SANDBERG
Table 1: IAA contents of protonema (nmol/g dry weight) and protoplasts (nmol/20 x 106 protoplasts) as determined by EIA and GCMS. protonema crude extract I semi-purified extract 2 HPLC-purified extract
EIA
GC-MS
28.79 14.34 8.19
8.93
protoplasts semi-purified extract 1.24 HPLC-purified extract I methanolic extracts after ether partitioning 2 PVP slurry and passing through CIS cartridges 3 not determined
c .2 U ...c
0.96 Discussion
IAAme
•
(5
E
0..
o~----aL--~-----+------~
o
10
retention time [min
amount of endogenous IAA per 20 million protoplasts (isolated from 30 g protonema) was 1.24 nmol (217 ng) determined by EIA and 0.96 nmol (168 ng) determined by GCMS. Also the control by internal standardisation as described for the protonema gave a slope near 1.0 (Fig. 1). The IAA-concentration of the protoplasts calculated in respect to a mean diameter of 20 p.m, was 15 p.M. Related to IAA-contents measured in the tissue, the amounts of IAA per gram fresh weight in protoplasts are in the same order.
I
20
Fig. 2: Immunoreactivity of HPLC-fractions of an extract of Fu· naria hygrometrica. extracts and semipurified samples of moss protonema contain substances which influence the assay system in a manner which can lead to overestimations. This is also the case for samples when fractions co-chromatographing with IAAme after thin-layer chromatography were analysed (data not shown). Internal standardization of extract aliquots by adding increasing amounts of IAAme (Fig. 1), according to Pengelly and Meins (1977), resulted in a slope of 1.323, which indicates the presence of nonspecific inhibitors of the antigen-antibody reaction. However, no unspecific interferences could be observed after HPLC-purification (Fig. 1). The results are in good agreement with GC-MS determinations (Table 1). Immunological analysis of fractions of a complete HPLC run shows that apart from IAA no considerable amounts of immunoreactive material could be detected (Fig. 2). Although for a single HPLC-EIA determination of IAA in Funaria a minimum of 0.1 g tissue (in terms of fresh weight) is sufficient, for each analysis between 1 and 3 g fresh weight were used (immunoassay), whereas 15 g protonema was extracted for GC-MS analysis extracted. For the protoplast extract, HPLC purification was omitted. Despite this fact, the levels of IAA determined by EIA are in good agreement with the GC-MS data: The total
This is the first report of IAA quantification in Funaria hygrometrica by enzyme immunoassay which is verified by mass spectrometric data. For this type of tissue, it shows that a combination of HPLC and EIA represents a reliable method for analyzing IAA. It is suitable for routine quantification because assay sensitivity is very high and the number of purification steps is limited. There are only a few reports about determination of endogenous auxins in mosses: Jayaswal and Johri (1985) detected levels of 2 - 5 p.g IAA per g fresh weight in suspension cultures of Funaria hygrometrica which would be equal to about 20 - 45 pmol per g dry weight. They performed the quantifications by a fluorimetric assay of fractions separated by paper chromatography. Perhaps the more than hundredfold lower levels compared to our results can be explained by the fact that liquid cultures were used instead of protonema raised on agar plates. We observed that even slight modifications of the growing conditions (especially light intensity and moisture) can cause considerable changes in the endogenous auxin content (data not shown). Ashton et al. (1985) analyzed IAA by GC-MS in gametophytes of the moss, Physcomitrella patens, and they found about 0.5 nmol IAA per g dry weight in 19-day-old protonema cultivars. Despite that this concentration is also below our estimates, it is in a similar order of magnitude, keeping in mind that there may be a broad concentration range of endogenous IAA in different species and that it could be dependent on the different age of the plant material. Compared to IAA contents in vegetative tissues of higher plants (e.g. Cohen et al. 1987), mosses show much higher concentrations of endogenous auxins. Since IAA seems to have a dominating influence on developmental processes in Funaria (Bhatla and Bopp 1985, Bopp et al. 1986), relatively high endogenous concentrations can be expected, which is even the case in some auxin-resistant mutants (Atzorn et al. 1989). In protoplasts, reliable IAA quantification was possible without HPLC purification. Only a minimum of one million protoplasts is necessary for a single immunoassay determination, including the extract dilution series. For experiments dealing with IAA location, it is very useful to know the endogenous concentration of the hormone within the cytoplasm of the protoplasts. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft.
Physiology of auxins in Funaria
References ASHTON, N. W., A. SCHULZE, P. HALL, and R. S. BANDURSKY: Estimation of indole-3-acetic acid in gametophytes of the moss Physcomitrellapatens. Planta 164,142-144 (1985). ATZORN, R., M. Bopp, and U. MERDES: The physiological role of indole-acetic acid in the moss Funaria hygrometrica Hedw. II. Mutants of Funaria hygrometrica which exhibit enhanced catabolism of lAA. J. Plant Physiol. 135, 526-530 (1989). BHATLA, S. C. and M. Bopp: The hormonal regulation of protonema development in mosses III. Auxin-resistant mutants of the moss Funaria hygrometrica Hedw. J. Plant Physiol. 120, 233-243 (1985). Bopp, M. and U. GEIER: Protoplasts and transport. GLIME, J. M. (ed.): Methods in Bryology, Hattori Bot. Lab., Nichinan 1988, pp.89-97. Bopp, M., D. GERHAUSER, and U. KESSLER: On the hormonal system of mosses. Bopp, M. (ed.): Plant Growth Substances 1985, Springer, Berlin - Heidelberg - New York - Tokyo, pp. 13-21 (1986). COHEN, J. D., M. G. BAUSHER, K. BIALEK, G. BUTA, G. F. W. GOCAL, L. M. JANZEN, R. P. PHARIS, A. N. REED, and J. P. SLOVIN: Comparison of a commercial ELISA assay for indole-3-acetic acid at several stages of purification and analysis by gas chromatography
525
- selected ion monitoring - mass spectrometry using a 13C6-labeled internal standard. Plant Physiol. 84, 982-986 (1986). CROZIER, A., G. SANDBERG, A. M. MONTEIRO, and B. SUNDBERG: The use of immunological techniques in plant hormone analysis. Bopp, M. (ed.): Plant Growth Substances 1985, Springer, Berlin - Heidelberg - New York - Tokyo pp. 13-21 (1986). JAYASWAL, R., K. and M. M. JOHRI: Occurrence and biosynthesis of auxin in protonema of the moss Funaria hygrometrica, Phytochemistry 24, 1211-1214 (1985). PENGELLY, W. and F. MEINS: A specific radioimmunoassay for nanogram quantities of the auxin, indole-3-acetic acid Planta 136, 17h 180 (1977). RODBARD: Statistical quality control and routine data processing for radioimmunoassays and immunoradiometric assays. Clin. Chern. 20, 1255-1270 (1974). WEILER, E. W.: Plant hormone immunoassay based on monoclonal and polyclonal antibodies. LINSKENS, H. F. and J. F. JACKSON (eds.): Modern Methods of Plant Analysis. Vol. 4: Immunology in Plant Sciences, Springer, Berlin - Heidelberg - New York Tokyo, pp. 5-11 (1986). WEILER, E. W., P. S. JOURDAN, and W. CONRAD: Levels of indolel-3acetic acid in intact and decapitated coleoptiles as determined by a specific and highly sensitive solid-phase enzyme immunoassay. Plant 153, 561-571 (1981).
The Physiological Role of Indole Acetic Acid in the Moss Funaria Hygrometrica Hedw. 1. Quantification of Indole-3-Acetic Acid in Tissue and Protoplasts by Enzyme Immunoassay and Gas Chromatography-Mass Spectrometry RAINER ATZORN!, ULRIKE GEIER!, 1
2
and GORAN SANDBERG2
Botanisches Institut der Universitat, 1m Neuenheimer Feld 360, 6900 Heidelberg, Federal Republic of Germany Department of Forest Genetics and Plant Physiology, Swedish University of Agricultural Sciences, 90183 Umea, Sweden
Received July 15, 1989 . Accepted August 14, 1989
Summary A procedure for extraction, purification and quantification by reversed-phase high-performance liquid chromatography (HPLC) - enzyme immunoassay (EIA) was developed which allowed the specific and reliable determination of indole-3-acetic acid (IAA) contents in tissue and protoplasts of protonema of the moss Funaria hygrometrica Hedw. The results were validated by gas chromatography-mass spectrometry (GC-MS).
Key words: Indole-3-acetic acid, Funaria hygrometrica, protoplasts, enzyme immunoassay, gas chromatography-mass spectrometry. Abbreviations: BHT = Butyl hydroxy toluene; EIA = Enzyme immunoassay; GC-MS = Gas chromatography-mass spectrometry; HPLC = High performance liquid chromatography; IAA = indole-3-acetic acid; IAAme = Methyl ester of indole-3-acetic acid; PVP = Polyvinylpyrrolidone.
Introduction Immunoassays for the quantitative analysis of plant growth regulators are now widely applied (Weiler 1986, Crozier et a1. 1985). However, especially in the case of IAA determination, this method is strongly dependent on the kind of plant material and its grade of purification (Pengelly and Meins 1979, Crozier et a1. 1985). Cohen et a1. (1986) demonstrated that determination of IAA by enzyme immunoassay in various tissues led to overestimations when HPLC-purification was omitted, but in most of the analyses, the combined use of HPLC and EIA showed good agreement with results obtained by GC-MS analysis_ Therefore, it was necessary to validate IAA amounts quantified by EIA in moss protonema prior to routine application. This © 1989 by Gustav Fischer Verlag, Stuttgart
can be achieved by the use of physicochemical methods like GC-MS. In this paper, we report a convenient method using HPLC-EIA to determine the endogenous IAA in tissue and protoplasts of Funaria hygrometrica_
Materials and Methods Plant material Source and growth 0/ moss protonema. Protonema cultures of Funaria hygrometrica Hedw. were raised on cellophane sheets laid on Knop's medium with 2 % Difco-Bacto agar and maintained in a growth chamber (2W m -2,20 h light per day, 20°C) for 12 days, as described by Lehnert and Bopp 1983.
Physiology of auxins in Funaria
Isolation of protoplasts. The procedure is described in detail by Bopp and Geier (1988): Briefly, 15 g moss protonema was plasmolysed in 0.6M mannitol, pH 5.6, at 20°C for 1 h. Maceration (5% Cellulase TC, 5% Pectinase P5, 0.6M mannitol pH 5.6) occurred over night at 20°C. Protoplasts were isolated by centrifugation at < 500 g for 8 min. The pellet was resuspended in an isoosmotic medium, and the yield of intact protoplasts was determined in the usual way. Quantitative analysis of indole-3-acetic acid by HPLC and enzyme Immunoassay Extraction and purification. Up to 2 g moss protonema was frozen with liquid nitrogen and freeze-dried. This material (5 - 10% of the initial fresh weight) was extracted twice with 70 % methanol containing antioxidant butyl hydroxy toluene (BHT) at 4°C on an overhead shaker in darkness for 3 h. After centrifugation, 14C_lAA was added as internal standard to the combined supernatants, which were then stirred for 10 min with insoluble polyvinylpyrrolidone (PVP), filtered and passed through a CIS Sep-pak cartridge (Waters). The extracts were concentrated in vacuo, acidified with 0.1 M HCl and partitioned three times against an equal volume of diethyl ether. The combined organic phase was methylated with etherial diazomethane, dried under a nitrogen stream and redissolved in a small volume of methanol. The extracts were stored at - 20°C. Endogenous IAA of isolated protoplasts from 15 g moss protonema was extracted and purified in the same way. High-performance liquid chromatography. Solvents were delivered at a flow rate of 1 ml min - I by a Varian liquid chromatograph. Reversed-phase HPLC utilized a 10 ~m ODS Eurochrome (Knauer) column (250 mm long, 5 mm i.d.) eluted with a 20-min gradient of 20-100% methanol in 0.1 % glacial acetic acid. The retention property of 14C_IAAme was determined by liquid scintillation counting of aliquots of the collected 0.5 ml-fractions. Those fractions containing radioactivity and co-chromatographing with IAAme were combined and reduced to dryness in vacuo, then dissolved in 1 ml of trisbuffered saline (TBS), pH 7.8, and stored at - 20°C prior to analysis by enzyme immunoassay. Enzyme immunoassay. Raising of antibodies and synthesis of IAA-alkaline phosphatase was performed according to Weiler et al. (1981), as well as the immunoassay procedure. Percentages of crossreactivity of the antiserum with natural and synthetic auxins were in the following range: indole-3-acetone, 32.4 %; indole-3-acetaldehyde, 11 %; indole-3-acetamide, 9.7 %; indole-3-butyric acid, 9 %; indole-3-acetonitrile, 3.5 %: indole-3-pyruvic acid, 2 %; indole-3-propionic acid, 2.3 %; I-naphthyl acetic acid, 5.8 %; 5-hydroxyindole-3acetic acid, 0.2 %; 2,4-dichlorphenoxy-acetic acid, tryptamine and Ltryptophan did not cross-react with the antibodies. The assay had a linear detection range of 0.2 - 20 pmol IAAme. Data were calculated via logit-log trans-formation (Rodbard 1974).
523
model· 320 liquid chromatograph. A 250 x 5 mm i.d. 5 ~m ODS Nucleosil column eluted over 25 min with a gradient of 15-75% methanol in 1 % acetic acid was used. Column effluent was monitored with a Shimadzu fluorescence detector (excitation 280 nm, emission 350 nm). Gas chromatography-mass spectrometry. GC-MS analysis of the trimethylsilyl (TMS) derivatives was performed with an HP 5890 gas chromatograph linked via a direct capillary inlet to an HP 5970B mass selective detector equipped with an HP 9000 computer system. Samples were introduced in the splitless mode (2 min splitless time) at 225°C onto a 25 m x 0.31 mm i.d. cross linked methyl silicone capillary column with a 0.52/-1m film thickness. The column temperature was initially held at 60°C for 3 min, then programmed by 30 °C min - I to 130°C followed by a 7°C min - I gradient to 235°C. The interface temperature was maintained at 250 0C. IAAme-fractions were silylated at 70°C for 15 min, reduced to dryness and dissolved in 20 ~l n-hexane prior to GC-MS; 2 - 3 ~l was injected in the splitless mode, and relative intensities at m/z 202/208 (base peaks), 261/267 (M+) for TMS-IAAme were recorded. The M+ ratio was used to check peak homogenity, while the base peakratio was used to quantify the amount of endogenous substance by reference to a standard curve obtained by analysing increasing amounts of TMS-IAAme in the presence of a fixed amount of C3C 6)TMS-IAAme.
Results The sensitivity and specificity of the developed enzyme immunoassay for IAA is in a similar range as known for other IAA-antisera (e.g. Weiler et al. 1981). All data were corrected with internal standards for losses during purification. Comparing the amounts of IAA determined by EIA at different stages of purification (Table 1), it is evident that crude "0
c
40
::J
0
Q.I
E
« «
3
-0
E
Cl.
Quantitative analysis by combined gas chromatography-mass spectrometry (GC-MS) Purification. Freeze-dried moss protonema was extracted for 2 h at 4°C with 10 ml methanol containing 0.02 % DDC, 50.7 ng 13 ( C 6)lAA. After filtration, the methanolic extract was reduced to dryness in vacuo. The sample was dissolved in 5 ml 0.1 M phosphate buffer, pH 8.0, slurried with 0.3 g PVP, filtered through a Waters reversed-phase Sep pak, diluted with an equal volume of H 20 and partitioned three times against an equal volume of diethyl ether. The pH was then adjusted to 2.7 with 1 M HCl and the sample was partitioned three times against an equal volume of diethyl ether. The latter organic phases were combined, reduced to dryness and methylated prior to further purification by reversed-phase HPLC: Solvents were delivered at a flow rate of 1 ml min - I by a Beckman
o~-----+------+-----~-
10
20 30 pmol IAAme added
Fig. 1: Internal standardization of extract aliquots by adding increasing amounts of IAAme. A slope different from 1.0 indicates the presence of nonspecific inhibitors in the extract. 0 semi-purified protonema extract; slope = 1.323 .• HPLC-purified protonema extract; slope = 1.030.... semi-purified protoplast extract; slope = 0.996.
524
RAINER ATZORN, ULRIKE GEIER, and GORAN SANDBERG
Table 1: IAA contents of protonema (nmol/g dry weight) and protoplasts (nmol/20 x 106 protoplasts) as determined by EIA and GCMS. protonema crude extract I semi-purified extract 2 HPLC-purified extract
EIA
GC-MS
28.79 14.34 8.19
8.93
protoplasts semi-purified extract 1.24 HPLC-purified extract I methanolic extracts after ether partitioning 2 PVP slurry and passing through CIS cartridges 3 not determined
c .2 U ...c
0.96 Discussion
IAAme
•
(5
E
0..
o~----aL--~-----+------~
o
10
retention time [min
amount of endogenous IAA per 20 million protoplasts (isolated from 30 g protonema) was 1.24 nmol (217 ng) determined by EIA and 0.96 nmol (168 ng) determined by GCMS. Also the control by internal standardisation as described for the protonema gave a slope near 1.0 (Fig. 1). The IAA-concentration of the protoplasts calculated in respect to a mean diameter of 20 p.m, was 15 p.M. Related to IAA-contents measured in the tissue, the amounts of IAA per gram fresh weight in protoplasts are in the same order.
I
20
Fig. 2: Immunoreactivity of HPLC-fractions of an extract of Fu· naria hygrometrica. extracts and semipurified samples of moss protonema contain substances which influence the assay system in a manner which can lead to overestimations. This is also the case for samples when fractions co-chromatographing with IAAme after thin-layer chromatography were analysed (data not shown). Internal standardization of extract aliquots by adding increasing amounts of IAAme (Fig. 1), according to Pengelly and Meins (1977), resulted in a slope of 1.323, which indicates the presence of nonspecific inhibitors of the antigen-antibody reaction. However, no unspecific interferences could be observed after HPLC-purification (Fig. 1). The results are in good agreement with GC-MS determinations (Table 1). Immunological analysis of fractions of a complete HPLC run shows that apart from IAA no considerable amounts of immunoreactive material could be detected (Fig. 2). Although for a single HPLC-EIA determination of IAA in Funaria a minimum of 0.1 g tissue (in terms of fresh weight) is sufficient, for each analysis between 1 and 3 g fresh weight were used (immunoassay), whereas 15 g protonema was extracted for GC-MS analysis extracted. For the protoplast extract, HPLC purification was omitted. Despite this fact, the levels of IAA determined by EIA are in good agreement with the GC-MS data: The total
This is the first report of IAA quantification in Funaria hygrometrica by enzyme immunoassay which is verified by mass spectrometric data. For this type of tissue, it shows that a combination of HPLC and EIA represents a reliable method for analyzing IAA. It is suitable for routine quantification because assay sensitivity is very high and the number of purification steps is limited. There are only a few reports about determination of endogenous auxins in mosses: Jayaswal and Johri (1985) detected levels of 2 - 5 p.g IAA per g fresh weight in suspension cultures of Funaria hygrometrica which would be equal to about 20 - 45 pmol per g dry weight. They performed the quantifications by a fluorimetric assay of fractions separated by paper chromatography. Perhaps the more than hundredfold lower levels compared to our results can be explained by the fact that liquid cultures were used instead of protonema raised on agar plates. We observed that even slight modifications of the growing conditions (especially light intensity and moisture) can cause considerable changes in the endogenous auxin content (data not shown). Ashton et al. (1985) analyzed IAA by GC-MS in gametophytes of the moss, Physcomitrella patens, and they found about 0.5 nmol IAA per g dry weight in 19-day-old protonema cultivars. Despite that this concentration is also below our estimates, it is in a similar order of magnitude, keeping in mind that there may be a broad concentration range of endogenous IAA in different species and that it could be dependent on the different age of the plant material. Compared to IAA contents in vegetative tissues of higher plants (e.g. Cohen et al. 1987), mosses show much higher concentrations of endogenous auxins. Since IAA seems to have a dominating influence on developmental processes in Funaria (Bhatla and Bopp 1985, Bopp et al. 1986), relatively high endogenous concentrations can be expected, which is even the case in some auxin-resistant mutants (Atzorn et al. 1989). In protoplasts, reliable IAA quantification was possible without HPLC purification. Only a minimum of one million protoplasts is necessary for a single immunoassay determination, including the extract dilution series. For experiments dealing with IAA location, it is very useful to know the endogenous concentration of the hormone within the cytoplasm of the protoplasts. Acknowledgements This work was supported by the Deutsche Forschungsgemeinschaft.
Physiology of auxins in Funaria
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