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Auxin metabolism in mosses and liverworts¹

²Department of Cell Biology and Molecular Genetics, University of Maryland at College Park,College Park, Maryland 20742-5815; and ³Horticultural Crops Quality Laboratory, Beltsville Agricultural Center, Agricultural Research Service,United States Department of Agriculture, Beltsville, Maryland 20705-2350

Received for publication June 11, 1998. Accepted for publication March 23, 1999.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The plant hormone auxin (indole-3-acetic acid, IAA) is involved in the control of many phenomena during plant development. By characterizing steady-state free and conjugated IAA levels using a stable isotope dilution method coupled with gas chromatography- selected ion monitoring- mass spectrometry, this paper provides a detailed characterization of IAA metabolism in five liverworts, four mosses, and two tracheophytes. Long-term IAA conjugation patterns were monitored by incubating actively growing tissue with ¹⁴C-IAA and then analyzing the de novo synthesis of IAA conjugates with radioimaging techniques. The liverworts, mosses, and tracheophytes can be differentiated by the total amount of IAA metabolites, the proportion of free and conjugated IAA, the chemical nature of their IAA conjugates, and the rates of IAA conjugation. Our tentative conclusion is that the liverworts appear to employ a biosynthesis–degradation strategy for the regulation of free IAA levels, in contrast to the conjugation–hydrolysis strategy apparently used by the mosses and tracheophytes. Such alternative metabolic strategies may have profound implications for macroevolutionary processes in these plant groups.

Key Words: auxin • auxin conjugates • auxin metabolism • indole-3-acetic acid • liverworts • mosses • tracheophytes • vascular tissue

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The major structural characteristics used to differentiate the various plant divisions include embryo development, vascular tissue organization, meristem structure, and root initiation (Gifford and Foster, 1988 ). Considerable evidence suggests that underlying mechanisms responsible for regulating these developmental phenomena involve the metabolism, transport, and activity of the plant hormone auxin (indole-3-acetic acid, IAA) (Bandurski et al., 1995 ).

Recent efforts to construct land plant phylogenies with molecular traits have involved the use of convenient markers, such as rRNA genes (Mishler et al., 1992 ; Waters et al., 1992 ) and rbcL genes (Albert et al., 1994 ; Manhart, 1994 ; Lewis, Mishler, and Vilgalys, 1997 ). While such markers may help to distinguish among different land plant lineages, they do not provide any insight into the developmental mechanisms presumed to act in the macroevolutionary processes operating in those lineages. By contrast, other workers have exploited the modern analytical technology available for characterizing hormone metabolism in order to investigate developmental regulation in major land plant divisions. In particular, Sztein et al. (1995) explored the ability of land plants, ranging from liverworts to angiosperms, to carry out conjugation of exogenous ¹⁴C-IAA during a 22-h incubation. The liverworts appeared unable to synthesize IAA conjugates, the mosses and hornworts synthesized a few conjugates, and the tracheophytes were able to synthesize many conjugates, with indole-3-acetyl-L-aspartate (-glutamate) and indole-3-acetyl-ß-1-0-glucose being the predominant forms. Moreover, in a thorough review of the conjugation patterns of the hormone cytokinin, Auer (1997) discovered that green algae, mosses, and ferns contain relatively few isopentenyl adenine and zeatin conjugates, while gymnosperms and angiosperms present a more complex set of zeatin and dihydrozeatin N-glucoside and O-glucoside conjugates. Finally, Osborne et al. (1996) demonstrated that two lower land plants synthesize the hormone ethylene via a biosynthetic pathway that does not involve 1-amino-cyclopropane-1-carboxylic acid, in contrast to the angiosperms, which use this compound as the intermediate for ethylene biosynthesis.

Our previous paper focusing on auxin conjugates in the land plants (Sztein et al., 1995 ) presented only a partial picture of IAA metabolism, because endogenous IAA concentrations are regulated by three main processes: biosynthesis, degradation, and conjugation (Normanly, Slovin, and Cohen, 1995) . Biosynthesis can take place through either a tryptophan-mediated or a tryptophan independent pathway. Degradation can occur through a decarboxylative pathway (to indole-3-carboxylic acid) or through a nondecarboxylative pathway, involving the oxidation of the indolic ring. Conjugation can take place between IAA and sugar(s) to form ester conjugates or can be linked to amino acids or small peptides to form amide conjugates. These conjugates can be stored, hydrolyzed to release free IAA, or further processed in the degradation pathways (see Normanly, 1997 ). In higher plants, most IAA is present in the form of IAA conjugates (Bandurski et al., 1995 ), and some of the enzymes that catalyze IAA conjugation have recently been characterized (Bartel and Fink, 1995 ; Ludwig-Müller, Epstein, and Hilgenberg, 1996 , and references therein). It is widely believed that IAA is active in its free state, with the conjugates being short-term intermediates involved in the regulation of free IAA levels (Cohen and Bandurski, 1982 ; Kleczkowski and Schell, 1995 ). However, several recent reports suggest that IAA conjugates may also have specific physiological and/or developmental functions in the plant (Wodzicki, Pharis, and Wodzicki, 1987 ; Bialek and Cohen, 1992 ; Oetiker and Aeschbacher, 1997 ).

The literature contains a few scattered reports of endogenous IAA contents in lower land plants, with the IAA being detected by different techniques ranging from bioassays (Schneider, Troxler, and Voth, 1967 ), paper chromatography (Croxdale, 1976 ), and fluorometry (Jayaswal and Johri, 1985 ), to modern extraction and measurement protocols using gas chromatography-mass spectrometry (GC-MS) (Ashton et al., 1985 ; Schneider and Wightman, 1986 ; Li, Wurtele, and LaMotte, 1994 ). The relative amounts of free vs. conjugated IAA in lower land plants have not been investigated in large part because the methodologies most frequently used in the evaluation of IAA content of these plants could not accurately discriminate between free IAA and its various metabolites.

The overall objective of our work is to present a complete picture of IAA metabolism in the various divisions of land plants. Our specific goals were: (1) to establish axenic cultures of land plants in order to ensure the measurement of auxin metabolism in the absence of any confounding microbial metabolism, (2) to characterize their steady-state concentrations of free IAA and IAA metabolites using a stable isotope dilution method in conjunction with gas chromatography-selected ion monitoring-gas spectrometry (GC-SIM-MS), and (3) to make a provisional determination of the chemical identities and synthetic rates of IAA conjugates in land plants. Finally, auxin metabolism was examined in both vascular and nonvascular taxa in an attempt to identify significant aspects that might be attributed to the presence of vascular tissue. Our results demonstrate that the liverworts, mosses, and tracheophytes can be distinguished on the basis of several characteristic features of their IAA metabolisms. This approach should ultimately provide novel insights into the hormonal regulation of macroevolutionary events responsible for the origin of the different plant divisions.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Axenic culture
Except for those cultures generously supplied by other investigators (Table 1), representative members of the Hepatophyta, Bryophyta, Lycophyta, and Polypodiophyta were placed into axenic culture using the following procedures. One- to 1.5-cm long thallus tips of Pallavicinia lyellii were rinsed in tap water to dislodge soil particles, submerged in a sterilizing solution made of 7% commercial Clorox and 0.02% Triton X-405 solution (Sigma Chemical Co., St. Louis, Missouri) for 5 min, and then rinsed for 5 min with sterile double-distilled H₂O (DDH₂O). The thalli were again exposed to the Clorox-Triton solution for 5 min, rinsed once more for 10 min, blotted with sterile filter paper, and plated in solid medium as described below. One- to 1.5-cm long thallus tips of Marchantia polymorpha were similarly treated, except that they were exposed to 5% Clorox for 2 min and rinsed three times for 10 min, each time in sterile DDH₂O before being blotted and plated on solid medium as described below. Two-centimeter long stem tips of Selaginella kraussiana (including a bifurcation) were immersed 6 min in the sterilizing solution as described above for Pallavicinia, rinsed three times for 10 min in sterile DDH₂O and plated in solid medium, taking special care to place the cut surface below the surface of the medium. Capsules of Polytrichum ohioense were sterilized in 5% Clorox for 10 min, rinsed with sterile DDH₂O, and opened with sterile forceps in a sterile Petri dish, to which 2.5 mL of sterile DDH₂O were added. The resulting spore solution was spread among five plates with solid medium as described below. When the protonema cultures exhibited some buds, the material was distributed among several plates to allow for development into gametophores. Spores of Ceratopteris richardii were sterilized in a solution of 15% Clorox for 3 min, rinsed three times with sterile DDH₂O, and the resulting spore solution distributed in small sterile disposable Petri dishes on solid medium as described below. When the prothalli showed mature gametangia, the plates were opened and a few drops of sterile DDH₂O were added to facilitate fertilization. When the young sporophytes had two to three juvenile leaves they were transplanted to larger petri dishes (two per plate) to allow for better development of the plants. All cultures were subsequently tested in glucose-supplemented medium to ensure that they had no obvious latent microbial contaminants.

The effort was made to grow all these cultures under uniform conditions; however, it was observed that the different cultures required somewhat different conditions in order to achieve maximal growth with normal morphology. All the plants appeared like field-grown specimens, except for the Reboulia cultures, which grew in less compact mats than did wild populations. Reboulia, Orthotrichum, Selaginella, and Ceratopteris were grown in half-strength Murashige-Skoog modified basal salt mixture medium (catalog number M-8900: 1/2 NH₄NO₃ and KNO₃ prepared at half strength; Sigma Chemical Co.), while Marchantia, Pallavicinia, Plagiochila, Polytrichum, Funaria, and Sphagnum were grown in Knop's medium supplemented with 1% glucose, plus 1 mL/L of both iron and formulation VII micronutrient solutions (Basile, 1978 ). Sphaerocarpos was grown in Knop's medium supplemented with both iron and micronutrient solutions as above, but without glucose. All media were prepared with 1% Phytagar tissue culture grade agar (Gibco BRL, Grand Island, New York) and titrated to pH 6 prior to autoclaving. All plants were grown in a growth chamber at 23°C under continuous light (30 µmol·m^-2·s^-1), except for Ceratopteris, which was grown in a chamber kept at 27°C with a 16 h light: 8 h dark photoperiod, and for Sphaerocarpos, which was grown in a chamber kept at 14°C, with a 12 h light : 12 h dark photoperiod, under the same light intensity.

Stable isotope dilution method for endogenous IAA measurement
In all species except for Sphaerocarpos, the endogenous content of free IAA and IAA metabolites was measured using the stable isotope dilution method described in Chen et al. (1988) . In brief, shoot or thallus tips of actively growing plants kept in axenic conditions were harvested, weighed in a precision balance, immediately placed in a mortar previously cooled with liquid nitrogen, and homogenized with the addition of 2 mL of 65% isopropanol/ 35% 200 mmol/L cold imidazole buffer (pH 7) and 100 µL of ¹³C₆-IAA internal standard solution [1 ng/mL in isopropanol, synthesized according to Cohen et al. (1986) ]. Another 3–4 mL of cold buffer were added while the samples were further ground. In addition, 800 Bq of ³H-IAA (specific activity: 83.25 x 10¹⁰ Bq/mmol; American Radiolabeled Chemicals Inc., St. Louis, Missouri) were added as a radioactive tracer. This extract was placed in a glass Corex (Corning Inc., Corning, New York) tube in ice for 1 h to allow for full extraction of IAA and IAA metabolites. After the equilibration period, and three sequential centrifugations in a clinical table centrifuge at 2500 g, the resulting sample was divided into three identical subsamples for the determination of free, free + ester, and total amounts of IAA metabolites. Liquid scintillation counting was used at the various purification steps to check for adequate processing of the subsamples.

The free IAA fraction was diluted 10 times with DDH₂O and directly passed through a NH₂-Prep Sep SPE (solid phase extraction) column (Fisher Scientific, Fairlawn, New Jersey) that had been preconditioned by rinsing with a sequence of hexane, acetonitrile, water, one volume of 200 mmol/L imidazole buffer (pH 7), and three volumes of DDH₂O. Following sample application, the column was washed with a sequence of hexane, ethyl acetate, acetonitrile, and methanol, and the sample eluted with 1 mL of freshly made 5% acetic acid in methanol. The free + ester fraction was hydrolyzed in 1 mol/L NaOH for 1 h at 25°C, adjusted to pH 2.5, and then desalted through a C₁₈-Baker SPE column (J. T. Baker, Phillipsburg, New Jersey), which had been preconditioned by rinsing with a sequence of hexane, methanol, H₂O, and 1% acetic acid. Following sample application, the column was rinsed with DDH₂O, and the IAA metabolites eluted with 3 mL of acetonitrile. The total IAA fraction was hydrolyzed in 7 mol/L NaOH for 3 h at 100°C in a solid 30 mL-Teflon vial ("Tuftainer," Pierce, Rockford, Illinois) purged with oxygen-scrubbed N₂, titrated to pH 2.5, and desalted through a solvent-conditioned C₁₈ Baker SPE column (J. T. Baker) following the same procedure used for the 1 mol/L hydrolysis.

All three fractions were dried in vacuo prior to the final purification step and resuspended in 50% methanol. The high performance liquid chromatograph (HPLC) used in this procedure (Waters 501 pump, Waters Inc., Wilford, Massachusetts; Rheodyne 7125 valve, Rheodyne Inc., Cotati, California; and Gilson FC-205 fraction collector, Gilson Inc., Middleton, Wisconsin) was equipped with a C₁₈ Ultracarb column (50 mm long, 4.6 mm internal diameter, Phenomenex, Torrance, California) preceded by a precolumn filled with Whatman C₁₈ pellicular ODS (Upchurch Precolumn Kit C-135B, Upchurch Scientific Inc., Oak Harbor, Washington; Whatman Inc., Hillsboro, Oregon). The running solvent was a freshly made 25% methanol/ 1% acetic acid solution, and 100% methanol was used as the cleaning solvent. The fractions containing IAA were collected, dried in vacuo, and resuspended in 100 µL of 100% methanol.

Due to the small size of Sphaerocarpos thalli, another technique, modified from Chen et al. (1988) and Ribnicky, Cooke, and Cohen (1998) , was used to prepare the samples for quantifying their endogenous free IAA and IAA metabolites. Actively growing thalli (approximate area 0.5 cm²) kept in axenic conditions were harvested, weighed on a precision balance, and immediately placed in a blue Kontes tube (Kontes Glass Co., Vineland, New Jersey) previously cooled with liquid nitrogen. The material was homogenized with a plastic Kontes pestle (number 749520), with the addition of 200 µL of 65% isopropanol/35% 200 mmol/L cold imidazole buffer (pH 7), and 20 µL of ¹³C₆-IAA internal standard solution (1 ng/mL in isopropanol). Another 300 µL of cold buffer were added while the samples were further ground. In addition, 200 Bq of ³H-IAA (American Radiolabeled Chemicals Inc.) were used as a radioactive tracer. The tube was then covered with aluminum foil and incubated for 90 min in ice to allow for full extraction of the free IAA and IAA metabolites.

After the end of the equilibration period, the tube was centrifuged 5 min at 10 000 g (National Centrifuge model Z230M microcentrifuge, National Lab Net Co., Woodbridge, New Jersey), and the supernatant recovered. The sample was then divided in three identical subsamples for the determination of free, free + ester, and total amounts of IAA. As above, all subsamples were monitored in a liquid scintillation counter.

The free IAA sample was placed in a 1.5-mL microcentrifuge tube with the addition of ten volumes of water, and run directly through a Prep Sep Fisher NH₂-mini column previously rinsed with one and a half volumes each of n-hexane, acetonitrile, water, conditioned with one volume of 200 mmol/L imidazole buffer, and rinsed again with three volumes of water. Following sample application, the column was washed with a sequence of one volume each of n-hexane, ethyl acetate, acetonitrile, and methanol, and the sample eluted with 1 mL of freshly made 5% acetic acid in methanol.

The free + ester IAA fraction was placed into another microcentrifuge tube with the addition of one volume of water and two volumes of 2 mol/L NaOH and hydrolyzed for 1 h at 25°C. At the end of the hydrolysis, the pH of the sample was adjusted to 2.5 by using two drops of 42% H₃PO₄ and 100 µL of 3 mol/L HCl, and the pH was then checked by placing small drops of the sample onto pH indicator strips (number 9586, pH 0–6 range, EM Science, a division of EM Industries, Gibbstown, New Jersey). Finally, the titrated sample was then desalted through a Prep Sep Fisher C₁₈-mini column, which was conditioned by rinsing with a sequence of n-hexane, methanol, water and 1% acetic acid, and eluted with 1 mL of acetonitrile.

The total IAA fraction was placed in a solid 10-mL Teflon vial plus three volumes of water and four volumes of 14 mol/L NaOH. The vial was then sealed with a Teflon-lined cap, placed in a heating block at 100°C, and hydrolyzed for 3 h under a slow purge with oxygen-scrubbed nitrogen. At the end of the hydrolysis, the sample was allowed to cool for 15 min in ice while still attached to the nitrogen line. The sample was adjusted to pH 2.5 by the addition of two drops of 85% H₃PO₄ and 900 µL of 6 mol/L HCl and desalted as above.

All three fractions were dried in vacuo prior to the final purification step and resuspended in 100 µL of freshly made 20% acetonitrile/1% acetic acid solution. The HPLC used in this procedure was equipped with a C₁₈ Ultracarb 5 column (150 mm long, 2 mm internal diameter, Phenomenex) without a precolumn to reduce losses at this step. The running solvent was a freshly made solution of 20% acetonitrile/1% acetic acid, and 100% methanol was used as the cleaning solvent. The HPLC was operated at a flow rate of 0.2 mL/min, and the fractions containing IAA (typically, 14–16 min; 2.8–3.2 mL) were collected, dried in vacuo, and resuspended in 100 µL of 100% methanol.

All samples were methylated with an ethereal solution of diazomethane (prepared as described in Cohen, 1984 ) and resuspended in 20 µL of ethyl acetate prior to injection into the GC-MS. Selected ion spectra for samples of Funaria, Sphagnum, Sphaerocarpos, Marchantia, and Orthotrichum were determined using a Hewlett-Packard GC 6890/5973 MS (Hewlett–Packard Co., San Fernando, California) equipped with a 30 m x 0.250 mm HP 19091S-433 capillary column (5% phenyl methyl siloxane). Chromatographic parameters were as follows: injector temperature 250°C, initial oven temperature 70°C followed by a ramp at 20°C/min to 280°C. Selected ion spectra for samples of Pallavicinia, Plagiochila, Reboulia, Polytrichum, Selaginella, and Ceratopteris were determined using a Hewlett-Packard 5890 GC/ 5971 MS (Hewlett-Packard Co.) equipped with a 15 m x 0.237 mm DB-1701 fused silica capillary column (J & W Scientific, Folsom, California) as previously described (Chen et al., 1988 ). Chromatographic parameters were as follows: injector temperature 250°C and initial oven temperature 140°C, followed by a ramp at 20°C/min to 280°C. For all samples, the monitored ions were m/z 130, 136 (quinolinium ion, m + 6) and 189, 195 (molecular ion, m + 6). The results obtained with both systems were cross checked to ensure equivalent results.

IAA conjugation pattern analysis
Experiments were conducted as described in Sztein et al. (1995) with slight modifications. In summary, thallus or shoot tips of axenically grown plants were cut using sterile technique and placed in wells of 12-well plastic dishes (Costar, Cambridge, Massachusetts). Six to eight fronds of axenically cultured Lemna gibba L. var. G3 (a gift from Dr. J. P. Slovin, Climate Stress Laboratory, USDA, Beltsville, Maryland) were placed in other wells, while the remaining wells were left as medium-only controls. Each well contained 1.5 mL of Murashige and Skoog incubation medium (Sigma Chemical Co.) supplemented with 3% sucrose and 250 Bq of ¹⁴C-IAA (specific activity: 0.2109 x 10¹⁰ Bq /mmol, American Radiolabeled Chemicals Inc.) per milligram of plant material. The trays were then covered and sealed with Parafilm and kept at 26°C in a growth chamber under constant light (25 µmol·m^-2·s^-1). The plant samples were harvested at 24, 48, and 72 h, blotted on filter paper to remove excess medium, weighed on a precision balance, and rinsed with distilled water to remove any adsorbed ¹⁴C-IAA. The samples were then blotted again, placed in 1.5-mL Kontes microcentrifuge tubes, quick frozen in liquid N₂, ground in 80% acetone with Kontes pestles, and centrifugated for 10 min at 10 000 g. The preparation of the extracts, the thin layer chromatography (TLC), and the imaging with an AMBIS Model 4000 Radioisotope Image Acquisition and Analysis System (AMBIS Inc., San Diego, California) were performed as detailed in Sztein et al. (1995) . The abbreviations of the names of the IAA metabolite standards that comigrated with the IAA conjugates in the TLC plates are listed in Table 2, and the compounds with R_fs of 0.06 or less are ascribed to the "origin."

Tentative identification of the IAA metabolites was performed by comparing the R_f values of authentic IAA metabolite standards. While this procedure reflects genuine chemical differences, including the nature of the conjugating bond, the actual identity of the ¹⁴C-metabolites can only be ascertained unequivocally by rigorous analytical procedures, which were not performed in this work. In our reevaluation of earlier experiments on radiolabeled IAA conjugate levels published in Figs. 2–4 in Sztein et al. (1995) , it was determined that those values were erroneously high by a factor of 100, due to a simple calculation error.

View larger version (58K): [in this window] [in a new window]   Fig. 2. Characterization of IAA metabolites from four mosses and two tracheophytes following 24-, 48-, and 72-h incubations with 14C-IAA. For details, see legend of Fig. 1

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Endogenous free IAA and IAA metabolite concentrations
The quantitations below are based on stable isotope dilution, using GC-SIM-MS to measure the levels of free IAA. The levels of IAA conjugates are similarly determined as the IAA released following the hydrolysis of ester and/or amide conjugates. The concentrations of free IAA found in the five liverworts investigated in this study ranged from 10 ng/g FW in Plagiochila to 21 ng/g FW in Pallavicinia (Table 3). Thus, in liverworts, the free IAA concentrations averaged 14 ng/g FW, which represents 28% of the total IAA and IAA conjugates in these plants. In contrast, the levels of IAA-ester conjugates fell between 3 ng/g FW in Plagiochila, and 18 ng/g FW in Pallavicinia (a range of 7–36% of the total IAA and IAA conjugates), while IAA-amide conjugates ranged between 12 ng/g FW for Reboulia, and 53 ng/g FW for Marchantia, (a range of 31–70% of the total IAA and IAA conjugates). Therefore, the balance between amide and ester conjugates was variable in liverworts: Marchantia accumulated almost 90% of its conjugates in the amide form, whereas in Reboulia <50% of the conjugates were IAA-amides. The amount of total IAA and IAA conjugates varied from 35 ng/g FW in Sphaerocarpos to 75 ng/g FW in Marchantia. No consistent differences in the levels of various IAA metabolites occurred between the nonvascular (Marchantia, Plagiochila, Reboulia, and Sphaerocarpos) and vascular (Pallavicinia) liverwort taxa analyzed.

Of the two tracheophytes included in this study, the lycophyte Selaginella had 37 ng/g FW of free IAA, 64 ng/g FW of IAA-ester conjugates, and 307 ng/g FW of IAA-amide conjugates, representing 9, 16, and 75%, respectively, of the total IAA measured in this plant. The fern Ceratopteris exhibited 25 ng/g FW of free IAA, 1 ng/g FW of ester conjugates, and 87 ng/g FW of amide conjugates, for a total IAA metabolite content of 113 ng/g FW. When expressed as the percentages of the total IAA and IAA conjugates, these levels amounted to 22, 1, and 77%, respectively.

Provisional identification of IAA conjugates
The proportion of ¹⁴C radioactivity present at different R_fs on TLC plates is used below as the measure of the levels of IAA metabolites following 24-, 48-, and 72-h exposures to ¹⁴C-IAA in the same taxa analyzed above. The nature of IAA conjugates is provisionally identified by comparison to the R_fs of authentic IAA and a large number of authentic amide and ester conjugates. Thin layer chromatography was used to characterize the nature of IAA conjugates and the rate of their formation during 24-, 48-, and 72-h incubation periods in the same taxa analyzed above. These longer term experiments were initially performed to determine why Marchantia and Pallavicinia exhibited higher steady-state levels of IAA conjugation than expected from the 22 h ¹⁴C-IAA labeling experiments performed in Sztein et al. (1995) .

The liverwort Marchantia had high levels of free IAA at the 24-h measurement, but these levels were sharply decreased at 48 h and stayed at these low levels at 72 h (Fig. 1). No evidence of IAA conjugation was apparent at 24 h, but high amounts of an unknown amide conjugate at R_f 0.22, the degradation product oxIAA, and low amounts of the amide conjugates IAgly and IAval/nval, were all present at 48 h. Small increases in the levels of both IAgly and the more abundant amide conjugate at R_f 0.22 were evident following the 72-h incubation. The radioactive compounds residing at the origin are likely to consist of IAA directly complexed to insoluble molecules larger than simple sugars or amino acids, or are highly polar molecules containing the ¹⁴C from oxidized IAA. Their levels increased threefold from the 48- to the 72-h measurement. In Pallavicinia, very high initial levels of free IAA decreased by one-half every 24-h interval to the end of the experiment (Fig. 1). Pallavicinia was also able to synthesize increasing levels of IAala over the course of the experiment, in addition to some indole metabolites with side-chain modifications and simple esters at R_f 0.92 (IAN, IAAMe, and IAAEt) that first appear at 48 h. In Plagiochila, the moderate levels of free IAA present at 24 h declined over the next 48 h (Fig. 1). The concentration of the amide conjugates at R_f 0.28 almost tripled from the first to the second measurement, maintained its level at the 72-h data point, while the amide conjugate at R_f 0.22 remained at low amounts throughout the 72-h incubation period. The compound(s) at R_f 0.92 first appeared in the 48-h sample, and remained at low levels through the end of the experiment. In Reboulia, the low levels of free IAA present at 24 h remained relatively constant for the duration of the experiment (Fig. 1). Reboulia was also able to synthesize high amounts of an amide conjugate (R_f 0.18) that doubled every 24 h, and very small amounts of IAval/nval at R_f 0.46 that appeared at 48 h and remained low at 72 h. While all the other liverworts retained some labeled IAA in its free form, Sphaerocarpos did not show any labeled IAA in its free form at any sampling time in this experiment (Fig. 2). The IAA-amide conjugate at R_f 0.24 stayed at constant moderate levels for the full duration of the experiment, the unknown ester conjugate at R_f 0.63 dropped by one-half at 48 and 72 h, and IAacet (R_f 0.81) was present only at very low levels. As a general trend, the leafy and thalloid liverworts, except for Sphaerocarpos, exhibited a progressive reduction of the levels of free IAA over time and a concurrent increase in the levels of conjugated IAA.

View larger version (54K): [in this window] [in a new window]   Fig. 1. Characterization of IAA metabolites from five liverworts following 24-, 48-, and 72-h incubations with 14C-IAA. Data are expressed as the mean of three replicates (± SE) in nanograms of IAA equivalents per gram FW. All Rfs are normalized to the averaged Rf of free IAA. Designations such as UC-A and UC-E refer to unknown conjugates in which the nature of the chemical bond was identified by selective hydrolysis as amide and ester, respectively (see Sztein et al., 1995 ). Compounds with Rfs of 0.06 or less are ascribed to the "origin."

The lycophyte Selaginella exhibited an extremely low amount of free IAA only in the 24-h measurement (Fig. 2). The major IAA metabolite synthesized by this plant was an unknown amide conjugate (R_f 0.18), accompanied by much lower stable levels of IAgluc (R_f 0.64), and by another amide conjugate at R_f 0.22, the latter being undetectable after 24 h. Moderate levels of simple IAA esters (R_f 0.90) were also present in the last two measurements. The fern Ceratopteris showed no detectable levels of labeled free IAA at any measurement. It was able to synthesize very high levels of IAasp/IAglu throughout the experiment and moderate amounts of IAleu/ile at 48 and 72 h. An unknown ester conjugate at R_f 0.66 was present at 24 h, doubled its concentration by 48 h, and then decreased almost to its initial levels by the last sample. The two tracheophytes (Selaginella and Ceratopteris) synthesized mostly amide conjugates that reached very high levels within 24 h from the beginning of the experiment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Our current results on the endogenous free IAA and IAA metabolite concentrations and on the longer term IAA conjugation patterns will now be integrated with the short-term conjugation patterns presented in our earlier work (Sztein et al., 1995 ) in order to provide a more detailed picture of IAA metabolism at different levels of land plant evolution. Our work has several advantages over the few previous reports of IAA metabolism in lower land plants. One, all our observations are directly comparable to each other due to the use of almost uniform growth conditions, comparable shoot tissues, and identical experimental techniques. Two, the stable isotope dilution method used in conjunction with mass spectral analysis results in the accurate measurement of the actual levels of the IAA and IAA conjugates in the extracted tissues. The GC-MS isotope dilution methods used in this study offer the advantage that the results obtained are largely independent of sample size, purification method, and preparative losses (Chen et al., 1988 ; Ribnicky, Cooke, and Cohen, 1998 ). Thus, the modified technique for preparing Sphaerocarpos samples for GC-MS results in concentration values that are directly comparable to those obtained from larger samples of the other plants studied. However, the TLC methods also used in this paper do not use internal standards, and therefore, the results from those experiments may be affected by other factors such as ¹⁴C-IAA uptake rates, which are likely to vary among plants. Three, endogenous IAA levels reported in previous work were frequently determined in nonaxenic material. This situation may lead to erroneous measurements of endogenous IAA levels due to the ability of various microbes to synthesize or metabolize IAA (Libbert et al., 1968 ; Yamada, 1993 ; Patten and Glick, 1996 ). Four, the concentrations of the various IAA metabolites may vary greatly among different structures at various stages in the plant life cycle. For example, in one study performed in a lower plant, Jayaswal and Johri (1985) reported a two- to threefold difference between IAA levels in Funaria hygrometrica caulonema and chloronema. Similar considerations apply to seed plants, where seeds store very high concentrations of conjugated forms relative to the levels measured in growing vegetative tissues (Phaseolus vulgaris: Bialek and Cohen, 1989, 1992 ; Zea mays: Bandurski and Schulze, 1977 ; Epstein, Cohen, and Bandurski, 1980 ). In conclusion, the auxin literature has many other examples where marked discrepancies exist due to the use of diverse methodologies with varying analytical accuracy, nonuniform and/or uncontrolled growth conditions, different tissue samples, and frequent use of nonaxenic material, as noted by Bandurski and Schulze (1977) .

Although the methods used in our study circumvented the aforementioned problems, we could not transcend one particular limitation. Even though the sample sizes were as small as 50 mg FW, our shoot-tip explants included apical meristems together with subapical tissues extending far below the region of organogenesis. Thus, no conclusions about the levels of IAA or its metabolites in the shoot meristematic regions can be drawn from our data.

IAA metabolism in liverworts
In the liverworts, the GC-MS measurements indicate that the levels of free IAA in thallus or shoot tips average 14 ng/g FW, which represents 28% of the total IAA and IAA conjugates, whereas the levels of IAA conjugates correspond to an average of 36 ng/g FW (Table 3). However, our previous work on several liverworts showed no observable conjugate synthesis following a 22-h incubation with ¹⁴C-IAA (Sztein et al., 1995 ). In the present study, the wider range of liverwort species and longer incubation periods appears to have resolved this conundrum: liverworts are indeed able to metabolize exogenous IAA into moderate amounts of amide and ester conjugates, but at a much slower rate than in other plants (cf. Figs. 1, 2). Conjugated IAA was found either as predominantly amide conjugates, as is the case in Marchantia and Plagiochila, or was divided more or less equally between amide and ester conjugates. We did not examine enough species to determine whether specific liverwort classes have unique patterns of IAA conjugation. Law, Basile, and Basile (1985) have previously reported that they could not detect IAasp in Plagiochila arctica gametophytes. Earlier, Zenk (1964) summarized his initial analysis of IAA conjugates in the lower land plants, but his data are difficult to interpret in the light of our current knowledge of IAA conjugation.

IAA metabolism in mosses
In the four mosses used in this study, free IAA averaged 6 ng/g FW, which represents 10% of the total IAA found in gametophores. The rest of the IAA metabolites were found almost exclusively as amide conjugates rather than as ester conjugates (Table 3). In the long-term ¹⁴C-IAA incubation experiments, diverse conjugates were often present at 24 h. This implies that the conjugation rate is more rapid in mosses than in liverworts. Although the literature has no definitive reports of IAA conjugates in mosses, Ashton et al. (1985) did mention that the low levels of IAA (2.1 ng/g FW) detected using GC-MS in Physcomitrella patens chloronema could possibly be attributed to the presence of IAA conjugates, which could not be measured with their methodology.

IAA metabolism in tracheophytes
We chose Selaginella and Ceratopteris as representatives of two evolutionary lineages in tracheophytes. The lycophyte Selaginella contained 9% of free IAA (37 ng/g FW of the total 408 ng/g FW), with most of the IAA metabolites being present as amide conjugates. By contrast, although the fern Ceratopteris had free IAA levels roughly equivalent (25 ng/g FW) to those present in Selaginella, the free IAA in the fern represented a much higher portion of the total IAA and IAA conjugates (22% of the total 113 ng/g FW). Almost all of the conjugates in Ceratopteris were also amides (Table 3). The levels of total IAA and IAA conjugates in Ceratopteris were quite low for the vascular plants, where levels between 400–600 ng/FW are usually measured (Normanly, Cohen, and Fink, 1993 ). In the labeling experiments, remarkable levels of IAA conjugates quickly accumulated in these two plants, which demonstrates that conjugation occurred at very rapid rates (Fig. 2).

There are only a few reports of endogenous free IAA content in lycophytes and fern sporophytes. Using HPLC followed by an enzyme-linked immunosorbent assay, Pilate, Sossountzov and Miginiac (1989) measured 23 and 39 nmol of free IAA/gDW in subapical and apical buds of axenic Marsilea plants, respectively. Other studies have presented data on IAA measurements of nonaxenic material. Expanding Davallia croziers had 30 ng/g FW of free IAA as determined by bioassay (Croxdale, 1976 ); Matteuccia croziers and young rachises had 21 ng/g FW and 38 ng/g FW, respectively, as determined by GC-MS (Schneider and Wightman, 1986 ); and Regnellidium leaflets had 13 ng/g FW of free IAA as measured by spectrofluorometry (Walters and Osborne, 1979 ). However, we found no references in the literature regarding endogenous IAA conjugates in ferns or in lycophytes.

Free IAA levels in seed plants range from 4.7 ng/g FW in axenic Daucus hypocotyls (Ribnicky et al., 1996 ) to 35 ng/g FW in nonaxenic Pisum vegetative tissue (Bandurski and Schulze, 1977 ), with typical values ranging between 10 and 20 ng/g FW. The percentage of free IAA to the total IAA and IAA conjugates present in plants is usually between 5 and 10% (Cohen and Bandurski, 1982 ). In general, vegetative tissues of monocotyledonous plants have a higher percentage of ester conjugates (Bandurski and Schulze 1977 ), while dicotyledonous plants usually show a higher percentage of amide conjugates (Bialek and Cohen, 1992 ; Normanly, Cohen, and Fink, 1993 ).

Developmental significance
It is well documented that auxin is the primary regulator of vascular tissue development in seed plants, including primary vascular tissue development (Roberts, Gahan, and Aloni, 1988 ), positioning of primary vascular strands (Sachs, 1991 ), and the activity of vascular cambia (Uggla et al., 1996 ). Thus, it is of interest to determine whether or not the vascular species of liverworts and mosses show any distinctive features in their IAA metabolisms that can be assigned to the presence of vascular tissue. Since the differences in the IAA metabolisms of liverworts vs. mosses are far greater than those between vascular and nonvascular species within each division, we cannot identify any metabolic feature unequivocally associated with vascular tissue in these plants. However, because the apical meristems represent only 5% of the tissues sampled in the IAA metabolite measurements, the observed concentrations may not reflect localized levels at the sites of vascular tissue initiation. It is clear that we need to adopt a different approach toward apex dissection in order to understand whether or not auxin regulates the formation of vascular tissue in the liverworts and mosses.

Evolutionary patterns in the IAA metabolism of land plants
Another objective of this study was to provide more informative characters that might reveal evolutionary relationships among these plants. Too little is known about IAA metabolism in the green charophycean algae related to the land plants to discuss the evolutionary origins of IAA metabolism. All previous work on IAA metabolism in algae is plagued by the use of nonaxenic cultures (Jacobs, Falkenstein, and Hamilton, 1985 ; Bradley, 1991 ; Evans and Trewavas, 1991 ). Among the Charophyceae, Nitella showed no conjugation products following a 22-h incubation with ¹⁴C-IAA, where 95% of the total IAA and IAA conjugates was in the form of free IAA, and the rest was in the form of putative degradation products (Sztein et al., 1995 ). Chara has IAA uptake carriers capable of mediating transmembrane IAA transport, but its carriers for IAA efflux are not inhibited by the same phytotropins that block IAA transport in higher plants (Dibbfuller and Morris, 1992 ). Given the paucity of reports, there is an obvious need for a systematic characterization of IAA metabolism in Charophyceae.

In the land plants, the picture that emerges from both the published literature and the present study is that the liverworts, mosses, and tracheophytes can be distinguished on the basis of the characteristics of their IAA metabolism. Figure 3 plots some of the quantitative features of the IAA metabolism of these three groups, with the percent free IAA being a precise measure of steady-state conditions, and with the percent conjugated IAA/total IAA being a rough measure of conjugation rates of applied radiolabeled IAA. In essence, the liverworts have adopted a different strategy for regulating their endogenous free IAA levels than the mosses and the tracheophytes. While the liverworts have low absolute levels of both free IAA and IAA conjugates, a high proportion (30%) of the total IAA and IAA conjugates is represented by free IAA (Fig. 3). In addition, since the liverworts tend to exhibit slow or intermediate rates of IAA conjugate formation (Fig. 3), it suggests that their free IAA concentrations are primarily regulated via the balance between the biosynthesis of new IAA molecules and the degradation of existing molecules. By contrast, the mosses and tracheophytes have low free IAA and high IAA conjugate levels under steady-state conditions, and these plants also exhibit intermediate to very rapid rates of conjugate formation (Fig. 3). Thus, these mosses and tracheophytes must primarily regulate their IAA concentrations via the equilibrium between conjugate synthesis vs. conjugate hydrolysis (e.g., Epstein, Cohen, and Bandurski, 1980 ; Bartel and Fink, 1995 ; Jensen and Bandurski, 1996 ).

View larger version (16K): [in this window] [in a new window]   Fig. 3. Relationship between the percentage of free IAA at steadystate levels measured with GC-SIM-MS and the ratio of the percentage of conjugated IAA vs. total IAA following 22- or 24-h incubation with 14C-IAA in representative land plants. L1: Marchantia polymorpha, L2: Pallavicinia lyellii, L3: Plagiochila sp., L4: Reboulia hemisphaerica, L5: Sphaerocarpos texanus. M1: Funaria hygrometrica, M2: Polytrichum ohioense, M3: Sphagnum angustifolium, M4: Orthotrichum lyellii. T1: Selaginella kraussiana, T2: Ceratopteris richardii, T3: Arabidopsis thaliana, T4: Daucus carota, T5: Zea mays. All the data except for those from the angiosperm taxa were obtained in this study. The references for the steady-state values of the angiosperms were: Normanly, Cohen, and Fink (1993) for Arabidopsis thaliana, Ribnicky et al. (1996) for Daucus carota, and Bandurski and Schulze (1977) for Zea mays. The reference for the conjugation rates of these plants was Sztein et al. (1995)

We believe that these interpretations about hormone metabolism in the land plants can now be placed into an explicit evolutionary framework following Kenrick and Crane (1997) . In so far as liverworts represent a green plant lineage that has retained many primitive structural features, it is consistent that this lineage would employ a less sophisticated biosynthesis-degradation strategy for regulation of auxin and other hormones. The sister moss and tracheophyte clades appear to have evolved a conjugation-hydrolysis strategy, which should, in theory, allow for more precise spatial and temporal regulation of free IAA levels with a reduced need for synthesizing new IAA moieties. The absence of typical tracheophyte conjugates in the mosses provides further evidence for the early divergence of these two lineages. Finally, we speculate that these alternative metabolic strategies for IAA metabolism may have profound implications for the developmental phenomena, such as embryo development and meristem structure, which underlie the macroevolutionary differences among the land plants.

	FOOTNOTES

 
¹ The authors thank James Reveal and David Ribnicky for technical guidance and helpful discussions, and Richard Pharis, Peter Davies, and an anonymous reviewer for their constructive criticisms and comments on the manuscript. This work was supported in part by DOE grant DE–A102–94ER20153.

Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.

² Author for correspondence.

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