Endogenous Auxin and Ethylene in Pellia (Bryophyta)' Received for Publication January 25, 1983 and in Revised Form May 19, 1983

Plant Physiol. (1983) 73, 395-397 0032-0889/83/73/0395/03/$00.50/0

Endogenous Auxin and Ethylene in Pellia (Bryophyta)' Received for publication January 25, 1983 and in revised form May 19, 1983

ROBERT J. THOMAS2, MARCIA A. HARRISON, JANE TAYLOR, AND PETER B. KAUFMAN Departments ofBotany and Cellular and Molecular Biology, Division ofBiological Sciences, University of Michigan, Ann Arbor, Michigan 48109; and Department ofBiology, University ofMichigan, Flint, Michigan 48503

ABSTRACT MATERIALS AND METHODS The occurrence of endogenous indole-3-acetic acid and ethylene in Plant Material. Thalli of the liverwort Pellia epiphylla (L.) bryophyte tissue was tentatively demonstrated using gas chromatography, Corda were collected between April and May, 1981-82 along the high performance liquid chromatography, and double-standard isotope banks of the Swift River near Tamworth, NH. Plant material dilution techniques. Rapidly elongating stalks (or setae) of Pellia epi- was either used fresh, or was stored before use at 0 to 4°C in phylla (L.) Corda sporophytes contain approximately 2.5 to 2.9 micro- darkness for up to 4 weeks without adverse effects on growth grams per gram fresh weight of putative free IAA. Ethylene released by response (22). Sporophytes (Fig. 1) were dissected from thalli setae increases during growth from 0.027 to 0.035 nanoliter per seta per prior to experimental treatment. Whole seta segments were pre- hour. Application of 5 microliters per liter ethylene inhibits auxin- pared by cutting away the sporophyte capsule and foot. stimulated elongation growth of this tissue, a result which suggests that Growth Studies. Setae (1 cm in length) were washed thoroughly both endogenously produced compounds act in tandem as natural growth with sterile distilled H20 and then placed in Petri dishes (100 x modulators. 15 mm) lined with Whatman No. 1 filter paper saturated with 6 ml of incubation medium. Eight setae per dish were incubated in the dark at 20C. Experimental treatments included incubation in 10 gM IRA (Sigma) and 5 to 75 ul/l C2H4 (Applied Sciences Laboratories), with pure C2H4 gas diluted by injection into a 5-L desiccator containing the setae in Petri dishes. Length and width of setae were measured before and after 24 h treatment. Experiments were repeated four times. Morphogenetic processes in bryophytes appear to be regulated C2M4 Measurements. One to two hundred whole seta segments by hormones that are quite similar to those found in higher (excised as described above) were placed in a 5-ml vial sealed plants (3, 14). This conclusion is based largely upon the interpre- with a serum septum. Vacuum was then applied by three 10-cc tation of morphologic responses to exogenously applied growth syringes with plungers held out for 4 min, after which the syringes regulators. Very few studies have been directed toward isolation were released and removed. After 15 min, a l-ml air sample was and identification of endogenously produced hormones in these analyzed for C2H4 by flame ionization chromatography (10). plants. Two well-documented exceptions are the chemical char- IAA Extraction. Excised setae (20-35 g) were extracted by acterization of cytokinin from cultured mosses (2, 24), and the homogenization in 80% acetone as described by Bandurski and identification ofa bibenzyl compound that apparently substitutes coworkers (1, 16). A trace amount of [2-'4C]IAA (55 ,Ci/mmol; for ABA in liverworts (9, 17, 23). Less convincing experimental New England Nuclear) was added as an internal standard im- evidence exists for the endogenous occurrence of auxin in bry- mediately after homogenization, and acetone was removed by ophyte tissues (13, 18). Criticism (21) has been directed especially rotary evaporation at 48°C. For determination of free IAA, toward the adequacy ofethanolic extraction and paper chromat- concentrated aqueous aliquots were acidified with 1 M phos- ographic purification techniques used until now for characteri- phoric acid to pH 2.5 and transferred by solvent partitioning to zation, by bioassay, of the trace amounts detected. Ethylene diethyl ether. For determination of free IAA plus ester-IAA and released by Marchantia thalli, on the other hand, is readily amide-IAA conjugates, aqueous aliquots were first hydrolyzed detected with modern gas chromatographic techniques (6). The with 7 N NaOH for 3 h at 100°C before acidification (1). Acid- response of thalli and other liverwort tissues to this hormone, ether extracts from both treatments were further partitioned however, remain almost completely unknown. against 1 N NaHCO3, reacidified and repartitioned into ether, The object of this paper is to present further evidence for the flash evaporated to near dryness, and redissolved in 2 ml of 50% occurrence of endogenous free auxin in bryophytes using a aqueous ethanol for chromatography. modified Bandurski extraction method (1) and a double-standard Chromatographic Isolation and Quantitative Estimation of isotope dilution assay (4). The seta of Pellia sporophytes was UIA. Samples in 50% ethanol were eluted on a DEAE-Sephadex selected as experimental material because of its high sensitivity column (Sigma) with a linear gradient of 50% (v/v) aqueous to exogenously applied auxin (1 1, 20, 22). Seta tissue was also ethanol containing from 0 to 5% glacial acetic acid. Fractions used to measure endogenous ethylene production and to test for containing putative IAA were pooled, taken to near dryness in growth sensitivity to exogenously applied ethylene. vacuo, and redissolved in 100 ,d of 100% methanol for elution by C,8-reverse phase HPLC (4). Eluted radioactive fractions were 1 Supported in part by National Aeronautics and Space Administration treated as described by Cohen and Schulze (4), with appropriate Grant NAGW-34 to P. B. K and a Research Corporation Grant to R. J. addition oftrace [(ring-2)-'4C]IBA3 (0.508 uCi/mmol; a gift from T. R. S. Bandurski) to facilitate assay of IAA by double-standard 2Permanent address: Department ofBiology, Bates College, Lewiston, Maine 04240. To whom correspondence should be directed. 3Abbreviation: IBA, indole-3-butyric acid. 395 396 THOMAS ET AL. Plant Physiol. Vol. 73, 1983

X~~u ~IBA

0 2 4 6 8 minutes FIG. 2. Representative GC profile of free-1AA from Pellia. 1AA extracts were purified by solvent partitioning, DEAE-Sephadex, and Ci- reverse phase HPLC prior to methylation and nitrogen-specific GC detection. GC profiles for free and bound IAA samples are similar to that shown for free IAA alone.

Table I. Double-Standard Isotope Dilution Analysis ofFree JAA4 and JAA Conjugates in Pellia Setae Calculated Values (4) Free-IAAa Free-IAA + Conjugatesa FIG. 1. Scanning electron micrograph of an unelongated Pellia spo- a. ug IAA at methyla- rophyte. Basal foot, cylindrical seta, and apical spore capsule are evident. tion (peak ratio de- x 8. termination) 1.14 2.87 b. dpm in IAA col- isotope dilution. A Beckman LS-7000 liquid scintillation system lected (dpm ratio de- with microprocess controller was used for determination of ra- termination) 3720 9386 dioactivity. For peak area values of diazomethane-treated, N2- c. dpm/pg of reisolated dried, and tetrahydrofuran-redissolved IAA and IBA, a Varian IAA (b * a) 3260 3271 aerograph 1400 GC equipped with a nitrogen/phosphorus detec- d. pg/g fresh wt of IAA tor was used. in setae (initial dpm/ ,pg . c times pg IAA RESULTS added + g fresh wt) 2.89 2.88 a Representative assay, 35 g sample, 3.0 cm average seta length. Calculation ofputative endogenous IAA in elongating setae of Peilia sporophytes by the double-standard isotope dilution assay indicates the presence of approximately 2.5 to 2.9 pg/g fresh attributed to marked differences in fresh weight due to the weight of free IAA. Quantification was based on peak area ratios massive water uptake by setae during growth (22). derived from GC profiles (Fig. 2) and determinations of specific Exogenous C2H4 at 5 gl/l has no discernable effect on Pellia activity ofreisolated IAA. Values obtained for free-IAA plus IAA seta growth when compared to either length or width changes in conjugates were equivalent to values for free-IAA alone (Table control sections (Fig. 3). Only with the use ofhigh concentrations I). Thus, while free IAA was readily detected, bound IAA (defined of C2H4 (75 h1/) could significant effects be observed; under by this procedure as alkali labile conjugates) was not. these conditions, seta length was reduced to the extent of 50% from setae cm The amount of C2H4 released unelongated (0.5 to that of with no change in seta average length) was calculated to be 0.027 nl seta-' h-' (or 25.8 compared controls, significant nl g fresh weight h-'). For elongating setae (2.1 cm average diameter. IAA at 10 pM stimulates seta lengthening maximally length) the amount was slightly higher, namely 0.035 nl seta-' (a 70% increase is elicited by IAA at this concentration), but this h-'. Calculated on a fresh weight basis, the value for elongating stimulation is essentially abolished by application of C2H4 (Fig. setae is lower (6.7 nl g fresh weight' h-'), a disparity that can be 3). ENDOGENOUS AUXIN AND ETHYLENE IN PELLIA 397 that observed for ethylene inhibition (20). mm From this and previous studies, we can list auxin, ethylene, and lunularic acid (9) as being the three known growth regulatory substances found in Pellia setae. In light of the observed inhibition of auxin-stimulated growth by exogenous ethylene (Fig. 3), 20 it seems likely that both endogenously produced IAA and C2H4 act in tandem as natural growth modulators of seta elongation in Pellia. Lunularic acid (an ABA-like growth regulator in liverworts) could also act as a growth modulator by influencing auxin- oxidase activity (15). The manner in which these substances 15 interact to control seta growth and development needs further investigation. Acknowledgments-The authors thank Fred Behringer and Jim Ellis for assistance with the ethylene studies, and Craig Lombard, Doug Couper, Cathy Neale, 10 Julie Martel, Larry Lackey, and Don Home for assistance in collection and preparation of material for the auxin assay. The senior author carried out a portion of this research while on sabbatical leave. LITERATURE CITED 5 1. BANDURSKI RS, A SCHULZE 1977 Concentration of indole-3-acetic acid and its derivatives in plants. Plant Physiol 60: 211-213 2. BEUTELMANN P, L BAUER 1977 Purification and identification of a cytokinin from moss callus cells. Planta 133: 215-217 3. Bopp M 1981 Entwicklungsphysiologie der Moose. In W Schulte-Motel, ed, 0 Advances in Bryology, Vol 1. AR Ganter, Vanduz, pp11-77 4. COHEN JD, A SCHULZE 1981 Double-standard isotope dilution assay. I. Quan- control C2H4 IAA C2H4+ IAA titative assay of indole-3-acetic acid. Anal Biochem 112: 249-257 5. CROMBIE WM, JA PATON 1958 An age effect on seta elongation in Pellia FIG. 3. Effectof C2H4 (5 MI/1) and IAA (10 Mm) on growth of Pellia epiphylla. Nature 182: 541 setae. Setae were incubated in darkness at 20C for 24 h. Means ± SE; P 6. DEGREEF JA, M DEPROFr, F VEROUSTRAETE, H FREDERICQ 1981 Case studies <0.05 (Student test) for comparisons of seta lengths between control of ethylene release in higher and lower plant systems. In B Jeffcoat, ed, Aspects and Prospects of Plant Growth Regulators. Wessex, Oxfordshire, pp and IAA-treated, and between IAA versus C2H4 + IAA-treated setae. 9-18 7. EPSTEIN E, JD COHEN 1981 Microscale preparation ofpentafluorobenzyl esters. DISCUSSION Electron-capture $as chromatographic detection of indole-3-acetic acid from plants. J Chromatogr 209: 413-420 Auxin assay data presented here provide tentative evidence for 8. GOLDWIN GK, RL WAIN 1973 Studies on plant growth-regulating substances. XXXV. Ethylene production by coleoptiles treated with auxin type chemi- the occurrence of endogenous free IAA in bryophytes at levels cals. Ann AppI Biol 75: 71-81 25 to 29 times greater than that reported in the only previous 9. GORHAM J 1978 Recent research on lunularic acid. Bull Brit Bryol Soc 31:11- quantitative study (18). Levels of auxin detected also fall within 12 the upper range reported for higher plants using similar extrac- 10. HARRISON MA, PB KAUFMAN 1982 Does ethylene play a role in the release of lateral buds (tillers) from apical dominance in oats? Plant Physiol 70: 81 1- tion and purification procedures (1, 7). As with any analytical 814 technique applied to previously uninvestigated material, how- 11. KAUFMAN PB, P DAYANANDAN, RJ THOMAS, J TAYLOR, RJ UMBERFIELD 1982 ever, it would be prudent for future efforts to be directed toward Comparative analysis of rapid growth responses using three model systems: additional physical characterization of the auxin presumably Conocephalum carpocephalum-stalk, Pellia seta, and Avena internode. J Hattori Bot Lab 51: 195-201 isolated by HPLC and GC. Definitive characterization would 12. LANG JM, WR EISINGER, PB GREEN 1982 Effects ofethylene on the orientation depend on mass spectrometric analysis. of microtubules and cellulose microfibrils of pea epipcotyl cells with poly- It is not clear why chromatographically isolated IAA in elon- lamellate cell walls. Protoplasma1 10: 5-14 gating Pellia setae is found almost exclusively in the free form 13, LARuE CD, S NARAYANASWAMI 1957 Auxin inhibition in the liverwort Lun- ularia. New Phytol 56: 61-70 (TableI). We suspect that derivative storage forms of IAA would 14. MARAVOLO NC 1980 Control of development in hepatics. Bull Torrey Bot be more prevalent in dormant setae (5). Unfortunately, it was Club 107: 308-324 not feasible to collect unelongate dormant sporophytes in 15. MATO MC, R CALVO 1977 Effect of lunularic acid on auxin-oxidase activity. sufficient for such an auxin analysis. The appropriate Biol Plant (Prague) 19: 394-396 amounts 16. PENGELLY WL, PJ HALL, A SCHULZE, RS BANDURSKI 1982 Distribution of analysis awaits the application of a detection method (electron- free and ester indole-3-acetic acid in the cortex and stele of the Zea mays capture GC for example) which requires less plant material than mesocotyl. Plant Physiol 69: 1304-1307 the one employed here. 17. PRYCE RJ 1972 The occurrence of lunularic and abscisic acids in plants. C2H4 release in plants is a phenomenon typical of tissues high Phytochemistry 11: 1759-1761 18. SCHNEIDER MJ, RF TROXLER, PD VOTH 1967 Occurrence of indoleacetic acid in auxin and undergoing rapid elongation (8). It is therefore not in thebryophytes. Bot Gaz 128: 174-179 surprising to find that increased C2H4 production (compared on 19. SCHNEPF E, G DEICHGRABER 1979 Elongation growth of setae of Pellia (Bry- a per seta basis) is associated with rapidly elongating Pellia setae. ophyta): fine structural analysis. Z Pflanzenphysiol 94: 283-297 The typical swelling response seen in dicot seedlings as a result 20. SCHNEPF E, W HERTH, DJ MORRt 1979 Elongation growth of setae of Pellia (Bryophyta): effects of auxin and inhibitors. Z Pflanzenphysiol 94: 221-217 of C2H4 application, however, is not detected in Pellia (Fig. 3). 21. SHELDRAKE AR 1971 The occurrence and significance of auxin in the substrata Presumably, the disrupted orientation of cellulose microfibrils of bryophytes. New Phytol 70: 519-526 that would normally result in this response (12) is little affected. 22. THOMAS RJ 1980 Cell elongation in hepatics: the seta system. Bull Torrey Bot The arrangement of microfibrils during seta growth is known to Club 107: 339-345 23. VALIO IF, RS BURDON WW SCHWABE 1969 New natural growth inhibitor in be unaffected by colchicine treatment (19). Both colchicine and the liverwort Lunularia cruciata (L.) Dum. Nature 223: 1176-1178 cellulose synthesis inhibitors, however, inhibit auxin-induced 24. WANG TL, R HORGAN, D COVE 1981 Cytokinins from the moss Physcomitrella elongation of Pellia setae by amounts somewhat greater than patens. Plant Physiol 68: 735-738