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 incuba- tion 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 treat- ment. 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 ex- tracts 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 inhibi- tion 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 liver- worts) 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.