rapidly changed by draining the chamber and refilling it from the fluid reservoir. All solutions contained 5 mM succinate buffer, pH 6.0. Coordinates of data points taken at 1-min intervals from the original recorder graphs were analyzed by an IBM 370 computer and graphed in a normalized form by an IBM 1130 line plotter. The graphs which appear in this paper are tracings of the computer output. RESULTSIn buffer alone, the coleoptile segments were found to elongate at a rate of about 0.05 mm/hr per 1-cm segment. ABA was not tested directly on this low endogenous growth rate; rather, in order to establish a growth rate of sufficient magnitude so that the subsequent inhibition could be easily demonstrated, auxin was added to the bathing solution. IAA at a concentration of 2 tuM was selected for these experiments, since it consistently produced a substantial growth response (Fig. 2). Following a latent period of about 12 min after the addition of the auxin, a new elongation rate of about 0.7 mm/hr per segment was established.In the inhibitor studies, the following procedure was used. The coleoptile segments were first placed in the measuring chamber in buffer for 1 hr in order to allow depletion of endogenous IAA. The segments elongated at a minimal rate until the 2 uM IAA was added, and the new higher elongation rate was established. One hr later, IAA at the same concentration plus the inhibitor to be tested were added simultanewww.plantphysiol.org on May 12, 2018 -Published by Downloaded from

The effects of cordycepin (3'-deoxyadenosine), an RNA synthesis inhibitor, on auxin-induced elongation in Avena coleoptile segments were studied with a position-sensing transducer. Cordycepin rapidly inhibited auxin-stimulated growth in the coleoptile segments whether added before, at the same time as, or after, the 2 jtM auxin treatment. Midcourse In mammalian cells, cordycepin has a much smaller inhibitory effect on the synthesis of heterogenous nuclear RNA than has actinomycin D (13), and it has been shown that the posttranscriptional addition of poly A (150-200 residues of adenylic acid) to heterogeneous nuclear RNA is much more sensitive to cordycepin than to actinomycin D (9). Experimentally, it has been found that cordycepin inhibits the arrival of mRNA to the polyribosomes in the cytoplasm. Cordycepin has also been reported (18) to inhibit the synthesis of ribosomal and transfer RNA in HeLa cells.Little use has been made of cordycepin with plant systems (2,7,12,19). Nooden (12) reported that 5 mm cordycepin caused a 50% decrease in growth of corn coleoptiles within I hr. Stockert et al. (19) found that 0.1 mm cordycepin caused nearly 80% of nucleoli to exhibit segregation within 2 hr in meristematic cells (22nd hr, 0.1% caffeine-induced binucleate cells) of A llium cepa root tips. They concluded that this nucleolar aberration was probably related to the effect of cordycepin on RNA synthesis. In unpublished studies (M. Cline) there is evidence that cordycepin inhibition of RNA synthesis in plant systems is general for all species of RNA.The present experiments were carried out with a high resolution continuous growth measuring apparatus to determine the effects of cordycepin, an RNA synthesis inhibitor, on auxin-promoted elongation in Avena coleoptile segments. MATERIALS AND METHODSAn angular position-sensing transducer was used to measure continuous elongation in 4-to 5-day-old coleoptile segments of Avenia sativa L. cv. Victory as described previously (15). Ten 1-cm segments, with primary leaves removed, were strung vertically on a wire and immersed in a measuring chamber containing 5 mM succinate buffer, pH 6.0. After a 1-hr equilibration period in the buffer solution, the coleoptile segments were treated with 2 fiM auxin and/or cordycepin in a buffered solution, as indicated under "Results." Cordycepin was purchased from Sigma Chemical Co.Mitochondria were isolated from 4-day-old etiolated mung bean shoots (Phaseolus aureus L.) by standard isolation procedures. Approximately 100 g of shoots were placed in 200 ml of grinding media containing: 0.4 M mannitol, 50 mM trisTricine (pH 7.4), 1 mg/ml BSA, 5 mm EDTA, and 4 mM cysteine. The shoots were disrupted with Moulinex mixer for 15 sec, and the solution was passed through a nylon mesh cloth. The suspension was centrifuged at 600g for 10 min, and 160 www.plantphysiol.org on May 10, 2018 -Published by Downloaded from

An angular position-sensing transducer was used to make continuous measurements of acid-induced elongation of Avena sativa coleoptile segments. Elongation rates at pH 4.5 (5 mM succinate buffer) were about 5-fold greater than those at pH 6.0. Buffered 0.1 mM abscisic acid produced a partial decrease of the growth rate. Pretreatments with abscisic acid buffered at pH 6.0 usualy caused a further reduction of the elongation response when the coleoptile segments were subsequently placed in buffer at pH 4.5 containing abscisic acid. Abscisic acid did not completely prevent the pH effect in any of these experiments, and the brief latent period of the pH response was not affected by abscisic acid treatments. At pH 4.5, where the inhibitory effect of ABA was maximum, low pH-induced elongation was also inhibited by KCN these segments were strung on a wire and inserted in a measuring chamber in which they were completely immersed in an aerated buffer. The segments were allowed to equilibrate in a succinate buffer, pH 6.0, for 1 hr or longer, after which time a low growth rate was established. The chamber was then quickly drained and refilled with the buffer to be tested. An angular position-sensing transducer was used to produce continuous elongation curves which were analyzed by computer and replotted by an IBM 1130 line plotter as previously described (5). A 5 mm succinate buffer was selected for use in these experiments, since it consistently maintained the pH required for the various trials. Citrate buffer was also tested but proved less satisfactory, since the citrate at pH 6.0 caused a large initial stimulation of growth during the hour equilibration period before switching to a low pH. An inorganic MOPS3 buffer gave pH responses comparable to succinate but was somewhat more variable, and the pH-induced elongation rates did not remain constant as long. RESULTSAfter 1-hr equilibration in succinate buffer at a pH of 6.0, a low, steady growth rate was established which was not altered by a fresh addition of the pH 6.0 buffer. However, as the pH of the new bathing solution was lowered below 6.0, the elongation rate of the coleoptiles increased (Fig. 1). About a 10-fold increase in elongation rate was observed after a change in pH from 6.0 to 3.2, but the increase persisted for only about 20 min, after which time elongation ceased, and, in some cases, slight shrinkage occurred.The effects of pH 4.5 were investigated most thoroughly, since this treatment produced about a 5-fold increase in elongation rate which persisted for longer than 1 hr. When 0.1 mM ABA was present in the pH 4.5 buffer, the stimulating effect of the low pH was reduced (Fig. 2). Pretreatment with 0.1 mM ABA at pH 6.0 usually caused further inhibition of the elongation rate, when the coleoptiles were subsequently placed in pH 4.5 buffered ABA. The magnitude of the pH responses and their inhibition by ABA were somewhat variable, but when an entire set of experiments was performed on 1 day with seedlings from the same planting, inhibitory effec...

An unexpected acceleration in elongation of Avena coleoptile segments has been observed with a position sensor transducer 2 to 2.5 h after excision when the segments are immersed in 5 mM succinate buffer (pH 6). The cause of the accelerated growth is unknown.

The effects of cyclic adenosine 3': 5'-monophosphate (cAMP) on the growth of Arena coleoptile segments over 4 to 10 hours were monitored with a position sensing transducer. At pH 6, cAMP (0.1 mM with and without 2.5 mM glucose; or 2 mM alone) or dibutyryl cAMP (0.1 mM) was added at the beginning of the experiment, or after about 1 hour or after about 6 or 7 hours. Under all conditions tested, cAMP compounds had little or no effect on coleoptile segment elongation. Inasmuch as cAMP does not duplicate the rapid and vigorous elongation obtained with 2 gM auxin, the hypothesis that cAMP is a mediator of auxin activity is not supported by experimental evidence in this system. This conclusion is dependent upon the assumption that the cAMP compounds penetrated the tissue.