"'C-tryptophan to 14C-indole-3-acetic acid and by a nearly 3-fold increase in indole-3-acetic acid oxidase activity. The increase in growth rate is also observed in ...
Plant Physiol. (1975) 55, 757-762
The Nature of Spontaneous Changes in Growth Rate in Isolated Coleoptile Segments1 Received for publication October 24, 1974 and in revised form December 17, 1974
MICHAEL L. EVANS AND MARK R. SCHMITT2 Department ofBotany, The Ohio State University, Columbus, Ohio 43210 ABSTRACT About 4 hours after they are cut from the seedling, corn (Zea mays L.) coleoptile segments mounted vertically show a strong increase in growth rate. This increase occurs in water or various buffers near pH 7 and is not accompanied by the accumulation of a growth promoter in the medium. The increase in growth rate is prevented by mM p-fluorophenylalanine and is strongly inhibited by 0.1 mM p-chlorophenoxyisobutyric acid. The increased growth rate is accompanied by a 95% increase in the ability of tissue extracts to catalyze the conversion of "'C-tryptophan to 14C-indole-3-acetic acid and by a nearly 3-fold increase in indole-3-acetic acid oxidase activity. The increase in growth rate is also observed in segments from coleoptiles aseptically. The spontaneous increase in growth rate is completely but reversibly inhibited by 1 AM indole-3-acetic acid. Cytokinins have little effect on the spontaneous growth response, whereas gibberellic acid is observed to extend the latent period and reduce the magnitude of the response. It is tentatively concluded that the increase in endogenous growth rate may result from increased auxin production upon derepression of the auxin biosynthesis pathway after isolating the tissue from the normal supply of auxin from the tip. grown
When coleoptile segments are isolated from seedlings and placed on any of a variety of growth-recording devices, an initial period of rapid elongation (5, 6, 8, 10, 13) lasting 30 to 60 min is usually observed. This period is followed by a gradual decline in growth rate to a low steady value. Once the low steady growth rate is attained, it is commonly viewed as a basal rate of elongation, and the ability of various growthpromoting factors to increase the rate above that level can be tested. Inherent in this approach is the assumption that the low rate of elongation is constant once attained. Recent evidence (6, 10, 1 1) indicates that this is not the case but that the rate of elongation in isolated A vena coleoptile segments increases spontaIThis work was supported by a National Science Foundation Grant to M. L. E. Paper No. 876 from the Department of Botany, The Ohio State University. 2 Some of the material presented here is part of the Undergraduate Honors Program Thesis of M. R. S. Present address: Department of Botany, University of Wisconsin, Madison, Wis. 53706.
neously after a period of about 2.5 hr of slow growth. In corn the increase in growth rate occurs about 4 hr after cutting. The spontaneous increase in growth rate stands as a potential source of error in the interpretation of growth experiments. The previously reported (9) ability of acetylcholine to promote Avena coleoptile segment elongation has now been disclaimed (10) and attributed to the spontaneous increase in growth rate in such segments. Similar spontaneous growth responses have now been observed by various workers using different growth recording devices (personal communications from Drs. Purves, Hertel, Rubinstein, and Harmet). In this paper, we further characterize the spontaneous growth response in isolated corn coleoptile segments and describe some biochemical changes that accompany the response. The phrase "spontaneous growth response" is used in this context in reference to the increase in endogenous growth rate of isolated coleoptile segments and will be abbreviated SGR.3 The SGR is spontaneous in the sense that it occurs in the absence of any exogenous alteration of the system.
MATERIALS AND METHODS Plant Material. Nonsterile oat (Avenia sativa L., var. Victory) and corn (Zea inays L., hybrid WF 9X38, from Bear Hybrid Corn Co., Decatur, Ill.) seedlings were grown as previously described (12, 13). Sterile oat seedlings were obtained by rinsing seeds first in 70% ethanol for 2 min. The seeds were then shaken in 2% sodium hypochlorite for 25 min and rinsed with sterile H20 before planting in 10-cm Petri dishes on sterile nutrient agar. The seeds were exposed to red light for 24 hr and then allowed to grow in darkness at room temperature to a length of 3 to 4 cm before use in a growth experiment. Only seedlings in dishes with no evidence of bacterial contamination were selected for use in growth experiments, others were discarded. Sterile corn seedlings were obtained by rinsing the grains first in 95% ethanol for 5 min. The grains were then soaked in 2% sodium hypochlorite for 10 min and then rinsed with sterile H20 before planting on sterile nutrient agar in 500-ml Erlenmeyer flasks with cotton plugs. Seedlings for experimentation were selected as described for A vena seedlings. Growth Experiments. Growth experiments were done using a vertical column of 14 1-cm coleoptile segments. The segments were cut beginning 3 mm from the tip of the coleoptile, deleafed, and floated 30 to 45 min on distilled H20 before mounting on a shadowgraphic growth-recording device as previously described (13). 'Abbreviations: SGR: spontaneous growth response; PFPA: pfluorophenylalanine; PCIB: p-chlorophenoxyisobutyric acid; dimedone: 5, 5-dimethyl-1, 3-cyclohexanedione. 757
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EVANS AND SCHMITT
Preparation of Crude Extract for IAA Synthetase Assay. In order to measure the change in IAA synthesizing capacity ("IAA synthetase" activity) with time after removal of segments from corn coleoptiles, the following procedure was used. Forty 1-cm segments (about 1 g) were cut and strung on a stainless steel wire mounted vertically in a Teflon holding block. In order to simulate growth conditions, a weight was added to the top of the column and the assembly was submersed in distilled H20 under dim red light at room temperature. The H20 was continuously oxygenated as in growth experiments. After a specified period the segments were removed and ground in 0.2 ml of cold 0.5 M phosphate buffer (pH 6) in a chilled mortar and pestle with a small amount of washed sand. The homogenate was strained through two layers of cheesecloth, and the residue was reground in 1 ml of cold distilled H20 and also strained. The combined extracts were centrifuged at 10,500g for 30 min. The supernatant was collected, and the pellet was resuspended in a 1.5-ml fraction of the supernatant and centrifuged at 10,500g for 20 min. The supernatants were combined and brought to a volume of 3.5 ml with H20. Assay of IAA Synthetase Activity. The ability of this extract to catalyze the conversion of "4C-tryptophan to '4C-IAA was determined using a procedure similar to that described by Cheng (3). One ml of crude extract was added to a mixture of 0.5 ml of 10 mm a-ketoglutaric acid, 0.05 ml of 1 mM pyridoxal phosphate, 0.25 ml of 10 mm 1-tryptophan, 1 ,tCi of "4C-dl-tryptophan (57 mCi/mmole, Amersham/Searle, Arlington Heights, Ill.) and 1.2 ml of H20. The mixture was shaken at 30 C for 45 min, after which the samples were chilled in ice water and the pH was adjusted to 3 with HCl. Extraction and Identification of '4C-IAA. To the acidified reaction mixture, 3 ml of diethyl ether was added and the mixture was shaken vigorously. The mixture was then centrifuged, and the ether fraction was removed. This step was
1
3 TIME IN HOURS
5
FIG. 1. Effects of PFPA and PCIB on the SGR. Curves A and C: SGR in corn (A) and Avena (C) coleoptile segments growing in 3 mM phosphate buffer (pH 6.8); curves B and D: elimination of the SGR in corn (B) and Avena (D) by PFPA. Segments were treated with 1 mM PFPA plus 3 mM phosphate buffer (pH 6.8). IAA was added at the arrow in each curve to a final concentration of 0.01 mm. Curve E: inhibition of the SGR in corn by 0.1 mM PCIB.
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repeated, and the combined ether extracts were taken to dryness under vacuum. The residue was redissolved in two 100,ul aliquots of diethyl ether and spotted on Silica Gel F254 thin layer plates which were developed in chloroform-acetic acid (95:5) to 8.5 cm from the origin. Spots corresponding to authentic IAA were scraped from the plates into scintillation vials containing 10 ml of Bray's solution, and radioactivity was determined in a Beckman Model LS-230 scintillation spectrometer.
Preparation of Crude Extracts for IAA Oxidase Assay. In order to measure the change in IAA oxidase activity with time after removal of segments from corn coleoptiles, 25 1-cIm1 segments were cut and mounted as described above. At a specified time after Cutting, the segments were removed from the holding apparatus and ground in a chilled mortar and pestle in 3 ml of 0.05 M phosphate buffer (pH 6) containing 10 mM mercaptoethanol and 0.1 g/ml insoluble PVP (26). The homogenate was centrifuged at 5000g for 5 min. The pellet was discarded, and the supernatant was centrifuged at 20O000g for 20 min. Solid (NH4)2SO was added to the resultant supernatant over a 1-hr period to the point of saturation. Precipitated protein was collected at 40,000g for 30 min, and the pellet was resuspended in 3 ml of 0.05 M phosphate buffer (pH 6) for 24 hr. The dialyzed material was then centrifuged at 10,000g for 30 min, and the supernatant was used as the crude IAA oxidase preparation. Assay of IAA Oxidase Activity. IAA oxidase activity of the crude preparation was assayed spectrophotometrically by adding 0.5 ml of extract to 2.5 ml of a reaction mixture containing 0.286 mm IAA, 0.1 mm 2,4-dichlorophenol and 0.2 mM MnCl.., in 0.05 M phosphate buffer, pH 6. The oxidation of IAA was followed over a period of 30 min by measuring increasing absorption at 260 nm in a Gilford Model 240 spectro-
photometer (29). RESULTS AND DISCUSSION Changes in Endogenous Growth Rate. Figure 1 shows the time course of elongation of segments from Avena or corn coleoptiles mounted vertically and growing in 3 mm phosphate buffer (pH 6.8). When Avenia coleoptile segments are mounted on the growth recording device after floating about 30 min in distilled H20, their rate of elongation is initially quite high (up to 0.7 mm/hr- 10 mm segment). After about 60 min the rate of elongation declines to a low steady value (about 0.04 mm/hr 10 mm segment) and remains constant for 1.5 to 2 hr. The rate of elongation then increases again and remains high (usually between 0.1 and 0.3 mm/hr 10 mm segment) for abouLt 1 hr before dropping again to a low value (about 0.04 mm/hr- 10 mm segment), which remains constant for several hours. The initial rate of elongation of corn coleoptile segments treated in an identical manner is about 0.08 mm! hr 10 mm segment. After about 1 hr the rate of elongation drops to approximately 0.04 mm/hr- 10 mm segment. After an additional 2.5 hr, however, there is a dramatic increase in growth rate to about 0.25 to 0.50 mm/hr- 10 mm segment. We have recorded this rapid rate of elongation for 6 hr with no sign of a decline. We have not continued recording beyond 6 hr. Figure 1 also shows that 1 mM PFPA completely eliminates the SGR in both Avetna and corn coleoptile segments. The segments are still capable of responding to 0.01 mm IAA in the presence of 1 mm PFPA, but we have consistently found that, as reported by others (18, 27), the auxin response in the presence of PFPA is only about one-third as great as a normal response to auxin in the absence of PFPA (data not shown). We have attempted to study the SGR using multiple replicates by simply floating excised corn coleoptile segments
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SPONTANEOUS GROWTH RESPONSES
on appropriate media in Petri dishes and measuring their increase in length periodically using a millimeter ruler. We have never observed the SGR under these circumstances. When coleoptile segments are mounted on thread and submersed horizontally in oxygenated solutions in order to more closely approximate the growing conditions on the growth recording apparatus, the SGR also fails to develop. However, coleoptile segments treated in the same manner but held vertically consistently exhibit the SGR. We have no explanation for this apparent strong dependence of the SGR on the orientation of the tissue with respect to gravity. Since the SGR is stronger and longer lasting in corn coleoptile segments than in oat coleoptile segments, further attempts to characterize the nature of the phenomenon were made using corn. Among the possible explanations for the occurrence of the SGR are: (a) the production and accumulation in the medium to a growth-promoting level of some unknown growth factor, (b) the gradual release of hydrogen ions (H+) causing a decrease in pH to a growth-promoting level within the wall or within the medium itself, (c) a sudden release of bound auxin several hours after excision of the segments, and (d) the biosynthesis of auxin from endogenous precursors. The first alternative, an accumulation of some growthpromoting factor in the medium, can be ruled out since it was found that the SGR is unaltered when the medium is replenished at 45-min intervals during the latent period preceding the response. Furthermore, media bathing segments that have exhibited a SGR were found to have no effect on the growth rate of freshly prepared segments. It also seems unlikely that the increase in growth rate is due to accumulation of H+. Although coleoptile segments were observed to exude H+ when bathed in H20 (8 g of corn coleoptile segments reduced the pH of 100 ml of H20 from 6.9 to 5.9 in about 2 hr), buffered solutions were found to be without effect on the spontaneous increase in growth rate, i.e. the timing and magnitude of the SGR were the same whether the segments were grown in water, 3 mm phosphate buffer (pH 6.8), or 20 fM tris buffer (pH 7.2). Furthermore, the pH of the medium was the same at the beginning and end of each experiment. Further evidence against the involvement of H+ in the SGR is provided by the observation that the amino acid analogue, PFPA, eliminates the spontaneous growth response in both Avena and corn (see Fig. 1 and refs. 10 and 11) though it has no effect on the acid growth response nor on the normal loss of H+ from excised coleoptile segments in water (Evans, unpublished). The other alternatives listed involve changes in the effective auxin level in the tissue. It is well known that the uppermost portion of decapitated Avena coleoptiles develops the ability to synthesize auxin about 2.5 hr after decapitation (35), a time which corresponds very closely with the beginning of the SGR (Fig. 1). This "regeneration of a physiological tip" is thought not to occur in isolated coleoptile segments, since the process seems to depend upon a supply of cytokinins from the roots or seed (20, 32). We considered the possibility that the SGR in Avena segments might represent only a transient regeneration of a physiological tip and therefore may have gone undetected without high resolution measuring techniques. Regeneration of a physiological tip in corn segments, seems not to have been investigated. The observation that PFPA prevents the SGR in both oat and corn coleoptile segments (Fig. 1 and ref. 3) further suggests the possibility that regeneration of a physiological tip may be involved in the response, since Platt (28) has found that PFPA prevents the regeneration of a physiological tip in decapitated A vena seedlings. We have observed (Fig. 1) that the SGR is strongly inhibited by the
759
auxin antagonist, PCIB, suggesting that the SGR may be related to changes in endogenous IAA content. We tested the possibility that the spontaneous response in corn coleoptile segments is due to regeneration of a physiological tip (Fig. 2). Two sets of segments were prepared, and their growth was recorded for 4 hr, at which time the SGR was established. Both sets of segments were then removed from the chambers and the apical 2 mm were cut from each of one set of segments, while the basal 2 mm were cut from each of the other set of segments. The segments were then remounted in their original order and orientation and further growth was recorded. If rapid growth were due to auxin biosynthesis in the apical ends of the segments, one would expect the growth rate per mm of tissue to decline dramatically in the set of segments from which the apical 2 mm were removed but to remain relatively constant in the tissue from which the basal 2 mm were removed. As shown in Figure 2, the growth rate remained high for both sets of segments, indicating that localized apical or basal production of auxin is not responsible for the SGR. However, this observation does not rule out the possibility of auxin biosynthesis throughout the 1-cm coleoptile segment. Changes in IAA Synthetase Activity with Time after Cutfing. If the SGR is due to the initiation of uniform auxin biosynthesis throughout the tissue, it ought to be possible to detect an increase in IAA synthetase activity with time in excised coleoptile segments. This possibility was tested by measuring the ability of tissue segments 1 hr and 5 hr after excision to catalyze the formation of '4C-IAA from 14Ctryptophan in vitro (Table I). In the time period between 1 hr and 5 hr after cutting the segments, IAA synthetase activity increases about 95%. This fact may be enough to account for the SGR that occurs during that time period. Table I also shows that treatment of the tissue segments with 1 mm PFPA during aging has only a slight inhibitory effect on the development of IAA synthetase activity even though the same concentration of PFPA completely prevents the spontaneous growth response as shown in Figure 1. This result suggests that the inhibitory action of PFPA is not at the level of auxin biosynthesis. In contrast, treatment with 1 /M IAA during the aging period was found to reduce somewhat the time-dependent increase in IAA synthetase activity. This increase is con-
I 0 z
90
Lu
Tip Base 1
2
3
4
5
6
7
8
9
TIME IN HOURS FIG. 2. Test for localized IAA biosynthesis in corn coleoptile segments. Segments were grown in 3 mM phosphate buffer (pH 6.8) until establishment of the SGR. Segments were removed and 2 mm were cut from tip (upper curve) or base (lower curve) of each segment before remounting and continuing to record.
EVANS AND SCHMITT
760
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1975
corn coleoptiles. Segments were cut and allowed to grow in 100 [kg/ml penicillin G under otherwise nonsterile conditions with results as shown in Figure 4. Surprisingly, aseptically grown A vena coleoptile tissue shows a much stronger and longer lasting SGR than tissue grown normally. The SGR in aseptically grown Avena tissue resembles in magnitude and duration the SGR in corn coleoptile tissue. Aseptically grown corn coleoptile segments placed in penicillin G exhibit a somewhat weakened SGR compared to Radioactivity as IAA Increase Aging Mledium1 tissue grown normally. This tissue also exhibits a weaker auxin Tissue aged 5 hr Tissue aged 1 hr response compared to normally grown tissue. It is reported that penicillin G does not completely prevent tryptophan conversion to IAA by bacteria (25). Treatment with 100 yg/lml 95 8473 + 1128 4334 ± 901 Water streptomycin sulfate, on the other hand, is reported to reduce 80 8420 + 620 4675 ± 1025 1 mM PFPA bacterial conversion of tryptophan to IAA nearly 100% (25). 38 6250 + 763 4528 ± 599 1 /NI IAA Although we found that aseptically grown corn coleoptile -~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ segments placed in 100 ,tg/ml streptomycin sulfate showed 'Data for aging in water, PFPA, and IAA are averages of 20, 4, little or no SGR, the tissue also showed little or no growth and 3 experiments, respectively. response to exogenously supplied IAA, suggesting toxic side effects at this level of streptomycin (19). Since the timing of the SGR in A vena and corn is the same in both sterile and nonsterile tissue, and since the magnitude of the SGR in sterile tissue is nearly as great as in nonsterile tissue in the case of 600 corn and even greater in the case of Avena, it seems unlikely Table I. In Vitro Formatio,i of '4C-IAA from 14C-Tryptophan in Homogeniates of Corni Coleoptile Segmenits Segments were cut and mounted vertically in media indicated for aging. After the time indicated, the segments were ground, and extracts were tested for ability to catalyze the conversion of 14C-tryptophan to 14C-IAA during a 45 min incubation period at 30 C.
J lmm
400
200
1
2 TIME
AFTER
3
Sterile Aven
4
EXCISION (Hr.)
FIG. 3. Change in IAA oxidase activity with time in isolated coleoptile segments. IAA oxidase activity was determined spectrophotometrically in coleoptile segments cut and mounted vertically for various periods of time. Data for 0.5 hr and 3 hr points are averages of four experiments. Other points are averages of three experiments. corn
z UJ1
sistent with the observation described below that auxin treatment prevents the SGR. Changes in IAA Oxidase Activity with Time after Cutting. Changes in IAA oxidase activity with time after excising segments from coleoptiles of corn seedlings were followed as described in Materials and Methods. Figure 3 shows that the level of IAA oxidase activity may drop slightly during the 1st hr after cutting. The slight decrease shown did not occur in all experiments. However, during the 3rd hour we consistently observed a rise in IAA oxidase activity to a peak nearly three times greater than the initial level of activity. This peak in IAA oxidase activity somewhat precedes the SGR. Growth Experiments with Sterile Seedlings. There is disagreement concerning the role of tryptophan in auxin biosynthesis in higher plants (2, 25, 31, 36). Although there is evidence that coleoptile tissue is capable of converting "4Ctryptophan to 14C-IAA in vivo (2, 7, 24), there are indications that at least a portion of the ability of nonsterile coleoptile tissue to convert tryptophan to IAA during long term experiments can be attributed to epiphytic bacteria (24, 25). In order to examine the possibility that the SGR might be due to laccelerated auxin production by bacteria, growth experiments were done using tissue from aseptically grown A lenia and
No-trle
Avena
Non-sterile Corn
1
2
4 3 TIME IN HOURS
5
6
FIG. 4. Spontaneous growth response in aseptically and normally grown Avena and corn coleoptile segments. Curves A and D: SGR in Avtena (A) and corn (D) coleoptile tissue from seedlings grown aseptically. Segments were grown in 3 mm phosphate buffer (pH 6.8) plus 100 jLg/ml penicillin G. IAA was added at the arrow in curve D to make 0.01 mM IAA. Curves B and C: SGR in
Avenia (B) and corn (C) coleoptile tissue from seedlings grown under non-sterile conditions. Growth medium, 3 mM phosphate buffer (pH 6.8). IAA was added at the arrow in curve C to make 0.01
mM IAA.
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0
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SPONTANEOUS GROWTH RESPONSES
I
zI
z
z
9
LUJ IAA
1
3
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7
FIG. 5. Inhibition of the spontaneous growth response in corn by IAA. Upper curve: segments treated with 1 luM IAA at the first arrow. After 4 hr the IAA was thoroughly washed from the chamber (four rinses) and the segments were left in H20. Lower curve: corn coleoptile segments grown in H20. The arrow at the end of the curve indicates that this growth rate continued and was recorded an additional 2 hr.
that the SGR can be attributed to auxin production by epiphytic bacteria. It is also difficult to reconcile the strong dependence of the SGR on tissue orientation with the idea that the response is due to auxin production by bacteria. Repression of the Spontaneous Growth Response by Auxin. The delayed increase in both IAA synthetase and IAA oxidase activity suggests the following model for the phenomenon of the SGR. IAA synthetase activity in subapical tissue in situ may normally be repressed by auxin moving into the tissue from the tip. The excised segment while floating on H20 during the pretreatment period should lose IAA. The loss of IAA may lead to derepression of IAA-synthesizing enzymes with the gradual accumulation of IAA leading both to induction of IAA oxidase activity (14, 22, 23, 30, 33) and rapid growth. According to this model, it should be possible to show that IAA itself is capable of inhibiting the SGR. Figure 5 shows that this is indeed the case. Corn coleoptile segments were allowed to grow in 1 ,uM IAA for 4 hr at which time the SGR was well established in control segments. Upon removal of the auxin, the growth rate declined to a low value typical of isolated segments before they begin the SGR, i.e. in the presence of exogenously supplied auxin the SGR does not occur. About 3 hr after removal of auxin, however, the SGR appears, indicating that it is the removal of auxin that triggers events leading up to the increase in endogenous growth rate. Similar results have been reported by Anker (1) for auxin repression of regeneration of the physiological tip in decapitated A vena coleoptiles. Effects of Other Hormones on Spontaneous Growth Response. If the SGR is due to initiation of IAA synthetase activity in isolated segments, the IAA synthesis in this tissue must differ from IAA synthesis in the tip, since that reportedly depends upon a supply of cytokinins from the roots or seed. We considered the possibility that there may be enough cytokinin in isolated segments of this variety of corn to support IAA synthetase activity in the absence of roots or seed. However, we found that treatment of the coleoptile segments with 1 tM 7-isopentyl-3-methylpyrazolo[4, 3-pyrimidine (a specific cytokinin antagonist, see ref. 16) had no significant effect on the SGR. Furthermore, treatment with kinetin or benzylaminopurine in concentrations from 3 to 300 mg/l had no effect on the
1
2
3 4 TIME IN HOURS
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6
FIG. 6. Effect of kinetin and GA on the spontaneous growth response in corn. Curve B: segments grown in 30 mg/l kinetin plus 3 mM phosphate buffer (pH 6.8); curve A: control for curve B, segments grown in buffer only; curve D: segments grown in 7.5 mg/l GA3 plus 3 mm phosphate buffer (pH 6.8); curve C: control for curve D, segments grown in buffer only.
TIME
IN
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FIG. 7. Effects of dimedone and tryptophan on the spontaneous growth response in corn. Curve A: medium changed from 3 mM phosphate buffer (pH 6.8) to buffer plus 0.01 mM IAA at the arrow; curve B: segments grown in buffer; curve C: segments grown in buffer plus 5 mm dimedone. IAA was added to a final concentration of 0.01 mm at the arrow. Curve D: segments grown in buffer and transferred to buffer plus 5 mM tryptophan at the first arrow and back to buffer alone at the second arrow.
EVANS AND SCHMITT 762762 SGR except for a
slight shortening of the
latent period in
some
experiments (Fig. 6).
Treatment of the excised
coleoptile
segments with GA,,
on
the other hand, was found to reduce and delay the SGR significantly (Fig. 6). This result is surprising in view of the reported stimulatory effect of GA on the conversion of tryptophan to IAA in coleoptiles (21, 34). Other Factors Affecting Spontaneous Growth Response. If the SGR is due to increased auxin biosynthesis, one would expect inhibitors of auxin biosynthesis to interfere with the spontaneous response. The aldehyde complexing agent, dimedone, which is reported to prevent the enzymatic conversion of indoleacetaldehyde to IAA (4), was found to inhibit the SGR nearly completely at a concentration of 5 mm (Fig. 7). The tissue was still capable of responding to exogenous IAA at this concentration of dimedone (Fig. 7), although the response was about half as strong as a normal reponse to IAA for this tissue. A higher concentration (10 mm) of dimedone eliminated both the SGR and the response to exogenous auxin,
while
a
slightly
lower concentration (1 mm) inhibited but had no inhibitory effect on the
exogenous auxin
the SGR only response
to
(data not shown).
The auxin metabolite 3-methyleneoxindole (15. 17) was found to have no effect on the SGR. However, tryptophan, a added presumed auxin precursor, though without effect when increased prior to the SGR, was found to inhibit strongly the inhibition rate of elongation, once established (Fig. 7). This proved to be reversible upon removal of tryptophan. We have no explanation for this selective effect of tryptophan. Our data support the theory that the SGR is due to auxin biosynthesis possibly via derepression of the IAA synthetase system. However, the precise pathway of auxin biosynthesis under these conditions is not clear. Libbert and Silhengst (24) showed that sterile corn coleoptile tissue is capable of converting '4 C-tryptophan to 14C_IAA, but it may be that exogenously than supplied tryptophan is less effective as a precursor in vivo in vitro, since Black and Hamilton (2) have found that in
not coleoptile tissue, exogenously supplied tryptophan does that equilibrate with the internal pool of free tryptophanNeverappears to serve as a precursor in auxin biosynthesis. theless, the phenomenon of the SGR in isolated coleoptile ments seems ideally suited to the study of the auxin biosynthesis pathway. Toward this end we hope to investigate further the effects of other specific inhibitors of IAA biosynthesis, well as the effects of suspected precursors of IAA on the SGR. The system should also lend itself to studies of changes in the activities of enzymes suspected of playing a role in auxin biosynthesis and to studies of correlations between hormone seg-
4. CLARIK, A. J. A-ND P. J. G. MANN. 1957. The oxidation of tryptamine to 3indoleacetaldehyde by plant amine oxidase. Biochem. J. 65: 763-774. 5. CLELAND, R. 1972. e dosage-response curve for auxin-induiced cell elonga-
Th
tion: a reev,aluation. Planta 104: 1-9. 6. Mr MI. M. REHM. 1974. Accelerated endlogenous CLINE, . G., M. EDGERTON, AXND growth in Arena coleoptile segments. Planta 120: 213-214. 7. ERDMA-NN, N. AND U. SCHIEWER. 1971. Tryptophan-dependent IAA biosynthesis from indole, demonstrated by double labelling experiments. Plant a 97: 135-141.
8. EVANS, 'M. L. 1967. Kinetic sttidies of the cell elongation phenomenon in
9.
10. 11.
12. 13.
14.
Acknowledgments-We would like to thank Wendy Kline for excellent technical assistance and Maria Simon for performing the growth experiments with tryptophan and dimedone. We are also indebted to F. Skoog of the University of Wisconsin for a supply of synthesized by S. M. Hecht of the Department of Chemistry, Mlassachusetts
7-isopentyl-3-methylpyrazolo[4,3-d]pyrimidine
Institute of Technology.
LITERATURE CITED
1. AN-KER, L. 1973. The auxin production of the physiological tip of the Avena coleoptile and the repression of tip regeneration by indoleacetic acid (not by naphthylacetic acid and 2,4-dichlorophenoxyacetic acid). Acta Bot. Neerl. 22: 221-227.
R.
H. HAMILTON. 1971. Indoleacetic acid biosynthesis in 2. BLACK, R. C. AND Avena coleoptile tips and excised bean shoots. Plant Physiol. 48: 603-606. 3. CHENG, T. 1972. Induction of indoleacetic acid synthetases in tobacco pith explants. Plant Physiol. 50: 723-727.
Avenza coleoptile segmiients. Ph.D. thesis. Univ-ersity of California, Santa Cirtz. EV-ANS, M. L. 1972. Promiiotion of cell elongation in Avenia coleoptiles by acetylcholine. Plant Physiol. 50: 414-416. EVANS, 'M. L. 1973. Rapid stimulation of plant cell elongation by hiormional and non-hormonal factors. Bioscience 23: 711-718. spontaneous increases in growth rate in EVANS, A I. L. 1974. The nature ofPlant Physiol. 53: S-43. excised corn coleoptile segmi-enits. of coleoptiles EVA_NS, Ml. L. AND R. HOKANSO-N. 1969. Timing of the response to the application and withdrawal of various auxins. Planta 85: 85-95. in coleoptiles EVANS, 'M. L. AND P. 'M. RAY. 1969. Timing of the auxin response1-20. and its implications regarding auxin action. J. Gen. Physiol. 53: andi GALSTON, A. W. A-ND L. Y. DALBERG. 1954. The adaptive formation physiological significance of indoleacetic acid oxidase. Amer. J. Bot. 41: 373-380.
R. SCH'MIDT. 1968. Auxin Transport und Phototropisnmts. I. H. 15. HAGER,ANND fiu r den Transport v-on Die lichtbedingte Bilduing eines Hemmstoffes WVuchsstoffen in Koleoptilen. Planta 83: 347-371. 16. HECHT, A S. I., R. 'M. BocK, R. Y. SCHaMITz, F. SKOOG, AND N. J.LEON-ARD. 1971. Cytokinins: development of a potent antagonist. Proc. Nat. Acad. Sci. U.S. A. 68: 2608-2610.
17. H-IANMAN, R. L., C. BA-MA-N, AND J. LANG. 1961. The conversion 18. 19.
20. 21. 22.
23.
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