Plant Growth Regulation @ 1996 Kluwer Academic
19: 67-16,1996. Publishers. Printed
67 in the Netherlands.
Developmental and genotypic differences in the responseof pea stem segmentsto auxin Natalie M. Barratt’ & Peter J. Davies Section of Plant Biology, Cornell University, Ithaca NY 1483.5, USA (*present Department of Biology, Hiram College, Hiram OH 44234, USA) Accepted 24 October
Key words:
address:
215 Colton
Hall,
1995
auxin, ethylene, growth inhibition, stem segment, elongation, pea
Abstract The objective of this investigation was to examine the response to exogenous auxin (indole-3-acetic acid; 1AA)of stem segments at two developmental stages. The standard auxin response of excised stem segments and intact plants consists of an initial growth response and a prolonged growth response. We found that this biphasic response does not occur in internodes at very early stages. Stem segments of light grown pea of various genotypes were cut when the fourth internode was at 6-l 3% of full expansion (early-expansion) or at 18-25% of full expansion (mid-expansion). Length measurements of excised segments were made after 48 hours of incubation on buffer with or without auxin. An angular position transducer linked to a computerized data collection system provided high-resolution measurement of growth of stacks of segments incubated in buffer over 20 hours. Early-expansion segments of all genotypes deviated from the standard auxin response, while mid-expansion segments responded in a manner consistent with previous reports. Early-expansion segments of tall, light-grown plants were unique in showing an auxin-induced inhibition of growth. The auxin-induced inhibition correlated with high endogenous auxin content, as determined by HPLC and GUMS, across genotypes and between early-expansion and midexpansion segments of tall plants. Measurement of ethylene evolved from stem segments in response to auxin, and treatment of segments with the ethylene action inhibitor, norbomadiene, showed the inhibition to be mediated in part by heightened ethylene sensitivity. Growth of early-expansion segments of dwarf and severe dwarf plants was stimulated by exogenous auxin, but the growth rate increase was delayed compared to that in mid-expansion segments. This is the first time that such a growth response, termed the delayed growth response has been demonstrated. It is concluded that developmental stage and endogenous hormone content affect tissue response to exogenous auxin. Introduction Growth of both excised stem segments and intact plants is increased by exogenous auxin (indole-3-acetic acid; IAA) [38]. In this paper we used excised stem segments of pea to distinguish the responses of tissues at different developmental stages to IAA. The initial growth response (IGR) elicited by IAA is considered to be growth mediated by cell wall acidification [27]. The subsequent prolonged growth response (PGR) to IAA involves gene induction [2], wall synthesis and wall breakdown [5], and protein synthesis [9]. The two phases of the auxin response are classified as an early,
cytokinin independent increase in growth rate, followed by a prolonged augmentation of growth rate that was prevented by cytokinin application [35]. In both etiolated Avena coleoptile segments [24] and intact pea plants [38], the immediate IAA response is induced by low concentrations of the hormone, and the long-term auxin response has a higher IAA concentration maximum than does the initial response. In work with excised segments, questions arise as to the native hormone sensitivities and endogenous hormone content of tissues, both at the time of excision and as experiments progress. The basipetal decrease in the amount of auxin in the transport stream of expand-
68
ing internodes of pea [3 l] may bring about corresponding gradients in tissue sensitivity to auxin. There is a gradient of auxin response in mung bean hypocotyl that ranges from inhibition in apical segments to stimulation in basal segments [25, 121. Segments of various plants incubated on hormone-free buffer give greater growth responses to exogenous IAA than do freshly cut segments [21,37]. It is uncertain whether changes in auxin sensitivity during development or with time after excision are due to differing endogenous hormone levels or to other factors. Growth of some excised segments is inhibited by exogenous IAA [25, 361. In etiolated pea, ethylene mediates auxin-induced growth inhibition [6, 11, 301. Transgenic plants overexpressing genes for IAA biosynthesis have elevated auxin levels and produce unusually high amounts of ethylene as well [ 151, reinforcing the link between the two hormones. However, tomato plants transformed with a gene leading to the destruction of the ethylene precursor, ACC (1 -aminocyclopropane- 1-carboxylic acid), have much reduced ethylene levels but grow normally [ 161. The conclusion is that ethylene plays only a minor role in normal plant growth and development, but the possibility that it may be important under conditionsof stress is not excluded. Supraoptimal IAA concentrations may directly inhibit growth if two IAA molecules arrive simultaneously at a receptor, each making a single contact and preventing the other from making the two contacts critical to trigger a response [I]. However, little mention of this idea persists in recent literature. Light-grown pea sections, unlike the etiolated pea, are reported not to be inhibited by IAA concentrations as high as 10m3 M [ 171. At this high concentration, segments grow more than do those at lower auxin concentrations, and they also produce large amounts of ethylene [17]. Light-grown tissue seems to have lost sensitivity to ethylene. In this work, we used excised stem segments of light-grown pea to distinguish and characterize developmental differences in tissue response to auxin. We asked whether the differential sensitivity of early-expansion and midexpansion light-grown segments to IAA is mediated by another hormone, perhaps ethylene; whether changes in endogenous hormone level correlate with sensitivity to exogenous hormone; and whether the two phases of the auxin response are distinguishable in their developmental occurrence and with regard to IAA-induced inhibition.
Methods and materials Plant material
Seed was increased from genotypes (Table 1) supplied by James Reid, who developed and characterized the lines at the University of Tasmania. L1771 and L203 are a tall, dwarf pair on the same genetic background (Nordic Gene Bank 1771). L181 is of a different genetic background and provided a more severe IAAdeficient dwarf phenotype than L203. Commercial pea varieties, Progress #9 (dwarf) and Alaska (tall), were also used. Plants were grown on vermiculite in a chamber at 20 to 25 ‘C with fluorescent lights supplying 50 pmol s-imm2 (16 hours light/8 hours dark), and were watered every other day with tap water. Segments were excised from the uppermost portion of the fourth internode (counting the cotyledons as 0) at two stages prior to full expansion (Table I), 10 to 12 days after sowing. Segments 5 mm long were cut from intemodes of tall peas and from mid-expansion internodes of dwarf peas. Segments 2 mm long were cut from earlyexpansion internodes of dwarf plants. Treatments were started within 5 minutes of excision. Incubations were done under continuous fluorescent light ranging from 30 to 60 pmol s-‘mm2. Segment growth conditions
Growth experiments employed 17 mM potassium/ sodium phosphate buffer (pH 6.1) with 2% sucrose and 0.1 mg/L penicillin-G with or without hormones. Gibberellic acid (GAs; GA) and indole-3-acetic acid (Sigma Chemical Co.) were added to the buffer, each at 10d5M. GA has a relatively constant effect on pea segment growth over a concentration range from 0.01 to 100 ,ug/mL, and lo-‘M is an effective concentration [26]. Likewise, lo-‘M IAA was used as an effective concentration that facilitated comparison with the literature. Long-term growth studies
Segments were floated on buffer in petri plates under fluorescent light. Final segment length after 48 hours was measured under a dissecting microscope. Calculations were done according to the following formula: Growth relative to control = amount of growth with treatment ’ amount of control growth
69 Table 1. Genotype, hormonal content, and developmental stage of the pea lines used in this study. All genotypes are homozygous for the allele indicated, and all relevant genes not listed are present as wild type alleles (usually dominant). GA1 is the endogenous growth active GA in pea. Segments cut from internodes that were 6 to 13% of full expansion were designated“early”. Segments cut from internodes more than 20% fully expanded were designated “mid” Line
Phenotype and genotype
Hormonal content
Length of fully expanded fourth internode
L1771
Wild type or Tall, Le
High GA1 [18], high IAA [22]”
9cm
6-7
20-25
Alaska
Wild type or Tall, Le
Presumed similar to Ll77l
9cm
6-7
2625
L203
Dwarf le
Low GA1 [33], low IAA [22]
3cm
6-8
18-23
L181
Severe Dwarf IS
GA1 deficient [32], presumed low IAA
2cm
lo-13
% of final length when segments Early Mid
20-2
I
a IAA content presumed is similar to tall line L1769
Amount of growth is the total increase in segment length over 48 hours. Experiments were repeated at least three times and data were averaged. The number of segments represented in each experiment is specified in the results. Short-term growth kinetics
For high resolution growth kinetics, a system modified from that of Parrish and Davies [20] was used. Stem segments were stacked in lengths of Intramedic polyethylene tubing (Clay Adams, Parsippany NJ) having holes for aeration and exchange of buffer. This assembly was submerged in a larger plexiglass tube filled with buffer bubbled with air. A Masterflex cartridge pump (Cole Parmer, Niles, IL) maintained a constant flow of fresh buffer to the tubes. In-line syringes with three-way stopcocks allowed for rapid changes of hormone solution. The foot of a metal Ypiece rested on the top segment in the stack and held in its crook the arm of an angular position transducer (Gould Electronics, Cleveland, OH). Cumulative segment growth displaced the transducer arm, and the resulting voltage change was fed into a computer via a DAS-8 Analog to Digital Converter Board (KeithlyMetrabyte, Taunton, MA). Data collection and manipulation was performed using a program written in Basic. The transducers were linear over the growth range of the segments, and provided measurements
with 2 pm resolution. Measurements going off scale were automatically offset by a voltage produced by a CIO-DDA06 Digital to Analog Converter (Computer Boards, Inc., Mansfield MA) run by our Basic program. Data were collected at 1-minute intervals, and raw data were converted to growth rates using a first derivative calculation based on an 1 l-point convolution [4]. Graphs of growth rate are representative traces of from 3 to 10 repetitions of a treatment. Individual recordings showed considerable noise, but the described patterns were consistent between replications. Quantitation of IAA by GUMS
Segments 5 mm long of the fourth internode of Alaska peas were harvested at early-expansion and mid-expansion stages, weighed, and frozen in liquid nitrogen. Extracts were made from 1.5 g of tissue (approximately 300 segments). IAA isolation and quantitation were done after Law and Davies [18]. Internal standard, 70ng of [13Ce]-IAA (99% 13C, Cambridge Isotope Laboratories, Wobum, MA)[8], and tracer, 10,000 dpm of [t4C]-IAA equivalent to 14.5 ng (Amersham, specific activity 55 ,&i/pmol), were added to samples prior to grinding in 80% MeOH. Ground tissue was vacuum filtered through Whatman #l filter paper with a celite pad. Filtrate was reduced in volume by rotary film evaporation. Samples were centrifugally filtered through Whatman glass fiber filters
70 and 0.45 pm (Rainin, Nylon 66) filters. Separation of IAA by HPLC (high performance liquid chromatography) was done on C 18 column (25 x 4.6 mm, 5 pm Spherisorb ODS-2, Phase Sep, Norwalk, CT) with a gradient of acidified water (solvent A) to methanol (solvent B) (0% B for 4 minutes, 0 to 40% B over 2 minutes, 40 to 65% B over 15 minutes, and 65 to 100% B over 2 minutes). Loading was from a 5 mL loop. The IAA fraction eluted at 23 minutes as detected by an in-line radiodetector (Model 7140, Packard). IAA was dried and methylated with ethereal diazomethane. Methylated IAA was repurified by HPLC using a gradient as above except that the ramp from 40 to 65% B was done over 10 minutes. The methyl-IAA fraction was analyzed by GC-MS (gas chromatography-mass spectrometry) (Hewlett-Packard Model 5890A with a HP- 1,25 m x 0.02 mm x 0.022 pm column having a coating of bonded methyl silicone and a model 5970B mass selective detector) in the SIM mode. Values for endogenous IAA amount were calculated based on the ratio of ion 130 representing endogenous [‘*Cl IAA to ion 136 representing the 13Cg internal standard and corrected for [‘*Cl IAA introduced in the [14C] IAA used as a tracer. Ethylene treatments
Segments were incubated on buffer with or without IAA for 3 hours to allow the production of woundinduced ethylene to diminish, then transferred at 15 per each 5 mL well to 24-well tissue culture plates (Falcon - No. B047, Becton Dickinson, Lincoln Park, NJ) containing 0.25 mL of IAA or control buffer. Airtight seals were made on the plates by clamping a rubber gasket and a plexiglass cover over the tops, with the cover having openings for a syringe needle to be inserted into each well [7]. Plates were left in the light for 2 hours. One mL of air was withdrawn from each sample well with a syringe and injected immediately into a gas chromatograph with an alumina column at 110 “C, injector at 180 “C, and flame ionization detector at 250 “C. With carrier gas (Nz) flow at 20 mL/min, ethylene standards eluted at 0.68 to 0.7 1 min. Amounts of ethylene in samples were calculated from peak areas compared to the area of standard peaks. To test segment sensitivity to ethylene, open petri plates of segments on buffer were placed in transparent plastic containers (30 x 20 x 8 cm) modified to have gas inlets and outflows. Ethylene at 1 ppm, or air for controls, was bubbled through water and across the plates for 48 hours in the light.
Treatment of segments with norbomadiene (bicycle [2.2.l]hepta-2,5-diene, Aldrich Chemical Co., Milwaukee WI) was done in open petri plates inside 1 L sealed glass jars placed on their sides so segments received light from above. Norbomadiene (NBD) was applied as a liquid to a square of cheesecloth in each jar, and the gas-tight jar immediately sealed. The amounts NBD used (5 r&/L) was calculated on a gaseous basis assuming volatilization.
Results When comparing the growth of excised segments of various genotypes, it is more appropriate to compare the magnitudes of growth normalized relative to controls of the same genotype than to compare the final lengths of the segments of different genotypes. Differences in final length may be attributed to cell number, endogenous hormone content, and developmental age of the cells in the segments, factors that vary with developmental stage and genotype. In all experiments reported here, “early-expansion” segments were taken from the uppermost portion of the fourth internode at about 7% of full expansion, and “mid-expansion” segments were taken from the uppermost portion of the fourth internode of duplicate plants at about 20% full expansion. IAA uptake kinetics were measured and found to be similar in early and mid-expansion segments of both tall and dwarf genotypes (data not shown). Hence, the differences in the IAA response kinetics are inherent in the response process and not mediated by differential auxin uptake. In all experiments, the commercial tall and dwarf peas behaved similarly to tall L1771 and dwarf L203, respectively, indicating that the response phenomena are general and not specific to a particular genetic line of pea. Responses of a tall pea line to IAA
We found that early-expansion segments from tall, light-grown pea plants were inhibited by IAA whereas growth of mid-expansion segments was enhanced. Early-expansion segments of tall plants showed a unique overall inhibition of growth in the presence of IAA over 48 hours (Table 2A); while the growth of mid-expansion segments was enhanced in a manner typical of most previous reports. Inhibition was expressed in early-expansion segments of tall plants at levels of IAA as low as 10m8ikf, while mid-expansion segments of tall plants showed vigorous growth with
71 Table 2. Effects of exogenous hormone on growth of segments of three pea genotypes. Segments of the fourth internode of tall (A), dwarf (B), and severe dwarf (C) pea floated on petri plates of buffer in continuous light for 48 hours. Hormone concentrations were 10m5 M for both GA and IAA. Initial segment lengths were 2 mm for early-expansion dwarf segments and 5 mm for early-expansion tall segments and all mid-expansion segments. Data are averages of measurements from 30 to 60 segments with standard error. The tinal treatment shown for tall plants was a six hour preincubation on hormone-free buffer then transfer to IAA solution Early-expansion Treatment
A) L1771
Final length in mm(SE)
Control
8.82fO.l
GA IAA
C) L181
Final length in mm (SE)
Growth relative to control
1.41
7.07ztO.18 7.96f0.2 1
1.43
10.38f0.16 7.6f0.05
0.68
9.38f0.08
1.15
IAA
3.84f0.12 4.82f0.11
Control IAA
2.74f0.08 3.59f0.06
Con. j B) L203
Mid-expansion Growth relative to control
Control
IAA
as much as 10m3M IAA (Table 3). A 6-hour preincubation of segments on hormone-free buffer prior to IAA application removed the inhibition (Table 2A). Exogenous GA promoted growth at the early-expansion stage when exogenous IAA was inhibitory, showing that the tissue was fully capable of increased growth (Table 2A). The kinetics of IAA-induced growth over 20 hours differ between mid-expansion and early-expansion segments. Mid-expansion segments of both tall and dwarf plants gave the standard IGR peak within 10 minutes of application and then slowed to a steady state growth rate above controls (Figure 1A). By contrast, early-expansion segments showed no IGR or a diminishedIGR followed by a steady decline in growth rate to below controls after about 500 minutes (Figure 1B). Preincubation of early-expansion segments in hormone-free buffer for 6 hours restored the IGR and PGR absent in segments to which auxin is applied immediately after excision (Figure 1B). Responses of dwarf pea lines to IAA
Early-expansion segments from dwarf pea varieties showed no IAA-induced growth inhibition when measured after 48 hours incubation(Table 2B and C). Yet, these segments failed to give the standard IAA response, consisting of IGR and PGR, when measured with high-resolution auxanometry. Early-expansion
8.7f0.15
1.79
1.53
8.42f0.16 9.46f0.16
1.30
2.2
6.7f0.11 8.05f0.07
2.93
Table 3. Differential effects of IAA concentrations on earlyexpansion and mid-expansion, tall pea stem segments. Segments of 5 mm initial length were incubated for 48 hours on petri plates with the indicated concentration of IAA. Genotypes Alaska and L177 I behaved similarly at both developmental stages (data not shown) Early-expansion Alaska segments [IAA] in M Final length in mm f SE
Mid-expansion L1771 segments [IAA] in M Final length inmmfSE
none 10-7 10-6
9.45f0.02 8.48f0.12 7.76f0.08
none 10-5 10-4
7.02f0.16 8.17fO.l 10.5f0.16
10-5
8.14f0.14
10-3
10.07f0.36
segment growth rate in L203 dwarf remained at control levels for up to 700 minutes after IAA application and then started to rise to a higher rate (Figure 1C). Severe dwarf L18 1 showed an initial low level growth promotion followed by a more pronounced late rise in growth rate (Figure 1D). The late response, typical of early-expansion segments of dwarf plants when IAA is applied immediately, will be called the delayed growth response (DGR). Mid-expansion segments of dwarfs (Figure 1A) and even ultradwarfs (data not shown) demonstrate the standard IAA kinetics.
72
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L1771 Mid-Exp L203 Mid-Exp.
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i 11 ...... II ----
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Control IAA at 360
min,
0.6
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i E 0.2 P 5 0.0 a
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0.8
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0.6 -
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I I (3 P IAA at 30 min. 2 Oe7 - . . . . . . Control
750 I
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0
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.I
ii2 a 0.5 0.4 0.3 0.2 0.1 0.0
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800 1000 0 TIME IN MINUTES
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Figure I. Representative growth rate traces of stem segments. The concentration of IAA used in all experiments was 10-5.k4. All controls were treated with hormone-free buffer throughout the experiment. Growth rates are expressed as pm increase in length per mm of segment per minute. A) Response of mid-expansion segments of dwarf pea L203 and tall pea L1771 to IAA added at the time indicated by the arrows and applied continuously. Controls (not shown) maintained a low steady state growth rate. B) Response of early-expansion segments of tall pea Ll77l to IAA treatment begun immediately after excision and continued throughout (solid line); treatment with hormone-free buffer for the first 6 hours, then treatment with IAA for the duration of the experiment (dashed line); and control (dotted line). C) Response of early-expansion segments of dwarf pea L203 to continuous MA starting at 30 minutes and continued throughout (solid line) and to control buffer (dotted line). D) Response of early-expansion (dashed line) and mid-expansion (solid line) segments of severe dwarf pea Ll8 1 to IAA treatment beginning at 85 minutes and continued throughout. The control treatment shown (dotted line) is of early-expansion segments.
73 Endogenous MA levels in mid-expansion and early-expansion stem segments of tall pea
IAA content was higher in the apical 5 mm of early-expansion internodes of the tall pea, Alaska, than in the same portion of mid-expansion internodes (Table 4). Ethylene production and response in tall pea
Treatment with 10d5M IAA for 5 hours induced increased ethylene production in segments of tall pea at both growth stages (Table 5). Exogenous ethylene inhibitedgrowth of early-expansion tall stem segments but had no effect on growth of mid-expansion segments. Early-expansion segments of tall pea measured after a 48 hour incubation with 1 ppm ethylene were only 8.73 & 0.13 mm while controls were 9.43 f 0.08 mm. Mid-expansion segments given the same treatments were 7.47 & 0.11 mm (with ethylene) and 7.63 f 0.21 mm(contro1). Effect of norbornadiene on MA-induced inhibition
Early-expansion segments of tall pea treated simultaneously with 5mIJL NBD and 10m5M IAA, although still growth inhibited, elongated more than did segments treated with IAA alone. After 48 hours of treatment, control segments were 9.4 f 0.01 mm, those treated with NBD alone were 9.46 f 0.09 mm, those treated with IAA were 7.6 f 0.05 mm, and those treated with both NBD and IAA were 8.3 * 0.07 mm (data are averages of from 40 to 70 segments with standard error). NBD partially relieved the IAA-induced inhibition.
Discussion Previous work was consistent in demonstrating the following standard auxin response of isolated stem segments: the IGR consisting of a lag of approximately 20 minutes followed by a rise to an initial high peak of increased growth rate for about 40 minutes, then a drop in growth rate; and subsequently the PGR consisting of a rise over another 90 minutes to settle at an intermediate rate of growth [3,22,34]. The tissues used in these previously reported results were probably at least 25% fully expanded, so are equivalent to the “mid-expansion” segments reported here. By contrast, we found the IAA response of segments taken from
Table 4. Endogenous IAA content of 5 mm early-expansion and mid-expansion Alaska pea stem segments quantified by GUMS with [‘3Ce$AA internal standard. Numbers are averages of three experiments with standard error. Each experimentused 200 to 350 segments
IAA in rig/segment IAA in ng/gfw Segment weight(mg)
Early
Mid
0.076f0.02 19.35f4.1 4.5
0.06 1f0.005 11.27kl.2 6.1
Table 5. Ethylene production in responseto IAA as measured by GC in tall genotypes of pea at two developmental stages.Three hours after excision, IAA was applied to segments of tall pea and the ethylene produced over the next 2 hours was measured. Data represent single trials of 15 segments each. ND = none detected
Ethylene (nUmg plant tissue) produced in two hours Treatment
LI771 Early
Mid
Alaska Early
Mid
lO-5 M IAA Control
0.089 0.009
0.086 0.008
0.012 ND
0.225 ND
internodes at less than 8% of full expansion to differ markedly from the standard response. Further, the IAA response of early-expansion segments of dwarf plants differed from that of early-expansion segments of tall plants. Early-expansion segments of dwarf plants show little or no IGR but a delayed growth response (DGR) to auxin. This DGR may be the PGR normally seen in mid-expansion segments after the IGR, but with a considerable delay. Early-expansion, dwarf tissue does not show the part of the auxin response usually attributed to acid growth. Perhaps bonds in this tissue amenable to cleavage by acid-induced enzymes are not yet formed, or the system required for the acid growth response is not yet fully developed. Mid-expansion, dwarf segments do show both the IGR and PGR, indicating that acid growth is dependent on developmentally regulated factors. Early-expansion segments of tall plants are unique in showing IAA-induced inhibition of the PGR, combined with a reduced IGR compared to the “standard” response. Inhibition was partially relieved by a six hour preincubation on hormone-free buffer. The auxininduced inhibition of growth in other tissues has been shown to be lessened by such a preincubation
74 [21, 36, 371. The mechanism of IAA-induced inhibition remains enigmatic. The existence of an auxindependent inhibitor that alters tissue sensitivity to auxin has been suggested [36]. Further work using the auxin transport inhibitor, naphthylphthalamic acid, to maintain auxin levels in segments during a preincubation indicated that the inhibitory mechanism was independent of IAA content [37]. In contrast, we found IAA-induced inhibition to correlate with high endogenous IAA level. Relief of inhibition by a 6-hour preincubation on hormone-free buffer, time to allow for endogenous IAA levels to decrease by basipetal transport out of the segments, supports this correlation. IAA added after the preincubation may be received by tissues no longer primed to respond negatively to additional IAA. Our data support the hypothesis that the IAA-induced inhibition of early-expansion, tall pea segments is mediated by high endogenous IAA levels. Because the inhibition is dependent on developmental stage and is not titratable - it still exists at concentrations as low as lo-’ M - it seems unlikely that inhibition results from an overabundance of IAA competing for binding sites and making ineffective attachments to receptors. If inhibition were by a lack of two-point attachments of IAA to receptors, the degree of inhibition would decrease substantially with decreased exogenous IAA concentration, and inhibition would be seen in all segments. These conditions not being satisfied, it seems that high levels of endogenous IAA may prime early-expansion tissue to respond negatively to even low amounts of exogenous IAA through the action of another system. An inhibitory mechanism induced by exogenous IAA and specific to auxin-induced growth was indicated by the segments’ ability to grow in response to GA at the stage when IAA was inhibitory. Ethylene may provide such a mechanism. Ethylene production is triggered by IAA, but not by GA [ 191, and ethylene does not block GA action [33]. Because mid-expansion segments produced as much or more ethylene than did early-expansion segments after IAA treatment, differential ethylene production between the growth stages was ruled out as a cause for their distinct IAA responses. Differential ethylene sensitivity remained as a possibility. Ethylene’s role in IAA-induced inhibition of stem growth was demonstrated with norbomadiene (NBD), a competitive inhibitor of ethylene action [32]. NBD increased the growth of early-expansion segments exposed to IAA by 24.5% over those given IAA alone. NBD may not completely
overcome IAA-induced growth inhibition because of the time it takes NBD to penetrate the segments and diffuse to the site of ethylene action, and because of the competitive nature of the inhibitor. A previous report that ethylene is not inhibitory to excised stem segments of light-grown plants [17] was based on Alaska pea stem segments 10 mm long, taken from the apical portion of the fifth internode of unspecified length. As these were most likely beyond 25% of full expansion, their developmental stage may be the reason for their lack of response to ethylene. Our data show that neither ethylene nor high exogenous IAA concentrations inhibit elongation of midexpansion stem segments of tall plants, but either hormone alone inhibits early-expansion segments. We conclude that the growth inhibition seen with exogenous IAA in early-expansion segments of tall plants is due in part to a heightened ethylene sensitivity, and that ethylene sensitivity is conferred in part by high endogenous IAA. What physiological reason could there be for a developmentally dependent inhibitory system? It has been hypothesized that very young stem tissues are thickening and growing slowly before prolonged, rapid growth [30], and that ethylene may ensure this period of slower elongation [23]. During development, levels of GA or IAA may fluctuate, increasing growth rate abnormally and leading to distortion of form were it not for the feedback inhibition of growth by ethylene [ 191. Subapical tissue holds a unique position, as all of the IAA produced at the apex must be transported through it to reach more basal tissues. This tissue should have mechanisms to avoid overgrowth in light-grown as well as in etiolated plants. The magnitude and pattern of the growth response of segments of light-grown pea stem to exogenous auxin depend on both developmental stage and endogenous hormone content.
Acknowledgments Thanks to Dr. F. Behringer for developing the computerized data collection system used in this work, and to Dr. T. Owens and T. Silva for assistance in its modification. Funding for the first author was provided in part by the Cornell NSF Plant Science Center, a unit in the USDA/NSF/DOE Plant Science Centers program and a unit of the Cornell Biotechnology Program, which is sponsored by the New York State Science and Technology Foundation, a consortium of industries, and the US Army Research Office. Funding from Sigma Xi
75 is also acknowledged. The GC-MS used in this work was funded by NSF equipment grant DMB-8505974 and the College of Agriculture and Life Sciences at Cornell University.
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