Plant Physiol. (1 995) 109: 1435-1 440
lncreased 1-Aminocyclopropane-1-Carboxylic Acid Oxidase Activitv in Shoots of Flooded Tomato Plants Raises Ethvlene Production to Physiologically Active Levels' I
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J. English, Crantley W. Lycett, Jeremy A. Roberts, and Michael B. Jackson* IACR-Long Ashton Research Station, Department of Agricultura1 Sciences, University of Bristol, Long Ashton, Bristol BSI 8 9AF, United Kingdom (P.J.E., M.B.J.); and Department of Physiology and Environmental Science, Philippa
Faculty of Agriculture and Food Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough LE12 SRD, Leicestershire, United Kingdom (G.W.L., J.A.R.) 1978) of the gas in shoot tissues. In common with most higher plant species, ethylene synthesis in tomato proceeds from Met via severa1 intermediates. The last of these, ACC, is converted to ethylene by an oxidation reaction catalyzed by the enzyme ACC oxidase. It has been proposed that, in roots of flooded tomato plants, ACC synthesis continues or is enhanced, whereas oxidation to ethylene is blocked by the absence of oxygen (Bradford and Yang, 1980; Bradford et al., 1982; Wang and Arteca, 1992).Some of the accumulated and unmetabolized ACC is then transported, in the franspiration stream, to the aerial tissues. Here, the presence of oxygen allows ACC oxidase-mediated conversion to ethylene in amounts sufficient to promote epinasty (Bradford and Yang, 1980; Else et al., 1995). However, this may not be the complete story. A positive interaction between flooding and physical wounding on rates of ethylene biosynthesis by petioles has been observed (Jackson et al., 1978). Since both wounding and flooding increase ACC levels (Bradford and Yang, 1980; Botella et al., 19951, the interactive, rather than additive, effects of wounding and soil flooding on ethylene production suggest that flooding enhances the capacity of petioles to oxidize ACC to ethylene. Thus, ACC oxidase could regulate ethylene biosynthesis in the leaves of flooded tomato plants when ACC is plentiful. However, such a conclusion would be contrary to the generally accepted view of ACC oxidase as a highly expressed enzyme present in amounts that exceed those needed to oxidize endogenous ACC (Yang et al., 1985). Accordingly, in this paper we reexamine the role of ACC oxidase as a regulator of ethylene biosynthesis in flooded tomato plants. We also assess whether changes in ethylene production attributable to different levels of ACC oxidase activity are of physiological significance. The experimental approach we have taken is to compare responses to flooding by wild-type tomato plants with those of transgenic tomato plants containing an antisense construct to one member of the ACC oxidase gene family (ACO1). These plants (referred to as ACOl,,) were transformed with the ripening-related cDNA clone pTOM13 (Hamilton et al., 1990) inserted in reverse orientation. They have a reduced capacity to make ethylene (Hamilton et al., 1990). Regulation of ethylene production and epinastic curvature by
Soil flooding increased 1-aminocyclopropane-1-carboxylic (ACC) acid oxidase activity in petioles of wild-type tomato (Lycopersicon esculentum 1.) plants within 6 to 12 h in association with faster rates of ethylene production. Petioles of flooded plants transformed with an antisense construct to one isoform of an ACC oxidase gene (ACO1) produced less ethylene and had lower ACC oxidase activity than those of the wild type. Flooding promoted epinastic curvature but did so less strongly in plants transformed with the antisense construct than in the wild type. Exogenous ethylene, supplied to well-drained plants, also promoted epinastic curvature, but transformed and wild-type plants responded similarly. Flooding increased the specific delivery (flux) of ACC to the shoots (picomoles per second per square meter of leaf) in xylem sap flowing from the roots. The amounts were similar in both transformed and wild-type plants. These observations demonstrate that changes in ACC oxidase activity in shoot tissue resulting from either soil flooding or introducing ACC oxidase antisense constructs can influence rates of ethylene production to a physiologically significant extent. They also implicate systemic root to shoot signals in regulating the activity of ACC oxidase in the shoot.
In this paper we examine the possible role of ACC oxidation in regulating ethylene biosynthesis in vegetative shoots of flooded tomato (Lycopersicon esculentum) plants. We also assess whether changes in ethylene production attributable to different levels of ACC oxidase activity are of physiological significance for the shoots of flooded plants. During soil flooding, the root environment becomes depleted of oxygen (Grable, 1966). In response, shoot systems show a wide range of physiological and biochemical changes. Some of these changes, such as petiole epinasty in tomato, may be adaptive (Armstrong et al., 1994). Epinasty can be observed within 6 to 12 h of flooding (Jackson and Campbell, 1975) and is brought about by the action of ethylene, following enhanced accumulation (Kawase, 1972) and production (Bradford and Dilley, 1978; Jackson et al., This work was funded by the Science and Engineering Research Council (UK) as a CASE Award to P.J.E. IACR receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the UK. * Corresponding author; e-mail
[email protected]; fax 44-1275-394-281. 1435
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English et al.
ACC oxidase activity has been studied with the aid of this material. MATERIALS AND METHODS Plant Material
Wild-type and ACOl,, tomato (Lycopersicon esculentum Mill,, cv Ailsa Craig) seeds were provided by Dr. A.J. Hamilton (University of Nottingham, UK, Sutton Bonington Campus). Plants were grown in 90-mm-diameter pots containing Levington M2 peat-base compost and 2 kg m-3 of slow-release fertilizer (Osmocote; Sierra Chemical Europe BV, Heerlen, The Netherlands). The plants were grown under glasshouse conditions with minimum and maximum temperatures of 18 and 24"C, respectively, and a minimum day length of 16 h. Plants were used for experimental work at the five- to seven-leaf stage, i.e. approximately 5 weeks after sowing. Plants were flooded by placing each pot in a 1.2 X 10p3m3container with de-ionized water maintained at the leve1 of the cotyledonary node. Epinasty and Ethylene Exposure
Epinastic curvature was measured at intervals over 72 h in the third oldest leaf. Angles between the stem and the adaxial surface of the petiole were traced onto a white card using a fine pencil, taking care not to wound the plant. A tangent was drawn to the petiole curve using points at 20, 30, and 40 mm from the stem. The angle between this tangent and the stem was then measured using a protractor. Epinastic curvature was defined as any change in angle (O) after the initial reading. For ethylene treatment, plants were placed inside a 1.1-m3acrylic chamber. An appropriate amount of 99% ethylene was injected into the chamber, giving a final concentration of 10Psm3 m-'. If the chamber was opened part way through the experiment for epinasty measurements, the original concentration was restored by injecting an appropriate amount of ethylene to make up any shortfall assessed by GC. Ethylene Production and Xylem Sap Collection
Ethylene production was measured using petiole sections excised from the third and fourth oldest leaves of well-drained plants and plants flooded for 24 and 48 h. Two 20-mm-long proximal sections were placed together in 5 X 10-6-m3 conical flasks, each containing a small piece of damp filter paper. The flasks were immediately sealed using rubber puncture caps (Suba-seal; W.H. Freeman and Co., Barnsley, UK). Gas samples (1 X 10-6 m3) were removed at regular intervals for ethylene analysis, using a gas-tight syringe. Vials were aerated and resealed after each sampling. Estimates of ethylene production by petioles on the intact plants were obtained by measuring the amount of ethylene released during the first 30 min after excision, i.e. prior to the formation of significant amounts of wound-induced ethylene (Jackson and Campbell, 1976). Subsequent samples were taken every 90 min. Ethylene was measured with a Pye Unicam (Cambridge, UK) series 204 gas chromatograph fitted with a flame-ionization de-
Plant Physiol. Vol. 109, 1995
tector and a 1.8-m-longglass column, packed with alumina deactivated with sodium iodide and maintained at 1U0C with injection port and detector heated to 125°C. Fresh weight of the tissue was obtained after ethylene determinations, and ethylene production rates were calculatled as pmol kg-' s?. Xylem sap was collected from de-topped root systems, immediately after shoot removal, using purpose-built pressure chambers described by Else et al. (1994). Low pressures (maximum 0.4 MPa) were applied to the roots using compressed air for well-drained plants and nitrogm for flooded p1ant:s. Sap issuing from the cut stump was collected in Epplmdorf tubes or scintillation vials. Sufficient pressure was applied to cause sap to flow as close as possible to the transpiration rate of similar intact plants, measured by loss of weight over severa1 hours. The first 200 mm3 of sap were discarded to avoid transient contamination causetl by radial pressures applied while attaching tubing used to pass sap out of the pressure chambers for collection (Else et al., 1993,1994).The subsequent 300-mm3 volumes of sap were collected, weighed, and frozen for later ACC analysis. Sap collection took less than 20 min. Leaf areas of the de-topped plants were measured with a Delta-T Devices Ltd. (Cambridge, UK) measurement system. Analysis of ACC and ACC Oxidase
ACC concentrations were measured in sap from welldrained plants and from plants flooded for 24'48, and 72 h. Samples (300 mm3) were evaporated to dryness under vacuum and derivatized to N-benzoyl n-propyl ACC (Hall et al., 1993). HPLC was used to purify the extract. After drying, the residue was taken up in ethyl acetate to which 93 ng of N-benzoyl isobutyl ACC was added as an interna1 chromatography standard. The ACC-containing fraction was then analyzed using a Phillips PU4900 gas chromatograph (Pye Unicam) fitted with a nitrogen/phosphorus detector (Hall et al., 1993). ACC concentrations were calculated, and knowing the rate of sap flow, we estimated the amount of ACC delivered into the shoot per second, per square meter of leaf (pmol s-l m-'). ACC oxidase activity was assayed by supplying excised petioles with surplus (1 mo1 mP3) ACC and measuring the amount of ethylene evolved. Two 20-mm-long petiole sections excised from leaf node 3 were incubated for 15 min in a reaction mixture containing ACC and cofactors sodium ascorbate and ferrous sulfate (Ververidis and John, 1991). The reaction mixture was then discarded, vials were sealed with Suba-seal puncture caps, and ethylene was allowed to accumulate for 4 h. Gas samples (1 X 10-6 m3) were then removed, and ethylene concentration was determined by GC. Preliminary tests showed that 0.6 to 1 mo1 m-3 ACC were the jmallest concentrations required to give the largest release of ethylene and that ethylene production rates were maximal4 h following ACC treatment. RESULTS
In vivo ACC oxidase activity was estimated on five occasions during 48 h by measuring the amount of ethylene
Flooding-lnduced ACC Oxidase and Ethylene Production released from petiole sections 4 h after treatment with 1 mo1 m-’ ACC (Fig. 1). In well-drained plants, activity varied little during the experiment and was lower in ACOl,, plants at a11 times compared to the wild type. Flooding increased ACC oxidase activity in the petioles of wild-type plants within 6 to 12 h. The effect was fully expressed by 24 h. Flooding also increased ACC oxidase activity in ACOI, plants, but the effect was smaller than in the wild type and its onset delayed (Fig. 1). These differences in ACC oxidase activity were reflected in rates of ethylene production by petioles from flooded and welldrained wild-type and ACOIAs plants (Fig. 2). Prior to the onset of a significant wound reaction (0-30 min after excision), ethylene production was slow in well-drained plants (
