Characteristics of Ethylene Biosynthesis-Inducing Xylanase ... - NCBI

Received for publication April 24, 1992 Accepted August 24, 1992

Plant Physiol. (1992) 100, 2059-2065 0032-0889/92/1 00/2059/07/$01 .00/0

Characteristics of Ethylene Biosynthesis-Inducing Xylanase Movement in Tobacco Leaves' Amir Sharon, Bryan A. Bailey, John P. McMurtry, Rosannah Taylor, and James D. Anderson* Department of Horticulture, University of Maryland (A.S.), Weed Science Laboratory (B.A.B., R.T., J.D.A.), and Nonruminant Animal Nutrition Laboratory (1.P.M.), Beltsville Agricultural Research Center (West and East), Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland 20705 Induction of systemic resistance in plants by inoculation with fungi or application of elicitors has been known for many years (15, 21). The nature of the signal that travels through the plant has been intensively studied during the last years, and several compounds have been suggested as the communicating signal (10, 11, 17, 18, 21). Recent work in this laboratory with tissue-printing techniques showed that EIX and the vital stain Evans blue are translocated in the xylem both up and down from the petiole of application (4). When EIX is injected into the mesophyll intercellular spaces, induction of necrosis and elicitation of pathogenesis-related protein and capsidiol accumulation are restricted to the injected area (12, 16). Cryptogein, a proteinaceous elicitor isolated from Phytophthora cryptogea, was also reported to be translocated in the vascular tissues of tobacco plants (9). These findings raise the possibility that certain proteinaceous elicitors can induce systemic resistance without the involvement of intermediary compounds. It is not known whether cryptogein moves in the xylem or phloem to the intercellular spaces of the mesophyll, where it may interact with mesophyll cells. In the present study, we used 1251-labeled EIX to follow the movement and distribution of EIX in tobacco tissues and to map the areas of EIX accumulation.

ABSTRACT

1251-Labeled ethylene biosynthesis-inducing xylanase (EIX) was used to study the movement of this protein in tobacco (Nicotiana tabacum) tissues. A biologically active 1251-labeled EIX was obtained using chloramine-T as the oxidizing agent. Labeled EIX was detected in the far most edges of the leaf 5 min after it was applied to the petiole of a detached leaf. EIX was distributed uniformly throughout the leaf, including the mesophyll area within 5 to 15 min, after which there was only little change in the distribution of radioactivity in the leaf. 1251-Labeled EIX was extracted from treated leaves, and EIX translocation in the leaf was blocked by preincubation of labeled EIX with anti-EIX antibodies, indicating that the intact peptide moves in the leaf. Injection of anti-EIX antibodies into the intercellular spaces of the leaf mesophyll prevented induction of necrosis by EIX, suggesting the mesophyll as the site of EIX action. EIX was translocated both to upper and lower parts of the plant when applied to a whole plant through the petiole of a cut leaf. Radioactivity was found in all leaves and in the stem, although some leaves accumulated much more EIX than others; EIX was not found in the roots. There was no difference between the accumulation pattern of EIX in fresh and ethylene-treated leaves or between sensitive (Xanthi) and insensitive (Hicks) tobacco cultivars. These data support the hypothesis that intact EIX protein is translocated to the leaf mesophyll, where it directly elicits plant defense responses.

MATERIALS AND METHODS

Plant Material Two tobacco cultivars were used: Nicotiana tabacum cv Xanthi and N. tabacum cv Hicks. Plants were grown in a greenhouse until they were 25 to 35 cm tall. Fully expanded leaves 8 to 12 cm long from the Xanthi plants or 10 to 15 cm long from the Hicks plants were used for detached leaf experiments. Leaves were cut and used immediately unless otherwise mentioned.

EIX2 is a 22-kD protein isolated from the fungus Trichoderma viride (7, 8). When applied to tobacco (Nicotiana tabacum cv Xanthi) leaf tissue, EIX induces ethylene formation and hypersensitive necrosis, as well as other plant defense responses (1-3). There is evidence suggesting that the elicitor activity of EIX is independent of the endo-fl-1,4-xylanase activity, i.e. that the protein is an elicitor by itself (. Dean, personal communication). A number of enzymes and peptide elicitors have been isolated from fungi (5, 13, 19, 20, 22, 23). Proteinaceous elicitors with hydrolytic enzymic activity would generally be assumed to produce elicitors that are released from the plant or pathogen cell wall by action of the enzymic activity rather than the protein itself being the elicitor compound.

Preparation of 1251-Labeled EIX EIX was purified from xylan-induced cultures of Trichoderma viride as previously described (7). '25I-Labeled EIX was prepared by iodination of EIX with chloramine-T (Fisher Scientific3) as the oxidizing agent according to the procedure 3 Mention of trademark, proprietary product, or vendor does not constitute a guarantee of warranty by the U.S. Department of Agriculture and does not imply approval to the exclusion of other products or vendor that may also be suitable.

1

Scientific article No. A6324, Contribution No. 8500, of the Maryland Agricultural Experiment Station. 2 Abbreviation: EIX, ethylene biosynthesis-inducing xylanase. 2059

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SHARON ET AL.

described by Greenwood and Hunter (14). Briefly, EIX was added to 0.5 M phosphate buffer, pH 7.5 (15.6 ,g/25 AL), in a glass vial. Sodium [125I]iodide (Amersham; 10 nm, 0.1 mCi/ ,umol) was added (10 ,L) followed by 10 AL of chloramine-T (5 Mg/IL) in 50 mm phosphate buffer, pH 7.5, and the contents of the vial were mixed. The reaction was terminated after 30 s by the addition of 20 ML of sodium metabisulfite (5 Ag/,iL in 50 mm phosphate buffer, pH 7.5). A Sephadex G-75 column (0.7 x 50 cm) was equilibrated with 50 mm phosphate buffer, pH 7.5, and presaturated with crystalline 6% BSA (Sigma) in 50 mm phosphate buffer. The reaction mixture was transferred to the column and eluted with 50 mM phosphate buffer, pH 7.5, collected in 1-mL fractions. An aliquot was taken from each of the 1-mL fractions, and the radioactivity was determined with a gamma counter (model 1274, Pharmacia-LKB). The approximate specific activity of the labeled EIX was determined by the following formulas:

Specific activity = (% yield of protein/100)

*(mCi Nal25I added/mg of protein added)

1

(1)

% Yield = 100 - % yield of Na'251 (2) % Yield of Na'251 = (3) total counts in Na'251 fraction/total counts Aliquots of the fractions containing radioactivity were bioassayed for ethylene biosynthesis induction and xylanase activities as previously described (13). Fractions containing highly radioactive and biologically active EIX were pooled, loaded on an Excellulose GF-5 minicolumn (Pierce), and eluted with distilled water to separate the protein from remnants of salt (free 1251). The radioactivity was determined and the solution maintained at 40C for use within the next 4 weeks. Treatment of Detached Leaves

Fresh leaves averaging about 2.5 g were used. Leaves were placed on the a bench at room temperature, and a 5-,uL droplet of the "25I-labeled EIX (5 ng; 4 x 105 cpm) was applied as a hanging droplet to the cut petiole, which was absorbed within 4 to 6 min by transpiration-driven uptake. Larger volumes of solution were applied in halves; the second droplet was applied immediately after the first droplet was completely absorbed. Water droplets were added when needed to prevent the petiole from drying and to allow transpiration for the desired period of time. The first 5-mm portion of the petiole was removed, and transpiration was stopped by freezing the leaves on dry ice. Radiograms were produced by placing the frozen leaves on film (X-Omat AR, Kodak) for a 3-d exposure at -700C. Segments, 1 cm long, were cut along the leaf midrib (see diagram in Fig. 2B), and the distribution of the radioactivity in the leaf was recorded with a gamma counter.

Gel Electrophoresis and Western Blotting 1251-Labeled EIX (30 ng; 2.4 x 106 cpm) was applied to a detached leaf, and the proteins were extracted 1 h later to

Plant Physiol. Vol. 100, 1992

determine whether radioactivity in the leaves indicated the presence of EIX. The main vein and major side veins (major veins) were cut out and extracted separately from the other parts of the leaf (mesophyll). Protein was extracted by grinding in SDS-PAGE sample buffer (50 mm Tris-HCl, pH 6.8, 12% glycerol, 4% SDS, 0.01% Coomassie brilliant blue G250). Unlabeled EIX was added to the extracts (5 ng/AL) to prevent binding of the radioactive EIX to proteins on the gel during separation. The samples were boiled for 1 min, subjected to Tricine-SDS-PAGE, and electroblotted as previously described (2). Antibodies raised against the 22-kD EIX-polypeptide eluted from SDS-PAGE (8) were used for immunoblotting of one membrane, and the other membrane was placed on a film and exposed for 16 h. Treatment of Leaves with Antibodies Antibodies raised against native cellulysin EIX (13) were applied to the leaves either by application to the petiole as described above for EIX or by infiltration into the intercellular spaces of the mesophyll (16). The antibodies were applied to the leaf petiole, and 10 min later cross-sections of the leaf petiole were printed onto nitrocellulose membranes (6). Antibodies were immunolocalized with goat anti-rabbit immunoglobulin G alkaline-phosphatase conjugate (Bio-Rad). The antibodies against native EIX were also used to test whether they would interfere with the movement of EIX in detached leaves. Antibodies were applied in either of three methods: (a) "25I-labeled EIX (5 ng, 4 x 105 cpm) was mixed with antibodies (30 ,ug of protein), centrifuged, and then applied to the leaf petiole; (b) antibodies (20 Mg of protein) were first applied to the leaf petiole, followed by EIX (5 ng; 4 x 105 cpm) after absorption of the antibodies; and (c) 125labeled EIX (5 ng; 4 x 1i0 cpm) was premixed with antibodies (30 ,g of protein) together with 3 gg of unlabeled EIX, centrifuged, and then applied to the leaf petiole. For infiltration into the leaf mesophyll, antibodies were diluted in distilled water to give a concentration of 0.1 mg/mL and then injected to the mesophyll intercellular spaces through the lower side of the leaf. EIX (1 ug/mL) was injected into the same area immediately after injection of the antibodies. Treatment of a Whole Plant

Thirty nanograms (2.4 x 106 cpm) of '25I-labeled EIX and 10 Mg of unlabeled EIX in 100 uL of distilled water were applied to a leaf petiole located midway up the stem of normal or smaller (nine leaves) plants as previously described (4). The leaves were cut off the stem after 4 h and radioactivity was determined in the leaves or, in the case of the smaller plant, the leaves were removed and the soil was washed from the roots, and all of the plant parts were placed on a film and exposed for 16 h.

Ethylene Treatment We previously found that ethylene pretreatment greatly enhances the responsiveness of Xanthi leaves to EIX (2). Detached Xanthi leaves were incubated under an atmosphere

MOVEMENT OF ETHYLENE BIOSYNTHESIS-INDUCING XYLANASE IN TOBACCO

of 120 ML/L of ethylene for 14 h as previously described (4) to test the effect of ethylene pretreatment on EIX movement. Nonethylene-treated leaves were incubated in an atmosphere purged of ethylene. EIX was applied to the leaves through the petiole as described above.

2061

A5 min

I

RESULTS

1251-Labeling of EIX

1cm

EIX was labeled with 125I using chloramine-T as the oxidizing agent. Preliminary experiments showed that treatment of EIX with chloramine-T inactivated both xylanase and ethylene biosynthesis-inducing activities of EIX in a time- and concentration-dependent manner (data not shown). Both xylanase and ethylene biosynthesis induction activities of EIX were reduced by 60% when EIX was incubated for 60 s with 2.5 ,ug of chloramine-T. Incubation of EIX with 2.5 ,ug of chloramine-T for more than 4 min completely inactivated EIX. Based on the initial findings, optimal iodination was achieved with 5 ,ug of chloramine-T for 30 s. Under these conditions, a highly radioactive EIX (30-40 mCi/,ug of protein) was produced that retained a high level (70-80%) of EIX biological activity (Fig. 1A). Separation of fractions after G-75 column chromatography by SDS-PAGE showed that

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Figure 2. Time course of translocation of 1251-labeled EIX in detached Xanthi leaves. A droplet (5 gL) of water solution containing 5 ng of 1251-labeled EIX (4 x 105 cpm) was applied to the petiole of each leaf. A, The radiograms show the distribution of EIX in leaves 5, 15, and 60 min after application of 1251-labeled EIX. B, Leaf segments were cut at several times after application of 1251-labeled EIX, as demonstrated in the leaf diagram. C, The amount of radioactivity in different parts of the leaf was counted on individual plants.

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Figure 1. Separation of 1251-labeled EIX from [l251]iodide on Sephadex G-75 column. The iodination reaction mixture was loaded on a G-75 column (0.7 x 50 cm) and eluted with 50 mm phosphate buffer, pH 7.5, at 1 mL/min. A, The radioactivity of each fraction was counted, and the biological activity (induction of ethylene biosynthesis) was determined in each fraction. Four microliters of fractions 8 to 32 were loaded on an SDS-PAGE gel, and the proteins were electroblotted and placed on a film. B, The radiograms obtained after exposure for 24 h (only fractions 10-21 are shown).

no breakdown products of EIX were formed during the iodination process. Fractions 14 to 17, which contained high biological activity, were pooled and desalted, and a fraction was boiled and centrifuged for 10 min. No radioactivity was found in the supernatant, indicating that the solution did not contain free [125 ]iodide.

Translocation of EIX in the Leaves The movement and distribution of EIX in detached leaves studied with the 125I-labeled EIX. Radioactivity was detected throughout the vascular system of the leaf 5 min after application of a 5-jL droplet containing 5 ng EIX (4 x 105 cpm) to the leaf petiole (Fig. 2). The movement of EIX was continuous and reached a steady-state level within 5 to 15 min after application. At this time, the radioactivity was uniformly distributed throughout the leaf, including the internal mesophyll tissues (Fig. 2A). The amount of radioactivity taken into the leaf ranged from 20 to 90% of the applied material at 5 and 60 min, respectively. No significant difference was found in the distribution of '251-labeled EIX 15 to 120 min after EIX application (Fig. 2, A and C), and no

were


SHARON ET AL.

2062

detectable change in the distribution of the radioactivity was noted even 8 h after EIX application (not shown). Treated leaves were extracted after 1 h to ascertain whether the radioactivity detected in the leaves was associated with EIX. The major veins were extracted separately from the leaf mesophyll. A single radioactive band of 22 kD was found both in the major veins and in the remaining tissue extracts, indicating that all the radioactivity in the leaves was due to labeled EIX (Fig. 3, radiogram). A western blot with antibodies raised against an SDS-denatured form of the 22-kD EIX polypeptide showed that there was no difference between the EIX extracted from the leaves and the standard EIX, further indicating that there was no breakdown of the labeled EIX in the plant (Fig. 3, western blot). Antibodies raised against the native form of EIX were used to ascertain whether all of the tracer found in the leaves was due to intact EIX. "25I-Labeled EIX (5 ng) was incubated with 5 gL of antibody solution (30 gg of protein) for 5 min and centrifuged, and the supernatant was applied to the leaf petiole (Fig. 4B). Very small amounts of EIX were found in the leaf, as shown by the small amounts found in parts of the leaf distant from the petiole. Mixing 1251I-labeled EIX with the antibodies together with 3 ,g of unlabeled EIX left some free EIX, which was translocated in the leaf (Fig. 4C). In a separate experiment, antibodies applied to the petiole of detached leaves were found to be translocated in the leaf, confined to the leaf xylem (Fig. 5). When antibodies were applied to leaf petioles before application of EIX, they also inhibited EIX movement (Fig. 4D), suggesting that EIX moves through the xylem of the vascular tissue. In another experiment, antibodies were used to ascertain whether EIX was active in the leaf mesophyll. Antibodies against the native form of EIX were injected into the mesoRADOGRAM.

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Figure 3. Extraction of 1251-labeled EIX from leaves. Thirty nanograms (2.4 x 106 cpm) of 1251-labeled EIX were applied to a detached Xanthi leaf. Leaf proteins were extracted from the mesophyll and the major veins 1 h after application of EIX. Unlabeled EIX (5 ng/ ML) was added to the extracts, boiled for 1 min, and loaded (10 AL out of 500 ML) on an SDS-PAGE gel, and the proteins were electroblotted for western analysis. One membrane was immunoblotted using antibodies raised against an SDS-denatured form of EIX (StD, western blot); the other was placed on a film and exposed for 24 h (A-C, radiogram). St, Fifty nanograms of unlabeled EIX; A, mesophyll extract + unlabeled EIX; B, vein extract + unlabeled EIX; C, 1251 -labeled EIX + unlabeled EIX; D, untreated leaf.

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Figure 4. Blockage of translocation of EIX in detached Xanthi leaves by anti-EIX antibodies. Treatments were applied to the petiole of detached leaves, and radiograms were obtained after a 3-d exposure. A, Control '251-labeled EIX; B, 1251-labeled EIX incubated with 5 AL (30 Ag protein) of antibodies raised against the native form of EIX before application to the leaf; C, same as B except 3 ,ug of unlabeled EIX were added to the incubation tube; D, 5 ,L of antibodies were applied to the petiole and absorbed before application of EIX. The amount of 1251-labeled EIX was 5 ng (4 x 105 cpm) in all treatments.

phyll intercellular spaces, and EIX was injected into the same area. Preapplication of the antibodies completely inhibited the appearance of EIX-induced necroses (Fig. 6), indicating that active EIX was necessary for induction of symptoms.

Effect of EIX Concentration on the Movement In early experiments, we observed that a small amount of EIX moved very fast through the plant, whereas another component moved much slower (Fig. 2A). To study further EIX movement, 125I-labeled EIX was mixed with increasing amounts of unlabeled EIX and applied to leaf petioles. Addition of 0.5 ,g of EIX had no effect on the movement of 1251-labeled EIX, but 5 ,g EIX increased the amount of 1251_ labeled EIX that moved to the upper part of the leaf within the first 10 min after application (Fig. 7). After 1 h, there was no difference in the pattern of EIX accumulation among 0, 0.5, and 5 ,g EIX. These data may indicate that some of the protein is interacting with the internal surface of the xylem

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Figure 5. Localization of antibodies in the xylem of the leaf petiole. Five microliters of antibodies raised in rabbits against the native form of EIX (8) were applied to detached leaves. After 10 min, cross-sections were made 5 cm along the midrib and tissue printed onto nitrocellulose membranes. EIX-antibodies were detected with goat anti-rabbit immunoglobulin C alkaline phosphatase conjugate.


2063

and being irreversibly bound or its movement being retarded by interacting with the xylem. Thus, at high protein concentrations, EIX moved at a greater rate, as seen after 10 mn, but, after 1 h, when the slower moving fraction reached the upper parts, the labeling density was the same in all treatments. Movement of EIX in Different Tobacco Tissues

Figure 6. Inhibition of EIX-induced necrosis by anti-EIX antibodies. A water solution of EIX (7.5 ng/ttL) was injected into the mesophyll intercellular spaces alone or after the injection of antibodies (7.5 jig/jiL). Control leaves were injected with water or with antibody solution.

10 mins

60

Incubation of leaves in the presence of ethylene greatly enhances EIX-induced responses (2). In addition, the tobacco cv Hicks was found to be insensitive to EIX (B.A. Bailey, unpublished data). The movement of EIX in ethylene-treated and nontreated leaves as well as in leaves of cv Hicks were compared to ascertain whether the differences in the responses resulted from differences in the translocation of EIX. As shown in Figure 8, no obvious differences were found after 15 min in the translocation of EIX in fresh Xanthi leaves versus that in ethylene-treated or Hicks leaves. Also, there were few or no differences after shorter (5 min) or longer (2 h) translocation periods (not shown). Thus, the differences in sensitivity between Hicks and Xanthi and between ethylene-pretreated and nonethylene-pretreated plants are not caused by lack of or slower translocation of EIX. The movement of EIX in whole tobacco plants was studied with 125I-labeled EIX. As previously observed by tissue-printing techniques (4), '251-labeled EIX was translocated through the plant to leaves both above (+) and below (-) the point of application (Fig. 9). Some leaves accumulated high levels of EIX (e.g. +2 and +3, Fig. 9; +5 and -5, Table I), whereas others showed only weak or no signal after exposure of the film for 16 h. A longer exposure of the same plant (not shown) as well as counting of the radioactivity in another plant with a gamma counter (Table I) indicated that some EIX reached almost all of the leaves, but no EIX could be

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AMOUNT OF UNLABELLED EIX ([ig)

Figure 7. Effect of EIX concentration on EIX translocation in detached Xanthi leaves. Five microliters of a water solution of 1251labeled EIX (5 ng, 4 x 105 cpm) were mixed with water (5 AL) containing 0, 0.5, or 5 gg of unlabeled EIX. The solutions were applied to the petiole of detached leaves as described above. The leaves were frozen after 10 and 60 min and placed on a film for a 3-d exposure.

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Fresh \andih

Figure 8. Comparison between translocation of EIX in ethylenetreated and nontreated leaves and Xanthi and Hicks cultivars. Detached leaves were incubated for 14 h under an atmosphere of 120 AL/L of ethylene or no ethylene. 1251-Labeled EIX was applied to leaf petioles as described above. The leaves were frozen and placed on a film for a 4-d exposure. The radiograms show the distribution of EIX in the leaves 15 min after treatment.

Figure 9. Translocation of EIX in a whole plant. Plants were used after nine leaves had developed. The middle leaf was cut, and 100 AL of water solution containing 30 ng of '251-labeled EIX (2.4 x 106 cpm) and 10 Mig of unlabeled EIX were applied to the plant through the cut petiole. After 4 h, the leaves were cut, and plant parts were placed on a film and exposed for 16 h. Positive and negative numbers represent leaf position above and below, respectively, the petiole of EIX application. A represents the autoradiograph of the plant tissue (B) that was treated with 1251-labeled EIX.

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SHARON ET AL.

2064

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detected in the roots. EIX reached the root neck (Fig. 9, arrow) but did not enter the root system, as indicated by exposure of a film for 10 d (not shown) and by gamma counting (Table I).

DISCUSSION The data presented show that EIX is translocated to the leaf mesophyll via the xylem vascular system. The translocation of EIX through leaf tissue indicates that only a small amount of EIX interacts with the internal surfaces of the vascular tissue, whereas the rest of the protein moves as fast as the water transpiration flow and ends at the leaf mesophyll. Cryptogein, a proteinaceous elicitor isolated from Phytophthora cultures, is translocated through the vascular tissues when applied to the plant through cut roots (19). EIX was also found to be translocated to the leaves when applied through cut roots of tobacco seedlings (data not shown). Both EIX and cryptogein are relatively low mol wt polypeptides with high isoelectric points (9.4). The high isoelectric point may prevent adherence of the polypeptide to the xylem cells, enabling the movement of large proportions of the EIX after it penetrates the xylem vascular system. Thus, even a small amount of EIX that enters the vascular system of the roots or the stem is capable of being translocated to distant leaves. After EIX gets into the mesophyll, it elicits various plant responses. Small amounts of EIX are sufficient to elicit specific plant defense responses, such as the accumulation of pathogenesis-related proteins and phytoalexins and ethylene biosynthesis, but higher amounts cause necrosis in sensitive tobacco cultivars (12, 16). Migration of proteinaceous elicitors through the plant may be a mechanism of induction of systemic resistance, especially by soil-borne organisms that colonize the roots and penetrate the xylem. The fast movement of the protein assures that the signal will reach distant parts of the plant and trigger defense responses before the arrival of the pathogen. It can be expected that more proteinaceous elicitors will be isolated from soil-borne pathogens. This mode of signal transduction does

not exclude other mechanisms of induced systemic resistance and does not contradict the concept of secondary messenger compounds (10, 17, 18). The concept of secondary signals may well be relevant, e.g. in leaf pathogens for which the signal must travel via routes other than the xylem. The rapid up and down movement of EIX from the cut petiole (Fig. 9) indicates that there is a water deficit in other parts of the plant in relation to the cut petiole. Evans blue, a vital stain, follows a pattern of movement in the xylem similar to EIX when applied to a cut petiole (4), indicating that xylem transport is sufficient to explain the downward movement in

Table I. Distribution of '251-Labeled EIX in a Whole Plant after Application through a Petiole Midway Up the Stem of a Xanthi Tobacco Plant Negative (-) and positive (+) numbers represent leaves below and above the petiole of application, respectively. Percentage of cpm Part of the Plant Counts Recovered

cpm

Leaf -8 Leaf -7 Leaf -6 Leaf -5 Leaf -4 Leaf -3 Leaf -2 Leaf -1 Leaf +1 Leaf +2 Leaf +3 Leaf +4 Leaf +5 Leaf +6 Leaf +7 Tip of the plant Below leaf -8 Roots

56,000 19,500 16,500 540,000 700 304,000 103,000 8,500 100 138,000 238,000 50 434,000 5,600 4,400 28,500 27,000 0

2.91 1.01 0.86 28.07 0.04 15.00 5.35 0.44 0.10 7.17 12.37 0.10 22.56 0.29 0.23 1.48 1.40 0.00


the plant following petiole application. As shown by the radiograms of detached leaves, after EIX is translocated to the mesophyll, it reaches a steady-state level, and its distribution does not change significantly over long periods. Most likely it is either bound or free in the mesophyll, but penetration into the mesophyll cells cannot be excluded. There was no significant difference in the translocation of EIX in Xanthi and in Hicks, suggesting that the difference in the sensitivity to EIX is related to the interaction between the protein and the plant cells. A recent genetic study in this laboratory indicated that the sensitivity of tobacco plants to EIX is controlled by a single dominant gene (B.A. Bailey, unpublished data). The possibility of a specific binding site for EIX associated with the mesophyll cells is intriguing, and efforts are underway to ascertain whether or not such a site exists.

9.

10.

11. 12.

13.

ACKNOWLEDGMENT

The authors are thankful to Mr. David H. Clark for his excellent help in preparing the photographs.

14. 15.

LITERATURE CITED

1. Anderson JD, Bailey BA, Dean JFD, Taylor R (1990) A fungal endoxylanase elicits ethylene biosynthesis in tobacco (Nicotiana tobacum L. cv Xanthi) leaves. In HE Flores, RN Arteca, JC Shannon, eds, Polyamines and Ethylene: Biochemistry, Physiology and Interactions. American Society of Plant Physiologists, Rockville, MD, pp 146-156 2. Bailey BA, Dean JFD, Anderson JD (1990) An ethylene biosynthesis-inducing endoxylanase elicits electrolyte leakage and necrosis in Nicotiana tabacum cv Xanthi leaves. Plant Physiol 94: 1849-1854 3. Bailey BA, Korcak RF, Anderson JD (1992) Alterations in Nicotiana tobacum cv Xanthi cell membrane integrity following treatment with an ethylene biosynthesis-inducing endoxylanase. Plant Physiol 100: 749-755 4. Bailey BA, Taylor R, Dean JFD, Anderson JD (1991) Ethylene biosynthesis-inducing endoxylanase is translocated through the xylem of Nicotiana tabacum cv Xanthi plants. Plant Physiol 97: 1181-1186 5. Blein J-P, Milat ML, and Ricci P (1991) Responses of cultured tobacco cells to cryptogein, a proteinaceous elicitor from Phytophthora cryptogea. Plant Physiol 95: 486-491 6. Cassab GI, Varner JE (1989) Immunocytolocalization of extensin in developing soybean seed coats by immunogold-silver staining by tissue printing on nitrocellulose paper. J Cell Biol 105: 2581-2588 7. Dean JFD, Anderson JD (1991) Ethylene biosynthesis-inducing xylanase. II. Purification and physical characterization of the enzyme produced by Trichoderma viride. Plant Physiol 95: 316-323 8. Dean JFD, Gamble HR, Anderson JD (1989) The ethylene

16. 17. 18.

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