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Increased ability of transgenic plants expressing the bacterial


Journal of Biotechnology 81 (2000) 45 – 53 www.elsevier.com/locate/jbiotec

Increased ability of transgenic plants expressing the bacterial enzyme ACC deaminase to accumulate Cd, Co

, Cu, Ni, Pb, and Zn
Varvara P. Grichko, Brendan Filby, Bernard R. Glick *
Department of Biology, Uni6ersity of Waterloo, Waterloo, Ontario, Canada N2L 3G1 Received 28 June 1999; received in revised form 7 April 2000; accepted 12 April 2000

Abstract Transgenic tomato plants Lycopersicon esculentum (Solanaceae) cv. Heinz 902 expressing the bacterial gene 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, under the transcriptional control of either two tandem 35S cauliower mosaic virus promoters (constitutive expression), the rolD promoter from Agrobacterium rhizogenes (root specic expression) or the pathogenesis related PRB-1b promoter from tobacco, were compared to non-transgenic tomato plants in their ability to grow in the presence of Cd, Co, Cu, Mg, Ni, Pb, or Zn and to accumulate these metals. Parameters that were examined include metal concentration and ACC deaminase activity in both plant shoots and roots; root and shoot development; and leaf chlorophyll content. In general, transgenic tomato plants expressing ACC deaminase, especially those controlled by the PRB-1b promoter, acquired a greater amount of metal within the plant tissues, and were less subject to the deleterious effects of the metals on plant growth than were non-transgenic plants. 2000 Elsevier Science B.V. All rights reserved.
Keywords: 1-Aminocyclopropane-1-carboxylic acid; ACC deaminase; Transgenic tomato; Heavy metals; Stress; Phytoremediation

1. Introduction ACC is the immediate precursor of ethylene in plants (Abeles et al., 1992). The bacterial enzyme ACC deaminase is the only non-plant enzyme that metabolizes ACC; the enzyme converts ACC to a-ketobutyrate and ammonia (Honma and Shimomura, 1978). Transgenic tomato plants that
* Corresponding author. Tel.: + 1-519-8884567, ext. 2058; fax: + 1-519-7460614. E-mail address: glick@sciborg.uwaterloo.ca (B.R. Glick).

express ACC deaminase under the control of the 35S promoter from cauliower mosaic virus produce less ethylene and as a consequence tomato fruit ripening is delayed (Klee et al., 1991; Reed et al., 1995) and plants are less susceptible to damage from several different phytopathogens (Lunde et al., 1998). Consistent with the suggestion that transgenic tomato plants with decreased ethylene levels should be less susceptible to various types of stress (Klee, 1992), it was previously observed that the growth of canola and tomato seedlings treated with an ACC deaminase-containing plant

0168-1656/00/$ - see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 0 0 ) 0 0 2 7 0 - 4

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growth-promoting bacterium was partially protected from inhibition by nickel (Burd et al., 1998). In this case the bacterial plant growthpromoting effect was attributed to the ability of the bacterium to lower the level of stress ethylene induced by the nickel (Burd et al., 1998). Recently, considerable attention has been directed toward the possibility of using plants to remove heavy metals from the environment (Raskin et al., 1997; Salt et al., 1998). Phytoremediation, which may be dened as the use of plants to remove, destroy or sequester hazardous substances from the environment, is considered to be an attractive, although unproven, alternative to the approaches that are currently in use for dealing with heavy metal contamination. While considerable effort has been directed toward identifying plants that are metal tolerant and accumulate large amounts of metal from the soil and might, therefore, be useful in phytoremediation, an attractive alternative approach might include the genetic engineering of plants in an effort to specically increase their usefulness in phytoremediation. As a rst step in this direction, we have examined the ability of transgenic tomato plants that contain a bacterial ACC deaminase gene placed under the control of three different promoters, i.e. 2× 35S (Christopher et al., 1987), rolD (Elmayan and Tepfer, 1995), and PRB-1b (Eyal et al., 1992), to take up different metals from the environment and have compared the behaviour of these transgenic plants to non-transgenic tomato plants treated the same way.

m 2 s 1. Plants were germinated in 30×60× 10 cm3 boxes and were transferred to 5-inch pots after 17 days of growth. Plants were watered with tap water until the beginning of the experiment. Each of the three transgenic plants contained a single copy per genome of the ACC deaminase gene from Enterobacter cloacae UW4 (Shah et al., 1998) under the control of either a 2× 35S (Christopher et al., 1987), a rolD (Elmayan and Tepfer, 1995), or a PRB-1b (Eyal et al., 1992) promoter. All transgenic plants were homozygous for the ACC deaminase gene. ACC deaminase expression in the transgenic plants was conrmed by enzyme activity assays and Western blots (Robison et al., submitted for publication). As expected, larger quantities of ACC deaminase were produced in leaf and root material from the 35S plants, moderate levels were present in the roots of rolD plants (but not in the leaves), and low levels of ACC deaminase were detectable in PRB-1b plants provided that the plants were rst stressed, e.g. by wounding of the shoot or by disease inoculation.

2.2. Pouch assay
The 125× 157 mm growth pouches (Mega International, Minneapolis, MN, USA) were lled with 10 ml 1 mM 3CdSO48H2O, CoCl2, Cu(NO3)2, NiSO4, or Pb(NO3)2 or 10 ml 10 mM MgSO4 or ZnSO4 and were sterilized by autoclaving. Control pouches were lled with 10 ml of the corresponding acid with the pH and concentration adjusted to that of the metal solution. Seeds were sterilized in 70% ethanol for 1 min and in a 1% sodium hypochlorite solution for 10 min before being rinsed thoroughly with sterilized distilled water. Five seeds were placed aseptically in each growth pouch. Trays containing the upright growth pouches were incubated in a Conviron model CMP 32444 growth chamber (Controlled Environment, Winnipeg, Manitoba, Canada) for either 7 or 9 days with a photo period of 12 h and a photosynthetic photon ux of 12.9 mE m 2 s 1 at the bottom pouch level, at a temperature of 20°C.

2. Materials and methods

2.1. Plant material
Lycopersicon esculentum Mill cv. Heinz 902 (Stokes Seeds, Canada) and ACC deaminase transgenic plants (Robison et al., submitted for publication) were grown in Pro-Mix ‘BX’ general-purpose growth medium (General Horticulture, Red Hill, PA, USA) in a greenhouse at 259 5°C (day) and 2095°C (night) with an average day time light illumination of 250 mmol

V.P. Grichko et al. / Journal of Biotechnology 81 (2000) 45–53

47

2.3. Pot-grown plants
With Co, Cu, Ni, and Pb 34-day-old tomato plants were watered with 100 ml of a 1 mM salt solution for 2 days and were then treated with 50 ml of a 1 mM salt solution for 5 days up to a cumulative dosage of 0.45 mmol per 5-inch pot. At the end of the treatment, the plants were 40 days old and are subsequently referred to as 40day-old plants. With Cd and Zn 42-day-old tomato plants were treated with either (a) 10 ml 10 mM 3CdSO48H2O for 10 consecutive days up to a cumulative dosage of 1 mmol per 5-inch pot or (b) 10 ml 100 mM Zn salt for 10 consecutive days up to a cumulative dosage of 10 mmol per 5-inch pot. At the end of the treatment, the plants were 51 days old and are referred to as 51-day-old

plants. In both cases, controls were done with the acid (either HCl, H2SO4, or HNO3) with an adjusted pH and were carried over in the same way.

2.4. Atomic absorption analysis (AAA)
Samples were digested with HNO3 (Soon, 1998) and the concentration of metals was measured by platform electrothermal AAS. A Varian model Spectra AA-600 atomic-absorption spectrophotometer equipped with a graphite furnace and an automated sampler was used as recommended by the manufacturer.

2.5. Chlorophyll determination
The amount of chlorophyll was determined as described previously (Arnon, 1949; Hiscox and Israelstam, 1978).

2.6. ACC deaminase assay
ACC deaminase activity was determined in total protein extracted from fresh plant tissue (Honma and Shimomura, 1978). Blanks were done with a specic reagent in the absence of the substrate.

3. Results and discussion In this study the effect of several different heavy metals on the growth of non-transgenic (NT) and transgenic tomato plants was examined. The short term effects of the presence of heavy metals was monitored following either 7 or 9 days in sterile growth pouches while the long term effects were measured after growth of plants in soil for either 40 or 51 days. For the most part, the growth of the transgenic plants was inhibited to a lesser extent by the presence of the heavy metals than was the growth of the non-transformed plants.
Fig. 1. Metal accumulation in the roots and shoots of ACC deaminase transgenic tomato plants. (A) Cd uptake by 7-day pouch-grown seedlings and 51-day-old potted tomato plants. (B) Co uptake by 9- (pouch) and 40-day-old (pot) tomato plants. (C) Cu uptake as in (B). Solid bar, roots; gray bar, shoots. NT, non-transformed.

3.1. Cd
Both roots and shoots of 1-week-old plants accumulated Cd (Fig. 1A). Transgenic seeds (35S

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V.P. Grichko et al. / Journal of Biotechnology 81 (2000) 45–53

Table 1 Effect of various on the root length in mm of 7- or 9-day-old non-transgenic and transgenic tomato plantsa Metal (1 mM) Growth in days Non-transgenic (n) ACC deaminase transgenic plants 35S (n) Cd +Cd Zn +Zn Co +Co Cu +Cu Ni +Ni Pb +Pb
a

rolD (n) 45 94 (24) 32 93b (21) 53 94 (24) 45 93 (23) 91 9 6 (21) 8895 (23) 95 95 (23) 90 9 4c (25) 100 9 5c (22) 89 9 3c (19) 94 9 5c (22) 9894c (23)

9 9 9 9 7 7 7 7 7 7 7 7

42 9 3 (24) 279 2b (23) 509 4 (24) 469 4 (20) 779 8 (13) 64 9 6 (15) 83 95 (14) 64 98b (11) 769 8 (15) 629 9 (13) 509 7 (15) 72 9 8 (15)

46 9 4 (24) 37 92b,c (24) 57 94 (20) 50 94 (21) 97 94c (23) 64 97b (21) 94 94 (22) 83 95c (24) 103 94c (21) 97 94c (23) 88 95c (22) 88 95 (19)

The values are means9S.E., PB0.05. Indicates signicantly different from metal control. c Indicates signicantly different from non-transgenic plants
b

and rolD) germinated faster in the presence of cadmium than did non-transgenic seeds (data not shown). Initial root growth was inhibited by cadmium as: NT\ rolD \35S (Table 1); PRB-1b seeds were not included in the short term experiments since they germinate much more slowly than non-transgenic seeds. The 35S plants treated with Cd developed roots that were signicantly longer than roots of non-transgenic plants treated with Cd and at the same time the 35S roots took up more Cd than non-transgenic plants. Roots of 51-day-old plants accumulated Cd as: PRB-1b \ rolD\35S \ NT (Fig. 1A); rolD plants did not accumulate Cd in their shoots, and apparently did not exhibit a decrease in shoot growth (Table 2). The rolD plants also had the highest leaf chlorophyll content after treatment with Cd (Table 3). The 35S and rolD plants took up a similar amount of Cd (approximately 50% more than NT plants) while PRB-1b plants acquired an almost 5-fold higher amount of Cd than did NT plants, mostly in their roots (Fig. 1A).

3.2. Co
Cobalt, which resembles iron in its chemical behavior, can inhibit ethylene synthesis by binding to ACC oxidase which relies on iron as a cofactor. When tomato seeds were germinated in the presence of Co the 35S seeds germinated faster than the non-transgenic seeds while the rolD seeds germinated at approximately the same rate as non-transformed seeds (data not shown). In these plants, the major target organ for cobalt accumulation is the roots for young transgenic plants and the shoots for older transgenic plants (Fig. 1B). The presence of Co results in a decrease in the fresh and dry weights of older plants (Table 2). In this case, the growth of rolD plants is inhibited to a lesser extent than is the growth of the other plants, and in fact, the fresh and dry weights of the rolD plants in the presence of Co is similar to the fresh and dry weights of non-transformed tomatoes in the absence of added metal. Cobalt accumulated in the roots and shoots of

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35S plants to a much greater extent than in the roots and shoots of the other plants; interestingly, ACC deaminase activity was also elevated considerably in the shoots, and to a lesser extent in the roots, of 35S plants treated with Co compared with control plants (data not shown).

3.3. Cu
The germination of tomato seeds in the presence of Cu was inhibited in both 35S and rolD transgenic seeds relative to non-transgenic seeds (data not shown). Young plants (especially non-

Table 2 Effect of metals on tomato plant fresh and dry shoot weights, in gramsa Treatment Plant age (days) Weight Non-transformed plants ACC deaminase transgenic plants 35S Control Control Cd Co Cu 40 51 51 40 40 Fresh Dry Fresh Dry Fresh Dry Fresh Dry Fresh Dry 2.899 0.32 0.1389 0.018 4.719 0.19 0.2179 0.006 5.229 0.91 0.16290.035 1.98 90.03b 0.09990.002 2.539 0.10 0.1239 0.005 3.36 9 0.18 0.163 90.014 10.56 9 1.34c 0.578 90.098c 6.43 9 1.20 0.426 90.072b 2.21 9 0.12b 0.117 9 0.012 2.37 9 0.21 0.112 9 0.013 2.15 9 0.19 0.108 90.013b
b

rolD 4.31 9 0.18c 0.220 90.008c 9.71 91.04c 0.598 90.047c 12.09 92.04c 0.631 9 0.094c

PRB-1b 3.24 9 0.21 0.168 90.006 7.01 9 0.52 0.435 90.121 4.35 91.96 0.279 90.062

2.75 9 0.25b,c 2.31 9 0.44 0.148 90.016b,c 0.125 90.026c 3.06 9 0.05b 3.34 9 0.12c b,c 0.152 90.004 0.161 90.004b,c 3.04 90.02 2.81 9 0.40 0.151 90.002b,c 0.141 90.022c
b,c

Ni Pb Zn

40 40 51

Fresh Dry Fresh Dry Fresh Dry

1.73 9 0.02 0.0899 0.010
b

2.03 9 0.22 0.0989 0.007 5.29 9 1.84 0.32990.051b

2.35 9 0.31b 0.120 90.015 10.289 1.43 0.487 90.073

2.85 9 0.33b 2.629 0.45 0.159 90.019b,c 0.134 90.024 13.08 91.30 0.603 90.048c 9.79 90.05b 0.492 9 0.014b

a

The values are means9S.E., n= 3, PB0.05. Indicates signicantly different from control. c Indicates signicantly different from non-transformed plants.
b

Table 3 The effect of Cd and Zn on tomato plant chlorphyll content, in mg g1 of fresh weight of leaf tissuea Metal Non-transformed ACC deaminase transgenic plants 35S None Cd Zn
a b

rolD 1.94 9 0.07 1.37 9 0.14 1.79 9 0.03

PRB-1b 1.71 9 0.08 0.79 90.07 1.67 90.11

1.559 0.07b 0.8490.21 1.3390.59

1.65 90.10b 0.81 90.07 1.00 9 0.03

The values are means9S.E., n= 2, PB0.05. Indicates signicantly different from rolD plants for PB0.05.

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V.P. Grichko et al. / Journal of Biotechnology 81 (2000) 45–53

3.4. Ni
In the presence of Ni, the germination of 35S and rolD transgenic seeds occurred at a rate that was similar to the non-transgenic seeds (data not shown). With 9-day-old plants, Ni was accumulated to the greatest extent in rolD plants, largely in the roots (Fig. 3A). In 40-day-old plants, Ni accumulated mostly in shoots as: PRB-1b \ 35S \ NT\ rolD (Fig. 3A). Root length was greater in 9-day-old 35S and rolD plants compared to non-transgenic plants both in the presence and absence of nickel (Table 1). Ni was more inhibitory to non-transgenic than to transgenic tomato plants as measured by its effect on both fresh and dry shoot weight in 40-day-old tomato plants (Table 2). Nickel substantially increased ACC deaminase activity in the leaves of 35S plants, and to a lesser extent in the roots of 35S plants (Fig. 2). This is similar to what was observed with Cu and may reect ACC oxidase inhibition by Ni.

Fig. 2. ACC deaminase activity in 40-day-old tomato plants treated with Cu, Ni or Pb. NT, non-transformed.

3.5. Pb
transgenic) accumulated Cu in their roots (Fig. 1C) concomitant with a decrease in root length (Table 1). On the other hand, 40-day-old tomato plants were able both to transport copper to the shoots (Fig. 1C) and develop normally (Table 2). PRB-1b plants accumulated 53 mg g 1 FW of Cu without a noticeable change in shoot weight (Table 2). The highest level of ACC deaminase activity in this study was observed in leaves of 35S plants treated with Cu (Fig. 2) although how the presence of this (or any other) heavy metal affects ACC deaminase activity is unclear. Perhaps surprisingly, the roots of 35S plants did not show any increase in enzyme activity despite the similar content of acquired copper (Fig. 1C). One possible explanation for the increase in ACC deaminase activity in the leaves of 35S plants treated with Cu is that Cu may prevent ACC oxidation by inhibiting the enzyme ACC oxidase which uses a radical-based mechanism (Pirrung et al., 1998) and thereby increase the amount of substrate (i.e. ACC) available to ACC deaminase. Of all of the metals tested lead had the most inhibitory effect on transgenic seed germination compared to non-transgenic seeds (data not shown). While 40-day-old PRB-1b plants accumulated Pb in both the roots and shoots, young plants did not transport lead from the roots to the shoots (Fig. 3B). In general, transgenic plants grew better than non-transgenic in the presence of lead (Tables 1 and 3), with rolD plants being the least affected by the presence of Pb. The leaves in PRB-1b plants and the roots in 35S plants responded to lead treatment by signicantly increasing ACC deaminase activity (data not shown).

3.6. Zn
While Zn inhibited the germination of 35S and rolD seeds relative to the non-transgenic (data not shown), Zn had little effect on the growth of seven- and 51-day-old plants (Tables 1 and 2). However, both 51-day-old transgenic plants and 7-day-old non-transgenic plants hyperaccumulated Zn (Fig. 3C). Importantly, all three types of

V.P. Grichko et al. / Journal of Biotechnology 81 (2000) 45–53

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transgenic plants accumulated considerably more zinc than did non-transformed plants. The presence of Zn was only slightly inhibitory to leaf chlorophyll levels especially for rolD and PRB-1b plants (Table 3). This data is entirely consistent with what was observed for the interaction of Zn with non-transformed canola plants in the presence and absence of plant growth promoting bacteria (Burd et al., 2000).

4. Conclusion The results of these studies are complex and not always easy to interpret. This probably is a reection of the fact that different heavy metals can affect tomato plants physiologically in different ways, in addition to stressing the plant and caus-

ing it to produce ethylene, and the sensitivity of tomato plants to a particular metal is likely to vary with the stage of development of the plant. Moreover, since the PRB-1b promoter requires ethylene in order to be induced, an ACC deaminase gene under the transcriptional control of the PRB-1b promoter will not be expressed unless the ethylene concentration becomes elevated. While the ACC deaminase enzyme that is subsequently expressed should ultimately lower the amount of ethylene that can be produced, in some instances the ethylene concentration that is required to turn on the PRB-1b promoter may also damage the plant. Thus, although PRB-1b plants may appear to be superior in the presence of some metals, the behaviour of these plants in different circumstances is difcult to predict. On the other hand, a study of the physiological responses of the three

Fig. 3. Metal accumulation in roots and shoots of ACC deaminase transgenic tomato plants. (A) Ni uptake by 9-day pouch-grown seedlings and 40-day-old potted tomato plants. (B) Pb uptake as in A. (C) Zn uptake by 7-day pouch-grown seedlings and 51-day-old potted tomato plants. Solid bar, roots; gray bar, shoots. NT, non-transformed.

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V.P. Grichko et al. / Journal of Biotechnology 81 (2000) 45–53

transgenic tomato plants used in this work indicated that in the absence of any physiological stress, PRB-1b plants are more similar to nontransformed plants than they are to 35S or rolD plants (Grichko and Glick, submitted). Ethylene plays a number of important roles in plant growth and development in addition to the apparently inhibitory effects of stress ethylene on plant proliferation (Abeles et al., 1992). In addition, stress ethylene is often reported to be synthesized in two peaks, a smaller one that occurs within several hours following the imposed physiological stress and a much larger peak that generally is observed around 3 days after the stress. It is thought that the rst stress ethylene peak acts as a trigger which initiates the synthesis of a number of plant defense mechanisms while the second peak may be deleterious for the plant. It would be nave to as¨ sume that tomato plants respond to all stresses, or even to all metals, in the same way with regard to the timing or amount of stress ethylene synthesis. It is, therefore, not surprising that the responses of the non-transformed and transgenic tomato plants used in this study responded differently to the presence of various heavy metals. These considerations notwithstanding, the expression of ACC deaminase resulted in: (i) constant stimulation of plant growth leading to a higher total amount of metal accumulated, (ii) in many cases an increase in metal uptake, and (iii) in some instances an increase in the shoot/ root metal ratio. Based on the results of the study reported here with transgenic tomato plants, genetically engineered plants that have lowered levels of stressed ethylene may be useful components of phytoremediation strategies to remove heavy metals from the environment. Tomatoes are neither hyperaccumulators of heavy metals nor is it likely that tomatoes will be used directly in the phytoremediation of metal contaminated soil. Nevertheless, the response of transgenic tomatoes to the presence of various heavy metals suggests approaches that could be useful in engineering other plants to be more effective in the phytoremediation of heavy metals in the environment. In this regard, the

transgenic tomato plants that were used in this study, originally constructed as part of an effort to develop phytopathogen tolerant cultivars, may be viewed as a model system and the approaches that were successful here may be successful with other ethylene sensitive plants that have been used in phytoremediation studies such as Brassica juncea (Indian mustard). In other experiments with the three transgenic tomato plants that were used in this study, it was found that these plants are also protected to some extent against the inhibitory effects of fungal pathogens (Robison et al., submitted for publication) and ooding stress (Grichko and Glick, submitted for publication), and the most effective transgenic plant in all of these studies included the ACC deaminase gene under the transcriptional control of the rolD promoter.

References
Abeles, F.B., Morgan, P.W., Salveit, M.E. Jr, 1992. Ethylene in Plant Biology, 2nd edn. Academic Press, San Diego, CA. Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphenooloxidase in Beta 6ulgaris. Plant Physiol. 24, 1 – 15. Burd, G.I., Dixon, D.G., Glick, B.R., 1998. A plant growthpromoting bacterium that decreases nickel toxicity in seedlings. Appl. Environ. Microbiol. 64, 3663 – 3668. Burd, G.I., Dixon, D.G., Glick, B.R., 2000. Plant growthpromoting bacteria that decrease heavy metal toxicity in plants. Can. J. Microbiol. 46, 237 – 245. Christopher, L.S., Byrd, A.D., Benzion, G., Altschuler, M.A., Hildebrand, D., Hunt, A.G., 1987. Design and construction of a versatile system for the expression of foreign genes in plants. Gene 61, 1 – 11. Elmayan, T., Tepfer, M., 1995. Evaluation in tobacco of the organ specicity and strength of the rolD promoter, domain A of the 35S promoter and the 35S2 promoter. Transgenic Res. 4, 388 – 396. Eyal, Y., Sagee, O., Fluhr, R., 1992. Dark induced accumulation of a basic pathogenesis-related (PR-1) transcript and a light requirement for its induction by ethylene. Plant Mol. Biol. 19, 589 – 599. Hiscox, J.D., Israelstam, G.F., 1978. A method for the extraction of chlorophyll from leaf tissue without maceration. Can. J. Bot. 57, 1332 – 1334. Honma, M., Shimomura, T., 1978. Metabolism of 1aminocyclopropane-1-carboxylic acid. Agric. Biol. Chem. 42, 1825 – 1831.

V.P. Grichko et al. / Journal of Biotechnology 81 (2000) 45–53 Klee, H.J., 1992. Control of fruit ripening and senescence in plants. International Patent to Monsanto, IPN WO 92/ 12249. Klee, H.J., Hayford, M.B., Kretzmer, K.A., Barry, J.F., Kishore, G.M., 1991. Control of ethylene synthesis by expression of a bacterial enzyme in transgenic tomato plants. Plant Cell 3, 1187 – 1193. Lunde, S.T., Stall, R.E., Klee, H.J., 1998. Ethylene regulates the susceptible response to pathogen infection in tomato. Plant Cell 10, 371 – 382. Pirrung, M.C., Cao, J., Chen, J., 1998. Ethylene biosynthesis: processing of a substrate analog supports a radical mechanism for the ethylene-forming enzyme. Chem. Biol. 5, 49–57. Raskin, I., Smith, R.D., Salt, D.E., 1997. Phytoremediation of metals: using plants to remove pollutants from the environment. Curr. Opin. Biotechnol. 8, 221–226.

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Reed, A.J., Magin, K.M., Anderson, J.S., et al., 1995. Delayed ripening tomato plants expressing the enzyme 1-aminocyclopropane-1-carboxylic acid deaminase. 1. Molecular characterization, enzyme expression, and fruit ripening traits. J. Agric. Food Chem. 43, 1954 – 1962. Salt, D.E., Smith, R.D., Raskin, I., 1998. Phytoremediation. Ann. Rev. Plant Physiol. Plant Mol. Biol. 49, 643 – 668. Shah, S., Li, J., Moffat, B.A., Glick, B.R., 1998. Isolation and characterization of ACC deaminase genes from two different plant growth promoting rhizobacteria. Can. J. Microbiol. 44, 833 – 843. Soon, Y.K., 1998. Determination of cadmium, chromium, cobalt, lead and nickel in plant tissue. In: Kalra, Y.P. (Ed.), Handbook of Reference Methods for Plant Analysis. CRC Press, New York, pp. 193 – 198.

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