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Journal of Biotechnology 81 (2000) 4553

Increased ability of transgenic plants expressing the bacteriaenzyme 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 ge

1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, under the transcriptional control of either two tandem 35

cauliflower mosaic virus promoters (constitutive expression), the rolD promoter from Agrobacterium rhizogenes (ro

specific expression) or the pathogenesis related PRB-1b promoter from tobacco, were compared to non-transgen

tomato plants in their ability to grow in the presence of Cd, Co, Cu, Mg, Ni, Pb, or Zn and to accumulate thes

metals. Parameters that were examined include metal concentration and ACC deaminase activity in both plant shoo

and roots; root and shoot development; and leaf chlorophyll content. In general, transgenic tomato plants expressin

ACC deaminase, especially those controlled by the PRB-1b promoter, acquired a greater amount of metal within thplant tissues, and were less subject to the deleterious effects of the metals on plant growth than were non-transgen

plants. 2000 Elsevier Science B.V. All rights reserved.

Keywords: 1-Aminocyclopropane-1-carboxylic acid; ACC deaminase; Transgenic tomato; Heavy metals; Stress; Phytoremediati

www.elsevier.com/locate/jbiot

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 thatmetabolizes ACC; the enzyme converts ACC to

a-ketobutyrate and ammonia (Honma and Shi-

momura, 1978). Transgenic tomato plants that

express ACC deaminase under the control of th

35S promoter from cauliflower mosaic virus pro

duce less ethylene and as a consequence tomat

fruit ripening is delayed (Klee et al., 1991; Reed

al., 1995) and plants are less susceptible to dam

age from several different phytopathogens (Lund

et al., 1998). Consistent with the suggestion th

transgenic tomato plants with decreased ethylen

levels should be less susceptible to various typ

of stress (Klee, 1992), it was previously observe

that the growth of canola and tomato seedlin

treated with an ACC deaminase-containing plan

* Corresponding author. Tel.: +1-519-8884567, ext. 2058;

fax: +1-519-7460614.

E-mail address: [email protected] (B.R. Glick).

0168-1656/00/$ - see front matter 2000 Elsevier Science B.V. All rights reserved.

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V.P. Grichko et al. /Journal of Biotechnology 81 (2000) 455346

growth-promoting bacterium was partially pro-

tected from inhibition by nickel (Burd et al.,

1998). In this case the bacterial plant growth-

promoting effect was attributed to the ability of

the bacterium to lower the level of stress ethyl-

ene induced by the nickel (Burd et al., 1998).

Recently, considerable attention has been di-

rected toward the possibility of using plants toremove heavy metals from the environment

(Raskin et al., 1997; Salt et al., 1998). Phytore-

mediation, which may be defined as the use of

plants to remove, destroy or sequester haz-

ardous substances from the environment, is con-

sidered to be an attractive, although unproven,

alternative to the approaches that are currently

in use for dealing with heavy metal contamina-

tion. While considerable effort has been directed

toward identifying plants that are metal tolerant

and accumulate large amounts of metal fromthe soil and might, therefore, be useful in phy-

toremediation, an attractive alternative approach

might include the genetic engineering of plants

in an effort to specifically increase their useful-

ness in phytoremediation. As a first step in this

direction, we have examined the ability of trans-

genic tomato plants that contain a bacterial

ACC deaminase gene placed under the control

of three different promoters, i.e. 235S

(Christopher et al., 1987), rolD (Elmayan and

Tepfer, 1995), and PRB-1b (Eyal et al., 1992),to take up different metals from the environ-

ment and have compared the behaviour of these

transgenic plants to non-transgenic tomato

plants treated the same way.

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 gen-

eral-purpose growth medium (General Horticul-

ture, Red Hill, PA, USA) in a greenhouse at

2595C (day) and 2095C (night) with an av-

erage day time light illumination of 250 mmol

m2 s1. Plants were germinated in 3060

10 cm3 boxes and were transferred to 5-inc

pots after 17 days of growth. Plants were w

tered with tap water until the beginning of th

experiment.

Each of the three transgenic plants containe

a single copy per genome of the ACC deam

nase gene from Enterobacter cloacae UW4 (Shaet al., 1998) under the control of either a 2

35S (Christopher et al., 1987), a rolD (Elmaya

and Tepfer, 1995), or a PRB-1b (Eyal et a

1992) promoter. All transgenic plants were h

mozygous for the ACC deaminase gene.

ACC deaminase expression in the transgen

plants was confirmed by enzyme activity assay

and Western blots (Robison et al., submitted fo

publication). As expected, larger quantities

ACC deaminase were produced in leaf and roo

material from the 35S plants, moderate levewere present in the roots of rolD plants (but n

in the leaves), and low levels of ACC deamina

were detectable in PRB-1b plants provided th

the plants were first stressed, e.g. by woundin

of the shoot or by disease inoculation.

2.2. Pouch assay

The 125157 mm growth pouches (Mega In

ternational, Minneapolis, MN, USA) were fille

with 10 ml 1 mM 3CdSO48H2O, CoClCu(NO3)2, NiSO4, or Pb(NO3)2 or 10 ml 10 mM

MgSO4 or ZnSO4 and were sterilized by aut

claving. Control pouches were filled with 10 m

of the corresponding acid with the pH and con

centration adjusted to that of the metal solutio

Seeds were sterilized in 70% ethanol for 1 m

and in a 1% sodium hypochlorite solution f

10 min before being rinsed thoroughly with ste

ilized distilled water. Five seeds were place

aseptically in each growth pouch. Trays contain

ing the upright growth pouches were incubatein a Conviron model CMP 32444 growth cham

ber (Controlled Environment, Winnipeg, Man

toba, Canada) for either 7 or 9 days with

photo period of 12 h and a photosynthetic pho

ton flux of 12.9 mE m2 s1 at the botto

pouch level, at a temperature of 20C.


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Table 1

Effect of various on the root length in mm of 7- or 9-day-old non-transgenic and transgenic tomato plants a

Metal (1 mM) Non-transgenic (n)Growth in days ACC deaminase transgenic plants

35S (n) rolD (n)

4293 (24)Cd 4694 (24)9 4594 (24)

2792b

(23) 3792b,c

(24)9 3293b

(21)+Cd

5094 (24) 5794 (20)9 5394 (24)Zn

4694 (20) 5094 (21)+Zn 4593 (23)9

7798 (13)Co 9794c (23)7 9196 (21)

7+Co 6496 (15) 6497b (21) 8895 (23)

8395 (14)Cu 9494 (22)7 9595 (23)

+Cu 7 6498b (11) 8395c (24) 9094c (25)

7698 (15) 10394c (21)7 10095c (22)Ni

6299 (13) 9794c (23)+Ni 8993c (19)7

5097 (15)Pb 8895c (22)7 9495c (22)

7+Pb 7298 (15) 8895 (19) 9894c (23)

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

and rolD) germinated faster in the presence of

cadmium than did non-transgenic seeds (data not

shown). Initial root growth was inhibited by cad-

mium as: NT\rolD\35S (Table 1); PRB-1bseeds were not included in the short term experi-

ments since they germinate much more slowly

than non-transgenic seeds. The 35S plants treated

with Cd developed roots that were significantly

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 chloro-

phyll 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 chemic

behavior, can inhibit ethylene synthesis by binding to ACC oxidase which relies on iron as

cofactor. When tomato seeds were germinated i

the presence of Co the 35S seeds germinate

faster than the non-transgenic seeds while th

rolD seeds germinated at approximately the sam

rate as non-transformed seeds (data not shown

In these plants, the major target organ for coba

accumulation is the roots for young transgen

plants and the shoots for older transgenic plan

(Fig. 1B). The presence of Co results in a decrea

in the fresh and dry weights of older plants (Tab2). In this case, the growth of rolD plants

inhibited to a lesser extent than is the growth o

the other plants, and in fact, the fresh and dr

weights of the rolD plants in the presence of Co

similar to the fresh and dry weights of non-tran

formed tomatoes in the absence of added meta

Cobalt accumulated in the roots and shoots


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Fig. 2. ACC deaminase activity in 40-day-old tomato plants

treated with Cu, Ni or Pb. NT, non-transformed.

3.4. Ni

In the presence of Ni, the germination of 35

and rolD transgenic seeds occurred at a rate th

was similar to the non-transgenic seeds (data no

shown). With 9-day-old plants, Ni was accumu

lated to the greatest extent in rolD plants, large

in the roots (Fig. 3A). In 40-day-old plants, Naccumulated mostly in shoots as: PRB-1b\

35S\NT\rolD (Fig. 3A). Root length w

greater in 9-day-old 35S and rolD plants com

pared to non-transgenic plants both in the pre

ence and absence of nickel (Table 1). Ni was mo

inhibitory to non-transgenic than to transgen

tomato plants as measured by its effect on bot

fresh and dry shoot weight in 40-day-old tomat

plants (Table 2). Nickel substantially increase

ACC deaminase activity in the leaves of 35

plants, and to a lesser extent in the roots of 35plants (Fig. 2). This is similar to what was o

served with Cu and may reflect ACC oxida

inhibition by Ni.

3.5. Pb

Of all of the metals tested lead had the mo

inhibitory effect on transgenic seed germinatio

compared to non-transgenic seeds (data n

shown). While 40-day-old PRB-1b plants accumu

lated Pb in both the roots and shoots, younplants did not transport lead from the roots to th

shoots (Fig. 3B). In general, transgenic plan

grew better than non-transgenic in the presence o

lead (Tables 1 and 3), with rolD plants being th

least affected by the presence of Pb. The leaves

PRB-1b plants and the roots in 35S plants r

sponded to lead treatment by significantly increa

ing ACC deaminase activity (data not shown).

3.6. Zn

While Zn inhibited the germination of 35S an

rolD seeds relative to the non-transgenic (data n

shown), Zn had little effect on the growth

seven- and 51-day-old plants (Tables 1 and 2

However, both 51-day-old transgenic plants an

7-day-old non-transgenic plants hyperaccum

lated Zn (Fig. 3C). Importantly, all three types o

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 g1 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. Per-

haps 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.


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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 promotingbacteria (Burd et al., 2000).

4. Conclusion

The results of these studies are complex and not

always easy to interpret. This probably is a reflec-

tion 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 o

tomato plants to a particular metal is likely

vary with the stage of development of the plan

Moreover, since the PRB-1b promoter requir

ethylene in order to be induced, an ACC deam

nase gene under the transcriptional control of th

PRB-1b promoter will not be expressed unless th

ethylene concentration becomes elevated. Whithe ACC deaminase enzyme that is subsequent

expressed should ultimately lower the amount o

ethylene that can be produced, in some instance

the ethylene concentration that is required to tur

on the PRB-1b promoter may also damage th

plant. Thus, although PRB-1b plants may appea

to be superior in the presence of some metals, th

behaviour of these plants in different circum

stances is difficult to predict. On the other hand,

study of the physiological responses of the thre

Fig. 3. Metal accumulation in roots and shoots of ACC deaminase transgenic tomato plants. (A) Ni uptake by 9-day pouch-grow

seedlings and 40-day-old potted tomato plants. (B) Pb uptake as in A. (C) Zn uptake by 7-day pouch-grown seedlings a

51-day-old potted tomato plants. Solid bar, roots; gray bar, shoots. NT, non-transformed.


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