Genetic Engineering of Resistance to Bleaching Herbicides Affecting Phytoene Desaturase and Lycopene Cyclase in Cyanobacterial Carotenogenesis

PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 57, 68–78 (1997) ARTICLE NO. PB972261

Genetic Engineering of Resistance to Bleaching Herbicides Affecting Phytoene Desaturase and Lycopene Cyclase in Cyanobacterial Carotenogenesis Ute Windho¨vel,* Gerhard Sandmann,† and Peter Bo¨ger* *Lehrstuhl fu¨r Physiologie und Biochemie der Pflanzen, Universita¨t Konstanz, D-78434 Konstanz, Germany; and †Botanisches Institut, FB Biologie, J. W. Goethe Universita¨t, D-60054 Frankfurt, Germany Received October 24, 1996; accepted March 20, 1997 Enzymes of the carotenoid biosynthetic pathway are target sites for bleaching herbicides affecting the pigment composition of photosynthetic organisms. Cyanobacteria represent convenient models for the genetic engineering of resistance to herbicides affecting carotenogenesis of higher plants. Therefore, the genes crtI and crtY of the nonphotosynthetic bacterium Erwinia uredovora were simultaneously inserted into the genome of the cyanobacterium Synechococcus PCC7942. The products of both genes, the enzymes phytoene desaturase and lycopene cyclase, respectively, catalyze the reaction sequence in carotenoid biosynthesis leading from phytoene to b-carotene via phytofluene, z-carotene, neurosporene, lycopene, and g-carotene. While plant-type phytoene desaturases are target sites of the bleaching herbicide norflurazon, the bacterial enzyme is highly resistant to this compound. Lycopene cyclases of plant and bacterial origin, on the other hand, have been shown to be inhibited by substituted trialkylamines. We demonstrate that the resulting decrease of cyclic carotenoids was accompanied by bleaching of chlorophyll in the light. The determination of molar I50 values for in vivo inhibition of carotenoid biosynthesis caused by norflurazon and by the trialkylamine compound BTS 6772 [2-(4-chlorophenylthio)methyldiethylamine hydrochloride] revealed a simultaneous resistance to these two herbicides in the cyanobacterial transformant strain. q1997 Academic Press

INTRODUCTION

the enzyme lycopene cyclase, which catalyzes the cyclization reaction at both ends of the molecule to form b-carotene with g-carotene as intermediate (5). The yellow b-carotene, an essential component of the photosynthetic reaction centers and antennae, is also a precursor of several oxygenated carotenoids like the xanthophyll zeaxanthin. The b-carotene series can be found in all plants, cyanobacteria, several fungi, and certain heterotrophic bacteria. So far, several genes coding for lycopene cyclase from different organisms, such as Capsicum annuum, Lycopersicum esculentum, Nicotiana tabacum, the cyanobacterium Synechococcus PCC7942, and the heterotrophic bacteria E. herbicola and E. uredovora, have been cloned and sequenced (6–12). In the two Erwinia species the lycopene cyclase gene belongs to a cluster of genes coding for enzymes of the whole carotenoid biosynthetic pathway in these organisms. As indicated by analysis of the Erwinia lycopene cyclase gene, one gene product is sufficient for the introduction of two b-ionone rings to form b-carotene (13–15). A single lycopene

Carotenoids, which are yellow, red, and orange pigments, are synthesized de novo by all photosynthetic and many nonphotosynthetic organisms. It is well established that in phototrophs carotenoids carry out several functions: besides light harvesting and serving as structural components of the photosynthetic apparatus, they are essential for photoprotection (1–3). The initial step of the carotenoid biosynthetic pathway is the condensation of two molecules of geranylgeranyl pyrophosphate (GGPP) to the C40 hydrocarbon phytoene, which is catalyzed by the enzyme phytoene synthase. Four desaturation reactions convert the colorless phytoene to the red-colored lycopene via phytofluene, zcarotene, and neurosporene. In plants and cyanobacteria these are catalyzed by the two enzymes phytoene desaturase and z-carotene desaturase, whereas in the nonphotosynthetic bacteria Erwinia uredovora and Erwinia herbicola there is only one enzyme, CRTI, catalyzing the four steps (4). Lycopene is the primary substrate of 68 0048-3575/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

HERBICIDE RESISTANCE IN CYANOBACTERIA

cyclase enzyme was also found in Synechococcus PCC7942 (8). Sequence comparison of the bacterial enzymes revealed that they share little sequence resemblance with the lycopene cyclase of Synechococcus (23% sequence identity and 47% similarity (9)), whereas the plant enzymes isolated from Lycopersicum esculentum and Nicotiana tabacum are more closely related to the cyanobacterial enzyme (35% sequence identity and 55% similarity (7)). In addition to phytoene desaturase and z-carotene desaturase, also lycopene cyclase had been shown to be a target of some so-called “bleaching compounds.” These compounds interfere with the carotenoid biosynthesis, and due to the loss or reduction of photoprotection by carotenoids this results in a destruction of chlorophyll when the respective organism is exposed to light. A number of substituted trialkylamines were found to inhibit the cyclization reaction, leading to an accumulation of lycopene at the expense of the formation of b-carotene. Especially CPTA [2-(4-chlorophenylthio)triethylamine hydrochloride] or MPTA [2-(4-methylphenoxy)triethylamine hydrochloride] had been used for several years for in vivo and in vitro inhibition studies (16). In recent studies performed with the cloned Synechococcus or tobacco enzymes, respectively, it was shown that MPTA acts directly on lycopene cyclase (7, 9). For the Aphanocapsa enzyme and recently for the enzyme of E. uredovora, a noncompetitive inhibition by CPTA or MPTA was found (16; Schnurr, personal communication). Alternative to the introduction of genes coding for enzymes that detoxify the herbicide or for enzymes that are resistant to the respective herbicide, also the overproduction of the target enzyme due to a strong promoter controlling the introduced gene may be a promising way to produce herbicide-resistant organisms by genetic engineering. It was shown previously that heterologous genes for carotenoid biosynthesis can be used to generate plants resistant to bleaching herbicides (17). It should be noted that insertion of the bacterial phytoene desaturase gene into the plant genome also changed the pattern of carotenoid composition (18). Studies have been conducted in our laboratory to relate

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such genetically engineered alterations to stability of the photosynthetic apparatus against strong light or UV-B irradiation. Since cyanobacteria share oxygenic photosynthesis with higher plants and can be easier manipulated genetically than the latter, they are often used as models for the manipulation of components of the photosynthetic apparatus in higher plants. Accordingly, the insertion of the phytoene desaturase gene of E. uredovora into the cyanobacterium Synechococcus PCC7942 led to a strain that was highly resistant to several bleaching herbicides affecting plant-type phytoene desaturases (19, 20). This study demonstrates the simultaneous insertion of the genes coding for the lycopene cyclase and the phytoene desaturase from E. uredovora into the “integration platform” residing in the genome of Synechococcus PCC7942, strain PIM8 (21), generating a transformant that is resistant to the trialkylamine BTS 6772 [2-(4-chlorophenylthio)methyldiethylamine hydrochloride], a lycopene cyclase inhibitor, as well as to the phenylpyridazinone norflurazon, an inhibitor of plant-type phytoene desaturases. MATERIALS AND METHODS

Organisms and growth conditions. The culture medium for Synechococcus PCC7942 strain PIM8 (21) and the transformants derived from it was composed essentially as described (19). Liquid batch cultures were grown in Erlenmeyer flasks with constant shaking and illumination with fluorescent white light at an intensity of 60 mEm22 s21 at 308C. Growth was followed by determination of the packed cell volume (pcv; in ml/ml culture; by centrifugation of 2-ml aliquots in graduated microcentrifugation tubes) and by measuring the chlorophyll content of the cultures. Escherichia coli DH5a (Bethesda Research Laboratories, Gaithersburg, MD), which was used for plasmid propagation, was grown as described (22). For selection of transformant strains, 50 mg/ml ampicillin or kanamycin was added to the growth medium. DNA manipulations. For the manipulation of DNA, standard techniques were followed (22,

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23). Transformation of Synechococcus was performed as described previously (19), taking advantage of the natural competence of strain PCC7942 for the uptake of DNA and its ability to integrate DNA by homologous recombination (24). DNA/DNA hybridization was carried out using the Nonradioactive DNA Labeling and Detection Kit from Boehringer (Mannheim, Germany) following the supplier’s protocol. Construction of plasmids. The construction of plasmids pBG0 and pBG1 and the generation of Synechococcus PCC7942-PIM8 strains -BG0 and -BG1 were described in a former study (19; refer to Fig. 1). To construct transformant PIM8YI an identical strategy was followed: Plasmid pCAR25 (12) was used as source for the two adjacent genes crtY and crtI from the bacterium E. uredovora, which code for lycopene cyclase

FIG. 1. Genomic region containing the “integration platform” in various Synechococcus PCC7942 strain PIM8 transformants. The sizes of fragments that are generated by restriction with enzymes used for Southern blot analysis (Fig. 2) are given. Arrowheads refer to transcriptional orientation. Cleavage sites for restriction enzymes are indicated as follows: B, BamHI; D, DraII; E, EcoRV. Abbreviations used: bla, gene coding for resistance to ampicillin; crtI, gene coding for the phytoene desaturase of Erwinia uredovora; crtY, gene coding for the lycopene cyclase of E. uredovora; metF8, gene involved in methionine biosynthesis of Synechococcus PCC7942, location of the integration platform of strain PIM8 (21); the dash at the notation designates the interrupted gene; nptII, gene coding for resistance to kanamycin; ori, origin of replication.

and phytoene desaturase, respectively. Both genes together were isolated on a 3-kb EcoRV fragment and inserted downstream of the kanamycin resistance gene nptII residing on pBG0 by blunt-end ligation to its SmaI site. The resulting plasmid pYI was used for transformation of Synechococcus PCC7942-PIM8, leading to strain PIM8-YI. Determination of herbicide resistance. Fresh precultures were diluted by culture medium to a density equivalent to 1 to 2 mg chlorophyll ml21, and 25-ml aliquots of the diluted cultures were dispensed in 250-ml Erlenmeyer flasks. Herbicides dissolved in methanol were added in various concentrations, and the cultures were cultivated for 2 days at 308C as described. The solvent concentration was kept below 0.1%. The molar concentrations of 50% inhibition (I50) were determined by a graphical procedure (25). To demonstrate growth on herbicide-containing solid medium cells from exponentially growing cyanobacterial liquid cultures were streaked on agar plates (1.5% (w/v) agar) and incubated for 10 days in an illuminated incubator (type Rumed; Rubarth Apparate GmbH, Hannover, Germany) at a light intensity of 30 mEm22 s21 and 308C. Pigment extraction and analysis. Cells used for the extraction of chlorophyll or carotenoids were harvested from 1-ml culture aliquots by centrifugation in an Eppendorf centrifuge at room temperature and maximum speed. To determine the chlorophyll a content of the transformants, the resulting pellets were resuspended in 1 ml methanol and incubated for 5 min at 658C. The chlorophyll concentration of the supernatant of a subsequent centrifugation step was determined by measuring the absorption at 665 nm using an extinction coefficient of 74.5 mg chlorophyll21 cm2 (26). For isolation of carotenoids, the pelleted cells were extracted (20 min, 658C) with methanol containing 6% (w/v) KOH, partitioned against 10% diethyl ether in petrol (bp 35–808C), and evaporated to dryness in a vacuum centrifuge. The dried carotenoids were redissolved in 1 ml petrolether. For cultures grown without a herbicide or in the presence

HERBICIDE RESISTANCE IN CYANOBACTERIA

of the phytoene desaturase inhibitor norflurazon the absorption at 445 nm was determined and the content of cyclic carotenoids was calculated using an overall extinction coefficient E1%1cm (i.e., optical density of 1 g carotenoids in 100 ml solution in a 1-cm light path spectrophotometer cuvette) of 2500 (27). For determining cyclic carotenoid contents of cells that had been grown in the presence of a lycopene cyclase inhibitor, the absorption at 445 nm and 505 nm was measured and the carotenoid concentration was calculated according to (28) using the following equation: carotenoids (mg ml21) 5 7.25 3 E445 2 3.31 3 E505. The carotenoid content was referred to the pcv of the culture, and analysis of carotenoids was performed by HPLC. For separation, the extracted and dried carotenoids obtained from 1 ml culture were resolved in 20 ml acetone, injected into a Spherisorb ODS-1 column (5-mm particle size) and eluted at a flow rate of 1 ml min21, employing an isocratic solvent system of acetonitrile/methanol/2-propanol (80/13/7, v/v). Carotenoid peaks generated were detected at 450 nm with a Waters 994 diode array detector and spectra were directly recorded on line. Carotenoids were identified by comparing retention time and absorption spectra to those of standard compounds.

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which codes for the phytoene desaturase of E. uredovora, was inserted downstream of nptII. In the latter strain a cotranscription of nptII and crtI starting from the promoter of the nptII gene has been detected. Strain PIM8-YI was generated analogously to strains -BG0 and -BG1 by inserting the plasmid pYI into the integration platform (Fig. 1). This new plasmid includes the following genes in the given order: nptII, crtY (which codes for the lycopene cyclase of E. uredovora), and crtI (phytoene desaturase). The correct insertion of the genes was checked by Southern hybridization using digoxygeninlabeled nptII-DNA as probe (Fig. 2). The molecular masses of the hybridizing fragments matched with the calculated ones, indicating the correct integration of pYI into the Synechococcus genome (refer to Figs. 1 and 2). Analysis of herbicide resistance in the transformant strains. To demonstrate that the trialkylamine compound BTS 6772, like CPTA and MPTA, inhibits the lycopene cyclase, the

Chemicals. Norflurazon (SAN 9789; 4-chloro5-methylamino-2-(3-trifluoromethylphenyl)pyridazin-3(2H)one) was a gift from Sandoz AG (Basel, Switzerland). BTS 6772 [2-(4-chlorophenylthio)methyldiethylamine hydrochloride] was from Schering AG (Berlin, Germany), and MPTA was from BASF AG (Ludwigshafen, Germany). RESULTS

Generation of transformant strains. The construction of Synechococcus PCC7942-PIM8BG1 and PIM8-BG0 by transforming strain PIM8 with plasmids pBG0 and pBG1 has been described in a previous paper (19). The control strain PIM8-BG0 harbored the nptII gene of transposon Tn5 (29), conferring resistance to kanamycin, in the “integration platform” that resides in the genome of Synechococcus PCC7942-PIM8 (21). In strain PIM8-BG1, crtI,

FIG. 2. Southern blot analysis of Synechococcus PCC7942 strain PIM8 transformants carrying various constructs in the integration platform (refer to Fig. 1). A 1% agarose gel was run with genomic DNA of the following strains: a, PIM8-BG0; b and c, PIM8-YI; d and e. PIM8BG1. The DNA was digested with EcoRV (a, b, and d) or double-digested with EcoRV and BamHI (c) and with EcoRV and DraII (e), respectively, blotted to a nylon membrane, and hybridized with digoxygenin-labeled nptII gene as probe. M, digoxygenin-labeled DNA molecular mass marker (HindIII, EcoRI double-digested phage l-DNA). Numbers indicate the sizes of molecular mass markers in kb.

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extracted carotenoids from a Synechococcus PCC7942 PIM8-YI liquid culture grown without and with 1025 M BTS 6772 present were analyzed by HPLC (Fig. 3). Chromatographic separation of the herbicide-free culture extract yielded only three carotenoids (Fig. 3A). By comparison of retention time and absorption spectra (Fig. 3C) to standard compounds they were identified as isomers of the dihydroxylated zeaxanthin (peaks 1 and 18), the monohydroxylated b-cryptoxanthin (peak 2), and isomers of b-carotene (peaks 3 and 38). When separating the carotenoids isolated from the culture grown in the presence of BTS 6772, additional peaks appeared in the HPLC elution profile (Fig. 3B). These were identified as isomers of lycopene (peaks 5 and 6) and g-carotene (peaks 7 and 78), representing an intermediate compound of the reaction sequence from lycopene to b-carotene. Since the absorption spectrum of the carotenoid

from peak 4 is identical to that of g-carotene (peak 7) and the retention time lies between that of zeaxanthin (peak 1) and that of b-cryptoxanthin (peak 2), we conclude that peak 4 represents hydroxylated g-carotene. Besides the appearance of new elution peaks, it is interesting to note the changed ratio between b-carotene and zeaxanthin in the two carotenoid extracts. While zeaxanthin nearly doubled b-carotene in the control, this ratio is reversed in the culture grown in the presence of BTS 6772. Liquid cultures of PIM8-BG0 and PIM8-YI were treated with different concentrations of BTS 6772 to show that the decrease of cyclic carotenoids in the photosynthetic apparatus and their replacement by the acyclic lycopene results in bleaching due to degradation of chlorophyll in the light, which may lead to a herbicidal effect. The residual contents of cyclic carotenoids and chlorophyll were determined (Table 1). In the

FIG. 3. HPLC analysis of carotenoids extracted from cells of Synechococcus PCC7942 strain PIM8-YI after growth without herbicide (A) or in the presence of 1025 M BTS 6772 (B). Absorbance spectra of the pigments that were detected at 450 nm were recorded on line (C). The numbered peaks were identified by comparison of retention time and spectra with standard compounds as follows: 1 and 18, zeaxanthin isomers; 2, b-cryptoxanthin; 3 and 38, b-carotene isomers; 4, hydroxy-g-carotene; 5 and 6, lycopene isomers; 7 md 78 g-carotene isomers.

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HERBICIDE RESISTANCE IN CYANOBACTERIA

TABLE 1 Content of Cyclic Carotenoids and Chlorophyll a (mg ml Culture21) in Synechococcus Transformants after Growth of Liquid Cultures in the Presence of Various Concentrations of BTS 6772 Strain PIM8-BG0 BTS 6772 (M) 0 1027 2.5 3 1027 5 3 1027 1026 5 3 1026 1025 2.5 3 1025 1024 a

Carotenoids

PIM8-YI Chlorophyll

1.3 1.1 1.0 0.6 0.4 0.2 0.3 n.d. n.d.

5.6 5.4 4.9 3.4 1.6 1.2 1.9 n.d. n.d.

Carotenoids

Chlorophyll

1.3 n.d.a n.d. n.d. 1.5 1.3 1.1 1.1 0.6

5.8 n.d. n.d. n.d. 7.0 7.2 5.2 6.8 3.6

n.d., not determined.

control strain Synechococcus PCC7942 PIM8BG0, 5 3 1027 M BTS 6772 resulted in the formation of only about half of the cyclic carotenoids. Under the same conditions, about 61% of the chlorophyll of a nontreated culture was found. An increase of the inhibitor concentration to 1025 M decreased the contents of cyclic carotenoids and chlorophyll to about 23 and 34% of the control, respectively. More or less the same inhibition properties were observed for the transformant PIM8-BG1. In contrast, the transformant PIM8-YI had to be treated with much higher concentrations of BTS 6772 to obtain a similar simultaneous decrease of cyclic carotenoids and chlorophyll. A concentration of 1024 M was necessary to lower the amounts of these pigments to about 46 and 62%, respectively. Application of MPTA instead of BTS 6772 yielded comparable results (data not shown). Since norflurazon and BTS 6772 affect the function of phytoene desaturase and lycopene cyclase, respectively, we analyzed to which extent the additional phytoene desaturase or lycopene cyclase genes influenced the sensitivity of Synechococcus PCC7942 PIM8-BG1 and PIM8-YI toward these herbicides. To obtain first information, the lethal concentration of either norflurazon or BTS 6772 for growth on solid medium was determined. Therefore, the two transformant strains containing the foreign

carotenoid biosynthesis genes and Synechococcus PCC7942 PIM8-BG0 as a control were streaked on agar plates containing the respective herbicide in several concentrations. In Fig. 4, the result of this analysis after 10 days of incubation under illumination is shown. On norflurazoncontaining medium, Synechococcus PCC7942 PIM8-BG1 exhibited the highest resistance: it grew well on plates with 1025 M (Fig. 4e) and still grew somewhat on 5 3 1025 M (Fig. 4f), while PIM8-YI could only grow on plates with up to 5 3 1026 M (Fig. 4d) norflurazon. The control strain PIM8-BG0 did not grow on any of the norflurazon concentrations tested; it only grew on plates free of this herbicide. Testing sensitivity to BTS 6772, the latter could grow with 5 3 1027 M (Fig. 4g), while strain PIM8YI still grew well at an 80 times higher BTS 6772 concentration (4 3 1025 M: Fig. 4i) and still showed some growth at 5 3 1025 M (Fig. 4j). Strain PIM8-BG1, on the other hand, grew worse than PIM8-BG0 on medium containing 5 3 1027 M BTS 6772 (Fig. 4g). Herbicide resistance of the transformants was quantified by the molar I50 values for the in vivo inhibition of carotenogenesis (Table 2). The cells were grown in liquid medium containing various concentrations of the three inhibitors, norflurazon, BTS 6772, or MPTA. Starting incubation with a chlorophyll concentration of 2 mg ml21,

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FIG. 4. Resistance to norflurazon (A) or BTS 6772 (B) in Synechococcus PCC7942 strain PIM8 transformants. Cells were taken from exponentially growing liquid cultures and streaked on agar plates as indicated (a) containing norflurazon in molar concentration: 5 3 1027 (c), 5 3 1026 (d), 1025 (e), and 5 3 1025 M (f); or BTS 6772: 5 3 1027 (g), 1026 (h), 4 3 1025 (i), and 5 3 1025 (j). (b) Control plate without herbicide. The figure shows the result after a 10-day incubation in white light (30 mEm22 s21) at 308C.

TABLE 2 I50 Values (Molar) for in Vivo Inhibition of Carotenogenesis in Synechococcus Transformants by Bleaching Herbicides Affecting (1) Phytoene Desaturase or (2) Lycopene Cyclase Strain

Norflurazon1

BTS 67722

MPTA2

PIM8-BG0 PIM8-BG1

3.4 3 1027 2.1 3 1024 (618)b 6.5 3 1025 (191)

8.3 3 1027 1.7 3 1027 (0.2) 3.1 3 1025 (37)

2.6 3 1027 n.d.a

PIM8-YI a

n.d.

n.d., not determined. Data in parentheses indicate the resistance factor of the transformant strains toward the bleaching herbicides with respect to the control strain Synechococcus PCC7942PIM8-BG0. b

the control liquid cultures of the three strains grown without herbicide reached about 5 to 6 mg chlorophyll ml21 after 48 hr and contained nearly identical amounts of colored carotenoids per cell (about 1.4 mg ml pcv21). In preceding papers (19, 20) we reported that strain Synechococcus PCC7942 PIM8-BG1 is highly resistant to norflurazon. The data presented in Table 2 confirm these results and show that strain -YI also exhibits a certain degree of resistance to this herbicide compared to the control strain, but about two-thirds less than -BG1. Compared to Synechococcus PCC7942 PIM8-BG0, strain -YI is more resistant to BTS 6772, while -BGI is even more sensitive to this herbicide than the control strain. The I50 value for MPTA of control strain PIM8-BG0 is about one-third of that for BTS 6772.

HERBICIDE RESISTANCE IN CYANOBACTERIA

DISCUSSION

In this investigation the two carotenoid biosynthesis genes crtI and crtY of the heterotrophic bacterium E. uredovora, which code for the enzymes phytoene desaturase and lycopene cyclase (12), respectively, were simultaneously introduced into the integration platform of the cyanobacterium Synechococcus PCC7942, strain PIM8 (21), resulting in strain PIM8-YI. This cyanobacterium already contains a complete set of genes coding for enzymes that catalyze the reactions of the carotenoid biosynthetic pathway leading to zeaxanthin. Since the Erwinia phytoene desaturase CRTI catalyzes all four desaturation reactions from phytoene to lycopene, the new transformant strain contains, in addition to the endogenous enzymes, heterologous catalytic activity to perform the reactions from phytoene to b-carotene. Like the introduction of only one gene, crtI, in transformant strain Synechococcus PCC7942-PIM8-BG1 (19), also the additional introduction of crtY had no effect on the overall synthesis of carotenoids in PIM8YI. All three transformant strains tested contained about 1.4 mg colored carotenoids ml pcv21 under the growth conditions used. In contrast, the introduction of an additional foreign phytoene synthase gene (29) or b-carotene hydroxylase gene (manuscript in preparation) resulted in an increase of the overall carotenoid content. While the amount of carotenoids in the transformant strains was not changed, the introduced genes influenced the sensitivity of transformants toward the bleaching herbicides norflurazon and BTS 6772, which were expected to inhibit phytoene desaturase and lycopene cyclase, respectively. The newly acquired resistances were manifested by the ability of the respective strains to grow on solid medium containing either of the two herbicides (Fig. 4). The results of our study are consistent with the findings for a mutant of Synechococcus PCC7942 (Mr-5 (8, 9)) in which the endogenous lycopene cyclase gene (lcy) was overexpressed due a point mutation resulting in a stronger promoter of the gene.

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On solid medium containing 5 3 1025 M triethylamine compound MPTA, which was shown to inhibit lycopene cyclase, this mutant grew well and still exhibited some growth in the presence of more than 1024 M of MPTA, while the lethal dose for the wild type was already reached at a concentration higher than 2 3 1026 M. The tendency of the results which had been obtained for herbicide resistance of the transformants on solid medium reflected the results from the determination of molar I50 values of cells grown in liquid medium (Fig. 4 and Table 2). The I50 value for in vivo inhibition of carotenogenesis by CPTA reported for the cyanobacterium Aphanocapsa (4.5 3 1025 M (16)) is about 50 times higher than that found for the analogous compound BTS 6772 in the control strain of this study (8.3 3 1027 M). Besides pointing to a different sensitivity of these two cyanobacterial genera to trialkylamine compounds, the difference may be due to different growth conditions, e.g., light regimes, under which cells for testing herbicide resistance had been cultivated. The action of BTS 6772 on the lycopene cyclase was clearly shown by HPLC analysis (Fig. 3): In addition to b-carotene, b-cryptoxanthin, and zeaxanthin, which represent the set of carotenoids in untreated Synechococcus PCC7942 cells, BTS 6772-treated cells contained lycopene and g-carotene. Since the latter intermediate compounds represent the substrates for lycopene cyclase, their accumulation demonstrate its inhibition by BTS 6772. Interestingly, the pigment extract contained a carotenoid identified as hydroxylated g-carotene, providing evidence that b-carotene hydroxylase may also hydroxylate g-carotene. A similar enzymatic activity to catalyze the respective reaction on only one-half of an unsymmetric compound had been shown for lycopene cyclase (9, 15). The latter could catalyze the cyclization of that part of the incompletely desaturated neurosporene that resembles lycopene to form the monocyclic b-zeacarotene. Another interesting by-product found by our HPLC analysis was the reversed ratio of b-carotene and zeaxanthin in BTS 6772treated cells compared to untreated cells. The

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finding that in the first case there is more bcarotene than zeaxanthin may indicate that the cells have to maintain a certain level of b-carotene in order to provide a functional photosynthetic apparatus. The parallel decrease of cyclic carotenoids and chlorophyll in cells grown in the presence of BTS 6772 (Table 1) illustrates that apparently this inhibitor of lycopene cyclase exerts its herbicidal effect on photosynthetic organisms by chlorophyll bleaching. Obviously, the acyclic lycopene accumulated under inhibitory conditions as the major carotenoid lacks sufficient potential to prevent chlorophyll degradation. This may be due to an inefficient insertion of this carotenoid into the photosynthetic apparatus. For strain Synechococcus PCC7942-PIM8BG1, which contains the bacterial phytoene desaturase (CRTI), the acquisition of resistance to herbicides inhibiting plant-type phytoene desaturases has already been demonstrated (19). The finding that strain PIM8-YI exhibited also a resistance to norflurazon indicates that the gene coding for phytoene desaturase, which lies downstream of the lycopene cyclase gene in this transformant, is cotranscribed together with the kanamycin and the lycopene cyclase gene (Fig. 1). The lower degree of resistance (resistance factor 191 vs. resistance factor 618) is probably the result of less CRTI protein produced in strain PIM8-YI. A lower amount of CRTI, which was detected by Western blotting (data not shown), might be due to the greater distance of the crtI gene from the nptII promoter in this strain compared to PIM8-BG1. In a previous study a lower amount of synthesized CRTI protein led to a lower resistance factor in a strain that contained a weaker promoter upstream of the crtI gene than PIM8-BG1 (19). While the norflurazon resistance of the two transformants Synechococcus PCC7942-PIM8BG1 and PIM8-YI is due to the synthesis of the highly resistant Erwinia phytoene desaturase, the Erwinia lycopene cyclase has been found sensitive to substituted triethylamines (G. Schnurr, personal communication). Thus, the resistance of strain PIM8-YI is obviously the

result of an overproduction of the sensitive bacterial lycopene cyclase in this strain. This is consistent with the similar degree of resistance to the triethylamine MPTA found for the Synechococcus PCC7942 mutant Mr-5, which obviously produced more strain. This is consistent with the similar degree of resistance to the triethylamine MPTA found for the Synechococcus PCC7942 mutant Mr-5, which obviously produced more lycopene cyclase due to a mutation in the promoter region of the endogenous lycopene cyclase gene (8). Interestingly, Synechococcus PCC7942-PIM8-BG1 was less resistant to BTS 6772 compared to the control strain. The reason is unclear, but it may be speculated that because of the very high amount of CRTI in strain BG1 (19) there is less space for the other enzymes of the carotenoid biosynthetic pathway, including lycopene cyclase, available in the thylakoids. Since cyanobacteria carry out plant-type oxygenic photosynthesis, the present investigation can serve as a model to generate crop plants that are resistant to substituted trialkylamines affecting the lycopene cyclase in these organisms. However, at present time the trialkylamines have no commercial importance as herbicides. The expression of the bacterial lycopene cyclase gene in plants may extend the use of carotenoid inhibitors for crops that are currently sensitive to these compounds. Like the bacterial phytoene desaturase (12) the bacterial lycopene cyclase shares little sequence homology with the respective plant-type enzymes (6, 7, 9). Thus, the introduction of crtY like that of crtI (17) avoids the problem of cosuppression of the endogenous enzyme (31) in the respective transgenic plant. ACKNOWLEDGMENTS We thank Dr. N. Misawa, Yokohama, for providing plasmid pCAR25, Dr. J. van der Plas, Utrecht, for providing Synechococcus PCC7942, strain R2-PIM8, and Reinhild Go¨bel for excellent technical assistance during part of this work. This study was supported in part by the Bundesministerium fu¨r Bildung, Wissenschaft, Forschung und Technologie, and the Fonds der Chem. Industrie.

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