JXB Advance Access originally published online on September 6, 2006
Journal of Experimental Botany 2006 57(14):3639-3645; doi:10.1093/jxb/erl103
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RESEARCH PAPER |
Ketocarotenoid formation in transgenic potato
Molecular Biosciences 213, J.W. Goethe Universität, PO Box 111932, D-60054 Frankfurt/M., Germany
* To whom correspondence should be addressed. E-mail: sandmann{at}em.uni-frankfurt.de
Received 16 January 2006; Accepted 4 July 2006
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Abstract |
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Potato has been genetically engineered for the production of commercially important ketocarotenoids including astaxanthin (3,3'-dihydroxy 4,4'-diketo-ß-carotene). To support the formation of 3-hydroxylated and 4-ketolated ß-carotene, a transgenic potato line accumulating zeaxanthin due to inactivated zeaxanthin epoxidase was co-transformed with the crtO ß-carotene ketolase gene from the cyanobacterium Synechocystis under a constitutive promoter. Plants were generated which exhibited expression of this gene, resulting in an accumulation of echinenone, 3'-hydroxyechinenone, and 4-ketozeaxanthin in leaves, as well as 3'-hydroxyechinenone, 4-ketozeaxanthin together with astaxanthin in the tuber. The amount of ketocarotenoids formed represent
10–12% of total carotenoids in leaves and tubers. Negative effects on photosynthesis due to the presence of the ketocarotenoids in leaves could be excluded by the determination of variable fluorescence. Key words: Astaxanthin, ketocarotenoids, ketolase gene crtO, transgenic potato, tuber carotenoids
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Carotenoids are pigments of red to yellow colour synthesized by micro-organisms and plants. Typical carotenoids consist of 40 carbon atoms and possess an extended conjugated polyene system (Goodwin, 1980). The ionone end groups may carry hydroxy, epoxy, or keto groups. Ketocarotenoids are especially photostable pigments with high antioxidative activities (Woodall et al., 1997). Due to their health-promoting properties, they are of pharmaceutical interest and are also used as feed additives for the coloration of salmon and trout (Bhosale and Bernstein, 2005). The 3,3'-dihydroxy 4,4'-diketo ß-carotene derivative astaxanthin is commercially one of the most important carotenoids. It was reported to stimulate immune functions (Jyonouchi et al., 1995) and reduce oral cancer and mammary tumour growth in animal models (Tanaka et al., 1995; Chew et al., 1999). The majority of its demand is met by chemical synthesis (Bernhard, 1989), but natural sources are becoming more and more important (Margalith, 1999; Guerin et al., 2003). Astaxanthin is derived from ß-carotene by 3-hydroxylation and 4-ketolation at both ionone end groups (Sandmann, 2001a). The hydroxylation reaction is widespread in many organisms, but ketolation is restricted to a few bacteria, fungi, and some unicellular green algae (Johnson and An, 1991). The genes involved in these oxygenation reactions are bacterial crtZ, cyanobacterial crtR, and bhy related to the latter. The bacterial ketolase gene crtW is closely related to the green algal bkt gene (Kajiwara et al., 1995). The crtO ketolase gene from cyanobacteria is a paralogous gene unrelated to all the others (Fernández-Gonzalez et al., 1997). Plants synthesize different hydroxy carotenoids but, apart from very few exceptions such as Adonis flowers (Seybold and Goodwin, 1959), they are devoid of ketocarotenoid. They lack the ß-carotene ketolase genes mentioned above. However, carotenogenesis in plants can be genetically manipulated (Giuliano et al., 2000; Sandmann, 2001b). A plant engineered with a microbial ketolase gene may be useful as a ketocarotenoid-producing system. Examples of transgenic plants genetically modified to synthesize ketocarotenoids already exist. For example, ketocarotenoid synthesis was achieved in tobacco nectary tissue and leaves (Mann et al., 2000; Ralley et al., 2004) and Arabidopsis thaliana seeds (Stalberg et al., 2003). Alternatively, ß-carotene-accumulating tomatoes were co-transformed simultaneously with a ß-carotene ketolase and a hydroxlyase gene (Ralley et al., 2004). In all cases, ketolase genes of the crtW/bkt family from the alga Haematococcus pluvialis (Kajiwara et al., 1995; Lotan and Hirschberg, 1995) or the bacterium Paracoccus (Misawa et al., 1995) have been used for genetic engineering of a ketolase pathway. In the case of vegetable plants transformed with a ketolase gene, the nutritional value is increased by the highly antioxidative ketocarotenoids. Therefore, potato tuber is a biological system of choice to use its carotenoid-synthesizing capacity (Iwanzik et al., 1983; Breithaupt and Bamedi, 2002) for ketocarotenoid production. The carotenoid biosynthetic pathway in potato tuber is shown in Fig. 1A. The main accumulating carotenoids are lutein and violaxanthin. The genetic manipulation of carotenoid biosynthesis in potato tuber has already been successful. It was possible to alter the carotenoid composition towards an accumulation of zeaxanthin by blocking the epoxidase reaction (Römer et al., 2002). As a consequence, zeaxanthin is formed at the expense of violaxanthin, and lutein formation was down-regulated (Fig. 1A). In another genetic approach, an increase of total tuber carotenoids was achieved by overexpression of phytoene synthase, the first enzyme of the biosynthetic pathway (Ducreux et al., 2005).
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In the present investigation, an attempt was made to modify carotenogenesis in potato tubers for the biosynthesis of astaxanthin and other ketocarotenoids. The strategy used was to start with the potato host Baltica 47-18 (Römer et al., 2002). This is a transgenic line with increased formation of zeaxanthin already carrying the 3- and 3'-hydroxy groups. It was transformed with the ketolase gene crtO from the cyanobacterium Synechocystis 6803 (Fernández-Gonzalez et al., 1997). The resulting transformants were analysed and the formation of different ketocarotenoids determined.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Biological material and growth conditions
The potato lines Solanum tuberosum L. cv. Baltica and Baltica 47-18 (Römer et al., 2002) were grown at 25 °C in a 16 h light and 8 h darkness cycle on MS20 medium for transformation. Transgenic plants were selected on hygromycin- (10 mg l–1) containing medium. After regeneration, transgenic in vitro potato plantlets were grown in a greenhouse at temperatures of
20–25 °C during the day and 10–15 °C at night. The potato tubers were harvested after 3 months and stored at 4 °C for further cultivation.
Agrobacterium tumefaciens strain LBA 4404 was used for potato transformation. Complementation experiments to evaluate the product specificity of crtO were carried out in Escherichia coli JM101. Escherichia coli DH5
was the host for subcloning and plasmid amplification. Both strains were grown on LB medium overnight at 37 °C with corresponding antibiotics, 20 mg l–1 ampicillin, 25 mg l–1 kanamycin, 20 mg l–1 tetracycline, or 34 mg l–1 chloramphenicol (Sambrook et al., 1989). The ketocarotenoid-producing transformant was grown for 2 d at 28 °C under constant gassing with air.
Plasmid construction and potato transformation
Plasmid pslr0088 (Fernandez-Gonzalez et al., 1997) was the source of the crtO gene from Synechocystis (accession no. NP_442491). For construction of the plant transformation vector containing the ketolase gene crtO and a hygromycin resistance gene, crtO was amplified by polymerase chain reaction (PCR) using primer SphI-O 5'-TTG CCT CAC CAC CGA TGT TGT C-3' (generating an SphI restriction site) and PstI-O 5'-TTC TGC AGT TAC CAA AAA CGA CGT TGT TG-3' (generating a PstI restriction site). The fragment was subcloned into SphI- and PstI-digested plasmid pTRA3XN (Wagner et al., 2002) which harbours the transit peptide signal from the small subunit of ribulose bisphosphate carboxylase from Phaseolus vulgaris. The transit–crtO fusion was cut out with XbaI and ligated into the XbaI-digested vector pGPTV-HPT-P35S, a pBIN19 derivate (Bevan, 1984), resulting in plasmid pGPTV-HPT-trcrtO (Wagner et al., 2002) with crtO expressed under the 35S cauliflower mosaic virus promoter, and hygromycin as selection marker. Agrobacterium-mediated transformation of the potato line Baltica 47-18 was performed according to Rocha-Sosa et al. (1989). It was transformed with a binary vector according to Nagel et al. (1990) and cultivated on LB medium supplemented with 50 mg l–1 kanamycin, 50 mg l–1 rifampicin, and 50 mg l–1 streptomycin at 28 °C.
The E. coli plasmids used were pACCAR16
X (Misawa et al., 1995) for ß-carotene formation and pBBRcrtZ for the hydroxlyase. The latter plasmid resulted from ligation of crtZ from pACCAR25crtX (Misawa et al., 1995) into the ApaI/HindIII site of plasmid pBBR1MCR2 (Kovach et al., 1994). The function of crtO was analysed by co-expression of plasmid pPQE30crtO in E. coli in a ß-carotene background. This plasmid was generated by PCR amplification from pslr0088 (Fernandez-Gonzalez et al., 1997) using the primers BamH1-O2 (5'- CCA TGG GAT CCA TCA CCA CCG-3') creating a BamHI restriction site and PstI-O (5'- TTC TGC AGT TAC CAA AAA CGA CGT TGT TG-3') creating a PstI restriction site. This fragment was cloned into the BamHI- and PstI-digested plasmid pPQE30 (Verdoes et al., 1999).
DNA isolation and analysis
Genomic DNA was isolated from leaf tissue using the GenElute Plant Genomic DNA Miniprep Kit from Sigma (Straubenhard, Germany). DNA analysis was performed using 20–30 µg of genomic DNA digested overnight with XbaI, EcoRI, and PstI. This DNA was separated via electrophoresis on a 1% agarose gel in TAE buffer. The DNA was transferred to a positively charged nylon membrane and hybridized with a digoxigenin-labelled probe from a section of the crtO gene. Post-hybridization washes were twice at room temperature with 2x SSC buffer containing 0.1% (w/v) SDS and then twice with 0.1% SSC buffer containing 0.1% SDS for 15 min at 65 °C. After immunological reaction with anti-DIG-AP (Fab-Fragments, Roche), chemiluminescence by CDP-Star (Boehringer/Roche) was detected on X-ray film (Hyperfilm ECL, Amersham Pharmacia Biotech).
RNA isolation and analysis
Total RNA was isolated from potato tubers according to the modified method of Verwoerd et al. (1989). The shock-frozen fresh plant tissues were powdered under liquid nitrogen. Then, 800 µl of 80 °C hot 0.2 M borate buffer pH 9.0 containing 1% (w/v) SDS, 30 mM EGTA, and 600 µl of phenol was added. The samples were centrifuged and the upper phase extracted twice with phenol/chloroform (25+25 ml) and once with chloroform. In the last upper phase containing the total RNA, 8 M LiCl (final concentration 2 M) was added and the precipitation occurred overnight at 4 °C. The RNA was sedimented via centrifugation and washed twice with 70% ethanol. The RNA was stored at –70 °C. The concentration was determined photometrically.
RNA analysis was performed using a 1.2% (w/v) denaturing agarose gel to separate 10–15 µg of total RNA (Rosen and Villa-Komaroff, 1990). The RNA was transferred via capillary transfer using 20x SSC (3 M NaCl, 0.3 M Na-citrate) to a positively charged nylon membrane and hybridized with the digoxigenin-labelled crtO probe at 42 °C overnight. Post-hybridization washes of the membranes were the same as for the RNA blot. As a reference, total rRNA was stained with ethidium bromide.
Pigment extraction and analysis
Chlorophyll was extracted from freeze-dried potato leaves (2 mg) with methanol at 60 °C for 15 min. The content was quantitated from the absorbance at 650 and 665 nm according to Mackinney (1941).
The carotenoids from freeze-dried tubers (150 mg) as well as from E. coli JM101/pACCAR16X/pBBRcrtZ/pPQEcrtO (10 mg) were extracted by heating with acetone for 15 min at 50 °C. In the case of freeze-dried leaf tissues (10 mg), either methanol or methanol containing 6% (w/v) KOH was used as solvent. The extracts were partitioned against 10% (v/v) diethyl ether in petrol. The carotenoids were determined by high-performance liquid chromatography (HPLC) analysis. The system used was a Hypersil HyPurity Elite C18, 5 µm column run with acetonitrile/2-propanol/methanol/water 83:10:5:2 (by vol.) at 32 °C and a YMC C30, 3 µm column run with acetone/methanol/acetonitrile/water 55:55:5:5 (by vol.) at 10 °C. Standards were isolated from Xanthophyllomyces dendrorhous (Andrewes et al., 1976) and E. coli transformed with appropriate carotenogenic genes for ketocarotenoid production, and their molecular weight confirmed by mass spectroscopy (Sandmann, 2002). All carotenoids mentioned were identified by co-chromatography with these reference compounds and comparison of their spectra.
Determination of photosynthetic efficiency
Chlorophyll fluorescence was measured with a PAM 101 fluorometer (Walz, Effeltrich, Germany). Leaves were measured without detachment from the plants. The initial fluorescence yield (F0) in weak modulated light was recorded, followed by the maximum fluorescence yield (Fm) after a saturating light pulse (4000 µmol m–2 s–1). The photosynthetic efficiency Fv/Fm ratio was calculated automatically.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Cultured plants are a cost-efficient source for secondary products (Giddings et al., 2000). Their yield of metabolites can be improved by genetic enhancement of biosynthetic capacity (Lessard et al., 2004). Furthermore, it is possible to genetically engineer novel pathways into plants (Daniell et al., 2001). In contrast to various micro-organisms, plants generally lack the biosynthetic potential for ketocarotenoid formation. Nevertheless, they are attractive as ketocarotenoid production systems, and several attempts have been made to modify carotenogenesis genetically towards the synthesis of astaxanthin and other keto derivatives.
Before the ketolase gene crtO gene was used to engineer potato plants, its potential to mediate ketocarotenoid formation was evaluated in a complementation experiment. Escherichia coli with two plasmids, pACCAR16crtX for ß-carotene formation and pBBRcrtZ for hydroxylation at positions 3 and 3', was used. This plasmid combination resulted in the accumulation of zeaxanthin, ß-cryptoxanthin, and ß-carotene (Fig. 2A). Upon co-transformation with the crtO-containing plasmid, several ketocarotenoids were formed (Fig. 2B). They include astaxanthin (3,3'-dihydroxy-ß,ß-carotene-4,4'-dione, peak 4), adonixanthin (3,3'-dihydroxy-ß,ß-carotene-4-one, peak 5), 3'-HO-echinenone (3'-hydroxy-ß,ß-carotene-4-one, peak 6), and echineneone (ß,ß-carotene-4-one, peak 7). These carotenoids represent the biosynthetic pathway of Fig. 1B. Although, in Synechocystis, the source of crtO, this gene mediates the formation of the monoketo product echinenone, it was demonstrated that higher oxygen supply and higher expression levels of the protein favour the formation of diketo products.
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In the tubers of wild-type potato, violaxanthin and lutein are the dominant carotenoids (Breithaupt and Bamedi, 2002; Römer et al., 2002). Both carotenoids are either formed in parallel to a potential pathway to astaxanthin or compete with it (Fig. 1). Therefore, the transgenic line Baltica 47-18 with a high accumulation of zeaxanthin is a better candidate as a host when astaxanthin formation is attempted than the wild type. The genetic background of line 47-18 is explained in Fig. 3A. For co-suppression of zeaxanthin epoxidase encoded by the zep gene, a zep construct under the tuber-specific potato granule-bound starch synthase (GBSS) promoter was integrated together with the kanamycin resistance gene nptII. For the second transformation with the ketolase gene, crtO was fused to the ribulose bisphosphate carboxylase small subunit transit sequence from pea and cloned behind the 35S promoter from cauliflower mosaic virus (Fig. 3B). The hygromycin resistance gene was introduced for selection.
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Leaves from young plants were prescreened for ketocarotenoid formation. Since crtO was under the ubiquitous 35S promoter, the presence of ketocarotenoids in leaves indicates that the ketolase gene is functionally expressed. Among the first 13 transgenic plants, eight positive ones were found. They were cultivated further for tuber production. Two of these lines exhibited formation of ketocarotenoid in tuber. The tuber carotenoid composition of lines 47-18crtO#51 and 47-18crtO#65 is shown in Table 1. Both lines exhibit the high zeaxanthin phenotype of 47-18 with low amounts of violaxanthin and lutein. In addition, three ketocarotenoids were detected: 3'-hydroxyechinenone, 4-ketozeaxanthin, and astaxanthin. In both lines, 4-ketozeaxanthin (adonoxanthin) shows the highest concentration of all keto derivatives, with 5.7% of total carotenoids in 47-18crtO#51and 8.5% in 47-18crtO#65. In the latter transgenic line, all the other ketocarotenoids are found in higher concentrations than in 47-18crtO#51. Although astaxanthin is the end-product of the complete 3-hydroxylation and 4-ketolation of ß-carotene (Fig. 1), 4-ketozeaxanthin lacking the second keto group is the major keto product. Ketozeaxanthin accumulation as the major ketocarotenoid in the tuber may suggest that 4-ketolation catalysed by CrtO preferentially converts ß-carotene and that 3-hydroxy products of ß-carotene like zeaxanthin are poor substrates. Therefore, ketolation competes with hydroxylation for ß-carotene and is the limiting reaction for astaxanthin formation. It should be pointed out that no canthaxanthin accumulated, which fits well into the picture of restricted ketolation and unrestricted hydroxylation. Although formally presented in Fig. 1 as intermediates in the reaction sequence to astaxanthin, products such as 3'-hydroxyechinenone should consequently represent end-products of side reactions instead. This limiting situation in the potato tuber resembles the finding in other transgenic plants when ketolase CrtW was used (Ralley et al., 2004). However, in Arabidopsis seeds, accumulation of adonirubin (3-HO-4,4'-diketolutein) and canthaxanthin indicates that in this transformant, hydroxylation is limiting and hampers the formation of astaxanthin (Stalberg et al., 2003).
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Integration of crtO was demonstrated by DNA analysis (Fig. 4A). Hybridization of EcoRI- and BamHI-digested genomic DNA implies that in 47-18crtO#51 and 47-18crtO#65, a single gene copy was inserted. In the host line 47-18, which was included as a negative control, no crtO signal was detected. The ketolase transcript was detectable by RNA analysis (Fig. 4B). In line 47-18crtO#65, the amount related to 16S rRNA was slightly higher than in line 47-18crtO#51. As expected, no signal was obtained for 47-18.
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Modification of the carotenoid composition in the photosynthetic apparatus may lead to a functional impairment of photosynthesis (Sandmann, 2001b). Therefore, pigment composition and photosynthetic efficiency of the leaves of the ketocarotenoid-producing lines were analysed. The amount of total carotenoids was very similar in all lines including the non-transgenic wild type (Table 2). Only in 47-18crtO#51 and 47-18crtO#65 were the ketocarotenoids 4-ketozeaxanthin, 3'-hydoxyechinenone, and echinenone formed. The latter was the most abundant ketocarotenoid. In all lines, the chlorophyll content was unchanged and the photosynthetic efficiency was also the same regardless of the presence or absence of ketocarotenoids. This finding indicates that ketocarotenoids of up to
12% of total leaf carotenoids do not interfere with the primary reactions of the photosynthetic apparatus. Ketocarotenoid composition was qualitatively and quantitatively different from that in the tuber. Echinenone was the major ketocarotenoid in leaves. This finding is similar to the situation in tobacco and tomato leaves transformed with a bacterial crtW gene (Ralley et al., 2004). Ketolutein, which is the main ketocarotenoid in Arabidopsis seed, was absent in potato leaves, although large amounts of lutein are present as a potential substrate (Table 1).
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In conclusion, it was possible to engineer potato tubers with a cyanobacterial ketolase gene for the production of astaxanthin and other ketocarotenoids. This is a successful example of the genetic manipulation of a staple crop improving its food quality by enrichment with antioxidative ketocarotenoids. Concerning potato as an astaxanthin production system, the present work is regarded as a proof of concept that such an approach is feasible. However, further attempts should be made to overcome the limitation in astaxanthin formation in potato tubers and to reach complete ketolation of both ß-ionone end groups of ß-carotene. A suggested strategy is to change the activities of ß-carotene 3-hydroxylation and 4-ketolation towards the latter.
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