JXB Advance Access originally published online on March 26, 2004
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Journal of Experimental Botany, Vol. 55, No. 399, pp. 975-982, May 1, 2004
© 2004 Oxford University Press
Cell and Molecular Biology, Biochemistry and Molecular Physiology |
Carotenogenesis during tuber development and storage in potato
Received 25 November 2003; Accepted 30 January 2004
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK
* To whom correspondence should be addressed. Fax: +44 (0)1382 562426. E-mail: mtaylo{at}scri.sari.ac.uk
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Abstract |
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Germplasm of Solanum tuberosum and Solanum phureja exhibit a wide (over 20-fold) variation in tuber carotenoid content. The levels of carotenoids during tuber development and storage were compared in a high carotenoid-accumulating S. phureja accession (DB375\1) with two S. tuberosum cultivars (Pentland Javelin and Desiree) that accumulate lower levels of tuber carotenoid. On a dry weight basis, total carotenoid levels were at a maximum early in tuber development. However, in the S. phureja accession, carotenoid levels remained at a high level throughout tuber development, whereas in the S. tuberosum accessions, carotenoid content decreased as dry weight increased. The carotenoid profiles of tissues during tuber development were analysed in greater detail by reverse phase HPLC. In S. phureja tubers at maturity the major carotenoids were zeaxanthin, antheraxanthin, and violaxanthin. Following 9 months storage at 4 °C the levels of zeaxanthin and antheraxanthin decreased, whereas the level of lutein increased; overall, however, there was only a small decrease in total carotenoid content. In order to explore the reasons for the wide variation in tuber carotenoid content, the expression patterns of the major genes encoding the enzymes of the carotenoid biosynthetic pathway were compared. Significant differences in the profiles were detected, suggesting that transcriptional control or mRNA stability gives rise to the large differences in tuber carotenoid content. In particular, there was an inverse trend between the level of zeaxanthin epoxidase transcript level and tuber carotenoid content in a range of potato germplasm, giving rise to an hypothesis for the regulation of carotenogenesis in potato tubers.
Key words: Carotenoid, gene expression, potato, storage, tuber, zeaxanthin epoxidase.
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Introduction |
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The flesh colour of potato tubers is due to the accumulation of two different classes of pigment. Whereas anthocyanin accumulation leads to red, blue or purple flesh colours (Hung et al., 1997), carotenoid levels determine whether the tuber flesh is white, yellow or orange. Several studies have characterized tuber carotenoid content from a range of potato germplasm. In diploid progeny of crosses of accessions of Solanum stenotomum and Solanum phureja, Brown et al. (1993) correlated the tuber orange flesh trait in potato with the content of zeaxanthin. In a different hybrid population of Solanum phureja crossed with Solanum stenotomum, progeny with yellow-fleshed tubers were obtained. In these tubers, lutein-5,6-epoxide and lutein were the major carotenoids with much lower levels of zeaxanthin (Lu et al., 2001). The total tuber carotenoid content reached up to 1435 µg 100 g–1 FW compared with typical carotenoid levels of c. 10 000 µg 100 g–1 FW in carrot taproot (Simon and Wolff, 1987).
Tuber carotenoid content in Solanum tuberosum cultivars has also been measured (Breithaupt and Bamedi, 2002; Iwanzik et al., 1983) and is generally much lower than that found for the high carotenoid S. phureja x S. stenotomum crosses. For example, yellow-fleshed cultivars contain 58–175 µg 100 g–1 FW carotenoid and white-fleshed cultivars contain 38–62 µg 100 g–1 FW carotenoid. The main carotenoids of Solanum tuberosum tubers are violaxanthin, antheraxanthin, lutein, and zeaxanthin, although the ratios of these carotenoids vary between cultivars. Carotenoid esters in tubers from some S. tuberosum cultivars can reach significant levels (up to 131 µg 100 g–1 FW; Breithaupt and Bamedi, 2002).
Genetic studies have attributed the orange flesh phenotype (that is, high tuber zeaxanthin) to the presence of an allele at the Y locus designated Or which is dominant over Y and y, which control yellow and white flesh, respectively. The Y gene has been mapped to chromosome three (Bonierbale et al., 1988). Other modifying genes are also important in controlling carotenoid content, as large variations in yellow-flesh intensity have been observed in populations that segregate for flesh colour (Bonierbale et al., 1988). The identity of the potato Y gene remains to be determined unequivocally. On the basis of the genetic map location of carotenoid biosynthetic genes in tomato and pepper, two candidate genes that map close to the Y locus have been identified; a gene encoding phytoene synthase and one encoding ß-carotene hydroxylase (Thorup et al., 2000). Although these two genes are good candidates for the Y gene, other unmapped alleles or linked regulatory elements may also be Y.
There is a growing awareness of the health benefits of carotenoids in the diet with different carotenoids having different beneficial properties. For example, lutein and zeaxanthin are the major pigments of the yellow spot in the human retina (Landrum and Bone, 2001). These pigments protect the retina from damage by blue light and possibly singlet oxygen. Age-related macular degeneration is the major cause of blindness in the elderly. However, a high dietary intake of zeaxanthin and lutein can protect against this disease (Seddon et al., 1994). The major dietary sources of lutein are dark green leafy vegetables, but zeaxanthin is found in significant levels in relatively fewer dietary components including some maize cultivars (Quackenbush et al., 1963) and yellow/orange pepper varieties (Minguez-Mosquera and Hornero-Mendez, 1994). The identification of significant levels of carotenoid within tubers from potato germplasm has provided the impetus to optimize carotenoid levels using both breeding (Brown et al., 1993; Lu et al., 2001) and transgenic strategies (Romer et al., 2002). Recently, tuber-specific down-regulation of the zeaxanthin epoxidase gene in Solanum tuberosum cultivars has been described (Romer et al., 2002). As well as increasing the amount of zeaxanthin that accumulated, surprisingly, the total levels of carotenoid increased by up to 5.7-fold compared with control tubers. These findings highlight the potential to manipulate both total carotenoid content and the type of carotenoids that are produced in potato tubers and are particularly significant in view of the relatively few sources of dietary zeaxanthin. In addition, they demonstrate that the molecular and biochemical mechanisms that control carotenogenesis remain to be clarified in detail and are an important prerequisite to both conventional breeding and further transgenic improvement. Attempts to manipulate transgenically, the carotenoid content of other plant species, whilst achieving success in many instances, (reviewed by Sandmann, 2001) have also raised awareness that control of carotenogenesis is not fully understood, for example, in tomato fruit (Romer et al., 2000) and rice endosperm (Ye et al., 2000).
In an attempt to gain insight into the key control factors in potato, carotenoid content has been measured during tuber development and storage. In addition, steady-state transcript levels of the major carotenogenic genes from a range of germplasm with low and high tuber carotenoid levels have been compared.
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Materials and methods |
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Growth of tubers and developmental stages
Solanum tuberosum L. (cultivars Pentland Javelin and Desiree) and Solanum phureja accession DB375\1 were grown from seed tubers in 30 cm diameter pots containing compost. Genotype DB375\1 was selected as a long-day adapted population and was subsequently named Inca Dawn (UK plant variety rights reference AFP 4/654). Seed stocks can be requested from the Scottish Agricultural Science Agency, Edinburgh. Plants were raised in a glasshouse maintained at a daytime temperature of 20 °C and a night-time temperature of 15 °C. The maximum irradiance was approximately 10 500 µmol m–2 s–1 and the mean day length was 16 h. Plants were harvested between 28 d and 80 d after planting to obtain stolons/tubers (tuber defined as a swelling at least twice the diameter of the stolon to which it is attached) at various developmental stages. The following developmental stages were harvested; non-swelling stolon tips (3–5 mm in length), swelling stolon tips (3–5 mm in length), small developing tubers (5–10 mm in diameter), and mature tubers (100–150 mm in diameter). Mature detached tubers that had been held at 4 °C in the dark for between 6 and 9 months were defined as stored tubers.
Expression profiling
Northern blot analysis: Northern blots were performed using total RNA from four tuber developmental stages of potato varieties S. tuberosum L., cv. Pentland Javelin, S. tuberosum L., cv. Desiree, and S. phureja DB375\1. Total RNA was isolated using a Qiagen RNA isolation kit (Qiagen GmbH, Hilden, Germany) and 10 µg underwent denaturing agarose gel electrophoresis. Nucleic acids were transferred to positively charged nylon membrane (Hybond-N+, Amersham Biosciences) as previously described (Sambrook et al., 1989). Filters were probed with random-primed (HiPrime, Boehringer-Mannheim) 
-32P dCTP labelled DNA for 16 h at 42 °C in NorthernMaxTM ultrahyb buffer (Ambion Inc., Austin, Texas). A potato 18S ribosomal RNA probe was used as a control. Filters were washed in low and high stringency buffer at 42 °C (according to Ambion protocol) until an acceptable signal-to-noise ratio was achieved. Relative gene expression was determined by autoradiography.
Quantitative RT-PCR: Total RNA (10 µg) was treated with DNase I (Ambion Inc., Austin, Texas) before undergoing reverse transcription, using random hexamers as primer and SuperScriptTM II reverse transcriptase (Invitrogen Life Technologies, Carlsbad, California, USA), to generate the first strand cDNA template. Potato 18S ribosomal RNA primers were used as a control. Samples were amplified using a Perkin Elmer ABI Prism 7700 sequence detector in conjunction with the Quantitect SYBR green PCR kit (Qiagen GmbH, Hilden, Germany). Thermal cycling conditions were: 15 min denaturation at 95 °C followed by 40 cycles (15 s at 95 °C, 30 s at 58 °C, 30 s at 72 °C). Relative expression levels were calculated and the primers validated using the 
Ct method (http://www.appliedbiosystems.com) using data obtained with the 18S ribosomal RNA-specific primers as an internal control.
cDNA cloning and sequencing
S. phureja DB375\1 tuber total RNA was treated with DNase I and reverse transcribed using Superscript II reverse transcriptase to generate a first strand cDNA template. Primers were designed to publicly available sequences of the carotenoid biosynthetic genes listed in Table 2 of the Results. PCR fragments were amplified in 20 µl PCR reactions containing 25–50 ng of template tuber cDNA, in the presence of 20 mM TRIS-HCl (pH 8.4), 2.5 mM MgCl2, 50 mM KCl, 200 nM of each gene specific primer, 100 µM of each dNTP, and 0.5 units of Taq DNA polymerase (Promega). Thermal cycling conditions were: 2 min denaturation at 95 °C followed by 32 cycles (30 s at 95 °C, 1 min annealing at the appropriate Tm, 1 min at 72 °C) followed by 5 min final extension step at 72 °C. PCR fragments were gel purified using the QIAquick gel extraction kit (Qiagen, GmbH, Hilden, Germany) and subsequently cloned into the T/A cloning vector pGEM-T easy (Promega). The authenticity of cloned PCR fragments was verified by sequencing. A full length phytoene synthase cDNA was cloned from a DB375\1 tuber cDNA library (Stratagene) using a PCR-based screening technique (Israel, 1993). Subsequent in vivo excision of the positive clone was carried out following the manufacturer’s protocol (Stratagene). Sequencing reactions were performed using an Applied Biosystems Perkin Elmer Big Dye Terminator cycle sequencing kit (PE Biosystems, Warrington, UK), M13 universal forward and reverse primers, and an ABI377 automated sequencer (PE Biosystems, LaJolla, California, USA).
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Carotenoid analysis
Extraction of carotenoids: Tissue samples (the apical 5 mm of stolon tips (pooled samples of 10–20 stolon tips) or peeled whole tuber samples (pooled samples of three tubers)) of the three varieties, at various stages of tuberization, were freeze-dried and stored at –80 °C prior to analysis. Approximately 250 mg of powdered freeze-dried material was mixed with
200 mg acid-washed sand (to aid in the release of cell contents), anhydrous sodium sulphate (to remove any residual water), and sodium bicarbonate (to neutralize any acids that may otherwise result in the production of undesirable artefacts). This mixture was ground in c. 5 ml acetone using a mortar and pestle and subsequently transferred, with washings (3x2 ml), to a 15 ml polypropylene centrifuge tube. Samples were centrifuged at 4 °C for 5 min at 4000 rpm and the supernatant passed through a 0.2 µm Whatman anotop-10 filter into a 15 ml graduated tube. The volume was recorded and a 1 ml aliquot taken for spectrophotometric determination at 450 nm. The remaining sample was dried under a gentle stream of oxygen-free nitrogen and stored at –80 °C prior to HPLC analysis. All manipulations were performed on ice and under subdued artificial light conditions. Saponification of carotenoid extracts: The dried carotenoid-containing extract, from approximately 250 mg freeze-dried material, was redissolved in 5 ml of methanolic potassium hydroxide solution (10%, w/v) and transferred to a 20 ml glass Quickfit tube. The solution was then allowed to stand overnight under nitrogen at 4 °C in the dark. Carotenoids were then purified by the addition of 5 ml degassed diethyl ether, followed by saturated aqueous NaCl (c. 5 ml) until two layers formed. The aqueous phase was re-extracted with ether and the combined ethereal extracts washed with water until free from alkali. The samples were then dried under a gentle stream of oxygen-free nitrogen (Britton, 1985).
Determination of total carotenoid concentration: Total carotenoid concentration was determined by spectrophotometry as described previously by Britton et al. (1995).
HPLC analysis: The carotenoid standards ß-carotene, violaxanthin, lutein, and neoxanthin were isolated from rocket lettuce by open column chromatography (Kimura and Rodriguez-Amaya, 2002). Zeaxanthin was kindly provided by Professor Andrew Young (Liverpool John Moores University). Separation was performed on a Gilson system controlled by Gilson Unipoint v2.10 software package using a 5 µm, 250x4.6 mm reverse phase C18 column (Phenomenex spherisorb ODS2). A binary solvent gradient of 0–40% B (0–20 min), 40–60% B (20–25 min), 60–100% B (25– 25.1 min), 100% B (25.1–35 min), 100–0% B (35–35.1 min) at a flow rate of 1.0 ml min–1 was used (A, acetonitrile/water (9:1 v/v); B, ethyl acetate). The column was allowed to re-equilibrate in 100% A for 10 min between injections. Peak responses were determined using a variable wavelength UV-Vis detector set at 460 nm. Compounds were identified by co-chromatography with standards and by elucidation of their spectral characteristics using a photo-diode array detector. Individual carotenoid concentrations were calculated by comparing their relative proportions, as reflected by integrated HPLC peak areas, to total carotenoid content determined by spectrophotometry. Commercially obtained carotenoid standards (Sigma-Aldrich) were also subjected to the preparation protocol described above to determine the percentage recoveries. The following recovery percentages were obtained; lutein, 84.22; trans-ß-apo-8'-carotenal, 84.65; astaxanthin, 92.04; and ß-carotene, 77.24.
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Results |
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Total tuber carotenoid content during tuber development and storage
Several studies have reported the carotenoid levels in mature tubers from a range of potato germplasm (Iwanzik et al., 1983; Brown et al., 1993; Lu et al., 2001; Breithaupt and Bamedi, 2002). In this study the authors wished to investigate carotenoid content and balance during tuber initiation, development, and storage. In order to gain insights into mechanisms that control tuber carotenoid accumulation, carotenoid profiles were compared from germplasm that accumulated carotenoids to different extents. As an example of a high tuber carotenoid plant an accession of S. phureja (DB375\1) was chosen. The carotenoid accumulation profiles in two S. tuberosum cultivars; Desiree, which has cream/yellow-fleshed tubers indicative of moderate tuber carotenoid content and Pentland Javelin which has white-fleshed tubers and low tuber carotenoid content, were also determined.
On a dry weight basis, total tuber carotenoid content decreased in Pentland Javelin and Desiree tubers as tuberization and tuber development progressed, whereas in the DB375\1 tubers, tuber carotenoid content increased slightly on tuberization and did not change significantly during tuber development (Table 1). Thus, in the Pentland Javelin and Desiree tubers, carotenoid accumulation proceeds at a lower rate than increases in tuber dry matter and the differences between the white-fleshed and orange-fleshed tuber carotenoid content are accentuated. The total tuber carotenoid content was also determined after 9 months of storage of detached tubers at 4 °C in the dark (Table 1). For DB375\1 tubers there was a small decrease in the mean carotenoid content, although analysis of variance showed that this decrease was not significant at the 95% level. Thus tuber carotenoids are stable during tuber storage. The levels of total carotenoid in tubers of Desiree and Pentland Javelin also showed relatively small changes during cold storage.
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Tuber carotenoid content was then investigated in more detail by reverse phase HPLC analysis in order to identify and quantify individual carotenoid levels during tuber development and storage (Fig. 1). Peaks were identified on the basis of retention time and spectra compared with standards. The main carotenoids in mature DB375\1 tubers (Fig. 1A) were zeaxanthin (51% of total), antheraxanthin (25%), violaxanthin (11%), an unidentified carotenoid (8%), and lutein (3%). In mature tubers c. 15% of the carotenoids were esterified. During 9 months of storage of detached tubers at 4 °C in the dark, whilst there was only a small decrease in total tuber carotenoid content, there were larger changes in the levels of the carotenoid components. Specifically, the quantity of carotenoid esters increased from 5.5 µg g–1 FDM to 9.5 µg g–1 FDM. Zeaxanthin and an unidentified carotenoid were the major esterified carotenoids, accounting for 47% and 36% of total esters, respectively. During tuber storage, zeaxanthin and antheraxanthin levels decreased by 11% and 7%, respectively, and the levels of lutein and an unidentified carotenoid both increased by 6%.
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In Solanum tuberosum cv. Desiree mature tubers, the major carotenoids were violaxanthin (51% of total), lutein (20%), neoxanthin (11%), and antheraxanthin (8%) with 23% present in the esterified form (Fig. 1B). In stored Desiree tubers there was also an increase in the level of esterified carotenoids (up to 45% of the total carotenoid). As with the DB375\1 tubers, there were also marked differences in the levels of individual carotenoids during tuber storage. The main changes were a decrease in the level of violaxanthin and an increase in lutein.
Profiling of gene expression during carotenogenesis in tubers
Mature tubers of DB375\1 contained 22-fold higher levels of total carotenoid than Pentland Javelin tubers. The authors wished to determine whether this large difference in carotenoid content was reflected in the transcript levels of the genes encoding the carotenogenic enzymes. Gene sequences exist for many of the carotenogenic genes, including many potato EST sequences with high degrees of sequence identity to known carotenogenic genes. In some plants, the carotenogenic genes form gene families with different members expressed in different tissues. For example, in tomato there are two genes for PSY; Psy-1 encodes a fruit- and flower-specific isoform whereas Psy-2 encodes a green tissue-specific form (Fraser et al., 1994). For the expression work in this study, sequences encoding the tuber-specific isoforms were required for probes for northern analysis or for primer design for quantitative RT-PCR analysis. For this reason sequences from potato tuber (S. phureja DB375\1) cDNA were amplified by RT-PCR using primers designed to conserved regions in available sequences (Table 2). The sequences of all the potato tuber cDNAs were very similar (at least 92% identical) to previously cloned gene sequences available in the EMBL database. A full-length cDNA encoding phytoene synthase was cloned from a DB375\1 tuber library. Its sequence was 96% identical to the tomato Psy-2 cDNA (Fraser et al., 1994).
Transcript levels were determined in stolon tips prior to any visible signs of tuberization, stolons that had visibly commenced tuberization, developing tubers (5–10 mm in diameter), and mature tubers (100–150 mm diameter) in each of the three potato types under study. It was not possible to detect some transcripts by northern analysis and so more sensitive quantitative real-time PCR was employed as an alternative (for DXS, GGPS, PSY, VDE, and NXS, Fig. 3). Transcript levels were similar in the three accessions, for many of carotenogenic genes including those encoding 1-deoxy-D-xylose-5-phosphate reductoisomerase, 
-carotene desaturase, lycopene-ß-cyclase, ß-carotene hydroxylase, and neoxanthin synthase (Figs 2, 3). Some genes were maximally expressed at different stages of tuberization. For example, phytoene desaturase was maximally expressed in swelling stolons of DB375\1 whereas, for Pentland Javelin and Desiree, the peak in transcript level occurred in developing tubers. For lycopene 
-cyclase however, higher transcript levels were detected in tissues of swelling stolons and developing tubers from the DB375\1 tubers than in either the Desiree or Pentland Javelin tubers (Fig. 2). Phytoene synthase was also expressed at higher levels than in Desiree or Pentland Javelin, with the greatest difference being apparent in the early stages of tuber development (swelling stolon stage; Fig. 3).
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Surprisingly, the transcript levels of some of the genes of the carotenogenesis pathway are higher in the lower carotenoid tubers than in the DB375\1 tubers. For example, DXS transcript level was much higher in the early tuberization stages of both Pentland Javelin and Desiree tissue than in the corresponding stages for DB375\1 (Fig. 3). The level of GGPS transcript was also higher in some Pentland Javelin and Desiree tissues than in that of DB375\1 (Fig. 3). Zeaxanthin epoxidase transcript level was also consistently lower in the DB375\1 tissues than in those from Pentland Javelin or Desiree (Fig. 2). The zeaxanthin epoxidase transcript level was also determined in tubers from a wider range of potato germplasm in order to determine the degree of correlation between transcript level and carotenoid content. An inverse relationship between the zeaxanthin epoxidase transcript level and the total tuber carotenoid content was detected (Fig. 4).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In this study, a detailed characterization of carotenogenesis has been carried out in potato tubers during tuber development and storage. The high tuber carotenoid S. phureja accession studied (DB375\1) predominantly accumulates zeaxanthin, characteristic of the orange-flesh phenotype. The total levels of carotenoid, when expressed on a fresh weight basis are similar to the total values obtained by Brown et al. (1993) and Lu et al. (2001) for their high tuber carotenoid lines obtained from crosses between S. phureja and S. stenotomum germplasm. These measurements have been extended to demonstrate that whilst the total tuber carotenoid content remains stable during tuber storage (an important nutritional consideration) there is a shift in the types of carotenoid that accumulate during tuber storage. Increases in the levels of lutein and an unidentified carotenoid occur as the levels of zeaxanthin and antheraxanthin decrease. Although there is no direct evidence for an interconversion of lutein and zeaxanthin, it has been speculated that such a mechanism may exist in some mammalian cells (Kachik et al., 1997). There were also significant increases in the proportion of esterified carotenoids during tuber storage. Thus the results presented here highlight the need to define tuber storage conditions when presenting results on tuber carotenoid content and it remains to be determined whether there are changes in the carotenoid balance on storage of other vegetables. The levels and types of tuber carotenoids measured in two S. tuberosum cultivars were typical for those found in yellow and white-fleshed S. tuberosum cultivars (4.91 µg g–1 DW for cream/yellow-fleshed Desiree mature tubers and 1.63 µg g–1 DW for mature tubers from white-fleshed Pentland Javelin). Again there were changes in the components of the total carotenoid content on storage, including an increase in the level of lutein, a decrease in the level of violaxanthin, and an increase in the level of carotenoid esters (Fig. 1b).
Recent epidemiological studies (http://www.aoanet.org/conditions/pdf/) have indicated that the inclusion of 5.8 mg of zeaxanthin in the diet is sufficient to provide significant protection against the onset of age-related macular degradation, a major cause of blindness in the elderly. The level of zeaxanthin in tubers of DB375\1 (at harvest) was c. 23 µg g–1 DW (therefore approximately 1 kg FW contains 5.8 mg zeaxanthin) and so including these tubers in a normal diet could provide biologically meaningful amounts of zeaxanthin. In two transgenic lines engineered for high zeaxanthin content, the zeaxanthin levels were even higher in mini tubers, reaching 40.1 µg g–1 DW (Romer et al., 2002). It should be noted that these transgenic manipulations were carried out in S. tuberosum cultivars Baltica and Freya which contain lower total carotenoid contents than the high carotenoid S. phureja lines. It is therefore important to carry out these genetic manipulations using parental material with an elevated carotenoid content.
Although genetic approaches to determine the key genes that control tuber carotenoid content are in progress, there are no reports of comparative transcript profiling of the carotenogenic genes as a means of indicating carotenogenic mechanisms in potato tubers. As germplasm with a wide variation in tuber carotenoid content had been identified, and the genes of the carotenoid pathway are available or were easily obtainable, this approach was considered to be timely. Some of the results obtained from this profiling study were not anticipated. For example, some of the genes encoding enzymes of the DOXP pathway (DXS and GGPS Fig. 3), prior to the committed step of carotenoid biosynthesis were more highly expressed in S. tuberosum tubers with the lower carotenoid content than in the high carotenoid DB375\1 tubers. The products of the DOXP pathway are precursors for a range of biosynthetic pathways (including chlorophylls, gibberellins, tocopherols, and quinones), providing a possible explanation for this result. Phytoene synthase is the first committed step in the carotenoid biosynthetic pathway and its activity is rate-limiting in several carotenogenic tissues (for example, tomato fruit, Fraser et al., 2002; canola seeds, Shewmaker et al., 1999; marigold petals, Moehs et al., 2001). The higher phytoene synthase transcript level in the early stages of tuberization in DB375\1 suggests that phytoene synthase activity is also an important determinant of final tuber carotenoid content levels. Interestingly, there was a large increase in the transcript level of the lycopene -cyclase in the DB375\1 tubers compared with those from Desiree and Pentland Javelin, and this may lead to the higher lutein level that accumulates in these tubers. Perhaps the most interesting outcome of the expression profiling study was the level of the zeaxanthin epoxidase transcript. This transcript level was significantly lower (c. 4-fold) in DB375\1 tubers than in white-fleshed tubers. The inverse relationship between zeaxanthin transcript level and total carotenoid content was evident in a range of potato germplasm (Fig. 4). Thus the effects of transgenic down-regulation of ZEP transcript level, that is, higher zeaxanthin and total carotenoid accumulation (Romer et al., 2002) are reflected in the relationship between ZEP transcript level and carotenoid content that occurs naturally in potato germplasm. The question remains as to the mechanism by which lowered ZEP transcript level stimulates carotenogenesis. One possibility is that reduced ZEP activity restricts the supply of precursors for ABA biosynthesis and the plant responds by increasing carotenogenic metabolic flux to compensate for this restriction. For tubers that accumulate predominantly lutein, a similar mechanism may operate, but, in this case, limiting activities may occur at other points in the carotenoid biosynthetic pathway (for example, at ß-carotene hydroxylase). These hypotheses are currently being tested.
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Acknowledgements |
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This work was funded by the Scottish Executive Environment and Rural Affairs Department. We gratefully acknowledge the help and advice of Professor A Young, and Dr D Phillip, John Moores University, Liverpool for assistance in measuring carotenoids, and Finlay Dale (SCRI) for providing DB375\1.
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References |
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