Carotenoids are a major group of isoprenoids, widely distributed in nature . Several of them are reported to have health benefits , most notably reduction in the incidence of prostate cancer from dietary lycopene  and alleviation of vitamin A deficiency by β-carotene, which is pro-vitamin A . Deficiency of vitamin A causes xerophthalmia, blindness and premature death, especially in children aged 1–4 . Since humans cannot synthesise carotenoids de novo, these health-promoting compounds must be taken in sufficient quantities in the diet. Consequently, increasing their levels in fruit and vegetables is beneficial to health. Tomato products are the most common source of dietary lycopene. Although ripe tomato fruit contains β-carotene, the amount is relatively low . Therefore, approaches to elevate β-carotene levels, with no reduction in lycopene, are a goal of plant breeders. One strategy that has been employed to increase levels of health promoting carotenoids in fruits and vegetables for human and animal consumption is genetic modification .
Lycopene, an acyclic carotenoid and precursor of β-carotene, is formed from the sequential desaturation of phytoene. These reactions are catalysed by the enzymes phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS). The desaturation sequence occurs in the cis geometric isomer configuration and the action of a carotene isomerase (CRTISO) converts either cis-neurosporene or poly-cis lycopene to all-trans lycopene prior to cyclisation . In green tissues, the action of light and chlorophyll are believed to overcome the necessity for CRTISO activity . Cyclisation of lycopene yields β-carotene, via the action of a β-cyclase. In tomato, two lycopene cyclase are present: LCYB is believed to predominate in the formation of vegetative carotenoids, whilst the CYCB gene shows ripening specific expression and CYCB is thus associated with β-carotene production during ripening . In vegetative tissues, ε-carotene is formed via the action of a ε-ring cyclase and β-ring cyclase, leading to the synthesis of lutein, via hydroxylation, which is the predominant vegetative carotenoid . The complete pathway is shown in Figure 1.
Since the herbicide 2-(4-chlorophenylthio) triethylamine (CPTA) is known to inhibit β-carotene formation in tomato fruit , the transgenic lines have been grown in its presence to determine if overexpression of LCYB can overcome this inhibition.
2 Materials and methods
2.1 Plant material
Tomato (Solanum lycopersicum cv Ailsa Craig) plants were grown in the glasshouse with supplementary lighting. Three plants per genotype were grown in a randomised manner concurrently with their respective backgrounds and fruit harvested at mature red ripe (7 days post breaker, dpb). Two or more fruits per plant were pooled to provide one replicate per plant and three per genotype.
2.2 Construction of the pVBLCYB vector
Digestion of pUC19 with Sma1 released the LCYB cDNA as a 1.6-kb fragment, including the sequence encoding the transit peptide, which was cloned into the Sma1 site of the pSP vector (Promega, Southampton, UK), containing the polyubiquitin promoter (UBQ3; At5g03240; UniProt KB-Q1EC66) from Arabidopsis thaliana, the nopaline synthase terminator, the nptII gene for kanamycin resistance, controlled by the AoPR1 promoter from Asparagus officinalis. The Age1 fragment was sub-cloned into the pVB6 plant binary vector and designated pVBLCYB (Figure 2). The vector/insert was sequenced to confirm the manipulations. E. coli strain XL1-Blue (Promega) was used in these experiments. Sequences of the tobacco and tomato LCYB are shown in Supplementary Material, Figure 1.
2.3 Transformation of tomato plants
Agrobacterium tumifaciens LBA 4404 was transformed with pVBLCYB by triparental mating, using the helper plasmid pRK2103 . Stem explants of tomato were transformed and plants regenerated as described previously . Kanamycin (50 mg/ml) and carbenicillin (250 mg/ml) were used to select resistant transformants.
2.4 DNA and RNA analyses
DNA was extracted for PCR analysis from leaf material (ca. 200 mg), as described previously . The presence of the transgene in the primary transformants (T0) was confirmed by PCR using primers designed to amplify a 500 bp internal fragment of LCYB. Southern blot analysis of the T0 and first-generation (T1) plants was carried out using the cetyltrimethylammonium bromide (CTAB) method . DNA was digested with the appropriate restriction enzymes, fragments separated in 1% (w/v) agarose gels and transferred to Hybond N+ (Amersham, Little Chalfont, UK) overnight in 20% saline sodium citrate (SSC) solution. 32P-labelled fragments (using the Stratagene Random primer labelling kit, Prime It II), corresponding to the LCYB and nptII regions of the transgene, were used to probe the filters. Hybridisation, washing and detection were performed as described in the Hybond N+ instructions. Northern blot analysis was carried out on tomato fruit of the To and T1 generations, harvested at 7 dpb . Total RNA (20 μg) from the fruit was electrophoresed on 1.4% agarose/formaldehyde gels and then transferred to Hybond-N+ in 20x SSC overnight. The 1.6-kb cDNA fragment was radiolabelled with 32P, as described above. Following pre-hybridisation, the filter was hybridised at 65°C in 7.5% SDS, 100 mM K phosphate buffer, pH 7.5. Each filter was washed for 15 min in 2× SSC, 0.5% SDS and then twice for 15 min with 1× SSC, 0.5% SDS and finally monitored for radioactivity for 24 h with a phosphorimager (MAcBAS V2.2, Fuji Film Ltd, Bedford, UK).
2.5 Treatment with CPTA
Sterilised seeds (ca. 30) from the homozygous T1 line C4S1-15 and azygous control were germinated on Murashige and Skoog (MS) agar containing the herbicide 2-(4-chlorophenylthio) triethylamine (CPTA, a gift from Prof. P. Böger) at 30 and 40 μM. The seedlings were grown in a controlled environment with a 16-h photoperiod and day and night temperatures of 23 and 19°C, respectively. They were assessed for germination and bleaching after 2 weeks and samples taken for isoprenoid analysis.
2.6 Analysis of isoprenoids
The isoprenoids of T0, T1 and T2 progeny of leaf and 7 dpb fruit were extracted, separated, identified and quantified by HPLC analyses, as described in .
3.1 Plants expressing the LCYB gene from tobacco showed increased levels of β-carotene in ripe fruit and changes to leaf carotenoids
Following transformation, progeny were selected on the basis of kanamycin resistance (nptII) and PCR analysis. During regeneration, growth and development of vegetative tissues from all transgenic lines appeared phenotypically normal. However, as fruit ripening commenced, virtually all transgenic lines showed a greater degree of orange colouration in their fruit compared to the control. Analysis of the T0 generation showed that increases in β-carotene content of the fruit were responsible for the altered colouration (Table 1). Of the To transformants, 85% exhibited a lycopene-to-β-carotene ratio within a range of 2:1 to 6:1, due to increases in β-carotene, with the wild-type (Ailsa Craig) ratio being 7.46:1. Analysis of the T1 and T2 generations established that the phenotype was inherited (Table 1).
Insertions at a single locus were determined by Southern blotting using a nptII probe. Of the 20 transformants, five lines were single copy, while the others had copy numbers ranging from 2 to 4 for the nptII gene (data not shown). Homozygous lines were generated from single-copy primary transformants, showing a range of β-carotene contents (603 C1S1, C1S2, C2S1 and C4S1, with lycopene-to-β-carotene ratios of 1.5, 2.99, 2.05 and 3.24, respectively; Table 1). Zygosity was assigned by Southern analysis using the nptII probe, co-segregation was assessed by expression of the transgene and inheritance of the phenotype by HPLC analysis. Co-segregation occurred in all cases and the high β-carotene phenotype was retained in the third generation (T2). As shown in Supplementary Material, Figure 2, the ratio of lycopene-to-β-carotene ratio differed between azygous, hemizygous and homozygous fruit. Although significant elevations in β-carotene were found in fruit of the transgenic lines, the content of biosynthetically related carotenoids such as phytoene, γ-carotene, δ-carotene, α-carotene, lutein, zeaxanthin, violaxanthin and neoxanthin was not altered. The pattern of β-carotene and lycopene geometric isomers found in the transgenic fruit were identical to the wild type (i.e. β-carotene 90–95% all-trans, 10–5% cis; all-trans lycopene 96–98%, 15-cis- lycopene 1–4% and 9-cis lycopene 0.6%). Alterations in other fruit isoprenoids were minor, with increases of 1.35-, 1.50- and 1.01-fold for tocopherols, plastoquinone and ubiquinone, respectively, in line 603 C4S1-15 (data not shown).
There were also changes to the carotenoid levels in transgenic leaves. For example, in the leaves of line 603 C4S1, all the carotenoids were increased, with lutein exhibiting the largest elevation (Table 2).
3.2 LCYB gene expression correlated with increased β-carotene levels in fruit
Northern blot analysis showed a positive correlation between the level of transgene expression and increased β-carotene formation (Figure 3). A comparison of expression between chloroplast-containing (leaf and green fruit) tissues and chromoplast-containing ripe fruit showed that although the UBQ3 promotor has been generally found to be constitutive in plants, LCYB transcript abundance was approximately 10-fold greater in ripe fruit (data not shown).
3.3 Transgenic lines showed tolerance to CPTA
A comparison of Ailsa Craig seedling and those of the transgenic line 603 C4S1-15, in the presence of CPTA, revealed dramatic differences in plant vigour. The control seedlings showed significant chlorosis, whilst those of the transformed line remained green (Supplementary Figure 3). Pigment analysis of the leaves revealed lower levels of carotenoids in the control (55% reduction at 40 μM CPTA), and qualitatively, β-carotene levels were some 4.3-fold less in the control leaves (Table 3). Increases in lycopene in these leaves were also observed. There was also an increase in lutein levels in 603 C4S1-15 in comparison to Ailsa Craig at both CPTA concentrations.
There have been several reports of using genetic modification for targeted and untargeted increases in carotenoid levels in crop plants, albeit with varying degrees of success . With respect to β-carotene, a modest, 1.5-fold, increase was achieved using the PSY-1 gene with a constitutive promoter (CaMV 35S) , whilst transformation with the Arabidopsis LCYB and PDS promoter elevated β-carotene some 7-fold . Alternatively, a bacterial PDS, CrtI resulted in a 3-fold increase, but a decrease in lycopene content . This was surprising, as CRTI catalyses lycopene, not β-carotene biosynthesis. Such an effect may be caused by a feed-forward mechanism for the upregulation of LCY or CYCB, as described previously in tomato fruit . Therefore, although increases in β-carotene were found, unintended perturbations to other carotenoids and related isoprenoids occurred, or else the total carotenoid content was reduced.
In contrast, in the present study, levels of β-carotene have been increased up to 6-fold, without a decrease in lycopene, perturbations to other fruit carotenoids, nor to the detriment of plant vigour. The phenotype was stable for at least three generations (Table 1). Therefore, the transgenic fruit contain an improved carotenoid profile compared with non-transgenic cultivars and introgression lines . This is the first report of using the UBQ3 promoter in tomato, although it has been used with other dicot plants . Although constitutive, the UBQ promoter gave differential expression levels of the transgene in vegetative and fruit tissues, with lower expression found in chloroplast-containing tissues that probably prevented the occurrence of gene silencing and/or co-suppression of the endogenous gene , since the homology of the tobacco and tomato β-cyclases is 87.2% (Supplemental Material, Figure 1). Since the endogenous LCYB gene is down-regulated during fruit ripening , co-suppression is not an issue. Despite the low expression in vegetative tissue, changes to carotenoids do occur (Table 2), most specifically a 4.4-fold increase in lutein, suggesting that LCYB catalyses the rate-limiting step in the formation of this xanthophyll.
Growth of the transgenic lines with CPTA did not cause changes to plant vigour, unlike the chlorosis shown with the wild type (Supplementary Material, Figure 3). Lycopene did not accumulate in the LCYB line treated with CPTA (Table 3). Tolerance to CPTA in the transgenic line may be due to the increase in LCYB protein levels, and/or because the tobacco LCYB is intrinsically more tolerant to CPTA than that of tomato. Therefore, such an approach is feasible for the production of a crop resistant to this herbicide and for screening putative bleaching herbicides that have different modes of action to CPTA . The increase in total carotenoids in the transgenic line is consistent with the report that CPTA modulates mRNA levels of carotenoid genes .
Nutritionally, an average ripe LCYB transgenic fruit contains virtually all the recommended daily allowance (RDA) for vitamin A compared to the wild type, which has about 39% of the RDA . For comparison, the crtI transgenic tomato contains similar levels of β-carotene (ca. 70% RDA), but only 50% of the wild type lycopene content. Thus, this new genotype not only has a significantly elevated provitamin A content, but maintains its level of lycopene, compared to the wild type.
In summary, expression of LCYB under the control of the UBQ3 promotor has resulted in the specific elevation of β-carotene in ripe tomato fruit, but no changes to other fruit carotenoids. There is much current debate concerning the acceptance of genetically modified plants. The present study has produced a tomato with the potential to reduce vitamin A deficiency and contribute towards a high antioxidant diet. Such germplasm will provide a valuable resource for further detailed analysis  and add to the genetic resources for nutritional benefit.
We are grateful to Dr Susanne Römer for providing the NtLCYB cDNA clone. The work was supported by the European Community FAIR Programme (# CT9616333). Syngenta Agrochemicals is gratefully acknowledge for the provision of glasshouse space and some laboratory facilities. We thank Karen Bacon and Carol Fisher for maintenance of the tomato plants and Mark Harker for some of the Southern analyses.
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Published Online: 2016-08-03
Published in Print: 2016-09-01