Z. Naturforsch. 2016; 71(9-10)c: 295–301 Louise Ralley, Wolfgang Schucha, Paul D. Fraser and Peter M. Bramley* Genetic modification of tomato with the tobacco lycopene β-cyclase gene produces high β-carotene and lycopene fruit DOI 10.1515/znc-2016-0102 and alleviation of vitamin A deficiency by β-carotene, Received May 18, 2016; revised July 4, 2016; accepted July 6, 2016 which is pro-vitamin A [4]. Deficiency of vitamin A causes xerophthalmia, blindness and premature death, espe- Abstract: Transgenic Solanum lycopersicum plants cially in children aged 1–4 [5]. Since humans cannot expressing an additional copy of the lycopene β-cyclase synthesise carotenoids de novo, these health-promoting gene (LCYB) from Nicotiana tabacum, under the control compounds must be taken in sufficient quantities in the of the Arabidopsis polyubiquitin promoter (UBQ3), have diet. Consequently, increasing their levels in fruit and been generated. Expression of LCYB was increased some vegetables is beneficial to health. Tomato products are 10-fold in ripening fruit compared to vegetative tissues. the most common source of dietary lycopene. Although The ripe fruit showed an orange pigmentation, due to ripe tomato fruit contains β-carotene, the amount is rela- increased levels (up to 5-fold) of β-carotene, with negli- tively low [1]. Therefore, approaches to elevate β-carotene gible changes to other carotenoids, including lycopene. levels, with no reduction in lycopene, are a goal of Phenotypic changes in carotenoids were found in vegeta- plant breeders. One strategy that has been employed to tive tissues, but levels of biosynthetically related isopre- increase levels of health promoting carotenoids in fruits noids such as tocopherols, ubiquinone and plastoquinone and vegetables for human and animal consumption is were barely altered. Transformants showed tolerance to genetic modification [6]. the bleaching herbicide β-cyclase inhibitor, 2-(4-chloro- Lycopene, an acyclic carotenoid and precursor of phenylthio) triethylamine. The phenotype was inherited β-carotene, is formed from the sequential desaturation of for at least three generations. phytoene. These reactions are catalysed by the enzymes Keywords: β-carotene; genetic modification; lycopene; phytoene desaturase (PDS) and ζ-carotene desaturase lycopene β-cyclase; Solanum lycopersicum. (ZDS). The desaturation sequence occurs in the cis geo- metric isomer configuration and the action of a carotene isomerase (CRTISO) converts either cis-neurosporene or Dedication: This work is dedicated to the memory of Professor Peter Böger, who kindly provided the opportunity for PDF and PMB to work poly-cis lycopene to all-trans lycopene prior to cyclisation in his laboratory on the mode of action of bleaching herbicides. [7]. In green tissues, the action of light and chlorophyll are believed to overcome the necessity for CRTISO activ- ity [8]. Cyclisation of lycopene yields β-carotene, via the 1 Introduction action of a β-cyclase. In tomato, two lycopene cyclase are present: LCYB is believed to predominate in the formation Carotenoids are a major group of isoprenoids, widely of vegetative carotenoids, whilst the CYCB gene shows distributed in nature [1]. Several of them are reported to ripening specific expression and CYCB is thus associated have health benefits [2], most notably reduction in the with β-carotene production during ripening [9]. In vegeta- incidence of prostate cancer from dietary lycopene [3] tive 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 veg- aPresent address: Fraunhofer Chile Research Foundation, Las etative carotenoid [10]. The complete pathway is shown in Condes, Santiago, Chile. Figure 1. *Corresponding author: Peter M. Bramley, School of Biological Since the herbicide 2-(4-chlorophenylthio) triethyl- Sciences, Royal Holloway University of London, Egham, amine (CPTA) is known to inhibit β-carotene formation in Surrey TW20 0EX, UK, E-mail: [email protected] Louise Ralley, Wolfgang Schuch and Paul D. Fraser: School of tomato fruit [11], the transgenic lines have been grown in Biological Sciences, Royal Holloway University of London, Egham, its presence to determine if overexpression of LCYB can Surrey TW20 0EX, UK overcome this inhibition. 296 Ralley et al.: Elevation of β-carotene in tomato fruit by genetic modification CH H3C CH3 3 CH3 x2 GGPP CH3 H3C PSY-1, PSY-2 H3C CH3 H3C CH3 15-cis Phytoene H3C CH3 CH H C 3 3 PDS CH3 H3C H3C CH3 H C CH 9. 15. 9′-tri-cis ζ-Carotene H3C 3 3 CH3 CH3 H3C CH3 H3C ZDS, CRTISO, light CH3 CH3 CH3 CH3 CH3 H3C All-trans Lycopene CH3 CH3 CH3 CH3 LCYE, LCYB LCYB, CYCB H C H C CH 3 H3C 3 CH CH3 3 H C CH CH 3 3 CH3 3 3 α-Carotene β-Carotene H C CH3 CH CH CH CH 3 CH H3C 3 3 3 3 CH3 CH3 3 CRTRB1, CYP97A, CYP97C CRTRB1, CYP97A, ZEP-1, NXS Lutein Zeaxanthin, Antheraxanthin, Violaxanthin, Neoxanthin Figure 1: Carotenoid biosynthesis in tomato. GGPP, geranylgeranyl diphosphate; PSY-1 and -2, phytoene synthase 1 and 2; PDS, phytoene desaturase; ZDS, ζ-carotene desaturase; CRTISO, carotene isomerase; LCYE, lycopene ε-cyclase; LCYB, lycopene β-cyclase; CYCB, fruit specific lycopene β-cyclase; CRTR-B1, β-carotene hydroxylase; CYP97A, P450 carotenoid β-ring hydroxylase; CYP97C, P450 carotenoid ε-hydroxylase; ZEP-1, zeaxanthin epoxi- dase; NXS, neoxanthin synthase. 2 Materials and methods the pSP vector (Promega, Southampton, UK), containing the polyubiquitin promoter (UBQ3; At5g03240; UniProt KB-Q1EC66) from Arabidopsis thaliana, the nopaline syn- 2.1 Plant material thase terminator, the nptII gene for kanamycin resistance, controlled by the AoPR1 promoter from Asparagus offici- Tomato (Solanum lycopersicum cv Ailsa Craig) plants were nalis. The Age1 fragment was sub-cloned into the pVB6 grown in the glasshouse with supplementary lighting. plant binary vector and designated pVBLCYB (Figure 2). Three plants per genotype were grown in a randomised The vector/insert was sequenced to confirm the manipula- manner concurrently with their respective backgrounds tions. E. coli strain XL1-Blue (Promega) was used in these and fruit harvested at mature red ripe (7 days post breaker, experiments. Sequences of the tobacco and tomato LCYB dpb). Two or more fruits per plant were pooled to provide are shown in Supplementary Material, Figure 1. one replicate per plant and three per genotype. 2.3 Transformation of tomato plants 2.2 Construction of the pVBLCYB vector Agrobacterium tumifaciens LBA 4404 was transformed Digestion of pUC19 with Sma1 released the LCYB cDNA as with pVBLCYB by triparental mating, using the helper a 1.6-kb fragment, including the sequence encoding the plasmid pRK2103 [12]. Stem explants of tomato were trans- transit peptide, which was cloned into the Sma1 site of formed and plants regenerated as described previously Ralley et al.: Elevation of β-carotene in tomato fruit by genetic modification 297 pVBLCY Age 1 Asc1 BamH1 Asc1 Age 1 UBQ3 LB AoPR1 nptIInos UBQ3 LYCB nonossRB Figure 2: Schematic diagram of the LCYB construct pVBLCY. UBQ3, ubiquitin three promotor; LCYB, N. tabacum lycopene β-cyclase; nptII, nopaline synthase terminator; ASe1, BamH1, Age1 restriction sites; LB, left border; RB, right border. [13]. Kanamycin (50 mg/ml) and carbenicillin (250 mg/ml) 2-(4-chlorophenylthio) triethylamine (CPTA, a gift from were used to select resistant transformants. 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. 2.4 DNA and RNA analyses They were assessed for germination and bleaching after 2 weeks and samples taken for isoprenoid analysis. DNA was extracted for PCR analysis from leaf material (ca. 200 mg), as described previously [13]. The presence of the transgene in the primary transformants (T0) was 2.6 Analysis of isoprenoids confirmed by PCR using primers designed to amplify a 500 bp internal fragment of LCYB. Southern blot analy- The isoprenoids of T0, T1 and T2 progeny of leaf and 7 dpb sis of the T0 and first-generation (T1) plants was carried fruit were extracted, separated, identified and quantified out using the cetyltrimethylammonium bromide (CTAB) by HPLC analyses, as described in [16]. method [14]. 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 3 Results citrate (SSC) solution. 32P-labelled fragments (using the Stratagene Random primer labelling kit, Prime It II), cor- 3.1 Plants expressing the LCYB gene from responding to the LCYB and nptII regions of the transgene, tobacco showed increased levels of were used to probe the filters. Hybridisation, washing and β + -carotene in ripe fruit and changes to detection were performed as described in the Hybond N leaf carotenoids instructions. Northern blot analysis was carried out on tomato fruit of the T and T generations, harvested at 7 dpb o 1 Following transformation, progeny were selected on the [15]. Total RNA (20 μg) from the fruit was electrophoresed basis of kanamycin resistance (nptII) and PCR analysis. on 1.4% agarose/formaldehyde gels and then transferred During regeneration, growth and development of veg- to Hybond-N+ in 20x SSC overnight. The 1.6-kb cDNA frag- etative tissues from all transgenic lines appeared pheno- ment was radiolabelled with 32P, as described above. Fol- typically normal. However, as fruit ripening commenced, lowing pre-hybridisation, the filter was hybridised at 65°C virtually all transgenic lines showed a greater degree of in 7.5% SDS, 100 mM K phosphate buffer, pH 7.5. Each filter orange colouration in their fruit compared to the control. was washed for 15 min in 2× SSC, 0.5% SDS and then twice Analysis of the T generation showed that increases in for 15 min with 1× SSC, 0.5% SDS and finally monitored 0 β-carotene content of the fruit were responsible for the for radioactivity for 24 h with a phosphorimager (MAcBAS altered colouration (Table 1).