PRESENCE OF TWO VARIANTS OF LYCOPENE β-cyclase GENE IN GENOMES OF CITRUS AND ITS RELATIVES

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1 Article DOI: /bbeq A&EB AGRICULTURE AND ENVIRONMENTAL BIOTECHNOLOGY PRESENCE OF TWO VARIANTS OF LYCOPENE β-cyclase GENE IN GENOMES OF CITRUS AND ITS RELATIVES Juan Xu 1, Nengguo Tao 2, Hongbo Cao, Qing Liu, Xiuxin Deng Huazhong Agricultural University, College of Horticulture and Forestry Science, Key Laboratory of Horticultural Plant Biology (Ministry of Education), Wuhan, P. R. China Correspondence to: Juan Xu Authors 1 and 2: equal contribution to this work ABSTRACT To illustrate the mechanism of lycopene accumulation in red-fleshed Cara Cara navel orange (Citrus sinensis Osbeck), the genes encoding citrus lycopene β-cyclases (CLCYbs) were compared among citrus accessions. Protein sequence alignment showed that CLCYbs could be classified into two types, CLCYb1s and CLCYb2s, based on the polymorphisms in the 4 th and the 50 th amino acids. Polymerase chain reaction - Restriction fragment length polymorphisms analysis (PCR-RFLP) further indicated that all studied eighteen accessions of citrus and its relatives (including four with lycopene accumulated flesh) could be classified into three groups. Pummelo (Citrus grandis), two grapefruits (C. paradisi), trifoliata orange (Pocirus trifoliata), two citranges (C. sinensis P. trifoliata) and kumquat (Fortunella crassifolia) are in Group-1, which are homozygous for CLCYb1s. Genomes in Group-2 and 3 are heterozygous for CLCYb1s and CLCYb2s. However, Group-2 includes two navel oranges and two sour oranges (C. aurantium), in which close amounts of Cslcyb1s and Cslcyb2s were amplified. Whereas Group-3 includes two lemons (C. limon), two mandarins (C. reticulata) and Calamondin, in which unequal and unstable levels of two variants were amplified. Southern blot analysis implied that CLCYbs had at least two copies in Cara Cara navel orange and other three tested genotypes. The data presented here indicated the irrelevancy of the two variants in their genomes to the accumulation of lycopene in the citrus fruits. Biotechnol. & Biotechnol. Eq. 2011, 25(3), Keywords: CLCYbs, citrus and its relatives, polymerase chain reaction - Restriction fragment length polymorphisms analysis (PCR-RFLP), southern blotting Abbreviations: CLCYbs: Citrus Lycopene β-cyclase genes; CPCYCb: Chromoplast Specific Lycopene β-cyclase gene in Carica papaya; CTAB: Cetyltrimethylammonium Bromide; Cycb: Chromoplast Specific Lycopene β-cyclase gene; Lcyb: Lycopene β-cyclase gene; Lcye: Lycopene ε-cyclase gene; PCR-RFLP: Polymerase Chain Reaction - Restriction Fragment Length Polymorphisms Analysis Introduction Carotenoids in fruit are important to human health, especially lycopene. As an effective antioxidant, lycopene plays a role in the prevention of cancer and chronic disease (24), while many other carotenoids serve as precursors of vitamin A (12). In the plastids of higher plants, lycopene is synthesized as an intermediate and a branch point in the carotenoids biosynthetic pathway. Genes encoding lycopene β-cyclase (LCYb) and lycopene ε-cyclase (LCYe) are involved in the biosynthesis of β-carotene and α-carotene (9, 10, 27). Downregulation of LCYb leads to the accumulation of lycopene in ripe tomato fruits (19). In tomato mutants like Beta and Oldgold, chromoplast specific lycopene β-cyclase (CYCb) was affected and lycopene production in fruits was largely altered (21). In watermelon (Citrullus lanatus (Thunb.) Matsum & Nakai), LCYb alleles may serve as genetic determinant for canary yellow or red flesh color (4, 5). Interestingly, in redfleshed papaya (Carica papaya), a frame-shift mutation and a premature stop codon happened in CpCYCb is the reason accounting for the fruit flesh color (7). Devitt et al. (11) also reported that a mutation in the lycopene β-cyclase 2 gene inactivates the enzyme activity, which results in lycopene accumulation in papaya fruit. However, in Crocus sativus, the strong expression of chromoplast-specific lycopene β-cyclases in flower stigmas activates and boosts beta-carotene accumulation (1). In citrus, diverse allelic LCYb and LCYe are reported tightly linked with the diversity of carotenoid accumulation (13, 14). According to our previous report, due to lycopene accumulation pink or red-fleshed fruits are found common in citrus, such as Cara Cara navel orange (26) and Honganliu sweet orange (17). Honganliu sweet orange is the pinkfleshed bud mutation of Anliu sweet orange, while Cara Cara navel orange is probably originated from Washington navel orange (23). Efforts have been made to elucidate the mechanisms underlying lycopene and other specific carotenoid accumulation in citrus fruits to enhance citrus fruit carotenoids production (2, 16, 17, 20, 25, 26). Lycopene accumulation in Star Ruby grapefruit during fruit maturation was associated with a substantial reduction in the expression of both lycopene β-cyclase 2 and β-carotene hydroxylase genes, and the former 2452 Biotechnol. & Biotechnol. Eq. 25/2011/3

2 Accessions of citrus and its relatives used in this study for different analyses TABLE 1 No. Common name Species Remarks 1 Newhall navel orange C. sinensis (L.) Osbeck 1 2 Skaggs Bonanza navel orange C. sinensis (L.) Osbeck 1 3 Cara Cara navel orange Citrus sinensis (L.) Osbeck 1 &2 Pink-fleshed 4 Washington navel orange C. sinensis (L.) Osbeck 2 5 Red Marsh grapefruit C. paradisi Macf. 2 Pink-fleshed 6 Star Ruby grapefruit C. paradisi Macf. 2 Pink-fleshed 7 Kao Pan pummelo C.grandis (L.) Osbeck 2 8 Variegated Pink Eureka C. limon Burmf. 2 Pink-fleshed 9 Eureka lemons C. limon Burmf Calamondin C. reticulata Blanco 2 11 Guoqing No.1 satsuma mandarin C. reticulata Blanco 2 12 Ponkan mandarin C. reticulata Blanco 2 13 Trifoliate orange Poncirus trifoliata (L.) Raf Carrizo Citrange C. sinensis (L.) Osb. P. trifoliata (L.) Raf Citrange C. sinensis (L.) Osb. P. trifoliata (L.) Raf Goutou sour orange C. aurantium L Sour orange C. aurantium L Meiwa kumquat Fortunella crassifolia Swingle 2 1. Southern blotting; 2, Polymerase chain reaction - Restriction fragment length polymorphisms (PCR-RFLP) analysis Primers used in this study for different analyses Primer code Nucleotide Sequence and theirs correspoding positions (GenBank Accession No. AY094582) Analysis that primers used in LCY1 5 -AGCTTCCCATGTATGACCCA Probe amplification LCY2 5 -GCAGCTAAAGTCCTTGCCAC Probe amplification LEFT 5 -ACGCAACACAAGCCTCATCT PCR-RFLP P2 5 -ACCGACAACTGCTAGGTCTAC PCR-RFLP TABLE 2 is chromoplast-specific with two alleles (3). Fanciullino et al. (15) reported that the carotenoid profiles of fruit of Cara Cara navel orange were irrelevant to changes in regulation of LCYb at the transcriptional level. In this study, two variants of LCYb were found in Cara Cara and Washington navel oranges respectively. Then a further investigation was made to find the relationship between lycopene accumultion and the presence of the two variants in citrus genomes. Materials and Methods Plant materials Eighteen accessions of citrus and its relatives were collected from the National Citrus Breeding Center, Huazhong Agricultural University, Wuhan, China (Table 1). Four of them, Cara Cara navel orange, Red Marsh grapefruit, Star Ruby grapefruit and Variegated Pink Eureka lemon, were pink-fleshed with high levels of lycopene (26). Biotechnol. & Biotechnol. Eq. 25/2011/3 Total DNA and RNA extraction and cdna synthesis Genomic DNA was extracted from leaves using a modified cetyltrimethylammonium bromide (CTAB) method (8). Total RNA was isolated using RNeasy Plant Mini Kit (QIAGEN, Hilden, Germany). First strand cdna was synthesized using RevertAid TM M MuLV KIT (MBI, Fermentas, Lithuania) according to the manufacturer s protocol. Primer synthesis and gene sequencing were carried out by Shenggong, Co. Shanghai, China. Polymerase chain reaction - Restriction fragment length polymorphisms (PCR-RFLP) analysis Polymorphism at the 4 th amino acid was found in deduced LCYbs encoded by the genes isolated from Cara Cara and Washington navel oranges genomes and a RsaI restriction site (GTAC) was generated due to the polymorphic change. PCR was performed with primers LEFT and P2 to amplify the region of 502 bp containing the polymorphisms from the genomes of 2453

3 sixteen accessions of citrus and its relatives (Skaggs bonanza and Newhall navel oranges were excluded). Thermocycles began at 95 C for 3 min, followed by 32 cycles of 1 min at 95 C, 40 s at 55 C and 1 min at 72 C. Final elongation was performed at 72 C for 5 min. The amplified products were then fully digested with five units of RsaI at 37 C for 10 h, seperated on agarose gel and visualized under UV light. Southern blotting Four citrus, Newhall navel orange, Cara Cara navel orange, Skaggs bonanza navel orange and Red Marsh grapefruit, were put for southern blot analysis. First, a partial coding region of LCYb was amplified from genomic DNA with primers LCY1 and LCY2 designed on the basis of conservative sequences of the gene (Table 2). PCR was programmed as follows: one cycle of 3 min at 95 C, 1 min at 65 C, 1 min at 72 C, followed by 28 cycles of 30 s at 95 C, 1 min at 65 C and 1 min at 72 C; and one final elongation at 72 C for 10 min. The amplified fragment (LCYF1) has no endonuclease restriction site of EcoRI and HindIII, and was electrophoresed in 1.5% (w/v) agarose gels, and purified with a DNA gel extraction kit (Shenggong, Co. Shanghai, China ) for sequencing and 32 p labeled as a probe in Southern blotting. Southern blotting was performed according to Sambrook et al. (22). Ten micrograms of genomic DNA were digested overnight with 50 units of EcoRI and HindIII, respectively. Digested DNA was resolved on an 0.8% agarose gel in 1 TAE buffer, and blotted onto a Hybond-N + membrane (Amersham, N J, USA) for 16~20 h. The blot was hybridized with 32 P labeled LCYF1 at 65 C overnight, washed with 0.1 SSC, 0.1% (w/v) SDS at 65 C for 2 h, and exposed to X-ray film at -70 C for 5~10 d. Bioinformatics analysis SEWER was employed in merging partial overlapping nucleotide sequences and in analyzing endonuclease restriction site. BLAST and ORF finder in GenBank were used for homology search and amino acid deduction. Sequence alignment of amino acids was carried out by using ClustalW ( Results and Discussion Amino acid polymorphisms in citrus LCYbs Two variants of LCYbs were previously isolated from Cara Cara navel orange (GenBank accession number: AY094582, CLCYb1) and Washington navel orange (GenBank Accession number: AY644699, CLCYb2) via thermal asymmetric interlaced PCR technology and normal PCR by our two team members, respectively. CLCYb1 (AY094582) is 3,313 bp long, whereas CLCYb2 is 1,900 bp; both contain an intact ORF, encoding 504 amino acids. Comparison between CLCYb1 and CLCYb2 revealed differences in the 4 th, 38 th, 50 th and 164 th amino acid residues (Fig. 1, aam and aau , respectively). The 50 th residue was a non-conservative replacement of arginine (R) in CLCYb1 by methionine (M) in CLCYb2, due to a single base change from T to G. The 50 th and 164 th residues were included in a putative N-glycosylation site and a tyrosine kinase phosphorylation site, respectively. As reported in lycopene ε-cyclase in lettuce (10) and in the light-harvesting complex of photosystem II in higher plants (18), amino acid substitution in a protein can significantly affect protein function. However, less β-carotene was detected in modified Escherichia coli that overexpressed CLCYb2 than in one that overexpressed CLCYb1 when supplied with an equal amount of substrates (J. C. Zhang, personal communication), implying different LCYb activities of the two variants. Amino acid alignment of the LCYb from seven citrus cultivars submitted to Genbank showed polymorphisms at multiple amino acid residues (Fig. 1). Interestingly, a consistent linkage between two amino acid residues (the 4 th and 50 th ) was found. A leucine (L) residue at the 4 th residue was always accompanied by an R at the 50 th, and a valine (V) residue at the 4 th by an M at the 50 th (indicated with arrows in Fig. 1). Accordingly, all the citrus LCYb genes (CLCYbs) compared in this study could be classified into two types based on the amino acid polymorphisms at the 4 th and 50 th residue: CLCYb1s (including CLCYb1, L at the 4 th and R at the 50 th ) and CLCYb2s (including CLCYb2, V at the 4 th and M at the 50 th ). The characteristics of CLCYbs at DNA level Under the high stringency conditions, using a probe of 910 b (LCYF1), southern blot hybridization of Cara Cara navel orange, Newhall navel orange, Skaggs Bonanza navel orange and Red Marsh grapefruit showed multiple bands (Fig. 2), indicating the existence of at lease two copies of the gene. As shown in EcoR I digestion products of Cara Cara navel orange, Skaggas Bonazza navel orange and Red Marsh grapefruit s genomes, two intensive bands were detected. The third intensive band presented in EcoR I digestion product of Newhall navel orange might have resulted from an extra copy of the gene or a base change within the restriction site in the genome. However, our result was at odds with Fanciullino et al. (14), in which different endonucleases were used and a single copy of LCYb gene was detected. PCR-RFLP analysis in genomes of different citrus accessions PCR-RFLP analysis was further employed to identify the two variants of the gene present in the genomes of sixteen accessions of citrus and its relatives (Fig. 3). Since CLCYb2(s) contains an RsaI restriction site, whereas CLCYb1s does not, after the endogenous enzymatic digestion, the PCR products of CLCYb1s remained as one band of 502 bp in length, while those of CLCYb2s yielded two close bands with about 230 and 270 bp. Based on the PCR-RFLP patterns, the sixteen genotypes could be divided into three groups. Group-1, including pummelo, two grapefruits, trifoliate orange, two citranges and kumquat, is homozygous for CLCYb1s. Group-2, including two navel oranges and two sour oranges, is heterozygous for CLCYb1s and CLCYb2s, in which nearly equal band intensities were detected. Group-3 includes two 2454 Biotechnol. & Biotechnol. Eq. 25/2011/3

4 Fig. 1. Amino acid sequence alignment of lycopene β-cyclases from seven citrus accessions aan (C. unshiu Miyagawa ), aar (C. maxima), and five C. sinensis of aaf , aau (Washington navel orange), aam21152 (Cara Cara navel orange), aau (Anliu sweet orange) and aau (Honganliu sweet orange). Different amino acid residues were marked with red or blue letters. Residues 4 and 50 are marked with arrows to indicate the amino acids linkage ( V accompanied by M, while L by A ). Biotechnol. & Biotechnol. Eq. 25/2011/3 2455

5 lemons, two mandarins (Guoqing No.1 Satsuma mandarin and Ponkan mandarin) and Calamondin, a hybrid of mandarin and kumquat (6), and is heterozygous for CLCYb1s and CLCYb2s. However, the band intensity ratio of CLCYb1s/ CLCYb2s was seldom close to 1, and was interestingly unstable and varied among many independent assays of PCR-RFLP analysis (data not shown). In those assays, either CLCYb1s or CLCYb2s was found sometimes with a trace amount, which implied the complicated structure of the two variants. the beginning of our research, whereas PCR-RFLP analysis revealed their coexistence in both genomes. Notably, according to our results, the character of lycopene accumulation in the four pink-fleshed citrus was found irrelevant to the structure of two variants in their genomes. However, like Honganliu sweet orange (17), the lycopene accumulation in the flesh might be caused by the downregulation of LCYb and/or LCYe, or other genes encoding enzymes with LCYb catalyzation ability. Cycb in tomato is a case in point, which encodes a protein showing a similar sequence to capsanthin capsorubin synthase (21), Alquezar et al. (3) recently emphasized its LCYb activity in Star Ruby grapefruit. Conclusions Two variants of lycopene β-cyclase gene were identified in the genomes of citrus and its relatives, CLCYb1s and CLCYb2s. The genomes of pummelo (Citrus grandis), two grapefruits (C. paradisi), trifoliata orange (Pocirus trifoliata), two citranges (C. sinensis P. trifoliata) and kumquat (Fortunella crassifolia) are homozygous for CLCYb1s, whereas the genomes of two navel oranges (C. sinensis), two sour oranges (C. aurantium), two lemons (C. limon), two mandarins (C. reticulata) and Calamondin are heterozygous for CLCYb1s and CLCYb2s. Fig. 2. Southern blot analysis of CLCYbs in citrus. Genomic DNA of Newhall (N), Cara Cara (Ca) and Skaggs Bonanza (SB) navel oranges and Red Marsh grapefruit (RM) was digested with EcoRI or HindIII, then hybridzed to probe LCYF1. M indicates λhind III DNA ladder. Acknowledgements This work was financially supported by the Natural Science Foundation of China (Grant Nos. of , and ). The primer sequences of LCY1 and LCY2 were kindly provided by Dr. Cristina D. Moore, at Citrus Research and Education Center, University of Florida, USA. We thank Dr. Jihong Liu for his critical review on the manscript. Fig. 3. PCR-RFLP analysis of CLCYbs in citrus (A) and its relatives (B). The gene was amplified with the primers LEFT and P2, and then fully digested with Rsa I. A: M-100 bp DNA ladder; Cara - Cara Cara navel orange; W - Washington navel orange; RM -Red Marsh grapefruit; SR -Star Ruby grapefruit; KP -Kao Pan pummelo; PE -Variegated Pink Eureka lemon; E - Eureka lemon; C -Calamondin; G -Guoqing No.1 satsuma mandarin and P -Ponkan mandarin; B: TO -Trifoliata orange; Cac -Carrizo citrange; Cit -Citrange; GT -Goutou sour orange; SO -Sour orange; KU -Meiwa Kumquat. Arrow shows the molecular weight of 500 bp. Interestingly, only one variant of the gene had been isolated from Cara Cara and Washington navel oranges respectively in REFERENCES 1. Ahrazem O., Rubio-Moraga A., Lopez R.C., Gomez- Gomez L. (2010) J. Exp. Bot., 61, Alquezar B., Rodrigo M.J., Zacarias L. (2008) Phytochemistry, 69, Alquezar B., Zacarias L., Rodrigo M.J. (2009) J. Exp. Bot., 6, Bang H., Kim S., Leskovar, D., King S. (2007) Mol. Breeding, 20, Bang H., Davis A.R., Kim S., Leskovar D.I., King S.R. (2010) J. Am. Soc. Hortic. Sci., 135, Bayer R.J., Mabberley D.J., Morton C., Miller C.H., Sharma I.K., Pfeil B.E., Rich S., Hitchcock R., Sykes S. (2009) Am. J. Bot., 96, Blas A.L., Liu Z.Y., Veatch O.J., Paull R.E., Moore P.H., Yu Q.Y., Ming R. (2010) Plant Physiol., 152, Cheng Y.J., Guo W.W., Yi H.L., Pang X.M., Deng X.X. (2003) Plant Mol. Biol. Rep., 21, Biotechnol. & Biotechnol. Eq. 25/2011/3

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