Article pubs.acs.org/JAFC
Simultaneous Silencing of Five Lipoxygenase Genes Increases the Contents of α‑Linolenic and Linoleic Acids in Tomato (Solanum lycopersicum L.) Fruits Tingzhang Hu, Hua Zeng, Zongli Hu, Xiaoxiao Qv, and Guoping Chen* Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044, People’s Republic of China ABSTRACT: α-Linolenic and linoleic acids are essential fatty acids (EFAs) for humans and required for maintenance of optimal health, but they cannot be synthesized by the human body and must be obtained from dietary sources. Using TomloxC fragment, TomloxD fragment, and partial TomloxA sequence that is highly identical with TomloxB and TomloxE, a RNAi expression vector was constructed. The construct was used to transform tomato cotyledon explants with the Agrobacterium-mediated co-cultivation method. The real-time reverse transcription polymerase chain reaction analysis showed that the expression of TomloxA, TomloxB, TomloxC, TomloxD, and TomloxE in transgenic tomato plants was drastically repressed, which led to a marked decrease in the levels of lipoxygenase activity. Finally, higher accumulations of the endogenous α-linolenic and linoleic acids were detected in the transgenic tomato fruits, which were 1.65−3.99 and 2.91−4.98 times that of the non-transformed tomato fruits, respectively. KEYWORDS: α-linolenic acid, essential fatty acids, linoleic acid, tomato (Solanum lycopersicum L.), lipoxygenase, transgenic tomato
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were detected in tomato fruit.11 TomloxA is expressed mainly in immature fruits, while both TomloxB and TomloxE are expressed principally in ripening fruits. TomloxC is expressed in fruits and leaves, and TomloxD is expressed in roots, leaves, flowers, and fruits.11,12 The ripening hormone ethylene can enhance the expression of TomloxB and TomloxC and repress TomloxA expression.13 TomloxD expression is upregulated by pathogen infection, wounding, jasmonate, and systemin.12,14 During growth and development of tomato plants, the individual LOX isoforms from tomato are differentially controlled and have distinct functions.11,13,15 In tomato fruit, the antisense suppression of TomloxA and TomloxB greatly reduced the LOX enzyme activity but caused no significant changes in the contents of the tomato flavor volatiles, as compared to wild-type plants.13 The specific depletion of TomloxC in transgenic tomato resulted in a marked reduction in the accumulation of flavor volatiles, including hexanal, hexenal, and hexenol, which was only 1.5% of those of wildtype tomato. Adding ALA or LA to fruit homogenates may significantly increase the contents of flavor volatiles, but the increase with the TomloxC-depleted transgenic fruit extracts was much lower than that with the wild-type fruits. The findings showed that TomloxC can use both ALA and LA as substrates to produce volatile C6 flavor compounds.11 TomloxC also has an essential function in synthesis of the important C5 flavor volatiles in both fruits and leaves, but this synthesis is not dependent upon hydroperoxide lyase (HPL). Large reductions in C5 and C6 volatiles in antisense TomloxC knockdown plants were observed, but those reductions did not alter the development of disease symptoms, which indidcate that these
INTRODUCTION Essential fatty acids (EFAs) cannot be synthesized by the human body, but they are required for maintenance of optimal health and must be obtained from dietary sources.1 Although humans and other mammals can synthesize saturated fatty acids and some monounsaturated fatty acids from carbon groups in proteins and carbohydrates, they lack the enzymes necessary to insert a cis double bond at the n-3 or n-6 position of a fatty acid. Consequently, ω-3 and ω-6 fatty acids are essential nutrients.2 α-Linolenic acid or ALA (18:3n-3) is an ω-3 fatty acid, and linoleic acid or LA (18:2n-6) is an ω-6 fatty acid. Both ALA and LA are EFAs for humans, which are the precursors of higher chain EFAs. ALA and LA can be metabolized to their respective long-chain metabolites: docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) from ALA and arachidonic acid (AA) and dihomo-γ-linolenic acid (DGLA) from LA.2,3 ALA and LA are required for eicosanoid synthesis, hormones (such as prostaglandins), and regulation of gene expression and control many physiological factors.4 Lipoxygenases (linoleate:oxygen oxidoreductase, EC 1.13.11.12; LOXs) are a enzyme family found ubiquitously in plants, animals, and microorganisms.5−8 Plant LOXs are widespread and encoded by multigene families. LOXs can be detected in all plant organs.9 LOXs can catalyze the regio- and stereospecific dioxygenation of polyunsaturated fatty acids (PUFAs) with a (1Z,4Z)-pentadiene structure, such as ALA and LA, to yield conjugated (1S,2E,4Z)-hydroperoxides,10 which can be further metabolized to yield antimicrobial and antifungal compounds, signaling compounds, a plant-specific blend of volatiles, etc.7,10,11 At the amino acid level, TomloxA, TomloxB, and TomloxE from tomato show high homology and are 72−77% identical to each other, while TomloxC and TomloxD are 46% identical to each other and only show 42 and 47% identity to the TomloxA protein, respectively. The expression of five tomato LOX genes © 2014 American Chemical Society
Received: Revised: Accepted: Published: 11988
August 6, 2014 November 15, 2014 November 22, 2014 November 22, 2014 dx.doi.org/10.1021/jf503801u | J. Agric. Food Chem. 2014, 62, 11988−11993
Journal of Agricultural and Food Chemistry
Article
Table 1. Nucleotide Sequences of Primers Used for PCR Amplification in the Present Study sequence number
name of the primer
sequence (5′−3′)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
TomA-f TomA-r TomC-f TomC-r TomD-f TomD-r NPTII f NPTII r TomA-qf TomA-qr TomB-qf TomB-qr TomC-qf TomC-qr TomD-qf TomD-qr TomE-qf TomE-qr leEF-1-f leEF-1-r
5′-GGTACCGCATGCATATGCCTCGAGAACTTTGC-3′ 5′-CTCGAGGATATCAGATCTCTCCACCAGCATTGATTAGG-3′ 5′-CACCAGATCTGGATCCTAGAAAATGAGCACCACAAG-3′ 5′-GTCGCTCGAGGTCGACCTAACGACTTCTCCGAGATC-3′ 5′-AGATCTGATATCCTCGAGGACAAGCAATAGCAGGAGTG-3′ 5′-GATCCTCTAGATAAGTGTGCCAACATCAGAC-3′ 5′-GTCACTGAAGCGGGAAGGG-3′ 5′-GGCGATACCGTAAAGCAC-3′ 5′-GAGGCGTGGGATAGGA-3′ 5′-GGATACGGGTAGTCAGCA-3′ 5′-TGCTACAATGACTTGGGTGAA-3′ 5′-CCTGTCCTGCCTCTACG-3′ 5′-ATGATCTCGGAGAAGTCG-3′ 5′-GTAGGGCGGAGTAAACG-3′ 5′-GACAAGCAATAGCAGGAGTG-3′ 5′-TAAGTGTGCCAACATCAGAC-3′ 5′-ATCTCAGTATCCGTATCCTCG-3′ 5′-TGTCCAAATCGCTCGTC-3′ 5′-GGAACTTGAGAAGGAGCCTAAG-3′ 5′-CAACACCAACAGCAACAGTCT-3′
On the basis of the TomloxA, TomloxC, and TomloxD cDNA sequences (GenBank accession numbers U09026, U37839, and U37840, respectively), the primers TomA-f, TomA-r, TomC-f, TomC-r, TomD-f, and TomD-r were designed. The appropriate restriction sites were added at the 5′ end of primers (Table 1). The fragments of TomloxA, TomloxC, and TomloxD genes were amplified from cDNA by PCR using PrimeSTAR HS DNA Polymerase (Takara, Dalian, China) with primers TomA-f and TomA-r, TomC-f and TomC-r, and TomD-f and TomD-r, respectively. The PCR products were cloned into pMD18-T vector (Invitrogen, Carlsbad, CA) and sequenced to obtain vectors TomloxA-T, TomloxC-T, and TomloxDT, respectively. Construction of RNA Interference (RNAi) Vector TomloxACDi-pBIN19 and Plant Transformation. RNAi expression vector TomloxACDi-pBIN19 was constructed as follows: (1) TomloxC gene fragment from the plasmid TomloxC-T (cut by BamHI and SalI) was cloned into the plasmid TomloxA-T in the sense orientation at BglII and SalI restriction sites of the plasmid TomloxA-T and obtained TomloxAC-T vector. (2) TomloxD gene fragment from the plasmid TomloxD-T (digested with Xho I and SalI) was cloned into the plasmid TomloxAC-T in the sense orientation at SalI restriction sites of the plasmid TomloxAC-T and obtained TomloxACD-T vector. (3) ACD fragment from the plasmid TomloxACD-T (cut by BamHI and SalI) was linked into the pDHG 5.2 plasmid at BamHI and SalI restriction sites in the antisense orientation and obtained plasmid pDHG5.2-rTomloxACD. (4) The ACD fragment from the plasmid TomloxACD-T (digested by KpnI and BamHI) was linked into the plasmid pDHG5.2-rTomloxACD at KpnI and BamHI restriction sites in the sense orientation and was called pDHG5.2-fTomloxACDrTomloxACD vector. (5) The double-stranded (ds) RNA expression unit from the plasmid pDHG5.2-fTomloxACD-rTomloxACD vector, including the cauliflower mosaic virus (CaMV) 35S promoter, TomloxACD fragment in sense orientation, GFP gene fragment, TomloxACD fragment in antisense orientation, and 35S terminator, was subcloned into the plant binary vector pBIN 19 (Takara, Dalian, China) with EcoRI to yield RNAi vector TomloxACDi-pBIN19 for Tomlox family genes silencing (Figure 1). The construct was confirmed by PCR, restriction digest analysis, and sequencing of the TomloxACD fragment. The binary vector TomloxACDi-pBIN19 was used to transform tomato cotyledon explants. After the construct was transferred into Agrobacterium tumefaciens LBA4404 by the freeze−thaw method,18 transformation was carried out according to the Agrobacterium-
volatiles do not have an important defensive function against this bacterial pathogen.16 The overexpression of TomloxD gene in transgenic tomato plants resulted in a marked increase in the LOX activity and jasmonic acid (JA) accumulation, which indicted that TomloxD may use ALA as a substrate to produce (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT), and 13HPOT was further metabolized to synthesize JA.15 In comparison to non-transformed tomato plants, the transgenic plants overexpressing TomloxD were more tolerant to Cladosporium fulvum and high-temperature stress.15 The overexpression of TomLoxD also leads to elevated woundinduced JA biosynthesis and increased expression of woundresponsive genes, thereby enhanced resistance to insect herbivory attack and necrotrophic pathogen infection.17 Therefore, TomloxD is involved in endogenous JA synthesis and tolerance to biotic and abiotic stress.16,17 Both ALA and LA in tomato are EFAs for humans. To improve the nutrition value of the tomato fruits, increasing the content of ALA or LA is advantageous. We produced some transgenic tomato plants silencing five LOX family genes. Transgenic tomato plants with greatly reduced TomloxA, TomloxB, TomloxC, TomloxD, and TomloxE were identified by quantitative real-time polymerase chain reaction (PCR) and LOX activity assays. Significant improvement in the contents of ALA or LA in tomato fruit was achieved.
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MATERIALS AND METHODS
Experimental Materials and Growth Conditions. All experiments were performed using a near-isogenic line of diploid tomato (Solanum lycopersicum cv. Ailsa Craig) plants. The inbred tomato was cultivated in soil. After 20 days of growth, seedlings were collected, frozen in liquid nitrogen, and used to extract RNA. Cloning of the Fragments of TomloxA, TomloxC, and TomloxD Genes. Total RNA was isolated from the seedlings using the Trizol reagent (Gibco-BRL, Grand Island, NY). Reverse transcription (RT) was conducted with 500 ng of total RNA using the SuperScript III RNase H Reverse Transcriptase kit (Invitrogen, Carlsbad, CA) and 10 mM anchored olig(dT)18 primer according to the instructions of the manufacturer. 11989
dx.doi.org/10.1021/jf503801u | J. Agric. Food Chem. 2014, 62, 11988−11993
Journal of Agricultural and Food Chemistry
Article
expression vector TomloxACDi-pBIN19 was constructed. The plasmid TomloxACDi-pBIN19 was introduced to A. tumefaciens LBA4404 and transformed into S. lycopersicum cv. Ailsa Craig plants by the Agrobacterium-mediated co-cultivation method. The transgenic plants were selected with kanamycin. The regenerated seedlings were acclimated for 2 weeks in zippy pot soil in a greenhouse, then transferred to soil, and cultured under field conditions. The transgenic lines were obtained and further verified by PCR. Because the NPTII gene is closely adjacent to the target gene in the plasmid TomloxACDipBIN19 and not present in the wild-type tomato genome, the positive transgenic plants should have a specific 493 bp amplified product, which did not appear in wild-type tomato plants (Figure 2). The transgenic tomato lines were used for further analysis.
Figure 1. Construct of RNAi vector TomloxACDi-pBIN19 for Tomlox family gene silencing. The fragments of TomloxA, TomloxC, and TomloxD genes in the sense and antisense orientations were linked with a GFP gene fragment and as a transcriptional unit for hairpin RNA expression, which was controlled by the CaMV 35S promoter and terminated by the 35S terminator. mediated co-cultivation method.19 Transgenic plants that rooted on kanamycin were transferred to compost and grown.11 Confirmation and Phenotype Analysis of Positive Transgenic Plants. The genomic DNA of transgenic and wild-type plants was isolated using Plant Genomic DNA Extraction kit (VOK-Bio, Beijing, China) according to the instructions of the manufacturer and used as templates. The forward primer NPTII f and reverse primer NPTII r were designed according to the NPTII gene sequence (Table 1). The positive transgenic plants were validated by PCR analysis. The phenotype of transgenic tomato plants was investigated. Real-Time RT-PCR Analysis of Transgenic Tomato Plants. Total RNA was isolated from the leaf tissues of transgenic and wildtype tomato plants using Trizol reagent and subsequently treated with DNase I. The RNAs were reverse-transcribed as described above. The specific primers of real-time RT-PCR analysis are listed in Table 1. The real-time RT-PCR analysis of TomloxA, TomloxB, TomloxC, TomloxD, and TomloxE genes was carried out using the quantitative SYBR Green PCR kit (Tianwei, China) as described by He et al.20 Real-time RT-PCR was performed using the CFX96 real-time system (C1000 thermal cycler, Bio-Rad, Hercules, CA). The gene expression was normalized to the LeEF-1 gene from the tomato plant.21 LOX Activity Assays. LOX activity from leaf tissues was determined spectrophotometrically. The extraction of tomato LOXs and LOX activity assay were performed using the methods as described by Hu et al.12 Proteins were measured by the Bradford method.22 A total of 1 unit of activity was defined as the quantity of enzyme catalyzing an increase in absorbance of 0.01 at 234 nm/min under assay conditions. Quantification of Endogenous ALA and LA in Tomato Fruits. Frozen samples of ripe fruits (8−10 days after breaker stage) were homogenized with a polytron apparatus, and 5 g of the homogenate was digested with 10 mL of HCl for 1 h at 70−80 °C. The mixture was cooled and extracted twice, each time being shaken for 2 min with 10 mL of ethanol, 10 mL of aether, and 25 mL of petroleum ether, followed by 10−20 min of quiescence. The supernatant liquid was transferred to another beaker and evaporated under reduced pressure. After saponification with 1 mL of 2 M NaOH−methanol for 15−20 min at 65 °C, the sample was methyl-esterified with 1 mL of 2 M HCl−methanol for 5 min at 65 °C. The resultant fatty acid methyl esters were recovered by adding 2 mL of saturated NaCl solution and 1 mL of hexane, vortexing, and centrifugation at 1000 rpm for 5 min to give the upper hexane layer. Fatty acid analysis was performed by the application of 100 μL aliquots to a gas chromatograph (LC-2010, Shimadzu, Japan) equipped with a TC-70 capillary column (30 cm × 0.25 mm, GL Sciences, Japan) under a temperature program of 180−220 °C at a rate of 4 °C/min. ALA and LA were calculated on the basis of the area of each peak, and the amount was obtained by comparison to the ALA and LA standards, respectively. Statistical Analysis. All experiments were repeated 3 times for two biological replicates. The data are means of six independent experiments ± standard deviation (SD). Student’s t test was used for statistical analysis to compare the data. A p value of
