ФИЗИОЛОГИЯ РАСТЕНИЙ, 2014, том 61, № 6, с. 864-872
ЭКСПЕРИМЕНТАЛЬНЫЕ СТАТЬИ
YM 581.1
FUNCTIONAL IDENTIFICATION OF ELO-LIKE GENES INVOLVED IN VERY LONG CHAIN FATTY ACID SYNTHESIS IN Arabidopsis thaliana1
© 2014 Q. Wang***, Q. Jiang***, J. P. Lian***, J. L. Sun***, H. Xu***, Z. L. Liu***, Y. Q. Yang***, H. X. Zhao***
*State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling, Shaanxi, P.R. China **College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, P.R. China ***State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresource; College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, P.R. China Received September 25, 2013
\fcry long chain fatty acids (VLCFAs) are essential lipid components in many plants. 3-Ketoacyl-CoA synthase (KCS) catalyzes the condensation reaction to form 3-ketoacyl-CoA in VLCFA synthesis. AtELO4 has been reported to be involved in VLCFA synthesis, functioning as a KCS in Arabidopsis. However, no studies on other three AtELO members have been reported. Here, we initially found by real-time PCR in Arabidopsis thaliana (L.) Heynh., that AtELOl, AtELO3, and AtELO4 displayed characteristic expression patterns, but AtELO2 was nearly expressed in any organ. Then the transient expression of ELO-like-eGFP fusions in Arabidopsis green leaf protoplasts showed that AtELOl, AtELO3, and AtELO4 were localized in ER, where VLCFA synthesis took place. Finally, we found that the contents of all fatty acids were decreased by 10—20% in seeds of atelol T-DNA insertion mutants. In seeds of Pro35S:AtELO1 plants, the levels of all remaining components, except C20:0 and C20:3, were significantly increased. Taken together, our study revealed biological functions of AtELO members and may lay the foundation for further genetic manipulations to generate oil crops with the high oil content.
Keywords: Arabidopsis thaliana — very long chain fatty acids — 3-ketoacyl-CoA synthase — AtELO
DOI: 10.7868/S0015330314060190
INTRODUCTION
Very long chain fatty acids (VLCFAs) refer to as fatty acids with chain lengths of 20 or more carbons. In plants, VLCFAs are essential components in seed storage triacylglycerols (TAGs), sphingolipids present in various cell membranes, and cuticular waxes accumulated on aerial surfaces [1]. In Arabidopsis seeds, up to 20% of total fatty acids in storage oil is C20:1 [2]. In addition, C22:1 has detrimental nutritional effects and is therefore undesirable in edible oils but widely used in the manufacture of lubricants, nylon, and plasticiz-ers [3].
1 This text was submitted by the authors in English.
Abbreviations: ECR — trans-2-enoyl-CoA reductase; ELO — elon-gase; ER — endoplasmic reticulum; FAE — fatty acid elongation; FAME — fatty acid methyl ester; HCD — 3-hydroxyacyl-CoA dehydratase; KCR — 3-ketoacyl-CoA reductase; KCS — 3-ketoacyl-CoA synthase; LFA — long chain fatty acid; TAG — triacylglycer-ol; VLCFA — very long chain fatty acid; WT — wild type. Corresponding author: Huixian Zhao. State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling, Shaanxi, 712100 P.R. China; fax: +86 29-8709-2262; e-mail: hxzhao212@nwsuaf.edu.cn
VLCFA synthesis, also called fatty acid elongation, is accomplished by sequential addition of C2 moieties from malonyl-CoA to C18 acyl groups derived from plastidial de novo fatty acid synthesis pathway. Four enzymatic reactions are carried out in the elongation process: condensation of a long-chain CoA with malonyl-CoA to form a 3-ketoacyl-CoA catalyzed by 3-ketoacyl-CoA synthase (KCS, also called condensing enzyme), reduction of 3-ketoacyl-CoA to 3-hydroxy-CoA by 3-ketoacyl-CoA reductase (KCR), dehydration to trans-2-enoyl-CoA by 3-hydroxyacyl-CoA dehydratase (HCD), and reduction of trans-2-enoyl-CoA by trans-2-enoyl-CoA reductase (ECR) [4].
Three very dissimilar gene families, FAE-like family, ELO-like family, and CER2-like family, have been reported to function as KCS to catalyze the first condensation step of VLCFA synthesis. FAE was first discovered as a seed-specific condensing enzyme involved in C20 and C22 fatty acid biosynthesis for seed storage lipids [5]. Another member of FAE-like family called CER6 was identified as a KCS required for the elongation of C24 fatty acyl-CoAs to provide substrates for cuticular wax biosynthesis in the epidermal
cells of Arabidopsis thaliana [6]. FAE-like family consists of 21 members, whose expression pattern in A. thaliana has been investigated by real-time PCR [2]. However, besides FAE and CER6, the functional analysis of the other FAE-like members has not been reported in A. thaliana. ELO-like family consists of four putative condensing enzymes in A. thaliana (At1g75000, AtELO1, At3g06460, AtELO2, At3g06470, AtELO3, At4g36830, and AtELO4) [7]. AtELO4, one member of ELO-like family was confirmed to complement yeast ELO2 and ELO3 mutants and reduced expression of AtELO4 resulted in approximately 15% increase in C22:0 level, 20% increase in C24:0 level, and 35% decrease in C26:0 level in leaves of atelo4 mutants as compared with those in Arabidopsis wild-type plants [8]. However, much information of ELO-like family members on developmental expression pattern, subcellular localization, and fatty acid profiles in AtELO knock-out/down mutants remains elusive. Recently, another novel CER2-like gene family has been identified as KCS involved in VLCFA synthesis beyond C28 for cuticular wax synthesis in A. thaliana, whose classification as a BAHD acyltrans-ferase based on sequence homology does not fit with CER2 catalytic activity in fatty acid elongation [9, 10]. In contrast to KCR, HCD, and ECR that have broad substrate specificities and are expressed in all cells, the condensing enzyme KCS has been reported to be rate-limiting as well as specific for tissues and chain length of VLCFA acyl-CoA synthesized [11].
In this study, we firstly studied the expression pattern of AtELO family by real-time PCR in various organs of A. thaliana. Then we investigated the subcellular localization of AtELO members in transiently expressed Arabidopsis leaf protoplast cells. Finally, we examined fatty acid profiles in T-DNA insertion atelos mutants and transgenic plants with AtELO overexpression. The main objective of this study was to reveal biological function of AtELO members and may be laid the foundation for further genetic manipulations to generate oil crops with the high oil content.
MATERIALS AND METHODS
Plant materials and growth conditions. The Arabidopsis thaliana (L.) Heynh. homozygous atelo1 mutant line (SALK_090523c) and atelo3 mutant line (SALK_108023c) from the ABRC (www.arabidopsis. org) and Columbia-0 (Col-0) ecotype (wild type, WT) were used in this study. Arabidopsis seeds were surface-sterilized, cold treated at 4°C, and imbibed in the dark for 4 days on 1% phytoagar plates containing 1% sucrose and 0.5-strength MS medium. The seeds were then germinated and grown in soil at 20°C under a 16-h photoperiod.
After transformation with the 35S-AtELO1 construct, T1 seeds were surface-sterilized, spread evenly onto agar plates containing kanamycin, stratified for
3 days in a cold room, and germinated at 20°C under continuous light. After 2 weeks, kanamycin-resistant plants were transferred to soil and grown in a controlled environment cabinet at a 16-h photoperiod.
Plasmid materials. Plasmid pUC18-35S-SP (signal peptide)-CFP-KDEL was kindly provided by Prof. Chuxiong Zhuang from South China Agricultural University, Guangzhou, and was used as ER marker.
RNA isolation, cDNA preparation, quantitative real-time PCR, and semiquantitive RT-PCR. The
15-day-old roots, 15-day-old seedlings, rosette leaves, stems, opened flowers, young siliques (green siliques containing developing seeds), and old siliques (dry and yellow siliques containing dry seeds) of wild-type Arabidopsis (Col-0 ecotype) and rosette leaves of homozygous SALK T-DNA insertion mutants were collected and frozen immediately in liquid nitrogen. With the exception ofyoung and old siliques, total RNA was isolated using Trizol reagent ("Invitrogen", United States) according to the manufacturer's protocol. Because young and old siliques containing seeds usually contain a high level of polysaccharides, a method involving acid phenol/LiCl was used to isolate RNA from young and old siliques [12]. By adding DNAse I ("Invitrogen") to each sample, possible residual DNA in the RNA sample was removed. For reverse transcription, 1—5 ^g of total RNA, oligo(dT) and M-MLV reverse transcriptase ("Promega", United States) were mixed to synthesize first-strand cDNA as specified by the manufacturer.
The primer pairs specific for each gene of the AtELO family were designed using Primer Premier 5.0 software, and validated to generate a single PCR product of expected size. The sequences ofAtELO1 specific primers are 5' -TCTTCTCTTCTCTGTATCTCCT- 3' and 5'-CGCCACGCAACCCAATCTC- 3'; the sequences of AtELO2 specific primers are 5'-AACTAT-GAGAAGACTACAACGT-3' and 5'-ACAACTAA-CAACACACATC-3'; the sequences ofAtELO3specific primers are 5'-GGCGATTCACCGGAGGCT- 3' and 5'-AAGGAGAGGAGACAGAGGAT- 3'; the sequences of AtELO4 specific primers are 5'-TTATG-GCGGCGGAGCAAAA-3'and 5' - CAGAGGAAA-GACGTGAAGG-3'. Real-time PCR was performed with an iCycler™ ("Bio-Rad", United States) system using SYBR green master mix ("Takara", Japan) according to the manufacturer's instructions, except that the reaction volume was 20 ^L. Cycling conditions were 95°C for 1 min followed by 40 cycles at 95°C for 5 s and at 60°C for 30 s. On cycling completion, melting curves were analyzed to distinguish true products from artifacts, such as primer dimers. Three technical replicates were performed for each of the three biological replicates assayed. Data analysis for real-time PCR was done using the iCycler™ iQ software (v. 3.0a, "Bio-Rad"), and the standard curve method was used for calculating the relative expression of experimental
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