Planta

, Volume 226, Issue 5, pp 1097–1108

Overexpression of glycerol-3-phosphate acyltransferase gene improves chilling tolerance in tomato

Authors

  • Na Sui
    • College of Life SciencesShandong Agricultural University, Key Lab of Crop Biology of Shandong Province
  • Meng Li
    • Shandong Academy of Agricultural Sciences
  • Shi-Jie Zhao
    • College of Life SciencesShandong Agricultural University, Key Lab of Crop Biology of Shandong Province
  • Feng Li
    • College of Life SciencesShandong Agricultural University, Key Lab of Crop Biology of Shandong Province
  • Hui Liang
    • The State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental BiologyChinese Academy of Sciences
    • College of Life SciencesShandong Agricultural University, Key Lab of Crop Biology of Shandong Province
Original Article

DOI: 10.1007/s00425-007-0554-7

Cite this article as:
Sui, N., Li, M., Zhao, S. et al. Planta (2007) 226: 1097. doi:10.1007/s00425-007-0554-7
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Abstract

A tomato (Lycopersicon esculentum Mill.) glycerol-3-phosphate acyltransferase gene (LeGPAT) was isolated. The deduced amino acid sequence revealed that LeGPAT contained four acyltransferase domains, showing high identities with GPAT in other plant species. A GFP fusion protein of LeGPAT was targeted to chloroplast in cowpea mesophyll protoplast. RNA gel blot showed that the mRNA accumulation of LeGPAT in the wild type (WT) was induced by chilling temperature. Higher expression levels were observed when tomato leaves were exposed to 4°C for 4 h. RNA gel and western blot analysis confirmed that the sense gene LeGPAT was transferred into the tomato genome and overexpressed under the control of 35S-CaMV. Although tomato is classified as a chilling-sensitive plant, LeGPAT exhibited selectivity to 18:1 over 16:0. Overexpression of LeGPAT increased total activity of LeGPAT and cis-unsaturated fatty acids in PG in thylakoid membrane. Chilling treatment induced less ion leakage from the transgenic plants than from the WT. The photosynthetic rate and the maximal photochemical efficiency of PS II (Fv/Fm) in transgenic plants decreased more slowly during chilling stress and recovered faster than in WT under optimal conditions. The oxidizable P700 in both WT and transgenic plants decreased obviously at chilling temperature under low irradiance, but the oxidizable P700 recovered faster in transgenic plants than in the WT. These results indicate that overexpression of LeGPAT increased the levels of PG cis-unsaturated fatty acids in thylakoid membrane, which was beneficial for the recovery of chilling-induced PS I photoinhibition in tomato.

Keywords

Chilling stressCis-unsaturated fatty acidGlycerol-3-phosphate acyltransferase (GPAT)Phosphatidylglycerol (PG)PhotoinhibitionTomato (Lycopersicon esculentum Mill.)

Abbreviations

DGDG

Digalactosyldiacylglycerol

Fv/Fm

Maximal photochemical efficiency of PS II

Fo

Initial fluorescence

Fv

Variable fluorescence

Fm

Maximum yield of fluorescence

GPAT

Glycerol-3-phosphate acyltransferase

LeGPAT

Lycopersicon esculentum glycerol-3-phosphate acyltransferase gene

MGDG

Monogalactosyldiacylglycerol

PPFD

Photosynthetic photon flux density

PG

Phosphatidylglycerol

PS I (II)

Photosystem I (II)

P700

PS I reaction center

PVDF

Polyvinylidene fluoride

SDS-PAGE

Sodium dodecyl sulfate poly acrylamide gel electrophoresis

SQDG

sulfoquinovosyldiacylglycerol

16:0

palmitic acid

16:1

Δ3-Trans-hexadecenoic acid

18:0

Stearic acid

18:1

Oleic acid

18:2

Linoleic acid

18:3

Inolenic acid

Introduction

Low temperature is a major factor limiting the productivity and geographical distribution of chilling-sensitive plant species, including important vegetable such as cucumber, tomato and sweet pepper crops. It has been suggested that the membrane is the primary location that was damaged under chilling stress (Kratsch and Wise 2000). Since Lyons and Raison (1970) considered that chilling stress could impair membrane permeability by the transition of membrane lipids from a liquid–crystalline phase to a gel phase, many experiments have suggested that chilling tolerance is related to the composition and structure of plant membrane lipids (Nishida and Murata 1996; Murata and Los 1997). In higher plants, the most abundant lipids of thylakoid membranes are glycolipids, including monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG). PG is the only phospholipid in thylakoid membranes. The role of glycerolipids in the function of thylakoid membranes has been studied in vitro and several of these studies were summarized in a review (Siegenthaler 1998). It was also demonstrated that tolerance to chilling stress is closely connected with the fatty acid unsaturation of plant membrane lipids (Somerville and Browse 1991; Moon et al. 1995; Sakurai et al. 2003; Szalontai et al. 2003).

The chilling resistance of higher plants is considered to closely correlate with the level of cis-unsaturated fatty acids in PG from chloroplast membranes (Murata et al. 1992; Nishida and Murata 1996). The biosynthesis of phosphatidylglycerol represents a central pathway in lipid metabolism in all organisms (Xu et al. 2006). Chilling-resistant plants contain a large proportion of cis-unsaturated fatty acids at the sn-1 position of PG and there are few cis-unsaturated fatty acids in chilling-sensitive plants (Murata et al. 1982). The Sn-2 position is occupied mainly by saturated and trans-unsaturated fatty acids (Bertrams and Heinz 1981), so the content of cis-unsaturated fatty acids at the sn-1 position of PG determines the chilling resistance. The dominant factor that determines the level of cis-unsaturated fatty acids in PG is the substrate selectivity of glycerol-3-phosphate acyltransferase(GPAT: EC2.3.1.15)in chloroplasts, which catalyzes the first step of glycerolipid biosynthesis by transferring the acyl group of acyl-(acyl-carrier protein) (ACP) to the sn-1 position of glycerol-3-phosphate to yield 1-acylglycerol-3-phosphate (lysophosphatidate; LPA; Roughan and Slack 1982). GPAT from chilling-resistant plants prefers oleoyl-ACP (18:1-ACP) to palmitoyl-ACP (16:0-ACP) as a substrate (Frentzen et al. 1983; Frentzen and Wolter 1998). Thus, a large proportion of oleic acid (18:1) occurs at the sn-1 position of PG in chilling-resistant plants, and palmitic acid (16:0) is esterified to the sn-2 position, making 3-phosphate-glyceride. Under chilling stress, oleic acid (18:1) of the sn-1 position desaturates further to the two cis-polyunsaturated fatty acids linoleic acid (18:2) and linolenic acid (18:3) by acyl-fatty acid desaturase in chloroplast membranes, while palmitic acid (16:0) of the sn-2 position desaturates into Δ3-trans-hexadecenoic acid [16:1 (3t)] (Frentzen et al. 1983; Frentzen and Wolter 1998), resulting in a high level of cis-unsaturated fatty acids in PG. The enzymes from chilling-sensitive plants hardly distinguish 18:1-ACP from 16:0-ACP. Both the sn-1 and sn-2 position are occupied by palmitic acid (16:0). Under chilling environments, the palmitic acid (16:0) of the sn-2 position is desaturated into Δ3-trans-hexadecenoic acid [16:1 (3t)] and the fatty acid of the sn-1 position remains unchanged, resulting in a low level of cis-unsaturated fatty acids at the sn-1 position of PG (Frentzen et al. 1983; Weber et al. 1991). In this way, fatty acids remain saturated, which renders the plants sensitive to chilling stress.

Chilling stress is known to inhibit photosynthesis by the process of photoinhibition (Aro et al. 1993). The crucial events of PS II photoinhibition are the turnover of protein D1 in the reaction center (Aro et al. 1993; Zhang et al. 2000). Because PS I is more stable than PS II under high light and the fast inactivation of PS II can protect PS I, the damage of PS II can easily be detected by using a chlorophyll fluorometer and an oxygen-electrode system. However, photoinhibition of PS I in chilling-sensitive plants did occur during chilling stress under low irradiance (Sonoike and Tereshima 1994; Li et al. 2004). It was reported that the activity of PS I decreased about 70–80%, when cucumber leaves were treated at 5°C with an irradiance of 100 μmol m−2 s−1 (Terashima et al. 1994).

The molecular characterization of GPAT has been widely studied in different kind of plants (Frentzen et al. 1987). Experiments in vivo showed that if the AtGPAT from Arabidopsis was transferred into tobacco, the degree of unsaturation of the fatty acids in PG and the tobacco’s resistance to chilling stress increased (Murata et al. 1992). An increase in unsaturation of fatty acids in PG transformed with cDNAs for AtGPAT improves the photosynthetic rates and growth at low temperatures in transgenic rice (Ariizumi et al. 2002). Tomato is an important vegetable crop that is different from the model plants Arabidopsis and rice. Whether the overexpression of tomato glycerol-3-phosphate acyltransferase (LeGPAT) could increase unsaturation fatty acids of PG and was relative to PS II and PS I photoprotection under chilling stress is still unknown. Clearly, considerable further work is required to explore the effect of LeGPAT on PS II and PS I photoinhibition under chilling temperature with low irradiance.

In the present work, we isolated and characterized the LeGPAT gene from tomato. It was interesting that GPAT in chilling-sensitive tomato exhibited selectivity to 18:1 over 16:0. Overexpression of chloroplast LeGPAT increased the content of PG unsaturation fatty acids and played an important role in alleviating chilling-induced photoinhibition of PS I.

Materials and methods

Plant materials and treatments

Seeds of tomato cultivar (Lycopersicon esculentum cv. Zhongshu 4) were used and germinated between moistened filter paper at 25°C for 3 day. Sprouted burgeons were then planted in 13.5 cm diameter plastic pots (one plant per pot) filled with sterilized soil and grown at 25–30/15–20°C (day/night temperature regime) under a 14 h photoperiod (300–400 μmol m−2 s−1 PPFD) in a greenhouse. When the sixth leaf was fully expanded, the plants were exposed to different temperatures. The treated roots, stems, leaves, petals and fruits were immediately frozen in liquid nitrogen and stored at −80°C until use.

Isolation and sequencing of LeGPAT

Total RNA was isolated from tomato leaves using the total RNA isolation system (Promega Corporation, Madison, WI, USA) and used for reverse-transcription polymerase chain reaction (RT-PCR) and RNA gel blot analysis. A 2 μg sample of RNA was denatured at 70°C for 5 min and 2 μl AMV reverse transcriptase (Promega) was added. The transcription reaction was mixed briefly and then incubated at 42°C for 1 h, and terminated at 85°C for 10 min.

To isolate the chloroplast GPAT gene from tomato, a 439 bp fragment was amplified from cDNA prepared from tomato leaves. Primers GP1: 5′-CA(C/T)CA(A/G)A(G/C) TGAAGC(A/T)GATCC-3′ and GP2: 5′-GGAGG(A/G/C)GGCAT(A/G/T)ATGTCAT(A/G)-3′, which contained a conserved sequence were designed based on the homology to the chloroplast GPAT gene from tobacco, sweet pepper, rice and Arabidopsis. The cDNA amplification products were cloned into the pMD-18T vector and sequenced.

The 5′- and 3′-ends of the gene were PCR-amplified from the cDNA. The 5′-RACE PCR was carried out by using the gene-specific primer: GP3: 5′-GCCTCCTCATGTTGTCTGTCGCAGA AG-3′ and an abridged universal amplification primer AAP according to the manufacturer’s instructions (GGBCO-BRL kit). The 3′-RACE PCR was carried out by using the gene-specific primers: GP4: 5′-GCATATGAATGATGACCCCGAACTTGC-3′ and B26: 5′-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3′.

The full-length DNA sequence of the putative LeGPAT gene was amplified with PCR using 5′ and 3′ specific primers: GP5: 5′-CATTCTGTGGTGGTGATGTTGAT-3′ and GP6: 5′-GCAAGCGTGAGGTAT GTGGAAAGAT-3′. The PCR amplification was as follows: initial denaturation at 94°C for 5 min, followed by 35 cycles, 94°C for 50 s, 48°C for 50 s, 72°C for 1 min, a final extension cycle of 72°C for 10 min and termination of the reaction at 4°C. All the primers were synthesized by Bioasia Bio-engineering Limited Company (Shanghai, China). Nucleotide and deduced amino acid sequences were analyzed using DNAman version 5.2 (Lynnon Biosoft, USA). Sequence data from this article have been deposited at GenBank under accession number DQ459433.

Intracellular targeting of putative LeGPAT

Two DNA conducts (p35S-GFP and p35S-LeGPAT-GFP) were prepared to investigate the intracellular targeting of LeGPAT using transient expression in cowpea mesophyll protoplasts. p35S-GFP was obtained from the Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences (Beijing). The complete coding region of LeGPAT was subcloned into the p35S-GFP vector between the NcoI and SalI sites, up-stream and in frame with the GFP coding region. Cowpea mesophyll protoplasts were isolated, transfected with the above two conducts (Shah et al. 2002), and examined by dual channel confocal microscopy (FV500 and I × 70, Olympus, Japan). The bright field image, GFP fluorescence and the red autofluorescence of chloroplast from protoplast expression were recorded simultaneously and compared. The potential colocalization of GFP fluorescence of chloroplast autofluorescence was further analyzed by checking the presence of yellow signals in the superimposed images.

RNA gel blot analysis

Twenty micrograms of total RNA were separated in a 1.2% agarose formaldehyde gel and transferred to nylon membrane as described by Sambrook et al. (1989). RNA was fixed on the membrane by cross-linking with UV light. Pre-hybridization was performed at 65°C for 12 h. The 3′ partial cDNA 0.5 kb of LeGPAT was used as gene-specific probe and labeled with [α-32P]-dCTP by the random prime labeling method (Prime-a-Gene-Labeling System, Promega). After 24 h hybridization, filters were washed subsequently in 2 × SSC (1 × SSC was 0.15 M NaCl, 0.015 M sodium citrate, pH 7) with 0.2% SDS and 0.2 × SSC with 0.2% SDS at 42°C. Autoradiography was performed at −80°C.

SDS-PAGE and immunological analysis

A coding region of LeGPAT in the pMD18-T vector at about 1,122 bp was subcloned into the pET-30a(+) vector between the NcoI and SalI sites. A recombinant of prokaryotic expression vector pET-LeGPAT was constructed and transformed to E. coli. BL21 and then expression was induced with IPTG. Depositions were solubilized in the presence of 2 × SDS loading buffer and separated by SDS-PAGE (Laemmli 1970) using 10% separate gels and 4% concentrated gels and containing 10% SDS. The strong induced fusion protein bands were collected into phosphate buffer solution (PBS) and were used to immunize white mice to obtain antiserum. The secondary antibody was peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, Inc.). The antibody was used at a dilution of 1:500 and the secondary antibody was used at 1:5,000. For immunoblotting, polypeptides were electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore), and proteins in the wild type and transgenic lines were detected with antibodies raised against LeGPAT. Protein content was determined by the dye-binding assay (Bradford 1976).

Plasmid construction and Agrobacterium-mediated transformation of tomato plants

The full-length LeGPAT cDNA was subcloned into the expression vector pBI121 downstream of the 35S-CaMV promoter to form sense constructs (pBI-LeGPAT). The 35S-CaMV LeGPAT constructs were first introduced into Agrobacterium tumefaciens LBA4404 by the freezing transformation method and verified by PCR and sequencing. Leaf disk transformation using wild-type tomato plants was performed as described by Horsch et al. (1985). Discs infected with A. tumefaciens were incubated on medium for inducing shoots. After a few weeks, the regenerated shoots were transferred to a root-inducing medium. Both media contained cefotaxime sodium (250 μg ml−1) and kanamycin (50 μg ml−1). Transgenic plants were screened using kanamycin selection generated by the incubation of transformed tomato leaf disks (Holsters et al. 1978). As a consequence, 25 individual kanamycin-resistant lines were obtained from tissue culture. Each transgenic line seemed to represent an independent integration event since a specific DNA fragment in each line was observed by genomic DNA gel blot analysis (data not shown).

Purification of the protein from E. coli cells

A recombinant of prokaryotic expression vector pET-LeGPAT was constructed and transformed to E. coli BL21, and then expression was induced with IPTG. Resuspended pellets of 80 ml bacterial culture in tubes were collected into 40 ml phosphate buffer (PBS) solution. The tubes were placed on ice and a sonication probe was immersed in each sample. The deposit after sonication was collected into 50 mM PBS (pH 7.4) containing 0.5 M NaCl. The suspension was filtered with a 0.45 μm filter and applied to a column of nickel gelose gel FF (1.6 × 20 m) with His label equilibrated in 50 mM PBS (pH7.4; buffer 1). The column was eluted with 50 mM PBS (pH7.4) containing 34 g imidazole (buffer 2). Finally, the column was eluted stepwise with 10, 20, 50, 100 and 200 mM imidazole in 50 mM PBS (pH 7.4; buffer 3). Peak fractions were pooled and stored at −20°C for use.

The content of the purified protein was determined by analysis of the A280 value using software obtained from http://www.au.expasy.org/tools/protparam.html.

Substrate selectivity of glycerol-3-phosphate acyltransferase from tomato

The substrate selectivity of LeGPAT was measured as described by Frentzen et al. (1987), using purified enzyme fractions of wild type and transgenic tomato leaves of T1-5 (Bertrams and Heinz 1981) and protein from the E. coli cells expressing LeGPAT. Each reaction mixture (80 μ;) contained 100 μM [1−14C]18:1-CoA (2.00 GBq mmol−1) and 100 μM [1−14C]16:0-CoA (2.07 GBq mmol−1), 0.3 mM glycerol-3-phosphate, 250 mM Hepes-NaOH buffer (pH 7.4) and 100 μg BSA. The reaction mixture was stopped by extracting the mixture with 2.5 ml of chloroform–methanol (1:1, v/v) and 1 ml 1.0 M KCl and 200 mM H3PO4. After mixing and phase separation by short centrifugation, reaction products were recovered in the subphase and quantified by liquid scintillation counting (XH-6925, Xian Nuclear Instrument Factory, China). To confirm the reaction products, the lipid extracts were subjected to TLC in a solvent system of chloroform/methanol/acetic acid/5% aqueous sodium bisulfite (100:40:12:4, by vol.; Zheng and Zou 2001). [1-14C]18:1-CoA and [1-14C]16:0-CoA were purchased from GE Healthcare (Chalfont St Giles, Bucks, UK), and glycerol-3-phosphate was purchased from the Sigma Chemical Company (St Louis, MO, USA).

Enzyme activity assays

The activity assay of LeGPAT was performed as described by Bertrams and Heinz (1981), using 300 ng enzyme fractions of wild type and transgenic tomato leaves of T1-5 and protein purified from E. coli cells. Glycerol-3-phosphate and [1-14C]18:1-CoA were used as substrates.

Fatty acid composition

Tomato leaf tissue was harvested from 3- to 4-month-old tomato plants and frozen immediately in liquid nitrogen. Lipids were extracted as described by Siegenthaler and Eichenberger (1984), and separated by two-dimensional TLC (Xu and Siegenthaler 1997). For quantitative analysis, individual lipids were separated by thin layer chromatography, scraped from the plates, and used to prepare fatty acid methyl esters. The fatty acid composition of individual lipids was determined by gas chromatography as previously described (Chen et al. 1994).

Measurement of relative electrolyte leakage

Six leaf discs (0.8 cm) were put into 10 ml distilled water and vacuumized for 30 min, and then surged for 3 h to measure the initial electronic conductance (S1). Then a cuvette was filled with leaf discs and distilled water, the mixture was cooked 30 min to determine the final electronic conductance (S2). The relative electrolyte leakage (REL) was evaluated as: REL (%) = S1 × 100/S2.

Measurement of net photosynthetic rate and chlorophyll a fluorescence

The net photosynthetic rate (Pn) was measured with a portable photosynthetic system (CIRAS-2, PP Systems, Hitchin, Hertfordshire, UK) under the condition of a concentration of ambient CO2 (360 μmol mol−1) and a PPFD of 600 μmol m−2 s−1.

Chlorophyll fluorescence was measured with a portable fluorometer (FMS2, Hansatech, King’s Lynn, UK) according to the protocol described by van Kooten and Snel (1990). The minimal fluorescence (Fo) with all PS II reaction centers open was determined by modulated light which was low enough not to induce any significant variable fluorescence (Fv). The maximal fluorescence (Fm) with all reaction centers closed was determined by 0.8 s saturating light of 7,000 μmol m−2 s−1 on a dark-adapted (adapted 15 min in darkness) leaf. The maximal photochemical efficiency (Fv/Fm) of PS II was expressed as: Fv/Fm = (Fm − Fo)/Fm.

The absorbance at 820 nm

Oxidation and reduction of P700 was measured at 820 nm with a Plant Efficiency Analyzer (PEA) senior (Hansatech) described by Schansker et al. (2003).

Results

Characterization of the tomato cDNA clone

A cDNA was isolated from tomato leaves. The full-length sequence of the cDNA consisted of 1,770 bp nucleotides and a 1,314 bp open reading frame at position 71-1,384 bp, encoding a 437-residue polypeptide. The deduced amino acid sequence of the cDNA showed that it encoded a polypeptide of approximately 48 kDa. This cDNA was designated as LeGPAT and was submitted to the GenBank database under accession number DQ459433 (the address is as follows: http://www.ncbi.nlm.nih.gov). A study performed by Lewin and co-workers revealed four regions of strong sequence homology, termed blocks I to IV, in various acyltransferases from species ranging from bacteria to mammals (Lewin et al. 1999). Amino acid sequence alignment revealed that the plant members contained the previously defined four acyltransferase domains (Fig. 1). The His and Asp residues in block I, the Gly residue in block III, and the Pro residue in block IV, all of which have been shown to form a catalytically important site in this family of acyltransferases, are absolutely conserved. The Phe and Arg in block II and the Ser in block III appear to be important in binding the glycerol 3-phosphate substrate. At the C terminus, several blocks of conserved residues extend beyond regions known to be implicated in acyltransferase enzyme function.
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Fig. 1

Deduced amino acid sequence alignment of GPAT from six plant species. Four conserved acyltransferase motifs are indicated above the alignment (I, II, III and IV). Identical and similar amino acid residues are shaded black, and dashes indicate gaps introduced to optimize alignment. The accession numbers of GPAT in GenBank are as follows: AtGPAT (AY093169), Arabidopsisthaliana; OsGPAT (NM_197897), Oryza sativa; PsGPAT (X59041), Pisum sativum; CtGPAT (L33841), Carthamus tinctorius; LeGPAT (DQ459433), Lycopersicon esculentum Zhongshu 4; LeGPAT-L402 (AY360170), Lycopersicon esculentum L402; CaGPAT (AY318749), Capsicum annuum. The alignment was done using DNAman version 5.2

Targeting of LeGPAT to chloroplast

To determine the subcellular localization of LeGPAT protein in plant cells, we performed targeting experiments in vivo in cowpea protoplasts derived from leaf tissue. In the protoplasts transfected with p35S-LeGPAT-GFP, which expressed LeGPAT-GFP fusion cistron, the green fluorescence was clearly associated with chloroplasts and colocalized with the red autofluorescence of chloroplasts (Fig. 2, top panel). In contrast, in the protoplasts transfected with the control construct p35S-GFP (expressing the GFP coding sequence alone) the green fluorescence was distributed in the cytoplasm surrounding the chloroplasts and was not colocalized with the red autofluorescence of chloroplasts (Fig. 2, bottom panel).
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Fig. 2

Intracellular targeting of LeGPAT in cowpea protoplast. The top panel shows cowpea protoplast transfected with p35S-LeGPAT-GFP, expressing LeGPAT-GFP fusion protein. The bottom panel shows a protoplast transfected with p35S-GFP, expressing GFP alone. Protoplasts were examined using dual channel confocal microscopy. Chloroplast targeting is demonstrated by colocalization of GFP fluorescence (green) and chloroplast (red), and the presence of chloroplast-associated yellow signals due to superposition of the green (GFP) and red (chloroplast) fluorescence

Expression of LeGPAT in tomato

The LeGPAT gene was constitutively expressed in stems, petals, fruits and leaves of wild type plants (Fig. 3) whether at 4 or 25°C. However, no expression signals from roots were detected, perhaps due to the low level of expression. The transcripts were relatively more abundant in leaves than in other organs, so leaves were used as materials in the following RNA gel blot.
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Fig. 3

Expression of LeGPAT in different organs at different temperatures. Total RNA was extracted from roots, stems, petals, fruits and leaves of wild type plants at 4 and 25°C. The probe was labeled with [α-32P]-dCTP by the random prime labeling method (Prime-a-Gene-Labeling System, Promega). The ethidium bromide staining of the RNA gel is shown as the control for loading (rRNA)

The expression of LeGPAT at different temperatures was measured with RNA gel blot after 4 h treatments (Fig. 4a). In the leaves of WT tomato, the highest transcript level of LeGPAT was observed at 4°C. A weak band of mRNA was noted at 40°C. When plants were treated at 4°C for different times, the relative LeGPAT expression in leaves changed markedly (Fig. 4b). Transcripts were low within the first 2 h, and the maximum level was observed after treatment for 4 h. Although the transcript level was still found in tomato leaves after treatment at 4°C for 48 h, the amount drastically decreased after 12 h of chilling stress. The expression of LeGPAT at 40°C for different times was low (Fig. 4c). These results indicated that LeGPAT was expressed extensively from 4 to 40°C in leaves and that low temperature induced the expression of LeGPAT.
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Fig. 4

Expression of LeGPAT in tomato leaves at different temperatures for 4 h (a) and for different periods of time at 4°C (b) and 40°C (c) after treatment for different times. Lane CK, expression of LeGPAT at 25°C. Total RNA was extracted from wild type plants. About 20 μg of total RNA was analyzed by RNA gel blot using 3′ partial cDNA of LeGPAT as a gene-specific probe

Molecular characterization of the transgenic plants

Transgenic plants infected with Agrobacterium tumefaciens carrying the LeGPAT gene were detected by PCR after the first screening with 50 μg ml−1 kanamycin (data not shown). Twenty-five individual kanamycin-resistant lines were obtained from tissue culture. These initial kanamycin-resistant plants were named T0 and the progeny obtained from T0 were named T1. Four lines named T1-5, T1-19, T1-41 and T1-58 were selected for RNA gel and Western blot analysis. From the four lines we selected T1-5 and T1-19 for physiological measurement. There were no obvious morphological differences between the transgenic and WT plants.

Kanamycin-resistant T1 plants were checked by PCR. The upstream primer of PBI121 and the 3′ primer of the LeGPAT gene were used in the amplification and an intense 1,770 bp band corresponding in size to the LeGPAT gene product was obtained from kanamycin-resistant plants, while nothing was obtained from WT plants (data not shown). RNA gel blot showed that all kanamycin-resistant plants had strong positive signals, and a weak signal was found in WT plants. Western blot analysis with an antiserum against LeGPAT revealed the presence of strong positive protein signals corresponding to LeGPAT in transgenic plant leaves, while a weak signal was found in WT tomato (Fig. 5).
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Fig. 5

RNA gel and Western-blot analysis of LeGPAT in transgenic tomato. Total RNA and protein were extracted from transgenic plants and WT, respectively. The probe of the RNA gel blot was labeled with [α-32P]-dCTP. About 20 μg of total RNA was analyzed by RNA gel blot. The antibody against LeGPAT was produced by immunizing white mice and used at a dilution of 1:500. The dilution of the secondary antibody was set at 1:5,000. About 35 μg of protein was analyzed by the dye-binding assay

Substrate selectivity of LeGPAT

To examine whether LeGPAT exhibited selectivity to 18:1 over 16:0, a mixture of [1-14C]acyl-CoAs was incubated with active fractions, and radioactive fatty acids incorporated into lysophosphatidate were determined. When protein purified from the E. coli cells was incubated with a mixture of 100 μM [1-14C]18:1-CoA and 100 μM [1-14C]16:0-CoA, 18:1 was incorporated into lysophosphatidate at an average ratio of 6.75 ± 0.043:1 ± 0.043 (= 3) over 16:0. Similarly, when enzyme fractions of wild type tomato leaves were incubated under similar conditions, 18:1 was incorporated into lysophosphatidate at an average ratio of 5.17 ± 0.029:1 ± 0.029 (= 3) over 16:0. And when enzyme fractions of T1-5 leaves were incubated under similar conditions, 18:1 was incorporated into lysophosphatidate at an average ratio of 6.95 ± 0.064:1 ± 0.064 (= 3) over 16:0. These results suggested that LeGPAT exhibited 18:1 selectivity over 16:0.

Activity of glycerol-3-phosphate acyltransferase

The activity of LeGPAT was also measured with the substrates of glycerol-3-phosphate and [1-14C]18:1-CoA, using 300 ng enzyme fractions of wild type and transgenic tomato leaves of T1-5 and protein purified from E. coli cells. Table 1 shows that the protein content from E. coli cells, transgenic plants and WT plants was different. There was no evident difference in the specific activity of GPAT between WT and transgenic plants, but overexpression of LeGPAT increased the total activity of GPAT in transgenic plants relative to that of WT plants (Table 1).
Table 1

Activity of glycerol-3-phosphate acyltransferase from tomato

Samples

Protein content (mg ml−1)

Specific activity (CPM min−1 μg−1 protein)

Total activity (CPM min−1 mg−1 FW)

Proteins from E. coli cells

0.124 ± 0.08

126 ± 1.1

Enzyme fractions of WT

0.049 ± 0.05

119 ± 1.2

58.31 ± 0.45

Enzyme fractions of T1-5

0.085 ± 0.06

134 ± 1.0

113.90 ± 1.29

Data are expressed as mean values ± SD (= 3; three measurements on each of the three plants)

Changes of fatty acid composition of WT and transgenic plants

Overexpression of LeGPAT in tomato had no significant effect on monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) or sulfoquinovosyldiacylglycerol (SQDG; data not shown). The fatty acid compositions of phosphatidylglycerol (PG) were significantly affected (Table 2). A higher content of 18:2 and 18:3 was detected in transgenic plants, and the saturated fatty acid content of 16:0 and 18:0 decreased in transgenic plants compared with that in WT plants. The relative levels of total cis-unsaturated fatty acids in PG increased from 51% in the wild type to 66% in T1-5 and 64% in T1-19. These results indicated that overexpression of LeGPAT in tomato leaves increased the content of cis-unsaturated fatty acids.
Table 2

Fatty acid composition of PG in WT and transgenic tomato leaves

Fatty acid (%)

 

16:0

16:1 (3t)

18:0

18:1

18:2

18:3

WT

25.46 ± 1.02

16.26 ± 0.09

7.20 ± 0.25

8.90 ± 0.04

24.69 ± 1.01

17.38 ± 2.23

T1-5

14.81 ± 0.80

18.93 ± 0.12

4.93 ± 0.99

35.61 ± 1.65

25.72 ± 1.09

T1-19

15.36 ± 1.76

19.69 ± 0.11

1.03 ± 0.23

3.10 ± 0.75

38.18 ± 0.21

22.64 ± 3.26

Present at trace levels (<0.1% of total fatty acid). Data are expressed as mean values ±SD (= 3; three measurements on each of the three plants) and are presented as the mole percentage. Standard deviations between triplicates was <3% of the indicated values

Effect of chilling stress on relative electrolyte leakage in WT and transgenic plants

Figure 6a shows that the relative electrolyte leakage increased both in WT and transgenic plants under chilling stress, but chilling treatment resulted in less ion leakage from the transgenic plants than from the wild type. After treatment at 4°C for 12 h, the relative electrolyte leakage of T1-5 and T1-19 increased to 21 and 19%, whereas it increased to 24% in the WT. This demonstrated that membrane damage was more serious in WT plants than that in transgenic plants under chilling stress.
https://static-content.springer.com/image/art%3A10.1007%2Fs00425-007-0554-7/MediaObjects/425_2007_554_Fig6_HTML.gif
Fig. 6

Effect of chilling stress on relative electrolyte leakage, Pn, Fv/Fm and the oxidizable P700 in WT and transgenic plants. Plants were exposed to chilling stress (4°C) for 12 h before measurement of relative electrolyte leakage (a). Plants were treated at 4°C and Pn was measured at 25°C under ambient CO2 (360 μmol mol−1; b). Before Pn measurement, tomato plants were kept about 30 min at 25°C and at the PPFD of 100 μmol m−2 s−1 to induce stomata opening, and then illuminated about 15 min at the PPFD of 600 μmol m−2 s−1. Recovery of Pn was measured at 25°C and a PPFD of 600 μmol m−2 s−1. After plants were treated, Fv/Fm was measured at 4°C and then recovered at 25°C (c). Before chilling stress, plants were adapted in darkness for more than 2 h to measure Fv/Fm. During chilling stress, plants were adapted in darkness for 15 min before Fv/Fm measurement. The oxidizable P700 was measured during chilling stress (4°C) and during recovery at 25°C under the low irradiance of 100 μmol m−2 s−1 (d). Before measurement of Pn, Fv/Fm and the oxidizable P700, plants were treated for 0, 1, 3, 6, 9 and 12 h at 4°C and recovered for 1, 2, 5, 8, 12 and 24 h at 25°C. Each point represents the mean ± SD of five measurements on each of the five plants Fig. 1

Responses of Pn to chilling stress in WT and transgenic plants

To evaluate the role of PG cis-unsaturated fatty acids in protecting the photosynthesis apparatus from low temperature, the net photosynthetic rates (Pn) were determined. The increase of PG cis-unsaturated fatty acids in transgenic plants did not significantly influence the Pn of the wild type under normal conditions (Fig. 6b). Although the Pn of WT and transgenic plants decreased markedly during chilling stress (4°C), the decrease in Pn was more obvious in WT than that in transgenic plants. After tomato plants were transferred to 25°C and the PPFD of 600 μmol m−2 s−1, the Pn of T1-5 and T1-19 recovered completely in 12 h, whereas the Pn of wild type plants recovered only 73% in 12 h and 86% in 24 h. These results indicated that elevated levels of cis-unsaturated fatty acid resulting from the overexpression of LeGPAT in tomato played a significant role in protecting the photosynthesis apparatus from chilling stress.

Overexpression of LeGPAT alleviates photoinhibition of PS II and PS I under chilling stress

Photoinhibition of PS II in WT and transgenic plants was estimated by measuring the maximal photochemical efficiency of PS II (Fv/Fm). Fv/Fm decreased obviously in wild type plants during chilling stress (4°C) relative to that in transgenic plants (Fig. 6c). At the end of 12 h chilling stress at 4°C, the Fv/Fm in wild type plants and the T1-5 and T1-19 transgenic plants decreased about 11, 5 and 4%, respectively. At the same time, it was found that the recovery of Fv/Fm in transgenic plants was also quicker than that in WT plants. The Fv/Fm of T1-5 and T1-19 recovered completely in 8 h, while the Fv/Fm of the WT only recovered 95% (Fig. 6c). This suggested that photoinhibition of PS II during chilling stress was alleviated in transgenic plants and the PS II reaction center was not as seriously damaged as in the WT.

It has been reported that PS I is more sensitive to chilling stress than PS II under low irradiance (Sonoike and Tereshima 1994). The oxidizable P700 decreased significantly both in WT and transgenic plants under chilling stress under low irradiance (Fig. 6d). There were no evident differences between WT and transgenic plants. When tomato plants were transferred to suitable conditions of 25°C and a PPFD of 100 μmol m−2 s−1, the oxidizable P700 of transgenic plants recovered more quickly than that of WT. After 24 h recovery, the oxidizable P700 could recover 99, 99 and 85% in the T1-5, T1-19 and WT plants, respectively. These results indicate that overexpression of LeGPAT was beneficial to the recovery of chilling-induced PS I photoinhibition.

Discussion

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the initial and rate-limiting step of glycerolipid synthesis and is therefore a potential site for regulating the synthesis of triacylglycerol and all of the glycerophospholipids. Previous research has indicated the existence of a relationship between the expression of GPAT and chilling tolerance in many model plants (Murata et al. 1992; Moon et al. 1995; Ariizumi et al. 2002). However, the function of the glycerol-3-phosphate acyltransferase gene in vegetable crops in response to chilling stress is still unclear.

In this study, a cDNA clone encoding GPAT was isolated from tomato leaves. The analysis of the predicted amino acid sequence of LeGPAT with GPAT from other higher plants showed that LeGPAT shared high sequence identity with GPAT in other plants and clearly identified the LeGPAT cDNA as encoding a GPAT protein targeted to the chloroplast (Fig. 1). The expression of p35S-LeGPAT-GFP conducts in cowpea mesophyll protoplasts, observed with confocal microscopy, confirmed this finding (Fig. 2).

Because the expression of GPAT is related to chilling stress (Moon et al. 1995) and the recovery process from photoinhibition, it was necessary to examine the expression pattern of LeGPAT in tomato. The expression of LeGPAT was observed in leaves, fruits, petals and stems of WT under chilling stress and at normal growth temperature. LeGPAT was found to be expressed constitutively and the expression level was higher in leaves than in other organs (Fig. 3). The expression of LeGPAT was also enhanced by chilling temperature and suppressed by high temperature (Fig. 4). The highest expression level in leaves was observed after 4 h exposure to 4°C (Fig. 4a, b). At 40°C, the expression of LeGPAT was hardly detectable at any time (Fig. 4a, c).

The strong positive signals of RNA gel and Western blot in transgenic plants compared with that in the WT showed that LeGPAT had been introduced into the tomato genome and the gene was expressed at both the RNA and protein levels (Fig. 5).

Phosphatidylglycerol (PG) is an integral component of photosynthetic membranes and is important for both the formation and function of photosynthesis apparatuses (Domonkos et al. 2004). Previous studies have indicated that in higher plants, PG contributes to the development of chloroplasts (Hagio et al. 2002). The majority of PG molecules are localized in the thylakoid membranes, which are the site of photosynthetic electron transport (Wada and Murata 1998). The main function of LeGPAT is to esterify glycerol-phosphate at the sn-1 position. Experiments that measured membrane fluidity in terms of the vsymCH2 vibration in the Fourier transform infrared (FTIR) spectrum showed that below 25°C the lipid molecules in thylakoid membranes from tobacco plants that expressed squash GPAT were more rigid than those from wild type plants (Szalontai et al. 2003). Their later study showed that an increase in the relative level of saturated and trans-monounsaturated molecular species in PG increased the sensitivity of tobacco plants to low temperature (Sakamoto et al. 2003). The positional distribution of fatty acids in PG from leaves indicated that 18:0 and 16:1 (3-trans) are localized at positions sn-1 and sn-2 of sn-glycerol, respectively, and 16:0 is at both positions (Murata et al. 1982). Although tomato is classified as a chilling-sensitive plant, the sum of palmitic (16:0), Δ3-trans-hexadecenoic acids (16:1) and stearic acid (18:0) in phosphatidylglycerol amounted to 49%, similar to the content in chilling-resistant plants (Table 2). As a result, the substrate selectivity of LeGPAT is similar to GPAT of chilling-resistant plants. Consistent with these presumptions, our experiment demonstrated a preference of LeGPAT for oleic acid (18:1). The overexpression of LeGPAT also increased the total activity of acyltransferase (Table 1), resulting in an increase of 18:1 content at the sn-1 position. The 18:1 fatty acid in the sn-1 position is finally desaturated to 18:2 and 18:3. Analysis in leaf fatty acid composition in transgenic plants (Table 2) revealed that the 18:2 and 18:3 content in PG increased while the PG saturated fatty acids content (16:0 and 18:0) decreased. The higher unsaturated fatty acid content of PG in transgenic plants (Table 2) alleviated membrane damage during chilling stress (Fig. 6a).

Previous research has showed that the characteristics of PS IIphotoinhibition are the loss of the oxygen-evolution activity, the breakdown of protein D1 in the PS II reaction center (Aro et al. 1993) and the replacement of the degraded D1 protein with newly synthesized D1 protein for recovery (Zhang et al. 2000). In all kinds of plants, D1 protein is continuously degraded, and hence, it must be continuously re-synthesized for the renewal of the reaction center, otherwise the plant will suffer serious damage under excess photon energy (Ariizumi et al. 2002). It has been proven by experiments that the unsaturation of fatty acids in thylakoid membrane lipids accelerates the recovery of the photosynthetic machinery from photoinhibition but does not affect the process of inactivation that leads to the photoinhibition of photosynthesis and the activity of PS II (Moon et al. 1995). In chilling-sensitive plants, a low level of cis-unsaturated fatty acids in PG prevents the renewal of the degraded D1 protein at low temperatures, resulting in the inhibition of photosynthesis. By contrast, chilling-resistant plants contain sufficient levels of cis-unsaturated fatty acids in PG to ensure the renewal of the degraded D1 protein at low temperatures, resulting in the decrease of photoinhibition. In the present study, the decrease of Fv/Fm suggested the possibility that LeGPAT affected the process of PS II damage (Fig. 6c). However, we could not exclude the possibility that LeGPAT also affected the recovery process. Photoinhibition in WT plants was more serious than that in transgenic plants. On the other hand, this was also supported by the changes of photosynthetic rate. Pn decreased more significantly and recovered more slowly in WT plants than in transgenic plants (Fig. 6b), which demonstrated that the increase of PG cis-unsaturated fatty acid content was helpful to protect the photosynthesis apparatus from photoinhibition.

There might be two reasons to explain the slow recovery and the decrease of oxidizable P700 during chilling stress under low irradiance. First, chilling temperature results in the accumulation of reducing power on the acceptor side of PS I (Terashima et al. 1994; Sonoike 1996). Second, some components of PS I are damaged by chilling stress under low irradiance, particularly the FeS centers of PS I, which are believed to be the active site of chilling stress under low irradiance (Tjus et al. 1998). Domonkos et al. (2004) demonstrated that the decrease of PS I activity is correlated with monomerization of the trimes of PS I in a mutant of Synechocystis PCC6803. They reported that PG depletion results in the degradation of PS I trimers and concomitant accumulation of monomer PS I. In vitro studies suggested that trimer formation is enhanced by the lipid environment (Kruip et al. 1999). It was reported that three PG molecules are bound to the reaction center of the PS I core complex, which suggests that PG has an important function not only in PS II but also in PS I, presumably in the assembly of the PS I core complex (Jordan et al. 2001; Sakurai et al. 2003). Under chilling stress the oxidizable P700 decreased significantly both in transgenic and wild type plants (Fig. 6d), but the oxidizable P700 recovered faster in the transgenic lines T1-5 and T1-19 than in the wild type. The slow recovery of oxidizable P700 in the WT is probably attributed not only to the limitation of electron acceptors at low temperature but also probably to the damage of PS I components. Terashima et al. (1994) concluded that chilling stress affects the recovery of PS II by influencing the transition of the membrane lipid phase. A more obvious decrease of Pn, Fv/Fm and oxidizable P700 in WT plants than in transgenic plants was observed when plants were treated at a chilling temperature and low light (Fig. 6b–d). The decrease of Pn in WT plants was about 80%, while decreases of Fv/Fm and oxidizable P700 were about 10 and 30%, respectively, after 12 h chilling stress. Apparently, limitation of carbon assimilation under chilling stress with low light led to excess light energy, causing severe PS I photoinhibition. Recovery of oxidizable P700 in WT plants was very slow and incomplete, which was probably the reason for the slow recovery of Pn.

In conclusion, we demonstrated that the expression of LeGPAT was regulated by chilling signals. Overexpression of LeGPAT increased cis-unsaturated fatty acid levels in PG. Increase of PG cis-unsaturated fatty acids in thylakoid membranes actually protected the photosynthesis apparatus against chilling stress under low irradiance by alleviating photoinhibition of PS II and PS I. The finding is of practical importance in improving the chilling sensitivity of tomato plants, an agriculturally important vegetable. Given that tomato is a chilling-sensitive plant, why does LeGPAT exhibit selectivity to18:1 over 16:0? What is the mechanism for the substrate selectivity? Such questions should be addressed in future studies.

Acknowledgments

This research was supported by the State Key Basic Research and Development Plan of China (2006CB100100) and the Natural Science Foundation of China (30471053).

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© Springer-Verlag 2007