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First published online June 4, 2004; 10.1104/pp.103.036376 Plant Physiology 135:947-958 (2004) © 2004 American Society of Plant Biologists Characterization of an Ultraviolet B-Induced Lipase in Arabidopsis1Department of Biology, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1
An Arabidopsis expressed sequence tag clone, 221D24, encoding a lipase has been characterized using an antisense approach. The lipase gene is expressed during normal growth and development of Arabidopsis rosette leaves but is down-regulated as the leaves senesce. When plants are exposed to sublethal levels of UV-B radiation, expression of the lipase is strongly up-regulated. The lipase protein is localized in the cell cytosol and is present in all organs of Arabidopsis plants. Recombinant lipase protein produced in Escherichia coli preferentially hydrolyzed phospholipids, indicating that the gene encodes a phospholipase. Transgenic plants in which lipase expression is suppressed showed enhanced tolerance to UV-B stress but not osmotic stress and were unable to up-regulate PR-1 expression when irradiated with UV-B. The observations collectively indicate that the lipase is capable of deesterifying membrane phospholipids and is up-regulated in response to UV-B irradiation.
Lipases are hydrolytic enzymes that catabolize complex lipids. They include phospholipases, lipolytic acyl hydrolases, galactolipases, and triacylglycerol lipases. Certain types of lipases, those that deesterify fatty acids, contain the consensus sequence [LIV]-X-[LIVAFY]-[LIAMVST]-G-[HYWV]-S-X-G-[GSTAC] encoding the esterase motif (Derewenda and Derewenda, 1991  ). Lipases are important agents of cellular metabolism in that they catabolize lipids, but they have also been implicated in signal transduction, mobilization of lipids during seed germination, and catabolism of membrane lipids during senescence (Thompson et al., 1998; Wang, 2001).
Phospholipase D (PLD) is the best characterized plant phospholipase. Five isoforms of PLD (
Lipolytic acyl hydrolase is a nonspecific lipase that cleaves fatty acids at the Sn1 and Sn2 positions from a broad range of substrates, including phospholipids, triacylglycerols, and wax esters (Galliard, 1971
Galactolipids, specifically monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), are the dominant lipids of thylakoids, accounting for more than 60% of total polar lipids in photosynthesizing tissues. Deesterification of galactolipids is a pronounced feature of natural leaf senescence (Engelman-Silvestre et al., 1989 In this study, we have characterized an Arabidopsis GenBank sequence (gene accession no. At2g42690) that encodes a protein (protein accession no. AAD21737.1) exhibiting phospholipase activity. The phospholipase is localized in the cytosol and induced by treatment with sublethal levels of UV-B. Transgenic plants in which expression of the UV-B-induced lipase is suppressed exhibited enhanced tolerance to sublethal UV-B stress and were unable to up-regulate pathogenesis related protein 1 (PR-1) in response to UV-B treatment.
Gene Isolation and Function
Full-length cDNA corresponding to the Arabidopsis sequence, GenBank accession number At2g42690 (encoding protein AAD21737.1), was obtained by reverse transcription (RT)-PCR using RNA isolated from the rosette leaves of 4-week-old plants. A comparison of the genomic and cDNA sequences revealed that the gene contains one intron and encodes a polypeptide composed of 412 amino acid residues with an approximate molecular mass of 47 kD. Analysis of the inferred amino acid sequence (Fig. 1) using the program Predotar indicated that the protein does not contain cleavable chloroplast- or mitochondria-targeting sequences. The protein does, however, contain the lipase consensus sequence [LIV]-X-[LIVAFY]-[LIAMVST]-G-[HYWV]-S-X-G-[GSTAC] (Fig. 1), which corresponds to an esterase motif and is a characteristic feature of all known lipases that deesterify fatty acids from complex lipids. Thus, the presence of this motif indicates that the cDNA encodes a lipase. The inferred amino acid sequence also contains predicted phosphorylation, glycosylation, and myristoylation domains (Fig. 1). A BLAST search revealed that the sequence matched most closely (55% identity with an e-value of 10–136) a cytosolic lipase (GenBank accession no. AAD01804.1) isolated from carnation petals (Hong et al., 2000
To confirm that the gene encodes a lipase, the full-length cDNA was ligated into pTrc 99a, which is isopropylthio-  -galactoside inducible, and overexpressed in Escherichia coli. SDS-PAGE and western-blot analysis indicated that the 47-kD recombinant protein was present in the pellet fraction, but not the supernatant fraction, isolated from E. coli (data not shown). This fraction also exhibited lipase activity (Fig. 2). Moreover, when the abilities of the recombinant lipase to deesterify fatty acids from soybean (Glycine max) phosphatidylcholine, monogalactolipid, and triacylglycerol were compared, the protein exhibited a strong preference for phosphatidylcholine substrate, indicating that it is a phospholipase. Specifically, its ability to hydrolyze monogalactolipid and triacylglycerol was only 58% and 22%, respectively, of its ability to hydrolyze phosphatidylcholine (Fig. 2). This distinction was further confirmed by testing commercially available fungal lipases with known substrate preferences, viz., Candida rugosa lipase, which is known to be a phospholipase, and Rhizomucor miehei lipase, which is known to be a galactolipase (Ishiguro et al., 2001; Fig. 2).
The possibility that the recombinant lipase exhibits specificity was examined by testing its ability to deesterify fatty acids from a range of purified molecular species of phosphatidylcholine that are available commercially. Lipid extracts of the reaction mixtures were fractionated by thin-layer chromatography (TLC), and deesterified fatty acids were quantified and identified by gas chromatography. For each of the 16:0/20:4 phosphatidylcholine, 20:4/20:4 phosphatidylcholine, 16:0/18:3 phosphatidylcholine, 16:0/18:2 phosphatidylcholine, 18:2/18:2 phosphatidylcholine, and 18:3/18:3 phosphatidylcholine substrates, only background levels of deesterified fatty acid, equivalent to those obtained for control reactions containing extract from E. coli transformed with empty pTrc 99a, were detectable over a range of substrate concentrations (data not shown). Yet the recombinant enzyme exhibited strong deesterification activity when soybean phosphatidylcholine, which contains an unknown mixture of molecular species, was used as substrate (Fig. 2). This suggests, although does not prove, that the lipase exhibits molecular species specificity and that the molecular species it is able to hydrolyze is not among those tested.
Extracts of total protein from different organs of 6-week-old Arabidopsis plants were analyzed by western blotting in order to determine the spatial localization of the native protein. An antibody (
When purified microsomal and cytosolic fractions isolated from rosette leaves of 3.5-week-old plants were analyzed by western blotting, a 47-kD band corresponding to the native protein was clearly present in the cytosolic fraction and only barely detectable in the membrane fraction (Fig. 3B). This indicated that the protein is soluble and localized in the cytosol. To confirm that it is not plastidial, chloroplasts from rosette leaves of 3.5-week-old Arabidopsis plants were subfractionated into broken chloroplasts, purified stroma, and chloroplastic membranes (thylakoids and envelope membranes), and the subfractions together with purified cytosol were analyzed by western blotting. The native protein was detectable in broken chloroplast (contaminated with cytosol) and cytosolic fractions but not in the intact chloroplasts, stroma, or chloroplastic membrane fractions, indicating that its presence in the cytosol is not attributable to leakage from broken chloroplasts (Fig. 3C).
Cytosolic localization of the lipase protein was further confirmed by confocal microscopy. These experiments were performed using leaf tissue in which chloroplasts are clearly discernible by chlorophyll autofluorescence. Rosette leaf tissue from 3.5-week-old Arabidopsis plants was cut into small pieces and labeled en bloc with
Northern-blot analysis indicated that transcript for the lipase protein is clearly present in leaves of plants 2 to 5 weeks of age but is substantially down-regulated by week 6 (Fig. 5). By week 3, the rosette leaves are fully expanded, and between weeks 4 and 5 the plants bolt and flower. By week 5, the rosette leaves have begun to senesce, and by week 6 leaf senescence is fully engaged and the leaves have turned yellow, reflecting loss of chlorophyll. Thus, expression of the gene is consistent during leaf development and begins to decline coincidently with the onset of senescence. Of particular interest, however, is the finding that its expression in leaves is strongly up-regulated when plants are subjected to sublethal UV-B stress (Fig. 6). The treatment was initiated when the plants were 3.5 weeks of age and was continued for 4 d. That the UV-B treatment was sublethal is indicated by the fact that the treated plants continued to develop and produce seed. Transcript levels began to increase within 2 d of treatment and by day 4 were pronounced (Fig. 6).
Antisense Transgenic Plants Homozygous transgenic lines of Arabidopsis expressing antisense lipase cDNA were obtained by screening seeds of three successive generations on kanamycin. Three lines, 4-2A-5, 1-4C-8, and 3-2C-5, were selected based on their increased tolerance to sublethal UV-B stress. Transgenic and wild-type plants grown under normal conditions were indistinguishable at all stages of growth and development. This is evident, for example, from the fact there were no significant differences in biomass accumulation throughout growth and development (Fig. 7A). However, when wild-type and transgenic plants were maintained under conditions of sublethal UV-B stress from 3.5 weeks of age until they produced seed, the transgenic plants fared better. Within 4 d of the initiation of treatment, the transgenic plants appeared visually larger than wild-type plants, whereas wild-type and transgenic plants of the same age that were maintained under control conditions were of comparable size (Fig. 8). By 5 weeks of age, when the plants had been exposed to UV-B for 1.5 weeks, the biomass of wild-type plants was on average 30% less than that of the transgenic plants under conditions of UV-B stress (Fig. 7B). The reduction in seed yield attributable to the sublethal UV-B stress was also less for the transgenic lines 4-2A-5, 1-4C-8, and 3-2C-5 than for wild-type plants (Fig. 7C). In addition, the formation of anthocyanin was much more pronounced in the wild-type plants than in any of the transgenic lines, indicating that the transgenic plants are less susceptible to UV-B stress. Indeed, after 13 d of UV-B stress, levels of anthocyanin were only 25% of the level in wild-type plants for line 1-4C-8, 30% for line 4-2A-5, and 43% for line 3-2C-5 (Fig. 9B). By contrast, anthocyanin was barely detectable in the leaves of wild-type and transgenic lines in the absence of UV-B stress (Fig. 9A). The UV-B stress also induced premature leaf senescence as evidenced by a reduction in leaf chlorophyll content, but again the effect was more pronounced in wild-type plants than in the transgenic lines. For example, a reduction in leaf chlorophyll was evident by 3 d after initiation of the stress for wild-type plants but not until 8 d for the transgenic lines (Fig. 9D). After 13 d of treatment, leaf chlorophyll levels in wild-type plants had declined by 50%, whereas those for the transgenic plants had declined by only 15%, 23%, and 30% for lines 1-4C-8, 4-2A-5, and 3-2C-5, respectively (Fig. 9D). During the same period, there was virtually no reduction in leaf chlorophyll levels of wild-type and transgenic plants that were not subjected to UV-B stress (Fig. 9C).
Wild-type and antisense transgenic plants were also subjected to sublethal salt stress and drought stress. For salt stress, each plant received 50 mL of 20 mM NaCl every other day, and for drought stress, each plant received 50 mL of 15% polyethylene glycol every second day. The treatments were initiated when the plants were 3.5 weeks of age and were continued until they matured and produced seed. Unlike their response to UV-B stress, the transgenic and wild-type plants proved to be equally susceptible to drought stress and salt stress (data not shown).
Yet another indication of reduced sensitivity of the antisense lipase transgenic plants to UV is the finding that they were unable to up-regulate PR-1 in response to UV-B treatment. Up-regulation of PR-1 is a well-characterized molecular response to UV radiation (Logemann et al., 1995
Southern analysis indicated that the genome of wild-type Arabidopsis contains only one copy of GenBank sequence AAD21737.1. Specifically, when genomic DNA was cut with EcoRI, a restriction enzyme that does not cut within the genomic sequence corresponding to GenBank sequence AAD21737, only a single restriction fragment was evident in Southern blots probed with the cDNA (data not shown). This finding is consistent with GenBank data indicating that the Arabidopsis genome only contains one copy of this gene. When genomic DNA from the transgenic lines 4-2A-5 and 1-4C-8 was digested with EcoRI, five restriction fragments in addition to the fragment corresponding to the endogenous gene were discernible in Southern blots (data not shown). This can be interpreted as indicating that these lines contain five copies of the antisense transgene. However, Southern blots for transgenic line 3-2C-5 featured only two copies of the transgene (data not shown).
To confirm that expression of the lipase had been suppressed in the transgenic plants, levels of cognate lipase protein were compared in wild-type and transgenic plants by western blotting. Protein was isolated from 3.5-week-old Arabidopsis rosettes, fractionated by SDS-PAGE, transferred onto a membrane, and probed with
Two lines of evidence indicate that the lipase AAD21737.1 encoded by GenBank sequence At2g42690 is capable of deesterifying fatty acids from complex lipids. First, the cognate protein contains the lipase consensus sequence [LIV]-X-[LIVAFY]-[LIAMVST]-G-[HYWV]-S-X-G-[GSTAC], which encompasses the active site for lipase-mediated fatty acid deesterification and is a characteristic feature of all known lipases that deesterify fatty acids. Second, the corresponding recombinant protein proved capable of deesterifying fatty acids from phospholipids, galactolipid, and triacylglycerol. However, the recombinant lipase exhibited a 2- to 4-fold higher preference for phospholipid substrate relative to galactolipid and triacylglycerol, indicating that it likely functions as a phospholipase in situ. Moreover, the putative phospholipase may possess molecular species specificity. This was evident from the finding that, although it readily deesterified fatty acids from purified soybean phosphatidylcholine, which contains a mixture of phosphatidylcholine molecular species, the lipase proved incapable of hydrolyzing fatty acids from selected individual molecular species of phosphatidylcholine, including 16:0/20:4 phosphatidylcholine, 20:4/20:4 phosphatidylcholine, 16:0/18:3 phosphatidylcholine, 16:0/18:2 phosphatidylcholine, 18:2/18:2 phosphatidylcholine, and 18:3/18:3 phosphatidylcholine. Although it is possible that this reflects the presence of some unknown factor required for lipase activity that is present in the soybean phosphatidylcholine substrate but not in the individual molecular species substrates, this seems unlikely inasmuch as all of the substrates were purchased as purified products. Moreover, the recombinant lipase proved to be active, although to a lesser degree, on trilinolein and MGDG, which, like soybean phosphatidylcholine, contain a mixture of molecular species. As well, 16:0/18:2 phosphatidylcholine substrate, one of the molecular species tested with lipase AAD21737.1, has been shown to be readily hydrolyzed by recombinant protein corresponding to the Arabidopsis lipase DEFECTIVE IN ANTHER DEHISCENCE1 after emulsification by the same protocol used in this study (Ishiguro et al., 2001 ). This further strengthens the contention that, as for soybean phosphatidylcholine, emulsifying individual molecular species by sonication in gum Arabic engenders the formation of liposomes. These observations, taken together, suggest, although do not prove, that the phospholipase only deesterifies specific molecular species of phosphatidylcholine.
Although the protein corresponding to Arabidopsis sequence AAD21737.1 appears to be a phospholipase capable of hydrolyzing membrane lipids, western-blot analysis and confocal microscopy indicated that it is localized in the cytosol. This is consistent with the fact that it is composed of mainly hydrophilic amino acids and does not contain any putative transmembrane domains. Nor does it exhibit a plastid- or mitochondrial-targeting sequence. Moreover, the lipase protein proved to be detectable in all organs of the Arabidopsis plant, although it is more abundant in leaves, stems, flowers, and siliques than in seeds. There are several previous reports of lipases that act on membrane lipids being localized in the cytosol, including lipolytic acyl hydrolases (Galliard, 1971
Transgenic plants with suppressed lipase expression were indistinguishable from wild-type plants, indicating that this lipase is not essential for normal growth and development. As well, transgenic and wild-type plants responded in a similar manner to salt and drought stress. However, the transgenic plants exhibited increased tolerance to sublethal UV-B stress in comparison with wild-type plants. Typical symptoms of UV-B stress include inhibition of growth (Day et al., 2001
There is increasing evidence that UV-B-induced changes in gene expression are mediated by a number of distinct signal transduction pathways (Jordan, 2002
Peroxidized linoleic acid and linolenic acid are also formed in membranes through the octadecanoid pathway. However, levels of these fatty acid hydroperoxides formed through the octadecanoid pathway are normally low, as it is a signal transduction pathway. Moreover, they are further metabolized by additional enzymes in the pathway and ultimately converted to volatile signaling compounds that do not accumulate in membranes (Gardner, 1995
Yet another indication of reduced sensitivity of the antisense lipase transgenic plants to UV is the finding that they were unable to up-regulate PR-1 in response to UV-B treatment. Up-regulation of PR-1 was clearly evident in the rosette leaves of UV-B-treated wild-type plants but was not discernible in UV-B-treated transgenic plants. UV irradiation is known to simulate some of the effects of pathogen ingression and wounding on gene expression (AH-Mackerness et al., 1999
Plant Material Seeds of Arabidopsis, ecotype Columbia, were sown in soil (Premier Pro-Mix BX; Premier Brands, Brampton, ON, Canada), cold treated at 4°C for 2 d, and germinated in a growth chamber at 23°C under 150 µmol m–2 s–1 photosynthetically active radiation in 16-h-light/8-h-dark photoperiods. Seedlings were grown to maturity using the same chamber conditions.
Full-length cDNA corresponding to the Arabidopsis GenBank protein sequence, accession number AAD21737.1, was obtained by RT-PCR. Template RNA was isolated from the rosette leaves of 4-week-old Arabidopsis plants according to Davis et al. (1986)
Recombinant protein was obtained by expressing pTrc 99a containing the cDNA insert corresponding to Arabidopsis GenBank protein sequence, accession number AAD21737.1, in Escherichia coli DH5
Lipase activity was assayed as described by Ishiguro et al. (2001)
The reaction mixture was incubated for 2 h at room temperature on a rotator. The lipids were extracted in 1 mL of chloroform and resuspended in 50 µL of water. Levels of nonesterified fatty acids were measured using the NEFA colorimetric kit (Wako Chemicals, Neuss, Germany). In some experiments, nonesterified fatty acids released during the lipase reaction were identified and quantified by gas chromatography. For this purpose, the chloroform extract of the reaction mixture was dried under nitrogen gas, resuspended in approximately 100 µL of 6:1 (v/v) chloroform:methanol containing 15 µg of heptadecanoic acid internal standard and fractionated by TLC (SIL G-25, 0.25-mm silica gel layer; Macherey-Nagel, Duren, Germany) in petroleum ether:diethylether:acetic acid (7:3:1). The fractionated lipids were identified using authentic standards (dioleoyl L-
Cytosolic and microsomal membrane fractions were isolated from the rosette leaves of 3.5-week-old Arabidopsis plants. The leaves (20 g) were homogenized at 4°C in 60 mL of homogenization buffer [50 mM 4-(2-hydroxyethyl)piperazine-1-propanesulfonic acid (EPPS; pH 7.4), 0.25 M sorbitol, 10 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol (DTT), 10 mM amino-n-caproic acid, 50 µg chymostatin, 4% polyvinylpolypyrrolidone (w/v)] containing 1 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, and a few drops of antifoam, using an Omni-mixer (eight 3-s bursts at a setting of 10), followed by treatment for 60 s with a Polytron homogenizer (setting of 6; Brinkmann Instruments, Westbury, NY). The homogenate was filtered through four layers of cheesecloth and centrifuged for 20 min at 12,000g. The pellet was discarded, and the supernatant was further centrifuged for 1 h at 305,000g. The resultant pellet of microsomal membranes was resuspended in 2 mL of resuspension buffer (50 mM EPPS [pH 7.0], 0.25 M DTT, 10 mM amino-n-caproic acid, 50 µg chymostatin), and the supernatant (cytosolic fraction) was centrifuged again at 305,000g for 12 h to sediment any residual membrane.
Intact chloroplasts were isolated essentially as described by Kunst (1998)
Suppression of the endogenous Arabidopsis sequence (GenBank protein accession no. AAD21737.1) was achieved by expressing the corresponding cDNA in the antisense orientation under the regulation of a constitutive promoter in transgenic plants. For this purpose, an expressed sequence tag (EST; 221D24) corresponding to the lipase protein was obtained from the Arabidopsis Biological Resource Center. The EST lacks 213 nucleotides of the 5' end of the coding region but is otherwise complete. It was subcloned into the binary vector pKYLX71 (Schardl et al., 1987 Transgenic plants were selected by germinating seeds from the vacuum-infiltrated plants on media containing kanamycin. The seeds were surface-sterilized in a solution of 1% sodium hypochlorite and 0.1% Tween 80, rinsed three times in sterile water, and plated on 0.7% agar containing one-half Murashige and Skoog salt (Sigma-Aldrich, Oakville, Canada) and 50 µg/mL of kanamycin. The plates were maintained at 4°C for 2 d and then transferred to a tissue culture chamber operating at 22°C ± 3°C with 16-h-light (150 µmol m–2 s–1 photosynthetically active radiation)/8-h-dark cycles. After 12 d, surviving seedlings were transferred to soil (Pro-mix BX) and grown to maturity in a growth chamber under conditions specified above. Seed was harvested, and the selection process was repeated until homozygous lines exhibiting 100% germination on kanamycin-containing media were obtained.
The number of antisense gene insertions in homozygous transgenic lines was assessed by Southern-blot analysis. Genomic DNA was isolated from the full complement of rosette leaves from 4-week-old wild-type and transgenic plants and digested with the restriction endonuclease EcoRI. The digested products (10 µg DNA) were fractionated on an agarose gel, immobilized on a nylon membrane, and hybridized with 32P-labeled lipase EST 221D24 according to the method described by Wang et al. (2001)
Wild-type and transgenic plants at 3.5 weeks of age were transferred to a UV-B treatment chamber (187.94 µmol m–2·s–1 photosynthetically active radiation, 2.46 µmol m–2·s–1 UV-B, and 15.04 µmol m–2 s–1 UV-A) operating on a 24-h cycle of 16 h of irradiation followed by 8 h of darkness. Any incidental UV-C radiation from the UV lamps was removed by screening with cellulose acetate. Plants were maintained in the treatment chamber for various periods of time and harvested for analysis at specified time points during the treatment.
For northern-blot analysis, total RNA (10 µg) was fractionated on 1.0% denaturing formaldehyde-agarose gels and immobilized on Hybond-N+ nylon membrane (Amersham Pharmacia Biotech, Uppsala). The membrane was probed with 32P-labeled cDNA corresponding to the Arabidopsis EST 221D24 using hybridization conditions described by Wang et al. (2001)
Sections (2 mm2) of rosette leaves were vacuum-infiltrated with 2% paraformaldehyde in 25 mM K-phosphate buffer (pH 7.2) for 1 h at 4°C, washed three times for 1 h in deionized water, and treated with 1% Tween 20 in phosphate-buffered saline (PBS) for 30 min. The fixed tissue was then washed three times for 20 min in a mixture of 0.2% Gly and 0.2% Tween 20 in PBS, blocked in 2% ovalbumin, 0.2% Gly, and 0.2% Tween 20 in PBS for 30 min, incubated in lipase antiserum (primary antibody; dilution of 1:200 in the blocking solution) for 1 h, and then washed three times for 30 min in blocking solution. The tissue was then incubated for 2 h in blocking solution containing Alexa 400 goat anti-rabbit fluorescein (dilution of 1:400), washed three times for 30 min in blocking solution and for 30 min in PBS. The labeled tissue was then viewed under a laser scanning spectral confocal microscope (Leica TCS SP2; Leica Microsystems Canada, Richmond Hill, Canada).
Chlorophyll and anthocyanin were measured as described by Porra et al. (1989) Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession numbers At2g42690 and AAD21737.1. Received November 18, 2003; returned for revision January 29, 2004; accepted January 30, 2004.
1 This work was supported by the Natural Sciences and Engineering Research Council of Canada.  Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.036376. * Corresponding author; e-mail jet{at}sciborg.uwaterloo.ca; fax 519–746–2543.
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) have been identified in Arabidopsis (Wang, 2001
