An Ethylene-Induced Cdna Encoding a Lipase Expressed at the Onset of Senescence

An ethylene-induced cDNA encoding a lipase expressed at the onset of senescence Yuwen Hong*, Tzann-Wei Wang*, Katalin A. Hudak, Frank Schade, Carol D. Froese, and John E. Thompson† Department of Biology, University of Waterloo, Waterloo, ON, Canada N2L 3G1 Communicated by S. J. Peloquin, University of Wisconsin, Madison, WI, May 11, 2000 (received for review December 1, 1999) A cDNA clone encoding a lipase (lipolytic acyl hydrolase) expressed fluidity accompanying senescence may alter the conformation of at the onset of petal senescence has been isolated by screening a membrane proteins, rendering them prone to proteolysis (10, 11). cDNA expression library prepared from carnation flowers (Di- Recent data suggest that free fatty acids arising from the anthus caryophyllus). The cDNA contains the lipase consensus metabolism of membrane lipids may be removed from the sequence, ITFAGHSLGA, and encodes a 447-amino acid polypeptide bilayer by blebbing of lipid particles highly enriched in free fatty with a calculated molecular mass of 50.2 kDa that appears to be a acids from the membrane surface into the cytosol (12–14). These cytosolic protein. Over-expression of the clone in Escherichia coli lipid particles appear to be structurally analogous to oil bodies. yielded a protein of the expected molecular weight that proved Indeed, there is growing evidence that the free fatty acids capable of deesterifying fatty acids from p-nitrophenylpalmitate, released from senescing membranes are metabolized by glyoxy- tri-linolein, soybean phospholipid, and Tween in both in vitro and late cycle enzymes also induced at the onset of senescence (15). in situ assays of enzyme activity. The abundance of the lipase However, it is also clear that free fatty acids accumulate in mRNA increases just as carnation flowers begin to senesce, and senescing membranes and induce lipid-phase separations. The expression of the gene is also induced by treatment with ethylene. resulting mixture of liquid-crystalline and gel phase lipid do- Southern blot analyses of carnation genomic DNA have indicated mains in the bilayer contributes to the leakiness of senescing that the lipase is a single copy gene. The lipase gene is also membranes because of packing imperfections at the phase expressed in carnation leaves and is up-regulated when the leaves boundaries (16). are treated with ethylene. Deesterification of membrane lipids and Deesterification of membrane lipids at the onset of senescence ensuing loss of membrane structural integrity are well established and the resultant release of free fatty acids can be attributed to early events of plant senescence, and the expression pattern of this lipolytic acyl hydrolase, an enzyme that has broad substrate lipase gene together with the lipolytic activity of its cognate specificity and can deacylate phospholipids directly (17). Rose protein indicate that it plays a fundamentally central role in petals, for example, exhibit an increase in lipolytic acyl hydrolase mediating the onset of senescence. at the onset of senescence that is accompanied by loss of membrane function (3). In the present study, we report the embrane deterioration is an early and characteristic fea- isolation and characterization of a cDNA clone from carnation petals that encodes a senescence-induced lipase exhibiting lipo- Mture of plant senescence engendering increased perme- PLANT BIOLOGY ability, loss of ionic gradients, and decreased function of key lytic acyl hydrolase activity. The carnation lipase contains the membrane proteins such as ion pumps (1). One of the clearest 10-amino acid consensus sequence characterizing animal, bac- manifestations of this is the onset of membrane leakiness terial, and fungal lipases. measurable as increased conductivity of diffusates from intact Materials and Methods tissue. This is detectable in carnation petals, for example, well before petal inrolling, the first morphological manifestation of Plant Material and Subcellular Fractionation. Rooted carnation senescence in this tissue, and also before the climacteric-like rise (Dianthus caryophyllus L. cv., Improved White Sim) cuttings in ethylene production (2). The decline in membrane structural were grown as described by Hudak and Thompson (18). Flowers integrity at the onset of senescence appears to be largely at four stages of development (19) were cut and used directly. attributable to accelerated metabolism of membrane lipids and The four stages are as follows: I, petals cream-colored, not fully ensuing change in the molecular organization of the bilayer. expanded from tight bud, outer petals about 30° from vertical; Indeed, loss of membrane phospholipid is one of the best II, flower open with outer petals about 60° from vertical, no documented indices of membrane lipid metabolism during se- ethylene production; III, flower fully open, petals white and nescence and has been demonstrated for senescing flower petals, about 90° from vertical, small amounts of ethylene produced; IV, leaves, cotyledons and ripening fruit (3, 4). petals turned from white to cream-colored, inrolling of petal tips, The selective depletion of phospholipid fatty acids from the loss of turgidity, flowers beginning to close, maximum ethylene membranes of senescing tissues results in an increase in the production (19). Cut flowers at stage I of development were also sterol:fatty acid ratio in the bilayer and a consequent decrease in placed in deionized water and were allowed to senesce under bulk lipid fluidity. This has been demonstrated by fluorescence post-harvest conditions at room temperature. In some experi- depolarization and electron spin resonance for microsomal ments, flowers at stage II of development were treated with 0.5 membranes from senescing cotyledons, flowers, leaves, and ppm of ethylene for 15 h in a sealed chamber to induce ripening fruit (5–8) and for plasmalemma of ripening fruit and premature senescence (2). senescing flowers (5). The decrease in lipid fluidity is engen- dered by an enrichment of free sterols relative to fatty acids in Data deposition: The sequence reported in this paper has been deposited in the GenBank the bilayer as fatty acids are cleaved from the membrane lipids database (accession no. AF026480). and selectively removed, reflecting the fact that free sterols are *Y.H. and T.-W.W. contributed equally to this work. known to restrict the mobility of phospholipid fatty acids (9). As †To whom reprint requests should be addressed. E-mail: [email protected]. well, in some senescing tissues, the decrease in bulk lipid fluidity The publication costs of this article were defrayed in part by page charge payment. This appears to be caused in part by a selective depletion of polyun- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. saturated fatty acids from membranes and an ensuing increase §1734 solely to indicate this fact. in the saturated-to-unsaturated fatty acid ratio (6). There are Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.140213697. also reports that the large changes in bulk membrane lipid Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.140213697 PNAS ͉ July 18, 2000 ͉ vol. 97 ͉ no. 15 ͉ 8717–8722 Downloaded by guest on October 1, 2021 Lipase polyclonal antibodies were obtained by immunizing fusion protein in Escherichia coli, thereby enabling purification rabbits with lipid particles purified from carnation petal cytosol of expression product that could be used for assaying enzymatic isolated just before the onset of visible flower senescence (stage activity. The lipase cDNA was subcloned into the fusion protein III of development). These particles exhibit lipase (lipolytic acyl expression vector, pMalc (New England Biolabs), using EcoRI hydrolase) activity, and they were purified from the petal cytosol and XbaI cloning sites, and the resultant construct was expressed by flotation centrifugation (18). in E. coli BL-21(DE3), yielding a fusion protein consisting of the lipase linked through a proteolytic cleavage site, Factor Xa, to RNA Isolation and Construction and Screening of a cDNA Library. maltose-binding protein. The lipase fusion protein was purified Total RNA for Northern blot analysis was isolated from carna- from E. coli extracts by amylose column chromatography (21, tion petals and leaves as described by Puissant and Houdebine 23). In some experiments, the fusion protein was denatured and (20). For construction of a cDNA library, poly(A)ϩ RNA for treated with Factor Xa (1 ␮g͞100 ␮g of fusion protein) as cDNA synthesis was purified from total RNA of petals at stage described by Sambrook et al. (21) to release the lipase. III of development by using a PolyA tract mRNA Isolation System (Promega). A cDNA library was prepared by using the Lipase Assays. Lipase (lipolytic acyl hydrolase) activity was mea- ZAP express cDNA Synthesis Kit (Stratagene). This library and sured by using both in vitro and in situ assays. For in vitro a cDNA library produced from petals of carnation flowers at measurements, p-nitrophenylpalmitate and soybean phospho- stage IV of development (kindly provided by R. Woodson, lipid (40% phosphatidylcholine and 60% other phospholipids) Purdue University) were both screened for lipase clones. were used as substrates, and the products of the reactions, Initially, Ϸ5 ϫ 105 clones of the stage IV cDNA library were p-nitrophenol and free fatty acids, respectively, were measured immuno-screened with lipid particle antiserum. Positive

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