An ethylene-induced cDNA encoding a lipase expressed at the onset of senescence

Abstract

A cDNA clone encoding a lipase (lipolytic acyl hydrolase) expressed at the onset of petal senescence has been isolated by screening a cDNA expression library prepared from carnation flowers (Dianthus caryophyllus). The cDNA contains the lipase consensus sequence, ITFAGHSLGA, and encodes a 447-amino acid polypeptide with a calculated molecular mass of 50.2 kDa that appears to be a cytosolic protein. Over-expression of the clone in Escherichia coli yielded a protein of the expected molecular weight that proved capable of deesterifying fatty acids from p-nitrophenylpalmitate, tri-linolein, soybean phospholipid, and Tween in both in vitro and in situ assays of enzyme activity. The abundance of the lipase mRNA increases just as carnation flowers begin to senesce, and expression of the gene is also induced by treatment with ethylene. Southern blot analyses of carnation genomic DNA have indicated that the lipase is a single copy gene. The lipase gene is also expressed in carnation leaves and is up-regulated when the leaves are treated with ethylene. Deesterification of membrane lipids and ensuing loss of membrane structural integrity are well established early events of plant senescence, and the expression pattern of this lipase gene together with the lipolytic activity of its cognate protein indicate that it plays a fundamentally central role in mediating the onset of senescence.

Figure 1

Nucleotide and inferred amino acid sequences of the senescence-induced lipase cDNA from carnation petals (GenBank accession no. AF026480). Nucleotide sequence: 5′ and 3′ noncoding cDNA sequence, lowercase letters; 19-bp oligonucleotide used for cDNA library screening, solid-line boxed region. Amino acid sequence: lipase consensus motif, solid-line boxed region; putative amidation site, single solid underline; putative protein kinase C phosphorylation site, broken-line boxed region; putative cAMP phosphorylation site, broken single underline; putative casein kinase II phosphorylation site, double solid underline; N-glycosylation site, double-line boxed region.

Figure 2

Northern blot analysis of total RNA isolated from carnation flowers at different stages of post-harvest development and carnation leaves. Each lane contained 10 μg of RNA. Upper panels, RNA blots probed with carnation lipase cDNA; lower panels, agarose gels of fractionated RNA. (A) Lanes 1–4, flowers at stages I–IV of post-harvest development, respectively. (B), lanes: 1, stage II flowers; 2, stage II flowers treated with ethylene; 3, a mixture of senescing and nonsenescing leaves from stems bearing flowers at stage II of development; 4, leaves from stems bearing flowers at stage II of development that were treated with ethylene.

Figure 3

Western blot analysis of purified lipase fusion protein from E. coli. The blot was probed with lipase antibody. Lanes: 1, maltose-binding protein control; 2, lipase fusion protein; 3, lipase fusion protein cleaved with Factor Xa. Molecular weight markers (kDa) are indicated.

Figure 4

In situ assay of lipolytic acyl hydrolase activity in E. coli cells harboring lipase fusion protein. Cells were cultured in basal salt medium supplemented with Tween 40 or Tween 60. ●, cells with maltose-binding protein cultured in medium containing Tween 40; ○), cells with lipase fusion protein cultured in medium containing Tween 40; ■, cells with maltose-binding protein cultured in medium containing Tween 60; □, cells with lipase fusion protein cultured in medium containing Tween 60; ▴, cells with maltose-binding protein cultured in the absence of Tween; ▵, cells with lipase fusion protein cultured in the absence of Tween. The assay was performed four times with comparable results.