Abstract
Sphingolipids, found in membranes of eukaryotic cells, have been demonstrated to carry out functions in various processes in plant cells. However, the roles of these lipids in fruit abscission remain to be determined in plants. Biochemical and fluorescence microscopy imaging approach has been adopted to investigate the accumulation and distribution of sphingolipids during mature-fruit abscission in olive (Olea europaea L. cv. Picual). Here, a lipid-content analysis in live protoplasts of the olive abscission zone (AZ) was made with fluorescent dyes and lipid analogs, particularly plasma membrane sphingolipid-enriched domains, and their dynamics were investigated in relation to the timing of mature-fruit abscission. In olive AZ cells, the measured proportion of both polar lipids and sphingolipids increased as well as endocytosis was stimulated during mature-fruit abscission. Likewise, mature-fruit abscission resulted in quantitative and qualitative changes in sphingolipid long-chain bases (LCBs) in the olive AZ. The total LCB increase was due essentially to the increase of t18:1(8E) LCBs, suggesting that C-4 hydroxylation and Δ8 desaturation with a preference for (E)-isomer formation were quantitatively the most important sphingolipids in olive AZ during abscission. However, our results also showed a specific association between the dihydroxylated LCB sphinganine (d18:0) and the mature-fruit abscission. These results indicate a clear correlation between the sphingolipid composition and mature-fruit abscission. Moreover, measurements of endogenous sterol levels in the olive AZ revealed that it accumulated sitosterol and campesterol with a concomitant decrease in cycloartenol during abscission. In addition, underlying the distinct sterol composition of AZ during abscission, genes for key biosynthetic enzymes for sterol synthesis, for obtusifoliol 14α-demethylase (CYP51) and C-24 sterol methyltransferase2 (SMT2), were up-regulated during mature-fruit abscission, in parallel to the increase in sitosterol content. The differences found in AZ lipid content and the relationships established between LCB and sterol composition, offer new insights about sphingolipids and sterols in abscission.
Highlights
A diverse set of lipids metabolically and structurally related, sphingolipids have many functions related to signaling as well as cell structure
To elucidate whether rising levels of lipids in abscission zone (AZ) and mature-fruit abscission induction were related events, neutral and polar lipid contents were located in live protoplasts from olive AZ in relation to the timing of maturefruit abscission by Bodipy and Nile red staining, respectively (Figures 1, 2)
The use of fluorescent Nile red for lipid measurement in olive AZ protoplasts revealed that abscission significantly increased the polar lipid concentration in the plasma membrane as well as in the intracellular membranes (Figures 2A,C,E), whereas the non-polar or neutral lipid concentration measured for olive AZ at the abscission stage was close to AZ at the pre-abscission stage levels (Supplementary Figure S1), as was detected with the Bodipy dye
Summary
A diverse set of lipids metabolically and structurally related, sphingolipids have many functions related to signaling as well as cell structure (reviewed in Huwiler et al, 2000; Hannun and Obeid, 2002; Pata et al, 2010; Markham et al, 2013; Pyne et al, 2014; Luttgeharm et al, 2016; Michaelson et al, 2016; Kraft, 2017). The amphipatic structural backbone of complex sphingolipids is ideal to form lipid bilayers. This backbone is composed of a long-chain base (LCB) that amidates a fatty acid and binds a polar head formed by a carbohydrate and, optionally, a phosphate group (Chen et al, 2009)
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