Localization of Sphingolipid Enriched Plasma Membrane Regions and Long-Chain Base Composition during Mature-Fruit Abscission in Olive

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.

Transcriptome and Hormone Analyses Revealed Insights into Hormonal and Vesicle Trafficking Regulation among Olea europaea Fruit Tissues in Late Development

“…Previously, we have reported that endocytosis, visualized by staining with fluorescent dye FM4-64, was strongly stimulated in AZ during olive ripe fruit abscission [ 50 ], which suggests that endomembrane trafficking likely modulates cell wall modifications during olive fruit abscission. Deposition changes in cell-wall material involve vesicle formation and transport, which is reflected by a high number of up-regulated genes found in ripe fruit and AZ tissues in olives ( Figure 6 , Table S3 ).…”

Regulation of sterol content and biosynthetic gene expression during flower opening and early fruit development in olive

“…A pool of 100 mg fresh weight of samples was used for each measurement, and divided into three independent biological replicates. Sterols were quantified by gas chromatography–mass spectrometry as described in (Parra‐Lobato et al ). As an internal standard, 5‐alpha cholestane (Sigma‐Aldrich) was added.…”

“…Purified cDNA (2 ng) was used as a template for qRT‐PCR. Quantitative RT‐PCR was performed and analyzed as described by Parra‐Lobato et al (). The qRT‐PCR assays were performed with gene‐specific primers.…”

Sphingolipid Distribution, Content and Gene Expression during Olive-Fruit Development and Ripening

“…Although sphingomyelin is not present as such in plants, the structure has the features of a complex sphingolipid. Plants have an enormous variety of these lipids and they show a physicochemical behavior expected for a complex sphingolipid, therefore, BD-SM molecules incorporate into the plasma-membrane and mixed with native sphingolipids ( Blachutzik et al, 2012 ; Parra-Lobato et al, 2017 ). Referring to information of the manufacturer, BD-SM features the same stereochemical conformation as biologically active sphingomyelins, despite the fluorescence labeling.…”

“…Experimental work has clearly established that sphingolipids are essential to sustain the vegetative growth of Arabidopsis ( Dietrich et al, 2008 ) but also for adequate pollen development and competence ( Chen et al, 2008 ; Rennie et al, 2014 ). Recently, we reported the first evidence for the potential involvement of sphingolipids in abscission processes as well as an increase in the relative content of sphingolipids during mature-fruit abscission in live protoplasts from abscission zone cells ( Gil-Amado and Gomez-Jimenez, 2013 ; Parra-Lobato et al, 2017 ). However, information regarding their possible involvement in fleshy-fruit development and ripening, a plant-specific process, is still lacking.…”