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

Fruit ripening and abscission are the results of the cell wall modification concerning different components of the signaling network. However, molecular-genetic information on the cross-talk between ripe fruit and their abscission zone (AZ) remains limited. In this study, we investigated transcriptional and hormonal changes in olive (Olea europaea L. cv Picual) pericarp and AZ tissues of fruit at the last stage of ripening, when fruit abscission occurs, to establish distinct tissue-specific expression patterns related to cell-wall modification, plant-hormone, and vesicle trafficking in combination with data on hormonal content. In this case, transcriptome profiling reveals that gene encoding members of the α-galactosidase and ß-hexosaminidase families associated with up-regulation of RabB, RabD, and RabH classes of Rab-GTPases were exclusively transcribed in ripe fruit enriched in ABA, whereas genes of the arabinogalactan protein, laccase, lyase, endo-ß-mannanase, ramnose synthase, and xyloglucan endotransglucosylase/hydrolase families associated with up-regulation of RabC, RabE, and RabG classes of Rab-GTPases were exclusively transcribed in AZ-enriched mainly in JA, which provide the first insights into the functional divergences among these protein families. The enrichment of these protein families in different tissues in combination with data on transcript abundance offer a tenable set of key genes of the regulatory network between olive fruit tissues in late development.

Cell Wall Composition and Ultrastructural Immunolocalization of Pectin and Arabinogalactan Protein during Olea europaea L. Fruit Abscission.

Cell wall modification is integral to many plant developmental processes where cells need to separate, such as abscission. However, changes in cell wall composition during natural fruit abscission are poorly understood. In olive (Olea europaea L.), some cultivars such as 'Picual' undergo massive natural fruit abscission after fruit ripening. This study investigates the differences in cell wall polysaccharide composition and the localization of pectins and arabinogalactan protein (AGP) in the abscission zone (AZ) during cell separation to understand fruit abscission control in 'Picual' olive. To this end, immunogold labeling employing a suite of monoclonal antibodies to cell wall components (JIM13, LM5, LM6, LM19 and LM20) was investigated in olive fruit AZ. Cell wall polysaccharide extraction revealed that the AZ cell separation is related to the de-esterification and degradation of pectic polysaccharides. Moreover, ultrastructural localization showed that both esterified and unesterified homogalacturonans (HGs) localize mainly in the AZ cell walls, including the middle lamella and tricellular junction zones. Our results indicate that unesterified HGs are likely to contribute to cell separation in the olive fruit AZ. Similarly, immunogold labeling demonstrated a decrease in both galactose-rich and arabinose-rich pectins in AZ cell walls during ripe fruit abscission. In addition, AGPs were localized in the cell wall, plasma membrane and cytoplasm of AZ cells with lower levels of AGPs during ripe fruit abscission. This detailed temporal profile of the cell wall polysaccharide composition, and the pectins and AGP immunolocalization in the olive fruit AZ, offers new insights into cell wall remodeling during ripe fruit abscission.

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

Phytosterols are lipophilic membrane components essential not only for diverse cellular functions but also are biosynthetic precursors of the plant hormone, brassinosteroid (BR). However, the interaction between phytosterol and BR during early fleshy-fruit growth remains largely uncharacterized. In olive, phytosterols are important lipids because they affect oil quality, but phytosterol composition during flowering and early fruit development has not been explored. Here, we first investigated the temporal changes in phytosterol composition, and biosynthetic gene expression that occurred during olive flower opening and early fruit growth. Next, we analyzed the interrelationship between phytosterol and BR, whose levels we manipulated through the application of exogenous BRs (24-epibrassinolide, EBR) or a BR biosynthesis inhibitor (brassinazole, Brz). In this report, the profiling of phytosterol measurement revealed that ß-sitosterol is the most abundant in olive reproductive organs. Our data demonstrate that both OeCYP51 and OeSMT2 genes are upregulated during floral anthesis in good agreement with the rise in cholesterol and ß-sitosterol contents in olive flower. By contrast, the OeCYP51 and OeSMT2 genes displayed different expression patterns during early olive-fruit development. Furthermore, our data show that exogenous EBR enhanced the early olive-fruit growth, as well as the OeSMT2 transcript and ß-sitosterol levels, but decreased the OeCYP51 transcript, squalene, campesterol and cholesterol levels, whereas the Brz treatment exerted the opposite effect. Overall, our findings indicate an up-regulation of ß-sitosterol biosynthesis by BR at the transcriptional level during early olive-fruit growth, providing a valuable tool to unravel the physiological function of SMT2 in future studies.

Modulation of sphingolipid long-chain base composition and gene expression during early olive-fruit development, and putative role of brassinosteroid.

Sphingolipids are abundant membrane components and signalling molecules in various aspects of plant development. However, the role of sphingolipids in early fleshy-fruit growth has rarely been investigated. In this study, we first investigated the temporal changes in sphingolipid long-chain base (LCB) content, composition, and gene expression that occurred during flower opening and early fruit development in olive (Olea europaea L. cv Picual). Moreover, the interaction between sphingolipid and the plant hormone, brassinosteroid (BR), during the early fruit development was also explored. For this, BR levels were manipulated through the application of exogenous BRs (24-epibrassinolide, EBR) or a BR biosynthesis inhibitor (brassinazole, Brz) and their effects on early fruit development, sphingolipid LCB content, and gene expression were examined in olive fruit at 14 days post-anthesis (DPA). We here show that sphingolipid with C-4 hydroxylation and Δ8 desaturation with a preference for (E)-isomer formation are quantitatively the most important sphingolipids in olive reproductive organs. In this work, the total LCB amount significantly decreased at the anthesis stage, but olive sphingosine-1-phosphate lyase (OeSPL) gene was expressed exclusively in flower and upregulated during the anthesis, revealing an association with the d18:1(8E) accumulation. However, the LCB content increased in parallel with the upregulation of the expression of genes for key sphingolipid biosynthetic and LCB modification enzymes during early fruit development in olive. Likewise, we found that EBR exogenously applied to olive trees significantly stimulated the fruit growth rate whereas Brz inhibited fruit growth rate after 7 and 14 days of treatment. In addition, this inhibitory effect could be counteracted by the application of EBR. The promotion of early fruit growth was accompanied by the down-regulation of sphingolipid LCB content and gene expression in olive fruit, whereas Brz application raised levels of sphingolipid LCB content and gene expression in olive fruit after 7 and 14 days of treatment. Thus, our data indicate that endogenous sphingolipid LCB and gene-expression levels are intricately controlled during early fruit development and also suggest a possible link between BR, the sphingolipid content/gene expression, and early fruit development in olive.

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

Plant sphingolipids are involved in the building of the matrix of cell membranes and in signaling pathways of physiological processes and environmental responses. However, information regarding their role in fruit development and ripening, a plant-specific process, is unknown. The present study seeks to determine whether and, if so, how sphingolipids are involved in fleshy-fruit development and ripening in an oil-crop species such as olive (Olea europaea L. cv. Picual). Here, in the plasma-membranes of live protoplasts, we used fluorescence to examine various specific lipophilic stains in sphingolipid-enriched regions and investigated the composition of the sphingolipid long-chain bases (LCBs) as well as the expression patterns of sphingolipid-related genes, OeSPT, OeSPHK, OeACER, and OeGlcCerase, during olive-fruit development and ripening. The results demonstrate increased sphingolipid content and vesicle trafficking in olive-fruit protoplasts at the onset of ripening. Moreover, the concentration of LCB [t18:1(8Z), t18:1 (8E), t18:0, d18:2 (4E/8Z), d18:2 (4E/8E), d18:1(4E), and 1,4-anhydro-t18:1(8E)] increases during fruit development to reach a maximum at the onset of ripening, although these molecular species decreased during fruit ripening. On the other hand, OeSPT, OeSPHK, and OeGlcCerase were expressed differentially during fruit development and ripening, whereas OeACER gene expression was detected only at the fully ripe stage. The results provide novel data about sphingolipid distribution, content, and biosynthesis/turnover gene transcripts during fleshy-fruit ripening, indicating that all are highly regulated in a developmental manner.

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

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.

Comparative transcriptional profiling analysis of olive ripe-fruit pericarp and abscission zone tissues shows expression differences and distinct patterns of transcriptional regulation.

BACKGROUND: In fleshy fruit, abscission of fully ripe fruit is a process intimately linked to the ripening process. In many fruit-tree species, such as olive (Olea europaea L. cv. Picual), there is a coupling of the full ripening and the activation of the abscission-zone (AZ). Although fully ripe fruit have marked physiological differences with respect to their AZs, dissimilarities in gene expression have not been thoroughly investigated. The present study examines the transcriptome of olive fruit and their AZ tissues at the last stage of ripening, monitored using mRNA-Seq. RESULTS: Roche-454 massive parallel pyrosequencing enabled us to generate 397,457 high-quality EST sequences, among which 199,075 were from ripe-fruit pericarp and 198,382 from AZ tissues. We assembled these sequences into 19,062 contigs, grouped as 17,048 isotigs. Using the read amounts for each annotated isotig (from a total of 15,671), we identified 7,756 transcripts. A comparative analysis of the transcription profiles conducted in ripe-fruit pericarp and AZ evidenced that 4,391 genes were differentially expressed genes (DEGs) in fruit and AZ. Functional categorization of the DEGs revealed that AZ tissue has an apparently higher response to external stimuli than does that of ripe fruit, revealing a higher expression of auxin-signaling genes, as well as lignin catabolic and biosynthetic pathway, aromatic amino acid biosynthetic pathway, isoprenoid biosynthetic pathway, protein amino acid dephosphorylation, amino acid transport, and photosynthesis. By contrast, fruit-enriched transcripts are involved in ATP synthesis coupled proton transport, glycolysis, and cell-wall organization. Furthermore, over 150 transcripts encoding putative transcription-factors (TFs) were identified (37 fruit TFs and 113 AZ TFs), of which we randomly selected eight genes and we confirmed their expression patterns using quantitative RT-PCR. CONCLUSION: We generated a set of EST sequences from olive fruit at full ripening, and DEGs between two different olive tissues, ripe fruit and their AZ, were also identified. Regarding the cross-talk between fruit and AZ, using qRT-PCR, we confirmed a set of TF genes that were differentially expressed, revealing profiles of expression that have not previously been reported, this offering a promising beginning for studies on the different transcription regulation in such tissues.

Mature fruit abscission is associated with up-regulation of polyamine metabolism in the olive abscission zone.

This study investigates whether, and how, polyamines (PAs) are involved in mature fruit abscission of olive (Olea europaea L.). Physiological abscission was studied in relation to the activation of the abscission zone (AZ), located between fruit and peduncle, from two olive cultivars where the breakstrength profiles and the scanning electron micrographs illustrated differences in the abscission program, under natural conditions, of mature fruit. The localization and activities of diamine oxidase (DAO), polyamine oxidase (PAO) and PA biosynthetic enzymes, together with PA content were investigated in the fruit AZ during development and abscission. The activities of arginine decarboxylase and S-adenosyl-l-methionine decarboxylase in the fruit AZ were significantly increased and decreased, respectively, by mature fruit abscission, in good agreement with the rise in free putrescine (Put), and content in uncommon PAs there, such as homospermidine and cadaverine, while no significant differences in free spermidine (Spd) and spermine (Spm) contents were detected. By contrast, an abscission-induced decrease was noted in the contents of insoluble conjugated Put, Spd and Spm. The maximum activity of PAO coincided with the maximum content of Spd and Spm, and it was localized mainly in parenchyma cells of pith, while DAO was present mainly in parenchyma cells of pith and cortex as well as at the base of the vascular tissue. These results suggest a clear correlation between the PA distribution and mature fruit abscission. The regulation of PA metabolism is discussed in relation to mature fruit abscission.

Tissue-specific expression of olive S-adenosyl methionine decarboxylase and spermidine synthase genes and polyamine metabolism during flower opening and early fruit development.

Polyamines (PAs) are required for cell growth and cell division in eukaryotic and prokaryotic organisms. The present study is aimed at understanding the developmental regulation of PA biosynthesis and catabolism during flower opening and early fruit development in relation to fruit size and shape. Two full-length cDNA clones coding for S-adenosyl methionine decarboxylase (SAMDC) and spermidine synthase (SPDS) homologs, key steps in the PA biosynthesis pathway, in the stone-fruit of olive (Olea europaea L.) were identified and the spatial and temporal organization of these genes were described. In olive flowers, OeSAMDC gene transcripts were highly expressed in ovary wall, placenta and ovules, while OeSPDS transcript was confined to the ovules of ovary at anthesis stage. A correlation was detected between the SAMDC enzyme activity/accumulation transcript and spermidine (Spd) and spermine (Spm) levels during flower opening, implying that the synthesis of decarboxylated SAM might be a rate-limiting step in Spd and Spm biosynthesis. OeSAMDC and OeSPDS transcripts were co-expressed in fruit mesocarp and exocarp at all developmental stages analyzed as well as in nucellus, integuments and inner epidermis tissues of fertilized ovules. In contrast, the OeSAMDC and OeSPDS genes had different expression patterns during early fruit development. The results provide novel data about localization of PA biosynthesis gene transcripts, indicating that transcript levels of PA biosynthesis genes are all highly regulated in a developmental and tissue-specific manner. The differences between the two olive cultivars in the fruit size in relation to the differences in the accumulation patterns of PAs are discussed.