Characterization of Transcriptome Dynamics during Early Fruit Development in Olive (Olea europaea L.).

In the olive (Olea europaea L.), an economically leading oil crop worldwide, fruit size and yield are determined by the early stages of fruit development. However, few detailed analyses of this stage of fruit development are available. This study offers an extensive characterization of the various processes involved in early olive fruit growth (cell division, cell cycle regulation, and cell expansion). For this, cytological, hormonal, and transcriptional changes characterizing the phases of early fruit development were analyzed in olive fruit of the cv. 'Picual'. First, the surface area and mitotic activity (by flow cytometry) of fruit cells were investigated during early olive fruit development, from 0 to 42 days post-anthesis (DPA). The results demonstrate that the cell division phase extends up to 21 DPA, during which the maximal proportion of 4C cells in olive fruits was reached at 14 DPA, indicating that intensive cell division was activated in olive fruits at that time. Subsequently, fruit cell expansion lasted as long as 3 weeks more before endocarp lignification. Finally, the molecular mechanisms controlling the early fruit development were investigated by analyzing the transcriptome of olive flowers at anthesis (fruit set) as well as olive fruits at 14 DPA (cell division phase) and at 28 DPA (cell expansion phase). Sequential induction of the cell cycle regulating genes is associated with the upregulation of genes involved in cell wall remodeling and ion fluxes, and with a shift in plant hormone metabolism and signaling genes during early olive fruit development. This occurs together with transcriptional activity of subtilisin-like protease proteins together with transcription factors potentially involved in early fruit growth signaling. This gene expression profile, together with hormonal regulators, offers new insights for understanding the processes that regulate cell division and expansion, and ultimately fruit yield and olive size.

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.

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.

Variations in Fruit Ploidy Level and Cell Size between Small- and Large-Fruited Olive Cultivars during Fruit Ontogeny.

Olive (Olea europaea L.) is one of the major oil fruit tree crops worldwide. However, the mechanisms underlying olive fruit growth remain poorly understood. Here, we examine questions regarding the interaction of endoreduplication, cell division, and cell expansion with olive fruit growth in relation to the final fruit size by measuring fruit diameter, pericarp thickness, cell area, and ploidy level during fruit ontogeny in three olive cultivars with different fruit sizes. The results demonstrate that differences in the fruit size are related to the maximum growth rate between olive cultivars during early fruit growth, about 50 days post-anthesis (DPA). Differences in fruit weight between olive cultivars were found from 35 DPA, while the distinctive fruit shape became detectable from 21 DPA, even though the increase in pericarp thickness became detectable from 7 DPA in the three cultivars. During early fruit growth, intense mitotic activity appeared during the first 21 DPA in the fruit, whereas the highest cell expansion rates occurred from 28 to 42 DPA during this phase, suggesting that olive fruit cell number is determined from 28 DPA in the three cultivars. Moreover, olive fruit of the large-fruited cultivars was enlarged due to relatively higher cell division and expansion rates compared with the small-fruited cultivar. The ploidy level of olive fruit pericarp between early and late growth was different, but similar among olive cultivars, revealing that ploidy levels are not associated with cell size, in terms of different 8C levels during olive fruit growth. In the three olive cultivars, the maximum endoreduplication level (8C) occurred just before strong cell expansion during early fruit growth in fruit pericarp, whereas the cell expansion during late fruit growth occurred without preceding endoreduplication. We conclude that the basis for fruit size differences between olive cultivars is determined mainly by different cell division and expansion rates during the early fruit growth phase. These data provide new findings on the contribution of fruit ploidy and cell size to fruit size in olive and ultimately on the control of olive fruit development.

Hormonal Content and Gene Expression during Olive Fruit Growth and Ripening.

The cultivated olive (Olea europaea L. subsp. europaea var. europaea) is one of the most valuable fruit trees worldwide. However, the hormonal mechanisms underlying the fruit growth and ripening in olives remain largely uncharacterized. In this study, we investigated the physiological and hormonal changes, by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS), as well as the expression patterns of hormone-related genes, using quantitative real-time PCR (qRT-PCR) analysis, during fruit growth and ripening in two olive cultivars, 'Arbequina' and 'Picual', with contrasting fruit size and shape as well as fruit ripening duration. Hormonal profiling revealed that olive fruit growth involves a lowering of auxin (IAA), cytokinin (CKs), and jasmonic acid (JA) levels as well as a rise in salicylic acid (SA) levels from the endocarp lignification to the onset of fruit ripening in both cultivars. During olive fruit ripening, both abscisic acid (ABA) and anthocyanin levels rose, while JA levels fell, and SA levels showed no significant changes in either cultivar. By contrast, differential accumulation patterns of gibberellins (GAs) were found between the two cultivars during olive fruit growth and ripening. GA1 was not detected at either stage of fruit development in 'Arbequina', revealing a specific association between the GA1 and 'Picual', the cultivar with large sized, elongated, and fast-ripening fruit. Moreover, ABA may play a central role in regulating olive fruit ripening through transcriptional regulation of key ABA metabolism genes, whereas the IAA, CK, and GA levels and/or responsiveness differ between olive cultivars during olive fruit ripening. Taken together, the results indicate that the relative absence or presence of endogenous GA1 is associated with differences in fruit morphology and size as well as in the ripening duration in olives. Such detailed knowledge may be of help to design new strategies for effective manipulation of olive fruit size as well as ripening duration.

A Yap-dependent mechanoregulatory program sustains cell migration for embryo axis assembly.

The assembly of the embryo's primary axis is a fundamental landmark for the establishment of the vertebrate body plan. Although the morphogenetic movements directing cell convergence towards the midline have been described extensively, little is known on how gastrulating cells interpret mechanical cues. Yap proteins are well-known transcriptional mechanotransducers, yet their role in gastrulation remains elusive. Here we show that the double knockout of yap and its paralog yap1b in medaka results in an axis assembly failure, due to reduced displacement and migratory persistence in mutant cells. Accordingly, we identified genes involved in cytoskeletal organization and cell-ECM adhesion as potentially direct Yap targets. Dynamic analysis of live sensors and downstream targets reveal that Yap is acting in migratory cells, promoting cortical actin and focal adhesions recruitment. Our results indicate that Yap coordinates a mechanoregulatory program to sustain intracellular tension and maintain the directed cell migration for embryo axis development.

Mutation of vsx genes in zebrafish highlights the robustness of the retinal specification network.

Genetic studies in human and mice have established a dual role for Vsx genes in retina development: an early function in progenitors' specification, and a later requirement for bipolar-cells fate determination. Despite their conserved expression patterns, it is currently unclear to which extent Vsx functions are also conserved across vertebrates, as mutant models are available only in mammals. To gain insight into vsx function in teleosts, we have generated vsx1 and vsx2 CRISPR/Cas9 double knockouts (vsxKO) in zebrafish. Our electrophysiological and histological analyses indicate severe visual impairment and bipolar cells depletion in vsxKO larvae, with retinal precursors being rerouted toward photoreceptor or Müller glia fates. Surprisingly, neural retina is properly specified and maintained in mutant embryos, which do not display microphthalmia. We show that although important cis-regulatory remodelling occurs in vsxKO retinas during early specification, this has little impact at a transcriptomic level. Our observations point to genetic redundancy as an important mechanism sustaining the integrity of the retinal specification network, and to Vsx genes regulatory weight varying substantially among vertebrate species.

Trap-TRAP, a Versatile Tool for Tissue-Specific Translatomics in Zebrafish.

Developmental and physiological processes depend on the transcriptional and translational activity of heterogeneous cell populations. A main challenge in gene expression studies is dealing with this intrinsic complexity while keeping sequencing efficiency. Translating ribosome affinity purification (TRAP) methods have allowed cell-specific recovery of polyribosome-associated RNAs by genetic tagging of ribosomes in selected cell populations. Here we combined the TRAP approach with adapted enhancer trap methods (trap-TRAP) to systematically generate zebrafish transgenic lines suitable for tissue-specific translatome interrogation. Through the random integration of a GFP-tagged version of the large subunit ribosomal protein L10a (EGFP-Rpl10a), we have generated stable lines driving expression in a variety of tissues, including the retina, skeletal muscle, lateral line primordia, rhombomeres, or jaws. To increase the range of applications, a UAS:TRAP transgenic line compatible with available Gal4 lines was also generated and tested. The resulting collection of lines and applications constitutes a resource for the zebrafish community in developmental genetics, organ physiology and disease modelling.

Analysis of gene network bifurcation during optic cup morphogenesis in zebrafish.

Sight depends on the tight cooperation between photoreceptors and pigmented cells, which derive from common progenitors through the bifurcation of a single gene regulatory network into the neural retina (NR) and retinal-pigmented epithelium (RPE) programs. Although genetic studies have identified upstream nodes controlling these networks, their regulatory logic remains poorly investigated. Here, we characterize transcriptome dynamics and chromatin accessibility in segregating NR/RPE populations in zebrafish. We analyze cis-regulatory modules and enriched transcription factor motives to show extensive network redundancy and context-dependent activity. We identify downstream targets, highlighting an early recruitment of desmosomal genes in the flattening RPE and revealing Tead factors as upstream regulators. We investigate the RPE specification network dynamics to uncover an unexpected sequence of transcription factors recruitment, which is conserved in humans. This systematic interrogation of the NR/RPE bifurcation should improve both genetic counseling for eye disorders and hiPSCs-to-RPE differentiation protocols for cell-replacement therapies in degenerative diseases.

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.

Transcriptomic events involved in melon mature-fruit abscission comprise the sequential induction of cell-wall degrading genes coupled to a stimulation of endo and exocytosis.

BACKGROUND: Mature-fruit abscission (MFA) in fleshy-fruit is a genetically controlled process with mechanisms that, contrary to immature-fruit abscission, has not been fully characterized. Here, we use pyrosequencing to characterize the transcriptomes of melon abscission zone (AZ) at three stages during AZ-cell separation in order to understand MFA control at an early stage of AZ-activation. PRINCIPAL FINDINGS: The results show that by early induction of MFA, the melon AZ exhibits major gene induction, while by late induction of MFA, melon AZ shows major gene repression. Although some genes displayed similar regulation in both early and late induction of abscission, such as EXT1-EXT4, EGase1, IAA2, ERF1, AP2D15, FLC, MADS2, ERAF17, SAP5 and SCL13 genes, the majority had different expression patterns. This implies that time-specific events occur during MFA, and emphasizes the value of characterizing multiple time-specific abscission transcriptomes. Analysis of gene-expression from these AZs reveal that a sequential induction of cell-wall-degrading genes is associated with the upregulation of genes involved in endo and exocytosis, and a shift in plant-hormone metabolism and signaling genes during MFA. This is accompanied by transcriptional activity of small-GTPases and synthaxins together with tubulins, dynamins, V-type ATPases and kinesin-like proteins potentially involved in MFA signaling. Early events are potentially controlled by down-regulation of MADS-box, AP2/ERF and Aux/IAA transcription-factors, and up-regulation of homeobox, zinc finger, bZIP, and WRKY transcription-factors, while late events may be controlled by up-regulation of MYB transcription-factors. SIGNIFICANCE: Overall, the data provide a comprehensive view on MFA in fleshy-fruit, identifying candidate genes and pathways associated with early induction of MFA. Our comprehensive gene-expression profile will be very useful for elucidating gene regulatory networks of the MFA in fleshy-fruit.