RNAseq reveals different transcriptomic responses to GA3 in early and midseason varieties before ripening initiation in sweet cherry fruits

AbstractGibberellin (GA) negatively affects color evolution and other ripening-related processes in non-climacteric fruits. The bioactive GA, gibberellic acid (GA3), is commonly applied at the light green-to-straw yellow transition to increase firmness and delay ripening in sweet cherry (Prunus avium L.), though causing different effects depending on the variety. Recently, we reported that GA3 delayed the IAD parameter (a ripening index) in a mid-season variety, whereas GA3 did not delay IAD but reduced it at ripeness in an early-season variety. To further explore this contrasting behavior between varieties, we analyzed the transcriptomic responses to GA3 applied on two sweet cherry varieties with different maturity time phenotypes. At harvest, GA3 produced fruits with less color in both varieties. Similar to our previous report, GA3 delayed fruit color initiation and IAD only in the mid-season variety and reduced IAD at harvest only in the early-season variety. RNA-seq analysis of control- and GA3-treated fruits revealed that ripening-related categories were overrepresented in the early-season variety, including ‘photosynthesis’ and ‘auxin response’. GA3 also changed the expression of carotenoid and abscisic acid (ABA) biosynthetic genes in this variety. In contrast, overrepresented categories in the mid-season variety were mainly related to metabolic processes. In this variety, some PP2Cs putative genes were positively regulated by GA3, which are negative regulators of ABA responses, and MYB44-like genes (putative repressors of PP2Cs expression) were downregulated. These results show that GA3 differentially modulates the transcriptome at the onset of ripening in a variety-dependent manner and suggest that GA3 impairs ripening through the modification of ripening associated gene expression only in the early-season variety; whereas in the mid-season variety, control of the ripening timing may occur through the PP2C gene expression regulation. This work contributes to the understanding of the role of GA in non-climacteric fruit ripening.

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RNA-seq analysis reveals different transcriptomic responses to GA3 in early- and mid-season varieties before ripening initiation in sweet cherry (Prunus avium L.) fruits

10.21203/rs.3.rs-189281/v1 ◽

2021 ◽

Author(s):

Nathalie Kuhn ◽

Jonathan Maldonado ◽

Claudio Ponce ◽

Macarena Arellano ◽

Alson Time ◽

...

Keyword(s):

Gene Expression ◽

Sweet Cherry ◽

Prunus Avium ◽

Gene Expression Regulation ◽

Dependent Manner ◽

Rna Seq ◽

Early Season ◽

Climacteric Fruit ◽

Transcriptomic Responses ◽

Ripening Index

Abstract Gibberellin (GA) negatively affects color evolution and other ripening-related processes in non-climacteric fruits. The bioactive GA, gibberellic acid (GA3), is commonly applied at the light green-to-straw yellow transition to increase firmness and delay ripening in sweet cherry (Prunus avium L.), though causing different effects depending on the variety. Recently, we reported that GA3 delayed the IAD parameter (a ripening index) in a mid-season variety, whereas GA3 did not delay IAD but reduced it at ripeness in an early-season variety. To further explore this contrasting behavior between varieties, we analyzed the transcriptomic responses to GA3 applied on two sweet cherry varieties with different maturity time phenotypes. At harvest, GA3 produced fruits with less color in both varieties. Similar to our previous report, GA3 delayed fruit color initiation and IAD only in the mid-season variety, and reduced IAD at harvest only in the early-season variety. RNA-seq analysis of control- and GA3-treated fruits of both varieties revealed that ripening related categories were overrepresented in the early-season variety, including 'photosynthesis' and 'auxin response.' GA3 also changed the expression of carotenoid and abscisic acid (ABA) biosynthetic genes in this variety. In contrast, overrepresented categories in the mid-season variety were related mainly to metabolic processes. In this variety, some PP2Cs putative genes were positively regulated by GA3, which are negative regulators of ABA responses. This was accompanied with downregulation of MYB44-like genes, putative repressors of PP2Cs expression. These results show that GA3 differentially modulates the transcriptome at the onset of ripening in a variety-dependent manner and suggest that GA3 impairs ripening through the modification of ripening associated gene expression only in the early-season variety; whereas in the mid-season variety, possibly a control of the ripening timing occurs through the PP2C gene expression regulation. This work contributes to the understanding of the role of GA in non-climacteric fruit ripening.

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Gibberellic Acid Modifies the Transcript Abundance of ABA Pathway Orthologs and Modulates Sweet Cherry (Prunus avium) Fruit Ripening in Early- and Mid-Season Varieties

Plants ◽

10.3390/plants9121796 ◽

2020 ◽

Vol 9 (12) ◽

pp. 1796

Author(s):

Nathalie Kuhn ◽

Claudio Ponce ◽

Macarena Arellano ◽

Alson Time ◽

Boris Sagredo ◽

...

Keyword(s):

Gibberellic Acid ◽

Fruit Ripening ◽

Sweet Cherry ◽

Prunus Avium ◽

Molecular Level ◽

Transcript Abundance ◽

Transcript Levels ◽

Early Season ◽

Rapid Transition ◽

Ripening Index

Several phytohormones modulate ripening in non-climacteric fruits, which is triggered by abscisic acid (ABA). Gibberellins (GAs) are present during the onset of ripening in sweet cherry fruits, and exogenous gibberellic acid (GA3) application delays ripening, though this effect is variety-dependent. Although an ABA accumulation delay has been reported following GA3 treatment, the mechanism by which GA modulates this process has not been investigated at the molecular level in sweet cherry. Therefore, the aim of this work is to analyze the effect of GA3 on the fruit ripening process and the transcript levels of ABA pathway orthologs in two varieties having different maturity time phenotypes. The early-season variety had a rapid transition from yellow to pink fruit color, whereas pink color initiation took longer in the mid-season variety. GA3 increased the proportion of lighter colored fruits at ripeness in both varieties, but it produced a delay in IAD—a ripening index—only in the mid-season variety. This delay was accompanied by an increased transcript abundance of PavPP2Cs, which are putative negative regulators of the ABA pathway. On the other hand, the early-season variety had increased expression of PavCYP707A2—a putative ABA catabolic gene–, and reduced transcript levels of PavPP2Cs and SnRK2s after the GA3 treatment. Together these results show that GA modulates fruit ripening, exerting its action in part by interacting with the ABA pathway in sweet cherry.

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Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab136 ◽

2021 ◽

Author(s):

Yu Takahashi ◽

Yu Inoue ◽

Keitaro Kuze ◽

Shintaro Sato ◽

Makoto Shimizu ◽

...

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Gene Expression Regulation ◽

Intestinal Epithelial Cells ◽

Three Dimensional ◽

Culture Conditions ◽

Dependent Manner ◽

Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

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Proinflammatory Interleukin-33 Induces Dichotomic Effects on Cell Proliferation in Normal Gastric Epithelium and Gastric Cancer

International Journal of Molecular Sciences ◽

10.3390/ijms22115792 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5792

Author(s):

Laura Francesca Pisani ◽

Gian Eugenio Tontini ◽

Carmine Gentile ◽

Beatrice Marinoni ◽

Isabella Teani ◽

...

Keyword(s):

Gene Expression ◽

Gastric Cancer ◽

Cell Cycle ◽

Cell Proliferation ◽

Cell Line ◽

Cell Lines ◽

Gene Expression Regulation ◽

Gastric Epithelium ◽

Cell Cycle Gene ◽

Dependent Manner

Interleukin (IL)-33 is a member of the interleukin (IL)-1 family of cytokines linked to the development of inflammatory conditions and cancer in the gastrointestinal tract. This study is designed to investigate whether IL-33 has a direct effect on human gastric epithelial cells (GES-1), the human gastric adenocarcinoma cell line (AGS), and the gastric carcinoma cell line (NCI-N87) by assessing its role in the regulation of cell proliferation, migration, cell cycle, and apoptosis. Cell cycle regulation was also determined in ex vivo gastric cancer samples obtained during endoscopy and surgical procedures. Cell lines and tissue samples underwent stimulation with rhIL-33. Proliferation was assessed by XTT and CFSE assays, migration by wound healing assay, and apoptosis by caspase 3/7 activity assay and annexin V assay. Cell cycle was analyzed by means of propidium iodine assay, and gene expression regulation was assessed by RT-PCR profiling. We found that IL-33 has an antiproliferative and proapoptotic effect on cancer cell lines, and it can stimulate proliferation and reduce apoptosis in normal epithelial cell lines. These effects were also confirmed by the analysis of cell cycle gene expression, which showed a reduced expression of pro-proliferative genes in cancer cells, particularly in genes involved in G0/G1 and G2/M checkpoints. These results were confirmed by gene expression analysis on bioptic and surgical specimens. The aforementioned results indicate that IL-33 may be involved in cell proliferation in an environment- and cell-type-dependent manner.

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Molecular investigation of Tuscan sweet cherries sampled over three years: gene expression analysis coupled to metabolomics and proteomics

Horticulture Research ◽

10.1038/s41438-020-00445-3 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Roberto Berni ◽

Sophie Charton ◽

Sébastien Planchon ◽

Sylvain Legay ◽

Marco Romi ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Analysis ◽

Sweet Cherry ◽

Prunus Avium ◽

Phenylpropanoid Pathway ◽

Primary Metabolism ◽

Differential Abundance ◽

Sweet Cherries ◽

Targeted Gene Expression ◽

Cell Wall Expansion

AbstractSweet cherry (Prunus avium L.) is a stone fruit widely consumed and appreciated for its organoleptic properties, as well as its nutraceutical potential. We here investigated the characteristics of six non-commercial Tuscan varieties of sweet cherry maintained at the Regional Germplasm Bank of the CNR-IBE in Follonica (Italy) and sampled ca. 60 days post-anthesis over three consecutive years (2016-2017-2018). We adopted an approach merging genotyping and targeted gene expression profiling with metabolomics. To complement the data, a study of the soluble proteomes was also performed on two varieties showing the highest content of flavonoids. Metabolomics identified the presence of flavanols and proanthocyanidins in highest abundance in the varieties Morellona and Crognola, while gene expression revealed that some differences were present in genes involved in the phenylpropanoid pathway during the 3 years and among the varieties. Finally, proteomics on Morellona and Crognola showed variations in proteins involved in stress response, primary metabolism and cell wall expansion. To the best of our knowledge, this is the first multi-pronged study focused on Tuscan sweet cherry varieties providing insights into the differential abundance of genes, proteins and metabolites.

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Enhancer Deregulation in Myeloma

Blood ◽

10.1182/blood-2018-99-109523 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. SCI-38-SCI-38

Author(s):

Paola Neri

Keyword(s):

Gene Expression ◽

Transcriptional Repression ◽

Gene Expression Regulation ◽

Therapeutic Targets ◽

Regulatory Elements ◽

Dependent Manner ◽

Cellular Viability ◽

Direct Role ◽

Myeloma Cells ◽

Bet Inhibitors

Abstract The complexity of gene expression regulation is the result of a composite interplay between promoters, enhancers and other cis-acting regulatory elements bound by transcription factors (TFs) that controls the transcriptional activity of genes. Primary tumor cells, in comparison to their healthy counterparts, are known to display altered enhancer repertoires that are associated with tumor-specific transcription. Large groups of transcriptional enhancers cluster together to form super-enhancers (SEs). These elements have been shown to control genes that are important for maintaining cell identity but are also frequently associated with oncogenes as well as translocations that result in aberrant gene expression in cancer. Immunoglobulin (IGH, IGL, IGK) and non-immunoglobulin (PVT1, FAM46C, DUSP22, etc.) enhancers hijacking by variable genes (MYC, MAF, CCND1/2/3, MMSET, IRF4) is a recognized oncogenic driver event in multiple myeloma (MM). However, the identity of the TFs or transcriptional regulatory complexes binding and regulating the activity of these enhancers remains to be fully elucidated and may yield valuable therapeutic targets. In this regard, the bromodomain and extra-terminal (BET) inhibitors have emerged as promising molecules for the treatment of hematologic malignancies. BET family proteins are chromatin adaptors, functionally linked to important pathways for cellular viability and cancer signaling. In particular, BRD4 has a direct role in the transcription regulation of different genes involved in the cell cycle progression and cellular viability. The BET inhibitor JQ1 selectively inhibits BRD4 by competitively binding to the acetyl-lysine recognition pocket of BET bromodomains from chromatin leading to the inhibition of MYC transcription in a dose- and time-dependent manner. Thus, BRD4 has been recently described as a therapeutic target for MM, among other hematologic diseases. Constitutive activation of MYC signaling is detected in more than 60% of patient-derived MM cells and can be involved in the pathogenesis of MM through different mechanisms. One of the most common somatic genomic aberrations in early and late-stage MM is rearrangement or translocation of MYC. Regardless of whether MYC rearrangements occur at early or late stages of MM pathogenesis, MYC rearrangements may provide one of several critical events contributing to increased autonomy and a more aggressive phenotype. Moreover promiscuous rearrangements of the MYC locus are known to hijack enhancers and super-enhancers to dysregulate MYC expression in MM and are involved in its pathogenesis. The development of the immunomodulatory drugs (IMiDs) has contributed significantly to improve the outcomes of MM patients. They possess pleiotropic anti-MM properties and through CRBN binding they induce Ikaros and Aiolos ubiquitylation and proteasomal degradation with an ensuing transcriptional repression of MYC and IRF4, two essential factors for myeloma cells survival. However, is not clear how IKZF1/IKZF3 regulate MYC transcription and how myeloma cells acquire resistance to IMIDs, "beyond CRBN". In addition, acquired resistance to IMIDs and the loss of the transcriptional repression of MYC are nearly universal and occur in spite of sustained IKZF1/3 degradation suggesting that transcriptional rewiring may be sustaining hijacked enhancers activity and transcription of driver oncogenes. In this contest we have recently demonstrated that IMiDs are repressors of IKZF1/3-depedent oncogenic enhancers. Transcriptional plasticity with expression of extra-lineage TFs such as the ETS family member ETV4 sustains oncogenic enhancers in MM overcoming IKAROS and AIOLOS dependency and promoting IMiDs resistance. Therefore defining TFs occupancy and their circuitry at enhancers identifies "non-canonical" (aberrant) myeloma TFs dependency that may be linked to potential therapeutic targets. Disclosures Neri: Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria.

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Characterization of ethylene-inducible pedicel-fruit abscission zone formation in non-climacteric sweet cherry (Prunus avium L.)

10.1101/2020.09.05.284497 ◽

2020 ◽

Author(s):

Seanna L. Hewitt ◽

Benjamin Kilian ◽

Tyson Koepke ◽

Jonathan Abarca ◽

Matthew Whiting ◽

...

Keyword(s):

Differential Expression ◽

Time Course ◽

Sweet Cherry ◽

Prunus Avium ◽

Developmental Time ◽

Abscission Zone ◽

Dependent Manner ◽

Zone Formation ◽

Treatment Groups ◽

Fruit Abscission

AbstractSweet cherry (Prunus avium L.) fruit harvest is a labor-intensive process. Mechanical harvesting of sweet cherry fruit is feasible; however, it is dependent on the formation of an abscission zone at the fruit-pedicel junction. The natural propensity for pedicel-fruit abscission zone (PFAZ) formation varies by cultivar, and the general molecular basis for PFAZ formation is not well characterized. In this study, ethylene-inducible change in pedicel fruit retention force (PFRF) was recorded in a developmental time course with a concomitant analysis of the PFAZ transcriptome from three sweet cherry cultivars. In ‘Skeena’, mean PFRF for both control and treatment fruit dropped below the 0.40kg-force (3.92N) threshold for mechanical harvesting and indicating the formation of a discrete PFAZ. In ‘Bing’, mean PFRF for both control and treatment groups decreased over time. However, a mean PFRF conducive to mechanical harvesting was achieved only in the ethylene-treated fruit. While in ‘Chelan’ the mean PFRF of the control and treatment groups did not meet the threshold required for efficient mechanical harvesting. Transcriptome analysis of the PFAZ followed by the functional annotation, differential expression analysis, and gene ontology (GO) enrichment analyses of the data facilitated the identification of phytohormone-responsive and abscission-related transcripts as well as processes that exhibited differential expression and enrichment in a cultivar-dependent manner over the developmental time-course. Additionally, read alignment-based variant calling revealed several short variants within essential ethylene- and auxin-responsive genes, likely explaining differences in phytohormone-responses between the cultivars. These results provide genetic targets for induction or inhibition of PFAZ formation, depending on the desire to harvest the fruit with or without the stem attached. Understanding the genetic mechanisms underlying the development of the PFAZ will inform future cultivar development while laying a foundation for mechanized sweet cherry harvest.

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Tudor staphylococcal nuclease acts as a docking platform for stress granule components in Arabidopsis thaliana

10.1101/2020.02.20.955922 ◽

2020 ◽

Author(s):

Emilio Gutierrez-Beltran ◽

Pernilla H. Elander ◽

Kerstin Dalman ◽

Jose Luis Crespo ◽

Panagiotis N. Moschou ◽

...

Keyword(s):

Gene Expression ◽

Arabidopsis Thaliana ◽

Cell Biology ◽

Gene Expression Regulation ◽

De Novo ◽

Staphylococcal Nuclease ◽

Fine Tuning ◽

Dependent Manner ◽

Stress Dependent ◽

And Function

SUMMARYAdaptation to stress depends on the modulation of gene expression. Regulation of mRNA stability and degradation in stress granules (SGs), - cytoplasmic membraneless organelles composed of messenger ribonucleoprotein (mRNP) complexes, - plays an important role in fine-tuning of gene expression. In addition, SG formation can modulate stress signaling pathways by protein sequestration. Molecular composition, structure, and function of SGs in plants remain obscure. Recently, we established Tudor Staphylococcal Nuclease (TSN or Tudor-SN; also known as SND1) as integral component of SGs in Arabidopsis thaliana. Here, we combined purification of TSN interactome with cell biology, reverse genetics and bioinformatics to study composition and function of SGs in plants. We found that under both normal (in the absence of stress) and stress conditions TSN interactome is enriched in the homologues of known mammalian and yeast SG proteins, in addition to novel or plant-specific SG components. We estimate that upon stress perception, approximately half of TSN interactors are recruited to SGs de novo, in a stress-dependent manner, while another half represent a dense protein-protein interaction network pre-formed before onset of stress. Almost all TSN-interacting proteins are moderately or highly disordered and approximately 20% of them are predisposed for liquid-liquid phase separation (LLPS). This suggests that plant SGs, similarly to mammalian and yeast counterparts, are multicomponent viscous liquid droplets. Finally, we have discovered that evolutionary conserved SNF1-related protein kinase 1 (SnRK1) interacts with TSN in heat-induced SGs and that SnRK1 activation critically depends on the presence of TSN and formation of SGs. Altogether, our results establish TSN as a docking platform for SG-associated proteins and important stress signal mediator in plants.

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Yolk platelets impede nuclear expansion in Xenopus embryos

10.1101/2021.01.13.426473 ◽

2021 ◽

Author(s):

Sora Shimogama ◽

Yasuhiro Iwao ◽

Yuki Hara

Keyword(s):

Gene Expression ◽

Endoplasmic Reticulum ◽

Gene Expression Regulation ◽

Early Embryogenesis ◽

Dependent Manner ◽

Nucleus Size ◽

Free System ◽

Dependent Cell ◽

A Cell ◽

Nuclear Expansion

ABSTRACTDuring metazoan early embryogenesis, the intracellular properties of proteins and organelles change dynamically through rapid cleavage. In particular, a change in the nucleus size is known to contribute to embryonic development-dependent cell cycle and gene expression regulation. Here, we compared the nuclear sizes of various blastomeres from developing Xenopus embryos and analyzed the mechanisms that control the nuclear expansion dynamics by manipulating the amount of intracellular components in a cell-free system. There was slower nuclear expansion during longer interphase durations in blastomeres from vegetal hemispheres than those from animal hemispheres. Furthermore, upon recapitulating interphase events by manipulating the concentration of yolk platelets, which are originally rich in the vegetal blastomeres, in cell-free cytoplasmic extracts, there was slower nuclear expansion and DNA replication as compared to normal yolk-free conditions. Under these conditions, the supplemented yolk platelets accumulated around the nucleus in a microtubule-dependent manner and impeded organization of the endoplasmic reticulum network. Overall, we propose that yolk platelets around the nucleus reduce membrane supply from the endoplasmic reticulum to the nucleus, resulting in slower nuclear expansion in the yolk-rich vegetal blastomeres.

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Tuscan Varieties of Sweet Cherry Are Rich Sources of Ursolic and Oleanolic Acid: Protein Modeling Coupled to Targeted Gene Expression and Metabolite Analyses

Molecules ◽

10.3390/molecules24081590 ◽

2019 ◽

Vol 24 (8) ◽

pp. 1590 ◽

Cited By ~ 4

Author(s):

Roberto Berni ◽

Mubasher Zahir Hoque ◽

Sylvain Legay ◽

Giampiero Cai ◽

Khawar Sohail Siddiqui ◽

...

Keyword(s):

Gene Expression ◽

Structure Prediction ◽

Sweet Cherry ◽

Prunus Avium ◽

Expression Profiles ◽

3D Structure ◽

Gene Expression Profiles ◽

Reaction Products ◽

Functional Studies ◽

Targeted Gene Expression

The potential of six ancient Tuscan sweet cherry (Prunus avium L.) varieties as a source of health-promoting pentacyclic triterpenes is here evaluated by means of a targeted gene expression and metabolite analysis. By using a sequence homology criterion, we identify five oxidosqualene cyclase genes (OSCs) and three cytochrome P450s (CYP85s) that are putatively involved in the triterpene production pathway in sweet cherries. We performed 3D structure prediction and induced-fit docking using cation intermediates and reaction products for some OSCs to predict their function. We show that the Tuscan varieties have different amounts of ursolic and oleanolic acids and that these variations are related to different gene expression profiles. This study stresses the interest of valorizing ancient fruits as alternative sources of functional molecules with nutraceutical value. It also provides information on sweet cherry triterpene biosynthetic genes, which could be the object of follow-up functional studies.

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