Biology subjects

Amato, A.

Publications and source records attributed to Amato, A..

12 recordsLinked to original sources

bioRxiv2026

GRASP: A PLANT TRANSFORMATION-INDEPENDENT CRISPR-BASED SYSTEM FOR AFFINITY PURIFICATION OF SPECIFIC CHROMATIN LOCI

Chromatin organization regulates genome stability and gene expression by controlling DNA accessibility to transcription factors and regulatory complexes. DNA-protein interactions are commonly investigated using chromatin immunoprecipitation (ChIP), which relies on specific antibodies often involving technically demanding protocols. CRISPR-Cas technologies have enabled sequence-specific targeting of genomic loci using catalytically inactive Cas9 (dCas9), but most CRISPR-based chromatin capture approaches in plants require transient or stable transformation to express the CRISPR machinery, limiting their applicability across species, tissues and physiological contexts. Here, we present GRASP (Genomic Region Affinity Sequestration by CRISPR-Purification), a transformation-independent strategy for sequence-specific chromatin isolation operating directly on purified plant nuclei. In GRASP, dCas9-gRNA ribonucleoprotein complexes are used to capture predefined genomic regions from chromatin under native conditions, bypassing the need for transgene expression. Using grapevine and tomato as model systems, we demonstrate efficient and highly specific enrichment of target loci, including telomeric repeats as well as low-copy and single-copy genomic regions, with qPCR and NGS validation. These results establish GRASP as a robust and broadly applicable platform for locus-specific chromatin isolation in plants. Beyond sequence-specific DNA isolation, GRASP establishes a versatile platform for potential downstream analyses of locus-associated chromatin components, including protein complexes, distal DNA-DNA interactions and chromatin-associated RNAs, providing new opportunities to investigate regulatory architecture in plant genomes. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=79 SRC="FIGDIR/small/712347v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@1d63d52org.highwire.dtl.DTLVardef@53e7f3org.highwire.dtl.DTLVardef@a11b4eorg.highwire.dtl.DTLVardef@107c94a_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

The effect of cold exposure on energy expenditure of mice fed an obesogenic diet

ObjectiveCold exposure is one of the most powerful physiological stimuli for thermogenic adipose tissue activity and may positively impact metabolic homeostasis. An important gap in knowledge is whether mice with diet-induced obesity respond to cold similarly to their lean counterparts. The goal of the present study was to compare the response to cold exposure between mice fed a standard (normal-fat) diet and a higher-fat diet. MethodsMale C56BL/6J mice fed a standard diet (13.1% fat) or a higher-fat diet (21.6% fat) during adulthood and exposed to cold (4-6{degrees}C) following different approaches. Body weight, body composition, food intake, rectal temperature, energy expenditure, and respiratory exchange ratio were assessed. ResultsCold exposure significantly increased energy expenditure and limited weight gain despite elevated food intake in mice fed either a standard or a higher-fat diet, while body composition, body temperature, and respiratory exchange ratio remained stable across both dietary groups. Moreover, energy expenditure measured at 4-6 {degrees}C was comparable between mice fed standard and higher-fat diets, demonstrating that cold-induced thermogenesis may elicit a consistent metabolic response independent of dietary fat content. ConclusionsOur results demonstrated that cold exposure increased energy expenditure in both lean and obese animals, highlighting thermogenesis as a promising target for obesity treatment beyond current approaches focusing on appetite suppression. HighlightsO_LICold exposure increases energy expenditure and limits weight gain in both control diet and higher-fat diet-fed mice, despite increased food intake. C_LIO_LICore body temperature and body composition remain stable during cold exposure, regardless of dietary fat content. C_LIO_LICold-induced thermogenesis elicits a comparable metabolic response in control diet and higher-fat diet-fed mice, supporting its potential as a therapeutic strategy for obesity. C_LI

physiology↗

bioRxiv2025

Decoupling of plastid and endomembrane homeoviscous response to low temperature and darkness in Phaeodactylum tricornutum

Understanding how marine diatoms adapt to fluctuating environmental conditions is critical for deciphering their ecological success. Here, we investigated the lipidomic responses of the model diatom Phaeodactylum tricornutum to low temperatures (10 {degrees}C), darkness, and combined stress. Using a detailed lipidomics approach, we revealed significant remodeling of plastidial and extraplastidial lipid species. Plastidial lipids showed increased unsaturation levels, particularly the 16:3-to-16:4 fatty acid switch, only under light conditions. This light-dependent adaptation suggests a tightly regulated mechanism that aligns lipid remodeling with photosynthetic activity. Phospholipids in the endomembrane system exhibited less dependency on light, with notable changes even under extended dark periods. These findings highlight distinct strategies employed by diatoms to maintain membrane fluidity and protect cellular processes during stress. Unlike plants, P. tricornutum lacks polygalactosyldiacylglycerols, underscoring a divergent adaptation pathway. Our results demonstrate that P. tricornutum adapts to cold and dark environments through lipid composition changes, supporting survival in variable marine habitats. This study provides critical insights into diatom resilience, paving the way for understanding their role in global biogeochemical cycles. HighlightLow temperature and darkness trigger distinct lipidomic remodeling in Phaeodactylum tricornutum, revealing species-specific adaptive mechanisms that maintain membrane integrity and enhance resilience under extreme environmental conditions.

physiology↗

bioRxiv2025

A Grapevine MYC2-MYB24 Regulatory Module Activates Terpenoid Biosynthesis Upon Methyl Jasmonate Elicitation

Terpenes contribute to the characteristic flavor and aroma of grapes and their derived products while serving protective roles in plants against radiation, oxidative stress, and biotic challenges. The phytohormone methyl jasmonate (MeJA) mediates many of these processes and enhances terpene content in grape berries, but its underlying regulatory mechanism remains unclear. To address this, we treated Vitis vinifera cv. Gamay Freaux berry cell suspensions with MeJA (100 M) and cyclodextrins (50 mM), generating transcriptomic data. Upregulated genes (URGs) were enriched in jasmonic acid biosynthesis and signaling pathways, and transcription factors (TF) that represent novel candidates modulating MeJA responses. Inspection of these TFs in terms of their co-expressed genes allowed us to focus on MYC2, the sole bHLH IIIe subgroup member in grapevine. Using DAP-seq, we identified MYC2-bound genes and integrated them with MeJA-URGs and MYC2 co-expressed genes (CEGs) to produce a high-confidence target list. These targets were bound via a conserved G-box motif and included jasmonate-related genes (e.g., LOX and JAZ) and TFs such as MYB24, previously found to interact with MYC2 to activate terpenoid biosynthesis genes. Consistently, MeJA treatment induced 15 terpene synthase genes (TPS), eleven of which were bound by MYB24, MYC2, or both. Terpenoid compounds associated with these induced TPSs accumulated both intra- and extracellularly following treatment with MeJA and cyclodextrins, but not with abscisic acid (ABA). Our findings suggest that MYC2 regulates the jasmonate pathway and cooperates with MYB24 to mediate MeJA-induced terpene biosynthesis, shedding light on mechanisms that may extend to flower and fruit development, where this MYB-bHLH complex is also expressed.

plant biology↗

bioRxiv2024

Functional study of Phaeodactylum tricornutum Seipin homolog highlights unique features of lipid droplets biogenesis in diatoms.

Diatoms are a major phylum of microalgae, playing crucial ecological roles. They derive from secondary endosymbiosis of a red alga by an unknown heterotrophic eukaryote, leading to a complex intracellular organization. In response to unfavorable conditions (stress), diatoms store oil in lipid droplets (LD), raising interest for applications, in particular biofuels. In spite of numerous investigations aiming to increase their oil content, LD biogenesis mechanisms in these organisms remain poorly understood. In this study, we functionally characterized the homolog of Seipin, a major actor of LD biogenesis, in the diatom Phaeodactylum tricornutum. PtSeipin shares conserved structural features with other Seipins, yet presents unique characteristics, that appear common to diatoms and more broadly Stramenopiles. We provide evidence that Stramenopiles Seipins were inherited from the host during secondary endosymbiosis. The localization of PtSeipin highlights that LD biogenesis can arise simultaneously from the endoplasmic reticulum (ER) and the plastids most external membrane. Finally, the knock-out of PtSeipin leads to a strong increase of TAG accumulation, a feature that is not observed in other organisms and is greatly enhanced following high light exposure. Our results suggest a redirection of lipid fluxes towards TAG synthesis, reduced TAG recycling or a combination of both.

plant biology↗

bioRxiv2024

Delta-5 elongase knockout reduces DHA and TAG synthesis coupled with an increase of heat sensitivity in a marine diatom

Recent global marine lipidomic analysis reveals a strong relationship in the ocean between temperature and phytoplanktonic abundance of omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are essential for human nutrition and primarily from phytoplankton in marine food webs. In phytoplanktonic organisms, EPA may play a major role in regulating the phase transition temperature of membranes, while the function of DHA remains to be explored. In the oleaginous diatom Phaeodactylum tricornutum, DHA is distributed mainly on extraplastidial phospholipids, which is very different from the EPA enriched in thylakoid lipids. Here, CRISPR/Cas9-mediated knockout of ptELO5a, which encodes a delta-5 elongase catalyzing the elongation of EPA to synthesize DHA, led to a substantial interruption of DHA synthesis in P. tricornutum. The ptELO5a mutants show significant alterations in transcriptome and glycerolipidomes including membrane lipids and triacylglycerols under normal temperature (22{degrees}C), and are more sensitive to elevated temperature (28{degrees}C) than wild type. We conclude that the PtELO5a-mediated synthesis of small amounts of DHA has indispensable functions in regulating the membrane lipid, and indirectly contributing storage lipid accumulation and maintaining thermomorphogenesis in P. tricornutum. This study also highlights the significance of DHA synthesis and lipid composition for environmental adaptation of P. tricornutum.

plant biology↗

bioRxiv2023

SPHINGOLIPIDS AND DELTA8-SPHINGOLIPID DESATURASE FROM THE PICOALGA O. TAURI AND INVOLVEMENT IN TEMPERATURE ACCLIMATION

Sphingolipids are crucial components of cell membranes. Sphingolipid {Delta}8-unsaturation is more specific to plants and is involved in the regulation of stress responses. The structure and functions of sphingolipids in microalgae are still poorly understood. Ostreococus tauri is a minimal microalga at the base of the green lineage, and is therefore a key organism for understanding lipid evolution. The present work reports the characterisation as well as the temperature regulation of sphingolipids and {Delta}8-sphingolipid desaturase from O. tauri. Complex sphingolipids are glycosylceramides with unique glycosyl moieties encompassing hexuronic acid residues, reminiscent of bacterial glucuronosylceramides, with up to three additional hexose residues. In contrast, the ceramide backbones show limited variety, with dihydroxylated C18/C18:1E{Delta}8 sphingoid bases and C16:0 fatty-acyl chain being the main compounds. The sphingolipid {Delta}8-desaturase from O. tauri, although phylogenetically related to plant homologues has a substrate preference similar to the diatom homologue. Both sphingolipid {Delta}8-desaturase transcripts and sphingolipid {Delta}8-unsaturation are regulated in a temperature- dependent manner being higher at 14{degrees}C than 24{degrees}C. Overexpressing the sphingolipid {Delta}8- desaturase in O. tauri at 24{degrees}C results in higher sphingolipid unsaturation and impairs the increase in cell size, structure and chlorophyll. In particular, the cell-size defect is not detected in cells acclimated to 14{degrees}C and is furthermore suppressed upon transfer from 24{degrees}C to 14{degrees}C. Our work provides the first functional evidence for the involvement of sphingolipid {Delta}8-unsaturation for temperature acclimation in microalgae, suggesting that this function is an ancestral feature in the green lineage.

biochemistry↗

bioRxiv2023

NAC61 regulates late- and post-ripening associated processes in grapes involving a NAC60-dependent regulatory network

During late-and post-ripening stages, grape berry undergoes profound biochemical and physiological changes whose molecular control is poorly understood. Here, we report the role of NAC61, a grapevine NAC transcription factor, in regulating different processes featuring the berry ripening progression. NAC61 is highly expressed during post-harvest berry dehydration and its expression pattern is closely related to sugar concentration. The ectopic expression of NAC61 in Nicotiana benthamiana leaves determines low stomatal conductance, high leaf temperature, tissue collapse and a higher relative water content. Transcriptome analysis of grapevine leaves transiently overexpressing NAC61, and DNA affinity purification and sequencing analyses allowed us to narrow down a list of NAC61-regulated genes. Direct regulation of the stilbene synthase regulator MYB14, the osmotic stress-related gene DHN1b, the Botrytis cinerea susceptibility gene WRKY52 and the NAC61 itself, is validated. We also demonstrate that NAC61 interacts with NAC60, a proposed master regulator of grapevine organ maturation, in the activation of MYB14 and NAC61 expression. Overall, our findings establish NAC61 as a key player in a regulative network that governs stilbenoid metabolism and osmotic, oxidative and biotic stress responses in grape berry during late-and post-ripening. HighlightsNAC61 regulates stilbene biosynthesis and abiotic/biotic stress responses that hallmark late-and post-ripening developmental stages in grapevine berry. NAC61 participates in a NAC60-dependent regulatory network, also triggering its self-activation.

genetics↗

bioRxiv2023

A physicochemical cause of betaine lipid evolutionary loss in seed plants?

Phosphate is vital for plant and algae growth, yield, and survival, but in most environments, it is poorly available. To cope with phosphate starvation, photosynthetic organisms used their phospholipids as a phosphate reserve. In microalgae, betaine lipids replace phospholipids whereas, in higher plants, betaine lipid synthesis is lost, driving plants to other strategies. The aim of this work was to evaluate to what extent betaine lipids and PC lipids share physicochemical properties and could thus substitute each other. Using neutron diffraction and molecular dynamics simulations of two synthetic lipids, dipalmitoylphosphatidylcholine (DPPC) and dipalmitoyl-diacylglyceryl-N,N,N-trimethylhomoserine (DP-DGTS), we show that DP-DGTS bilayers are thicker, more rigid, and mutually more repulsive than DPPC bilayers. The different properties and hydration response of PC and DGTS provide an explanation for the diversity of betaine lipids observed in marine organisms and for their disappearance in seed plants.

plant biology↗

bioRxiv2022

A molecular phenology scale of fruit development

Fruit growth and development consists of a continuous succession of physical, biochemical, and physiological changes driven by a genetic program that dynamically responds to environmental cues. Establishing recognizable stages over the whole fruit lifetime represents a fundamental requirement for research and fruit crop cultivation. This is especially relevant in perennial crops like the grapevine to scale the development of its fruit across genotypes and growing conditions. In this work, molecular-based information from several grape berry transcriptomic datasets was exploited to build a molecular phenology scale (MPhS) and to map the ontogenic development of the fruit. The proposed statistical pipeline consisted in an unsupervised learning procedure yielding an innovative combination of semiparametric, smoothing and dimensionality reduction tools. The transcriptomic distance between fruit samples was precisely quantified by means of the MPhS that also enabled to highlight the winding dynamics of the transcriptional program over berry development through the calculation of the rate of variation of MPhS stages by time. The MPhS allowed the alignment of time-series fruit samples proving to be a step forward in mapping the progression of grape berry development with higher precision compared to classic time- or phenotype-based approaches and inspiring the use of the transcriptional information to scale the developmental progression of any organ in any plant species.

plant biology↗

bioRxiv2021

The grape MYB24 mediates the coordination of light-induced terpene and flavonol accumulation in response to berry anthocyanin sunscreen depletion

The presence of naturally-occurring color mutants in plants has permitted the identification of many regulatory genes implicated in the synthesis of discrete metabolic compounds, mostly anthocyanins and carotenoids. Conversely, transcription factors that coordinate more than one specialized metabolic pathway seem challenging to screen from a forward genetics perspective. We explored the relationship between different branches of the phenylpropanoid and isoprenoid pathways while examining an infrequent berry skin color variegation in grapevine. Red and white berry skin sections were compared at the genetic, transcriptomic and metabolomic levels showing that, as in most cultivated white grape varieties, the uncolored skin section convened the non-functional alleles of the anthocyanin regulators MYBA1 and MYBA2, explaining the lack of pigments. In contrast, light-responsive flavonols and monoterpenes increased in anthocyanin-depleted areas. We disclosed an enrichment of the flavonol, terpene and carotenoid pathways among up-regulated genes from white-skin sections, accompanied by increased expressions of flavonol regulators and the still uncharacterized MYB24 gene. We used DAP-seq to examine the in vitro binding of affinity-purified MYB24 protein to genomic DNA and demonstrated its binding in the promoter regions of terpene (22) and carotenoid (6) genes, in addition to more than 30 photosynthesis and light-response genes, including the flavonol-regulator HY5 homologue (HYH). We confirmed the activation of TPS35 and HYH promoter:luciferase reporters in the presence of MYB24 and the grape bHLH MYC2, all of which correlate in their higher expression in white skin variegated sections. The integration of several datasets allowed to define a list of high confidence targets, suggesting MYB24 as a modulator of light responses including the synthesis of flavonoids (flavonols) and isoprenoids (terpenes, and putatively carotenoids). The correspondence between MYB24 and monoterpenes in all conditions surveyed implies that this regulatory network is broadly triggered towards berry ripening, and that the absence of anthocyanin sunscreens accelerates its activation most likely in a dose-dependent manner due to increased radiation exposure.

plant biology↗

bioRxiv2021

Initiation of organ maturation and fruit ripening in grapevine is controlled by the CARPO-NAC transcription factor

Grapevine is a woody temperate perennial plant and one of the most important fruit crops with global relevance in both the fresh fruit and winemaking industries. Unfortunately, global warming is affecting viticulture by altering developmental transitions and fruit maturation processes. In this context, uncovering the molecular mechanisms controlling the onset and progression of ripening could prove essential to maintain high-quality grapes and wines. Through a deep inspection of previously published transcriptomic data we identified the NAC family member VviCARPO (Controlled Adjustment of Ripening and maturation of Plant Organs) as a key regulator of grapevine maturation whose induction precedes the expression of well-known ripening associated genes. We explored VviCARPO binding landscapes through DAP-seq and overlapped its bound genes with transcriptomics datasets from stable and transient VviCARPO overexpressing grapevine plants to define a set of high-confidence targets. Among these, we identified key molecular ripening markers. Physiological, metabolic and promoter activation analyses showed that VviCARPO induces chlorophyll degradation and anthocyanin accumulation through the up-regulation of VviSGR1 and VviMYBA1, respectively, with the latter being up-regulated through a VviCARPO-VviNAC03 regulatory complex. Despite showing a closer phylogenetic relationship to senescent-related AtNAP homologues, VviCARPO complemented the nor mutant phenotype in tomato, suggesting it may have acquired a dual role as an orchestrator of both ripening- and senescence-related processes. Our data supports CARPO as a master regulator of the grapevine vegetative-to-mature phase organ transition and therefore an essential target for insuring fruit quality and environmental resilience. SIGNIFICANT STATEMENTCARPO is a grape NAC transcription factor central to fruit ripening and tissue senescence. This regulator influences multiple biological pathways common to both processes including cell wall metabolism, chlorophyll degradation, pigment production and hormone synthesis/signaling through regulation of their key genes. As various external stresses and changing climatic conditions influence vegetative growth and berry ripening, CARPO could prove a useful genetic and breeding target towards maintaining necessary crop performance and fruit-quality characteristics.

plant biology↗