Biology subjects

Brotman, Y.

Publications and source records attributed to Brotman, Y..

13 recordsLinked to original sources

bioRxiv2026

Resolving Gene-Metabolism Links through Multi-Metabolite mQTL Commonality Analysis

High-throughput metabolomics detects so many compounds that most studies use only a fraction of them, focusing on the few metabolites with an annotated structure or substructure. Metabolite genome-wide association studies of specialized metabolism detect many metabolite quantitative trait loci (mQTLs), but most associated metabolite features remain unannotated, limiting gene discovery. In leaves of 362 tomato accessions under control and water-deficit conditions, only 13 of the 826 metabolite features associated with 77 reproducible mQTLs were annotated with good confidence. To study the mQTLs with the largest impact, we developed commonality analysis, an approach that does not depend on well-studied pathways. Commonality analysis uses biologically defined groups, such as the features associated with one mQTL, to infer relatedness between metabolites. By identifying the fragments, neutral losses or mass transformations that are common in a group, it links the group to a substructure, pathway or reaction, and can extend annotation within the group. Most high-impact mQTLs had a significantly distinct fragment commonality, whose outstanding fragments suggested the pathway involved and its candidate gene. We demonstrate the approach in three representative mQTLs: (i) common fragments pointed to a sinapoyl-hexose pathway and a serine carboxypeptidase-like candidate acyltransferase; (ii) common fragments marked the features associated with a purine permease as nitrogen-containing, which isotope labeling then supported; (iii) a common 2.02 Da transformation pointed to a double-bond reduction and a putative reductase. Overexpression of each gene reproduced the commonality patterns, supporting the candidate genes and showing that the approach also applies to experimentally defined metabolite groups.

plant biology↗

bioRxiv2026

Water stress adaptive responses in plants require movement of ABA and AB-aldehyde from vascular to target tissues

Vascular plants rapidly coordinate root and shoot responses to water stress. Abscisic acid (ABA) mediates these adaptations; however, it remains unclear which cells produce ABA, whether ABA synthesis shifts during stress, and whether ABA movement is required for its adaptive functions. Here, we map ABA biosynthesis at cellular resolution in Arabidopsis and report that water-stress adaptive responses in roots and shoots require movement of ABA and its precursor AB-aldehyde from vascular tissues to target cells. We suggest that ABA accumulation arises from two parallel routes: (i) ABA synthesized in the vasculature via ABA2 and AAO3, then moving to guard cells, and (ii) phloem-derived AB-aldehyde being converted to ABA in the epidermis or bundle sheath by AAO1 and AAO2. Finally, we predict that tightly packed cells beneath leaf veins facilitate efficient ABA delivery to guard cells, an anatomical arrangement that has enabled angiosperms to evolve the use of ABA to rapidly close stomata.

plant biology↗

bioRxiv2026

The mitochondrial chaperone HSPD1 folds MTHFD2 independently of its co-chaperone HSPE1

Acquiring new cellular states entails metabolic reprogramming driven by changes in the expression of cytosolic and mitochondrial metabolic enzymes. Most mitochondrial proteins are synthesized in the cytosol and imported into the mitochondria in a linear form, after which they are folded by a network of mitochondrial chaperones and co-chaperones. Which mitochondrial protein is dependent upon which chaperone for its folding is largely unknown. HSPD1/HSPE1 (HSP60/HSP10) are evolutionarily conserved mammalian homologues of the bacterial proteins GroEL/GroES, forming a chamber-and-lid chaperonin to facilitate the folding of client proteins. We used gene knockdown and SILAC-based proteomics to identify HSPD1 client proteins. We found that HSPD1 supports the expression of Methylenetetrahydrofolate Dehydrogenase 2 (MTHFD2), a key essential protein in the mitochondrial one-carbon (1C) pathway, in cells and tumors, and directly folds MTHFD2, independently of its co-chaperone HSPE1. HSPD1 interacts with MTHFD2 in mitochondria, and MTHFD2 is degraded by LONP1 in HSPD1 knockdown cells. Consequently, we observed reduced nucleotide and S-adenosylmethionine (SAM) levels in HSPD1 knockdown and found minimal overlap in the transcriptional and metabolic cellular responses to HSPD1 vs. HSPE1 depletion. In C. elegans, knockout of HSP60 triggers the mitochondrial stress response in the gut, while HSP10 knockout triggers the mitochondrial stress response in muscle tissue. Our data support that HSPD1 is an MTHFD2 chaperone and that, in addition to working together, HSPD1 and HSPE1 have distinct biological functions.

cell biology↗

bioRxiv2024

Bacteria adapt nutrient deprivation by reducing vacant 70S ribosomes via the silencing factor RsfS

Bacteria rapidly remodel protein synthesis to survive nutrient limitation, yet how inactive ribosomes are selectively regulated remains incompletely understood. Ribosome silencing factor S (RsfS) is the only known bacterial factor that selectively dissociates vacant 70S ribosomes, but its physiological function in bacterial adaptation has remained elusive. Here, we demonstrate that RsfS promotes bacterial survival during nutrient starvation by selectively dismantling non-translating vacant 70S ribosomes while sparing translating ribosomes. Biochemical analyses show that RsfS efficiently dissociates vacant 70S ribosomes but has little effect on programmed ribosomes in the pre-translocation or post-translocation states, thereby preserving ongoing protein synthesis. Ribosome profiling further reveals that loss of RsfS results in a global reduction in translational efficiency and widespread translational reprogramming during starvation. Mechanistically, dissociation of vacant ribosomes by RsfS suppresses futile EF-G-dependent GTP hydrolysis and prevents unnecessary energy expenditure associated with inactive ribosome cycling. Consistent with this mechanism, RsfS enhances bacterial adaptation to nutrient deprivation without compromising active translation. Together, our findings identify RsfS as a unique regulator of bacterial ribosome homeostasis that conserves cellular energy through selective dissociation of vacant 70S ribosomes, revealing a previously unrecognized strategy by which bacteria optimize translation under nutrient-limited conditions.

microbiology↗

bioRxiv2024

Chemoproteomic profiling of serine hydrolases reveals the dynamic role of lipases in Phaeodactylum tricornutum

Phaeodactylum tricornutum is a model oleaginous pennate diatom, widely investigated for the accumulation of triacylglycerols (TAG) in lipid droplets during nitrogen (N) starvation. However, lipid droplet breakdown, TAG catabolism, and remobilization upon N replenishment during growth restoration are less studied. Serine hydrolases (SH) constitute a diverse family encompassing proteases, amidases, esterases, and lipases. In this report, we adopted a chemoproteomic approach called Activity-Based Protein Profiling (ABPP) to explore the repertoire of active serine hydrolases to elucidate the mechanisms of lipid metabolism in P. tricornutum (strain Pt4). A superfamily-wide profile of serine hydrolases revealed a differentially active proteome (activome) during N starvation and after nutrient replenishment. We report 30 active serine hydrolases, which were broadly categorized into metabolic serine hydrolases and serine proteases. Lipases appeared to be the major metabolic linchpins prevalent during lipid remobilization. Global transcriptomics analysis provided a complementary insight into the gene expression level of the detected serine hydrolases. It revealed putative phospholipases as central players in membrane lipid turnover and remodeling involved in cellular lipid homeostasis and TAG accumulation. TAG remobilization and lipid droplet breakdown were impaired in the presence of phenyl mercuric acetate (PMA), whose activity as an SH inhibitor was validated by competitive ABPP. Lipid species profiling corroborated the impairment in TAG degradation and the buildup of structural lipids in the presence of PMA after nutrient replenishment. Collectively, our functional proteome approach, coupled with the transcriptome and lipidome data, provides a comprehensive landscape of bona fide active serine hydrolases, including lipases in this model diatom.

biochemistry↗

bioRxiv2024

Comparative analysis of the plasma metabolome of migrating passerines during stopover: Novel insights into flight metabolism

During long distance migration, many birds may experience periods of either prolonged fasting, during endurance flights, or extensive feeding during stopovers. It was previously shown that habitat selection during stopover can largely affect the migration outcome of an individual. Despite decades of research of the avian metabolism during stopover and migration, many questions have remained unanswered, as such research mainly focused on targeted metabolites and fat metabolism. Here, we examined the plasma-metabolome of migrating passerines prior to their crossing the Sahara Desert. Birds were sampled at two sites populated by Pistacia trees, bearing fat-rich fruits, and at an additional site dominated by blooming Eucalyptus trees. The blood samples were analyzed using both GC-MS and LC-MS, using an untargeted approach. We found that birds from one of the sites had a distinguish metabolic profile, suggesting recent landing. Examination of metabolic pathways activated during stopovers indicated a crucial role for cycling glucose through the Cori and Cahill cycles in resting and recovery processes. This novel perspective, conducted on free-ranging birds, suggests the evolution of avian insulin resistance due to factors such as endurance exercise, fasting, and a preference for fatty acid oxidation during migration, akin to cell trauma recovery. Additionally, we investigated inter-site variations in birds metabolic profiles. Significant variations were observed in both polar and lipophilic metabolites among the sites. Differences in polar metabolites were primarily attributed to variations in the physiological state of the birds between sites, while distinctions in the lipophilic profiles of rested birds were linked to variations in their primary food sources. This study underscores the challenge of interpreting commonly used indicators for assessing migrating birds physiological states and site quality, which are predominantly derived from lipid metabolism, in complex ecological systems.

ecology↗

bioRxiv2023

Non-consecutive enzyme interactions within TCA cycle supramolecular assembly regulate carbon-nitrogen metabolism

Enzymes of the core energy metabolism pathways tend to assemble into transient supramolecular complexes, yet the functional significance of the interactions within these complexes, particularly between enzymes catalyzing non-consecutive reactions, remains unclear. Here, by co-localizing two non-consecutive enzymes of the TCA cycle from B. subtilis, malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICD), in highly crowded liquid-liquid phase separated droplets we discovered that MDH-ICD interaction causes an enhancement of ICD catalytic rate and an apparent sequestration of its reaction product, 2-oxoglutarate. Theory suggests that the observed phenomena are explained by the MDH-mediating clustering of ICD molecules. In vivo validation with targeted GC-MS and 13C tracer analyses revealed that when B. subtilis is grown on glucose and ammonia, overexpression of MDH leads to accumulation of 2-oxoglutarate with a concomitant reduction of fluxes flowing through both the catabolic and anabolic branches of the carbon-nitrogen intersection occupied by 2-oxoglutarate, resulting in impeded ammonium assimilation and reduced biomass production. Our findings thus suggest that in B. subtilis the MDH-ICD interaction is an important coordinator of carbon-nitrogen metabolism, thereby expanding the list of types of functionally understood unconventional enzyme-enzyme interactions.

microbiology↗

bioRxiv2023

R2R3-MYB EVER links emission of volatiles with epicuticular wax biosynthesis in petunia petal epidermis

The epidermal cells of petunia flowers are the main site of volatile emission. However, data on the mechanisms underlying the release of volatiles into the environment are lacking. Here, using cell-layer-specific transcriptomic analysis, reverse genetics by VIGS and CRISPR, and metabolomics we identified EPIDERMIS VOLATILE EMISSION REGULATOR (EVER)--a petal adaxial epidermis-specific MYB activator that affects the emission of volatiles. Using a three-step viral-based CRISPR/Cas9 editing system, ever knockout lines were generated and together with transient suppression assays, revealed EVERs involvement in the repression of low-vapor-pressure volatiles. Internal pools and annotated scent-related genes involved in production and emission were not affected by EVER. RNA-Seq analyses of petals of ever knockout lines and EVER-overexpressing flowers revealed enrichment in wax-related biosynthesis genes. LC/GC-MS analyses of petal epicuticular waxes revealed substantial reductions in wax loads in ever petals, particularly of monomers of fatty acids and wax esters. These results implicate EVER in the emission of volatiles by fine-tuning the composition of petal epicuticular waxes. Thus, we reveal a petunia MYB regulator that interlinks epicuticular wax composition and volatile emission, thus unraveling a new regulatory layer in the scent-emission machinery in petunia flowers.

plant biology↗

bioRxiv2023

Self-eating while being eaten: Elucidating the relationship between aphid feeding and the plant autophagy machinery in Arabidopsis leaves

Autophagy, an intracellular process that facilitates the degradation of cytoplasmic materials, plays a dominant role in plant fitness and immunity. While autophagy was shown to be involved in plant response to fungi, bacteria, and viruses, its role in response to insect herbivory is as yet unknown. In this study, we demonstrate a role of autophagy in plant defense against herbivory using Arabidopsis thaliana and the green peach aphid, Myzus persicae. Following six hours of aphid infestation of wildtype plants, we observed high expression of the autophagy-related genes ATG8a and ATG8f, as well as NBR1 (Next to BRCA1 gene 1), a selective autophagy receptor. Moreover, the number of autophagosomes detected by the overexpression of GFP-fused ATG8f in Arabidopsis increased upon aphid infestation. Following this, atg5.1 and atg7.2 mutants were used to study the effect of autophagy on aphid reproduction and feeding behavior. While aphid reproduction on both mutants was lower than on wildtype, feeding behavior was only affected by atg7.2 mutants. Moreover, upon aphid feeding, the Phytoalexin-deficient 4 (PAD4) defense gene was upregulated in wildtype plants but not affected in the mutants. By contrast, the hydrogen peroxide content was much higher in the mutants relative to wildtype, which might have disturbed aphid reproduction and interfered with their feeding. Additionally, an analysis of the phloem sap metabolite profile revealed that atg7.2 mutant plants have lower levels of amino acids and sugars. These findings, together with the high hydrogen peroxide levels, suggest that aphids might exploit the plant autophagy mechanism for their survival.

plant biology↗

bioRxiv2023

Autophagy in maternal tissues contributes to Arabidopsis thaliana seed development.

Seeds are an essential food source, providing nutrients for germination and early seedling growth. Degradation events in the seed and the mother plant accompany seed development. One degradation mechanism is autophagy, facilitating cellular component breakdown in the lytic organelle. Autophagy influences various aspects of plant physiology, specifically nutrient availability and remobilization, suggesting its involvement in source-sink interactions. During seed development, autophagy was shown to affect nutrient remobilization from mother plants and function in the embryo. Yet, these studies examined autophagy-knockout (atg mutant) plants, making it impossible to distinguish between the contribution of autophagy in the source (i.e., the mother plant) and the sink tissue (i.e., the embryo). To address this, we employed a novel approach to differentiate between autophagy in source and sink tissues. We investigated how autophagy in the maternal tissue affects seed development by performing reciprocal crosses between WT and atg mutant Arabidopsis thaliana plants. Although F1 seedlings possessed a functional autophagy mechanism, etiolated F1 plants from maternal atg mutants displayed reduced growth. This was attributed to altered protein but not lipid accumulation in the seeds, suggesting autophagy differentially regulates carbon and nitrogen remobilization. Surprisingly, F1 seeds of maternal atg mutants exhibited faster germination, resulting from different seed coat development. Our study emphasizes the significance of examining autophagy in a tissue-specific manner, revealing valuable insights into the interplay between different tissues during seed development. It sheds light on the tissue-specific functions of autophagy, offering potential for new research into the underlying mechanisms governing seed development and crop yield.

plant biology↗

bioRxiv2022

Genome-wide association studies of seed metabolites identify loci controlling specialized metabolites in Arabidopsis thaliana

Plants synthesize specialized metabolites to facilitate environmental and ecological interactions. During evolution, plants diversified in their potential to synthesize these metabolites. Quantitative differences in metabolite levels of natural Arabidopsis thaliana accessions can be employed to unravel the genetic basis for metabolic traits using genome-wide association studies (GWAS). Here, we performed metabolic GWAS (mGWAS) on seeds of a panel of 315 A. thaliana natural accessions, including the reference genotypes C24 and Col-0, for polar and semi-polar seed metabolites using untargeted ultra-performance liquid chromatography-mass spectrometry. As a complementary approach, we performed quantitative trait locus (QTL) mapping of near-isogenic introgression lines between C24 and Col-0 for specific seed specialized metabolites. Besides common QTL between seeds and leaves, GWAS revealed seed-specific QTL for specialized metabolites indicating differences in the genetic architecture of seeds and leaves. In seeds, aliphatic methylsulfinylalkyl and methylthioalkyl glucosinolates associated with the GS-ALK and GS-OHP locus on chromosome 4 containing alkenyl hydroxyalkyl producing 2 (AOP2) and 3 (AOP3) and/or with the GS-ELONG locus on chromosome 5 containing methylthioalkyl malate synthase (MAM1) and MAM3. We detected two unknown sulfur-containing compounds that were also mapped to these loci. In GWAS, some of the annotated flavonoids (kaempferol 3-O-rhamnoside-7-O-rhamnoside, quercetin 3-O-rhamnoside-7-O-rhamnoside) were mapped to transparent testa 7 (AT5G07990), encoding a cytochrome P450 75B1 monooxygenase. Three additional mass signals corresponding to quercetin-containing flavonols were mapped to UGT78D2 (AT5G17050). The association of the loci and associating metabolic features were functionally verified in knockdown mutant lines. By performing GWAS and QTL mapping, we were able to leverage variation of natural populations and parental lines to study seed specialized metabolism. The GWAS data set generated here is a high-quality resource that can be interrogated in further studies.

plant biology↗

bioRxiv2020

Autophagy is required for lipid homeostasis during dark-induced senescence in Arabidopsis

Autophagy is an evolutionarily conserved mechanism that mediates the degradation of cytoplasmic components in eukaryotic cells. In plants, autophagy has been extensively associated with the recycling of proteins during carbon starvation conditions. Even though lipids constitute a significant energy reserve, our understanding of the function of autophagy in the management of cell lipid reserves and components remains fragmented. To further investigate the significance of autophagy in lipid metabolism, we performed an extensive lipidomic characterization of Arabidopsis (Arabidopsis thaliana) autophagy mutants (atg) submitted to dark-induced senescence conditions. Our results revealed an altered lipid profile in atg mutants, suggesting that autophagy affects the homeostasis of multiple lipid components under dark-induced senescence. The acute degradation of chloroplast lipids coupled with the differential accumulation of triacylglycerols (TAGs) and plastoglobuli indicates an alternative metabolic reprogramming towards lipid storage in atg mutants. The imbalance of lipid metabolism compromises the production of cytosolic lipid droplets and the regulation of peroxisomal lipid oxidation pathways in atg mutants. One-sentence summaryAutophagy is required for the mobilization of membrane lipid components and lipid droplet dynamics during extended darkness in Arabidopsis.

plant biology↗

bioRxiv2020

Hello darkness, my old friend: 3-Ketoacyl-Coenzyme A Synthase4 is a branch point in the regulation of triacylglycerol synthesis in Arabidopsis by re-channeling fatty acids under carbon starvation

Due to their sessile lifestyle, plants have evolved unique mechanisms to deal with environmental challenges. Under stress, plant lipids are important as alternative sources of carbon and energy when sugars or starch are limited. Here, we applied combined heat and darkness and extended darkness to a panel of [~] 300 Arabidopsis accessions to study lipid remodeling under carbon starvation. Natural allelic variation at 3-KETOACYL-COENZYME A SYNTHASE4 (KCS4), a gene encoding for an enzyme involved in very long chain fatty-acid (VLCFA) synthesis, underlies a differential accumulation of polyunsaturated triacylglycerols (puTAGs) under stress. Ectopic expression in yeast and plants proved that KCS4 is a functional enzyme localized in the ER with specificity for C22 and C24 saturated acyl-CoA. Allelic mutants and transient overexpression in planta revealed the differential role of KCS4 alleles in VLCFA synthesis and wax coverage, puTAG accumulation and biomass. Moreover, the region harboring KCS4 is under high selective pressure and allelic variation at KCS4 correlated with environmental parameters from the locales of Arabidopsis accessions. Our results provide evidence that KCS4 plays a decisive role in the subsequent fate of fatty acids released from chloroplast-membrane lipids under carbon starvation. This work sheds light on both plant response mechanisms and the evolutionary events shaping the lipidome under carbon starvation. One sentence summaryNatural variation at KCS4 underlies a differential accumulation of polyunsaturated triacylglycerols under carbon starvation, by acting as a regulatory branch point in the fate of fatty acids.

genetics↗