Riboflavin treatment triggers stress-responsive gene networks for enhanced adaptation in Arabidopsis

Riboflavin treatment triggers stress-responsive gene networks for enhanced adaptation in Arabidopsis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Riboflavin treatment triggers stress-responsive gene networks for enhanced adaptation in Arabidopsis Dikran Tsitsekian, Efstratios Kamargiakis, Dimitris Templalexis, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6377416/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Riboflavin is the precursor of the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) which are vital coenzymes to a wide array of plant metabolic processes. While the exogenous application of riboflavin has been well-documented to enhance plant stress tolerance, the molecular mechanisms underlying this protective effect remain largely unknown. Here, we present a comprehensive transcriptomic analysis of riboflavin-treated Arabidopsis seedlings, revealing significant changes in gene expression related to stress responses, signaling transduction and secondary metabolism. Riboflavin treatment altered the expression of genes within specific cellular functional categories, supporting the role of riboflavin in regulating plant metabolism and enhancing stress adaptation. The transcriptional changes indicate a shift from growth to stress management, potentially downregulating photosynthesis to preserve energy for immediate stress responses and protect against damage from excess light or oxidative stress. Further, we identified a feedback mechanism where elevated riboflavin levels regulate the expression of genes of its own biosynthetic pathway, controlling both its synthesis and chemical conversion processes. Our study provides novel and valuable insights into the gene expression mechanisms underlying riboflavin-mediated stress tolerance and highlights a potential application of exogenous riboflavin as a strategy for improving crop plasticity and adaptation in the face of environmental challenges. Biological sciences/Plant sciences/Plant genetics Biological sciences/Plant sciences/Plant molecular biology Biological sciences/Plant sciences/Plant stress responses Riboflavin Vitamin B2 flavins transcription factors gene expression stress response plant resilience Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Key Message Riboflavin induces significant transcriptional changes shifting cellular processes from growth to stress management by reprogramming the expression of transcription factors and stress-responsive genes thereby enhancing stress tolerance and plant adaptation Introduction Riboflavin, also known as vitamin B2, is a water-soluble vitamin that was first identified and isolated from milk in 1879. While animals depend on exogenous riboflavin sources, plants and most microorganisms are able to synthesize riboflavin through a conserved biosynthetic pathway 1 . Riboflavin is critical for primary and secondary metabolism, serving as a precursor to the essential coenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Flavins are indispensable components of photosynthesis, energy generation and redox metabolism in plants, thereby contributing to plant growth, development and stress responses 2 , 3 , 4 . Additionally, riboflavin regulates the corn grain filling process through coordination of the mitochondrial energy metabolism and cell cycle 5 . Enhancement of vitamin content in plants through genetic engineering or other breeding approaches could stimulate plant stress tolerance or improve human nutrition 6 . In this context, vitamin B2 content has been enhanced in rice endosperm via metabolic engineering of enzymes from the Saccharomyces riboflavin biosynthetic pathway 7 . For more than three decades, exogenous application of riboflavin has been recognized as a process to enhance pathogen resistance in plants. Formulas containing riboflavin have been demonstrated to be effective in controlling powdery mildew, crown gall, Phytopthora and other diseases in numerous crops 8 , 9 , 10 . Molecular analysis in Arabidopsis and tobacco showed that exogenous application of riboflavin could activate signaling pathways leading to systemic resistance by stimulating Pathogenesis-Relatedgenes, involving protein kinases and regulation by NIM1/NPR1 11 . In Arabidopsis, riboflavin primed defense responses against Pseudomonas infection and induced resistance that was associated with the expression of defense genes and induction of cellular defense mechanisms 12 . Application of riboflavin was able to trigger systemic acquired resistance through the accumulation of ROS and activation of hormonal signaling pathways, which in turn promoted the phenylpropanoid pathway and the accumulation of phenolic and antioxidant compounds 13 , 14 , 15 , 16 . The role of riboflavin in enhancing disease resistance has been reported for various plant species mainly through the activation of host defense responses and callose deposition in stomatal cells 14 , 15 , 17 , 18 . The interplay between riboflavin, enzyme cofactors and abiotic stresses has also been well-documented 19 . Exogenous application of riboflavin positively affected plant growth, pigment biosynthesis and stress tolerance across various plant species 20 , 21 . Particularly, foliar application of riboflavin enhanced Hibiscus resistance to salinity stress by increasing the activity of antioxidant enzymes and the stability of the plasma membrane 22 . Additionally, riboflavin application modified the expression of Na + transporters to enhance ionic stress tolerance and restrict Na + accumulation in the leaf blades of rice, maintaining a favorable Na + /K + balance 23 . Foliar application of riboflavin in rice increased yield and accumulation of 2-acetyl-1-pyrroline fragrance during the heading stage 24 . Moreover, rice seedlings pretreated with riboflavin demonstrated higher plant biomass, lower electrolyte leakage ratio and lower levels of hydrogen peroxide 25 . Significant progress has been made in understanding the physiological and biochemical effects of exogenous riboflavin application in plants. However, the genes involved in plant stress responses due to riboflavin treatment remain largely unknown. In this study, we investigated the gene expression patterns in Arabidopsis seedlings treated with riboflavin through transcriptome analysis. We identified changes in key genes and pathways related to stress responses. Additionally, we identified a set of differentially expressed transcription factors and examined at the transcriptional level the effect of exogenous riboflavin application on its own biosynthetic pathway. Our analysis reveals the gene networks and stress-signaling responses triggered by riboflavin treatment, providing insights into the transcriptional regulation involved in flavin metabolism. Materials and Methods Plant material and growth conditions Arabidopsis thaliana seeds of the Col-0 ecotype were surface-sterilized and sown on Petri dishes containing 0.5x Murashige and Skoog (MS) medium (Duchefa), pH 5.7, supplemented with 1% sucrose and solidified with 0.6% Agarose (Sigma). After 24 h of stratification at 4 o C, plants were positioned to grow vertically at 22°C in a Fitotron (Weiss Gallenkamp) growth chamber with 100 µmol m − 2 s − 1 light intensity under 16 h light/8 h dark photoperiod for 6 days. After 6 days seedlings were transferred to the same media composition supplemented with 0.2 mM riboflavin (Applichem) during the 8 h of the darkness cycle to avoid photodegradation of riboflavin. The concentration of 0.2 mM riboflavin was selected based on previous studies applying a range of 0.01 mM to 2.5 mM (Supplementary Table S1 ). Petri dishes devoid of riboflavin were used as control. After 8 h of treatment with riboflavin, plants were retransferred to Petri dishes containing 0.5x MS medium and placed under a continuous light regime for 24 h. Samples were collected at 0, 4, 8, 12 and 24 h of continuous light treatment and histochemical staining for ROS detection was performed at each sampling point. For the transcriptomic analysis, plant samples were collected right after the 8 h Riboflavin treatment and were stab frozen in liquid nitrogen and stored at -80 o C until RNA extraction. Histochemical staining for ROS detection In situ detection of superoxide and hydrogen peroxide were performed by nitro blue tetrazolium (NBT) and 3,3’ diaminobenzidine (DAB) staining respectively, as previously described 26 . Briefly, seedlings were collected, and transferred in NBT (Applichem) staining solution (2 mM NBT, 20 mM potassium phosphate buffer pH 6.1, 100 mM NaCl), or DAB (Sigma-Aldrich) staining solution (DAB 1 mg/mL dissolved in water with the pH adjusted to 3.8 with 1N KOH), vacuum infiltrated for 5 min and incubated for 15 min (NBT) and 2 h (DAB) at room temperature in the dark. Stained plantlets were bleached in acetic acid-glycerol-ethanol (1/1/3) (v/v/v) solution and then stored in 20% glycerol (v/v) solution until photographed with a Leica M205 FCA stereomicroscope equipped with a Leica DFC7000 T digital camera (Leica Microsystems, Wetzlar, Germany). Extraction of RNA and transcriptome analysis The RNA preparation and RNA-seq analysis were performed as previously described 27 . Briefly, total RNA was extracted from Col-0 (wild-type) seedlings and Col-0 seedlings treated with 0.2 mM riboflavin using the Direct-zol RNA Miniprep kit (Zymo Research, Irvine, CA, USA) with an on-column DNase treatment according to the manufacturer’s instructions from control and PepMV-infected tomato tissue. The quantity and quality of RNA were assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA) and agarose gel electrophoresis. RNA-seq libraries were generated using the polyA Strand-Specific Transcriptome Library method of BGI for DNBSEQ platform. Sequencing was performed on DNBSEQ platform instrument at BGI (Beijing Genomics Institute) in three biological replicates for each sample. Raw reads were filtered into clean reads and aligned to the Arabidopsis genome (GCF_000001735.4_TAIR10.1). RNA-seq data were analyzed using SOAPnuke (version 1.5.2) with parameters “-l 15 -q 0.2 -n 0.05" and the HISAT2 pipeline (version 2.0.4) with parameters “--sensitive --no-discordant --no-mixed -I 1 -X 1000 -p 8". For the detection of differentially expressed genes (DEGs), clean reads were mapped to the reference genome with Bowtie2, then the gene expression level was calculated using RSEM with default parameters. Statistical analysis of differential gene expression was conducted utilizing DESeq2 28 . A multiple-test corrected p-value of 0.05 was adjusted using the Benjamini and Hochberg’s approach, resulting in adjusted p-value (Padj). Transcripts with fold change greater than 2 and a Padj value < 0.05, identified by DESeq2, were assigned as differentially expressed. Heatmaps and volcano plots of the DEGs were constructed with Perseus software (version 1.6.8.0) using Euclidean distancing with average linkage and without any constraints in the algorithm. Reverse-transcription qPCR analysis Total RNA was isolated from plant tissues using the phenol-sodium dodecyl sulfate (SDS) extraction method as previously described 29 . RNA concentrations were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA) and verified by ethidium bromide staining on agarose gels. DNA was eliminated with RQ1 RNase-free DNase (Promega, Madison, WI, USA). Reverse transcription (RT) was performed on 1 µg total DNA free RNA using Invitrogen SuperScript IV VILO Master Mix (Thermo Fisher Scientific). Quantitative gene expression analysis was performed in the PikoReal Real-Time PCR System (Thermo Fisher Scientific) using SYBR Green I as the DNA-binding dye provided in SYBR Select Master Mix (Applied Biosystems, Waltham, MA, USA) and applying the following cycler conditions: 2 min at 50 o C, 2 min at 95 o C, followed by 40 cycles of 15 s at 95 o C, 1 min at 60 o C. All quantitative PCR reactions were performed as triplicates of three biological repeats. At the end of each reaction, the cycle threshold (Ct) was automatically set up at the level that reflected the best kinetic PCR parameters by the PikoReal Software 2.1 and melting curve analysis was performed to monitor primer specificity. Τhe specificity of the amplification was verified on agarose gel. Negative controls included samples without a template and those without prior reverse transcription. The primers and amplicon length per gene are listed in Supplementary Table S2 . Quantification of gene expression was calculated as expression of the gene of interest relative to glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ) and polyubiquitin 10 ( UBQ10 ) gene expression based on the 2 −ΔCt method 30 . The fold change of transcripts between Riboflavin treated (CR) and untreated (Col) samples was calculated with the 2 −ΔΔCt method as previously described 31 , using the untreated (Col) as the normalizing samples. Bioinformatics resources for in silico analysis Functional enrichment analysis of the differentially expressed genes was performed as previously described 30 . Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed utilizing the Database for Annotation, Visualization, and Integrated Discovery (DAVID) 32 , 33 . GO analysis was conducted to elucidate significant genetic regulatory networks by organizing genes into hierarchical categories based on Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). Functional annotation to identify key biological pathways was carried out using KEGG. The enriched KEGG pathways were visualized with Pathview as previously described 34 , a toolset designed for pathway-based data integration and visualization, which maps and renders diverse biological data onto relevant pathway graphs from KEGG and related databases 35 , 36 . Results Exogenous riboflavin treatment results in alterations to Arabidopsis gene expression Extensive research has demonstrated the beneficial effects of exogenous application of riboflavin on plants, highlighting the protective role against abiotic and biotic stresses, as well as the ability to reduce post-harvest decay (Supplementary Table S1 ). To elucidate the transcriptional responses to exogenous riboflavin application, we transferred 7-day-old Arabidopsis seedlings to a growth medium supplemented with riboflavin in darkness. Riboflavin pretreatment has been shown to significantly mitigate oxidative stress by enhancing antioxidant enzyme activity and reducing lipid peroxidation 37 . Hence, we tracked the production of ROS, specifically superoxide anion and hydrogen peroxide, over a 24-hour period under continuous light conditions using nitro blue tetrazolium (NBT) and 3,3′-diaminobenzidine (DAB) staining. Seedlings treated with riboflavin (CR) displayed less ROS accumulation between 4 and 12 hours after treatment compared to untreated wild-type plants (Col) aligning with previous studies 38 , 39 (Fig. 1 ; Supplementary Table S1 ). Since transcriptional responses to exogenous stimuli occur rapidly, we opted to collect samples for RNA sequencing (RNA-seq) after 8 hours of riboflavin treatment to capture early changes in gene expression. RNA-seq analysis resulted in an average of 45 M clean reads and an average mapping ratio of 98.32% (Supplementary Table S3 ). Correlation analysis showed a high consistency among the replicates, which clustered together, while the gene expression profile of CR differed significantly from that of the control Col (Fig. 2 a). The results were highly reproducible within each RNA-seq sample revealing a deep and satisfactory representation of the Arabidopsis transcriptome. The analysis revealed 3,780 DEGs of which 1711 genes were up-regulated and 2069 genes were down-regulated (Fig. 2 a-c; Supplementary Table S4 ). Interestingly, fold enrichment analysis of UniprotKB keyword ligands classification of the DEGs mainly revealed the terms “Chlorophyll”, “Pyridoxal phosphate”, “FAD” and “Flavoprotein” (Fig. 2 d). Pathway enrichment analysis of the DEGs using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database revealed that “Photosynthesis”, “Glutathione metabolism”, “Fatty acid metabolism”, “Glycine, serine and threonine metabolism”, “Glucosinolate biosynthesis” and “Plant hormone signal transduction” were included as significant prominent enriched terms (Fig. 2 e). Based on these observations, it is reasonable to suggest that the exogenous application of riboflavin resulted in a dynamic and coordinated reprogramming of the Arabidopsis transcriptome by altering the expression of genes involved in specific biological processes. Riboflavin modified the expression of specific gene categories involved in various defense mechanisms A Gene Ontology (GO) functional enrichment analysis was performed on the up- and down-regulated DEGs to highlight the main functional categories that were affected by riboflavin. GO consists of three ontologies describing the molecular function (MF), cellular component (CC) and biological process (BP) of the transcripts (Fig. 3 ). Significant enrichment of the upregulated genes in BP was evident mainly for the terms “Cellular response to hypoxia”, “response to water deprivation”, “glutathione metabolic process”, responses to “abscisic acid”, “salt stress”, “heat”, “hydrogen peroxide”, “karrikin”, “wounding” and “to other organisms” (Fig. 3 a). Molecular Function classification of the up-regulated genes primary involved “oxidoreductase activity”, “glutathione transferase activity” and “FAD binding”. Concerning the cellular component, the prime category genes were associated with “membrane”, “cytoplasm”, “chloroplast” and “mitochondrial membranes” (Fig. 3 a). Taken together, these findings strongly suggest that application of riboflavin on Arabidopsis seedlings primarily stimulates the expression of genes associated with stress responses within various defense mechanisms consistent with data of previous studies demonstrating the protective role of riboflavin against biotic and abiotic stresses 38 , 39 . On the other hand, functional enrichment analysis for the BPs of the down-regulated genes revealed critical terms, including “photosynthesis”, “cell division and cycle”, “response to auxin”, “response to light stimulus”, “cell wall organization” and “regulation of growth” (Fig. 3 b). Regarding MF analysis of the down-regulated genes, those exhibiting the highest abundance were “microtubule binding”, “microtubule motor activity” and “cyclin-dependent protein serine/threonine kinases”. In terms of CC analysis, the down-regulated genes were annotated to “chloroplast”, “apoplast”, “cell wall” and “microtubule” (Fig. 3 b). Remarkably, the functional classification analysis of up- and down-regulated genes revealed that the application of riboflavin affects the expression of specific gene categories across all ontologies, indicating a distinct pattern of differential gene regulation for the induction or repression of these genes. Riboflavin triggers the reprogramming of stress response transcriptional controllers Transcription factors (TFs) are central regulatory hubs of the organismal transcriptome mediating responses to external stimuli through a perplexed network of signaling cascades. We therefore conducted a comprehensive analysis of RNA-seq data to identify differentially expresses TFs (DETFs) with significant differential expression. (Fig. 4 a; Supplementary Table S5 ). The analysis revealed that the majority of upregulated TFs were categorized in the NAC , ERF , MYC , and bHLH families. Conversely, the downregulated TFs predominantly belonged to the bHLH , ERF , MYB , and B3 families, with only a small proportion falling into the NAC category (Fig. 4 b). This distinct categorization suggests divergent regulatory pathways and signaling mechanisms between upregulated and downregulated TFs in response to riboflavin treatment. Gene Ontology analysis of the Biological Process category of the upregulated transcription factors identified significant enrichment in processes related to stress responses, whereas the GO analysis concerning the downregulated transcription factors identified terms associated with either plant growth or control of cellular processes. (Fig. 4 ). This pattern indicates a shift in resource allocation from growth to stress management, reflecting a strategic prioritization of survival under possible adverse conditions. Further classification of the DETFs resulted in a list of the top upregulated and downregulated TFs, using a threshold of the gene expression value TPM (Transcripts per Million) > 1.00 as an additional selection criterion (Fig. 4 e). The upregulated TFs included DREB19 , DREB2C , ERF71 , ABR1 , MYB112 and AT4G28140 which are associated with responses to stresses. On the other hand, the list of downregulated TFs included DDF2 and PRE5 , which are involved in the response to gibberellic acid, as well as AT1G33760 , which is related to the ethylene-activated signaling pathway. Furthermore, transcription factor PIL1 , associated with shade avoidance together with the red/far-red light signaling pathway. MYB29 and DDF1 transcription factors, which respond to various biotic and abiotic stresses, are also included in this list. To further validate the accuracy and reproducibility of the transcriptome analysis, we performed reverse transcription quantitative polymerase chain reaction (RT-qPCR) on a subset of the DETFs identified upon riboflavin treatment. Notably, the expression pattern of both upregulated and downregulated DETFs, as assessed by RT-qPCR, was almost identical between the two experimental approaches (Fig. 5 a-b). Additionally, a direct comparison of fold change values using a log 2 scale, demonstrated a strong correlation between the two methodologies, with RNA-seq-derived expression levels aligning well with RT-qPCR measurements (Fig. 5 c). These results collectively validate the reliability of the RNA-seq analysis and indicate a strong and satisfactory correlation of RT-qPCR data with the outcome of transcriptome analysis, confirming the pattern of DETFs identified by RNA-seq analysis. Riboflavin-induced transcriptional modulations in stress and photosynthesis To comprehend in depth the way Arabidopsis plants responded to exogenous application of riboflavin, we annotated the DEGs to the KEGG PATHWAY database (Fig. 6 ). Certain genes of various stress responses were annotated, such as genes of the abscisic acid (ABA) pathway, the plant defensin gene PDF1.2 , the wounding response gene VSP2 and the PAD3 gene responsible for camaxelin synthesis, the characteristic phytoalexin of Arabidopsis , which is induced by a great variety of plant pathogens 40 . Additionally, the peroxisomal catalase gene CAT2 which codes for an important hydrogen peroxide (H 2 O 2 )-scavenging enzyme is upregulated, whereas the FSD3 ( SOD ) gene encoding for the iron superoxide dismutaseshows lower expression compared to the control plants. Genes related to pathogen attack were also upregulated including the transcription factors EFR, WRKY22/25/33 and WRKY29 characterized for their important role in plant immunity 41 , 42 (Fig. 6 ). Considering that glutathione constitutes a major component of the antioxidant system, we focused on key genes of its metabolism. The expression of GLUTATHIONE PEROXIDASE 6 (GPX6) that convert reduced glutathione (GSH) to oxidized glutathione (GSSG) is positively regulated upon riboflavin treatment and another important peroxide reductase, the 1-Cys Prx (PER1) is also upregulated. The enzymes 6-phosphogluconate dehydrogenase (PGD) and glucose-6-phosphate dehydrogenase (G6PDH) are involved in the conversion of Glc-6-P to ribulose-5-phosphate (Ru-5-P), yielding NADPH and to this extent contribute to glutathione-based redox homeostasis (GSH/GSSG ratio). Under riboflavin treatment PGD3, G6PD2 and G6PD3 (GSTF/GSTU) are upregulated showing a transcriptional demand for NADPH production. Intriguingly, riboflavin triggered the expression of genes involved in the catabolism of phytol which is a product of chlorophyll degradation. The genes HPCL and PAHX ( PHYH ) that encode for phytanoyl-CoA hydroxylase and 2-hydroxy-phytanoyl-CoA lyase, respectively, show higher expression level compared to the control (Fig. 6 ). Additionally, the gene KAT2/PKT3 ( ΑCAA1 ), which encodes for the enzyme 3-ketoacyl-CoA thiolase-2 and catalyzes β -oxidation of fatty acids, is upregulated. KAT2/PKT3 positively regulates ABA signaling and has been shown to be important for ROS production 43 . Regarding unsaturated fatty acid β -oxidation, the gene encoding for the peroxisomal short-chain dehydrogenase-reductase B (SDRB/PDCR) displays elevated expression levels, while on the contrary the genes encoding for long-chain acyl-CoA synthetases 2 and 9 (LACS2/9, ACSL) are downregulated. Further, the expression of genes of the oxidative phosphorylation (OXPHOS) system was altered including NADH dehydrogenases, Cytochrome c oxidases and reductases indicating a potential shift in cellular energy metabolism and redox balance in response to riboflavin treatment (Fig. 6 ). Regulating protein synthesis and post-translational modifications enables cells to quickly respond to environmental signals. It is not surprising that genes involved in the protein processing pathway of the endoplasmic reticulum (ER) such as SEC61, SC13/31, BIP and genes involved in the ER-associated degradation and Ubuiquitin ligase complex were upregulated implicating a biological need for protein recycling upon Riboflavin application (Fig. 6 ). While exogenous application of riboflavin activated the expression of various genes involved in antioxidant mechanisms and adaptation to abiotic and biotic stresses, the process of photosynthesis was downregulated (Fig. 6 ). In particular, the majority of the components of the light-harvesting chlorophyll protein complexes, transferring the energy of photons to the attached complexes of Photosystem II (PSII) and Photosystem I (PSI), respectively, showed reduced expression. Likewise, a notable effect was evident on the expression of genes encoding components of the photosynthetic apparatus. Especially, components of PSII and PSI, cytochrome b6/f complex and F-type ATPase were downregulated potentially activating retrograde signaling and transcriptional reprogramming. Further, exogenous riboflavin application led to the down-regulation of the genes encoding for Brassinosteroid signaling Kinases 2 and 5 ( BSK2 and BSK5 ) and the downstream transcription factors BES1 and BES1-Homolog 1 ( BZR1/2 ), which regulate the expression of brassinosteroid responsive genes and ultimately affect plant growth, development, and stress adaptation 44 , 45 . Overall, these data suggest that exogenous riboflavin application initiates a profound reprogramming of transcriptional stress responses and a coordinated alteration in photosynthetic machinery, reflecting a strategic shift in resource allocation that may enhance survival and adaptation under stress conditions. Exogenous riboflavin alters the flavoproteome at the transcriptional level Flavoproteins constitute a highly diverse group of proteins, primarily enzymes, which incorporate (FMN) and/or (FAD) as cofactors, and are capable of participating in a broad spectrum of physiological reactions mostly catalyzing redox reactions 46 . The list of Arabidopsis flavoproteome includes 249 genes encoding potential FAD/FMN-binding proteins of which 211 and 32 proteins exclusively bind FAD and FMN, respectively 47 . Interestingly, the exogenous application of riboflavin affected the expression of genes encoding flavoproteins, with 70 out of 249 genes in the flavoproteome group showing differential expression (Fig. 7 ; Supplementary Table S6 ). Approximately two-thirds of these genes exhibited increased expression. Notably, a large group of FAD-binding Berberine genes, or Berberine bridge enzyme-like (BBE) genes, including 11 genes, were predominantly upregulated, with the exception of two genes that showed a decreased expression pattern (Fig. 7 ). In plants, BBE-like enzymes are implicated in a variety of physiological processes, including defense mechanisms and secondary metabolism 48 . These enzymes, characterized by their berberine bridge enzyme activity, mainly catalyze the oxidation of carbohydrates at the anomeric center to the appropriate lactones playing a key role as oligosaccharide oxidases and reducing the activity of olygogalacturonanes 49 . Additionally, components of the alternative electron transport pathway, namely the mitochondrial alternative NAD(P)H dehydrogenases (NDA1-2 and NDB2-4) were upregulated (Fig. 7 ). Alternative NAD(P)H dehydrogenases and alternative oxidase (AOX) are upregulated in response to a wide range of environmental and chemical stresses to reduce ROS production in mitochondria 50 , 51 , 52 . Other upregulated genes encoding flavoenzymes included various FAD and FMN-linked oxidoreductases, acyl-CoA oxidases, the absicic aldehyde oxidase 3, the L-apsarate oxidase and the monooxygenase 1 gene. Particularly, the 12-oxophytodienoate reductases 1 and 2 (OPR1/2) that belong to the class of the FMN-dependent oxidoreductases, showed increased stimulation of expression upon riboflavin treatment. On the contrary, genes encoding ferric reduction oxidases or members of the YUCCA flavin monooxygenases family that play a key role in auxin biosynthesis, were downregulated (Fig. 7 ). Overall, these data suggest that exogenous riboflavin application acts as a modulator of cellular redox homeostasis, potentially by influencing the oxidative stress response pathways through the transcriptional regulation of flavoenzymes. Riboflavin regulates the expression of genes in its own biosynthetic pathway To determine the regulatory effect of exogenous riboflavin on the expression of the genes involved in the riboflavin biosynthetic pathway, we mapped the expression pattern of genes involved in this pathway. The expression of most pathway genes was downregulated or remained unaffected (Fig. 8 ). On the contrary, the PYRD and PYRP2 genes were upregulated. The PYRD gene encodes for a monofunctional pyrimidine deaminase and the PYRP2 gene is responsible for the dephosphorylation of the intermediate 5-amino-6-ribitylamino-2,4(1 H ,3 H ) pyrimidinedione 5’-phosphate in plastids 53 , 54 , 55 . In addition, the expression of the FMN/FHY gene, which is responsible for converting riboflavin to flavin mononucleotide (FMN) in the cytosol 56 , was increased (Fig. 8 ). This upregulation profile potentially emanates from an increase in riboflavin concentration that in turn stimulates conversion of the excess riboflavin into FMN within the cell. In line with this hypothesis, the gene FHY1/PYRP1/At1g79790 , which encodes an FMN hydrolase located in the chloroplast 57 , was slightly downregulated contrary to FMN/FHY (Fig. 8 ). These results support the notion that exogenous riboflavin predominantly downregulates the expression of genes involved in its own synthesis, except for the PYRD , PYRP2 and especially the FMN/FHY gene, which is induced to convert riboflavin to FMN. In addition, the decreased expression of the FMN hydrolase highlights a possible endogenous regulatory mechanism that maintains a balance between riboflavin synthesis and conversion, in accordance with the cellular needs Discussion Numerous studies have demonstrated that riboflavin treatment enhances plant tolerance to both biotic and abiotic stresses across various species, including model plants like Arabidopsis and economically important crops such as rice, grapevine, soybean, sugar beet, and tobacco 38 , 39 (Supplementary Table S1 ). Riboflavin application has been shown to enhance abiotic stress tolerance and prime plant immune responses, leading to reduced disease severity against a wide range of pathogens (Supplementary Table S1 ). These findings highlight riboflavin as a potent modulator of plant immunity, reinforcing its potential role as a sustainable strategy for crop protection. While significant progress in understanding the physiological effects of exogenous riboflavin application in plants has been made, a critical gap remains regarding riboflavin effect on regulation of gene expression. To gain insights into the gene expression profiles underlying this enhanced tolerance, we analyzed the transcriptional profiles of plants grown in riboflavin-supplemented media. The application of exogenous riboflavin led to significant alterations in the gene expression profile of the treated seedlings, suggesting a possible role for riboflavin as a potent direct or indirect regulator of plant stress responses. The GO enrichment analysis of the DEGs demonstrated that the exogenous application of riboflavin results in induction of stress response mechanisms, as evidenced by the upregulation of genes associated with defense and stress adaptation. These results are in line with previous reports, which emphasize the protective role of riboflavin against various stresses, either abiotic or biotic 38 , 39 . The molecular function (MF) analysis revealed that products of the upregulated genes predominantly exhibit functions essential for maintaining cellular redox homeostasis and detoxification of reactive oxygen species, thereby supporting the plant's defense mechanisms. Conversely, the functional enrichment analysis of the downregulated genes revealed suppression of pathways linked to plant growth and development, indicating a downregulation of cellular processes critical for growth and cell proliferation. Additionally, the cellular components associated with these genes, including the "chloroplast", "apoplast", "cell wall" and "microtubule", further suggest a strategic reallocation of resources away from growth towards stress management. This shift in expression patterns reflects a trade-off between growth and stress responses, further supporting the idea that riboflavin uptake leads to activation of defense mechanisms and their prioritization over growth-related processes to enhance plant resilience. A comprehensive analysis of differentially expressed transcription factors revealed that the majority of upregulated TFs belong to the NAC , ERF , MYC , and bHLH families, while the downregulated TFs are predominantly members of the bHLH , ERF , MYB , and B3 families, suggesting the activation of specific regulatory networks involved in stress responses and adaptation. GO analysis of upregulated TFs indicated significant enrichment in stress-related processes, highlighting the role of riboflavin in enhancing stress adaptation and protective mechanisms. Conversely, downregulated TFs were associated with growth and cellular processes. This suggests a strategic reallocation of resources from plant growth to stress management, reflecting a prioritization of survival over growth under stress conditions. A recent study demonstrated that the transcription factor AtDREB2G is a novel regulator of riboflavin biosynthesis under low-temperature stress and abscisic acid treatment 58 . AtDREB2G was upregulated after FMN treatment and the dreb2g mutants exhibited reduced flavin levels and decreased expression of riboflavin biosynthetic genes compared to wild-type plants. Conversely, conditional overexpression of AtDREB2G led to an increase in the expression of riboflavin biosynthesis genes and elevated flavin levels. Interestingly, in our analysis, DREB19 and DREB2C were also identified among the top hits of transcription factors highly expressed upon riboflavin treatment. These factors share a relatively high degree of homology with AtDREB2G , belonging to the same group and clade in the phylogenetic tree of DREB transcription factors 59 , further supporting the involvement of this specific group of DREB transcription factors in the regulation of riboflavin biosynthesis. The KEGG analysis provides further insights into specific pathways affected by riboflavin treatment. Notably, the upregulation of genes of the ABA signaling pathway, including the plant defensin gene, the wounding response gene and the phytoalexin synthesis gene emphasizes the enhanced defense capabilities against biotic stressors. Further, the induction of transcription factors such as EFR , WRKY22/25/33 , and WRKY29 highlights a role in plant immunity, as these factors are crucial for pathogen recognition and defense signaling 41 . Specifically, Arabidopsis WRKY29 has been shown to act downstream of Flagellin Sensing 2 ( FLS2 ), where it plays a critical role in mediating resistance against bacterial and fungal pathogens 60 . Additionally, WRKY33 positively regulates the defense against necrotrophic fungi by regulating camalexin and ethylene biosynthesis 61 , 62 . In plant mitochondria, oxidative phosphorylation (OXPHOS) is a major source of ROS, especially under stress conditions, where disruption of the electron transport increases ROS production. Elevated ROS levels cause oxidative injury, but ROS can also act as signaling molecules to activate stress response pathways 63 , 64 , 65 . Riboflavin treatment resulted in an altered expression of genes associated with the OXPHOS system. This alteration indicates a potential reprogramming of energy metabolism, possibly to meet the increased energy demands associated with stress responses and ROS accumulation. Additionally, the upregulation of genes involved in protein processing within the endoplasmic reticulum (ER)suggests an enhanced capacity for protein recycling and post-translational modifications, further supporting the plant's adaptability to stress. The SEC61 complex, involved in protein translocation across the ER membrane, and the SEC13/31 complex, the key player in ER-Golgi trafficking, are critical for maintaining ER homeostasis during stress in plants 66 . Under stress conditions, Binding Immunoglobulin Protein (BiP) acts as a chaperone to assist in protein folding and alleviate ER stress, linking the activity of SEC61 and SEC13/31 with the unfolded protein response (UPR) 67 . Moreover, the effect of riboflavin uptake on the control of the antioxidant mechanisms is evident due to the upregulation of genes like CAT2 , which encodes for a peroxisomal catalase, a key enzyme in hydrogen peroxide detoxification. The modulation of the glutathione metabolism pathway further emphasizes the central role of riboflavin in maintaining cellular redox balance and enhancing oxidative stress tolerance. Interestingly, riboflavin treatment also influenced fatty acid metabolism and brassinosteroid signaling. The downregulation of genes encoding for brassinosteroid signaling kinases suggests a suppression of growth-promoting pathways, reinforcing the plant's shift towards stress response. The upregulation of genes involved in phytol catabolism and β -oxidationindicates an enhanced turnover of chlorophyll and fatty acids, potentially providing additional substrates for energy production and stress adaptation. Notably, the downregulation of components related to photosynthesispoints to a deliberate downshift in photosynthetic activity. This suppression may act as a protective mechanism, conserving energy and resources for more immediate stress responses, thereby preventing potential damage from excess light or oxidative stress. This observation is consistent with other reports supporting that photosynthesis is frequently and significantly impacted in many plant systems under stress conditions 34 , 68 . The analysis of transcriptional changes regarding the flavoproteome upon riboflavin application, demonstrates a substantial modulation of genes encoding flavoproteins. The genes encoding for BBE proteins, which are involved in carbohydrate oxidation and secondary metabolism, were prominently featured among the upregulated genes. BBEs play critical roles in plant defense and secondary metabolism, supporting the notion that riboflavin treatment may enhance defensive and metabolic responses 48 . Additionally, mitochondrial alternative NAD(P)H dehydrogenases, which are known to mitigate ROS production, were upregulated. This suggests that riboflavin treatment may alleviate oxidative stress by modulating mitochondrial electron transport and ROS management. Additional upregulated flavoproteins include various FAD and FMN-linked oxidoreductases, acyl-CoA oxidases, and the abscisic aldehyde oxidase 3. These enzymes are integral to redox reactions and stress responses. Particularly, the 12-oxophytodienoate reductases, which are inducible by environmental stress, were upregulated, indicating a potential enhancement in stress-responsive metabolic pathways 69 . On the contrary, downregulation was observed in genes encoding ferric reduction oxidases and YUCCA flavin monooxygenases, which are involved in auxin biosynthesis. This downregulation may reflect a shift in metabolic priorities, possibly towards stress management at the expense of plant growth-related processes. Exogenous flavin application has been documented to alter the intracellular flavin levels, which can either positively or negatively impact the expression of genes involved in flavin biosynthesis 70 . This suggests a potential negative feedback mechanism in response to elevated flavin levels within plant cells 70 . Additionally, exogenous riboflavin application has been shown to enhance salinity tolerance by stimulating riboflavin biosynthesis in rice seedlings under salinity stress 21 . Consistent with these findings, we observed a slight downregulation of most genes related to riboflavin synthesis. Nevertheless, the FMN/FHY gene, which encodes a bifunctional enzyme responsible for the hydrolysis of FMN to riboflavin and the phosphorylation of riboflavin to FMN 56 , exhibited increased expression. This increase suggests a cellular response to convert excess riboflavin into FMN, potentially to regulate riboflavin levels and maintain homeostasis. In contrast, the gene encoding the chloroplast FMN hydrolase was downregulated. This decrease may indicate a feedback mechanism where elevated riboflavin levels suppress its own synthesis or conversion processes. In conclusion, our findings provide a comprehensive overview of transcriptional reprogramming of Arabidopsis genes due to riboflavin exogenous application. Riboflavin has been shown to inhibit post-harvest decay and trigger resistance mechanisms against pathogens and abiotic stresses by priming defense responses. Our study provides compelling evidence that exogenous application of riboflavin stimulates a dynamic reprogramming of the Arabidopsis transcriptome, promoting stress adaptation through targeted changes in gene expression across a range of biological pathways. Riboflavin application facilitates key metabolic shifts essential for enhancing stress resistance, further strengthening plant’s capacity to cope with oxidative stress and maintain metabolic integrity under adverse conditions. The differentially expressed genes identified in this study could serve as potential targets for biotechnological applications aiming to at improve plant responses to biotic and abiotic stress factors, and improve fruit resistance to decay. Future studies may elucidate the precise molecular mechanisms and signaling networks through which riboflavin exerts these protective effects. Investigating the long-term effects of riboflavin on plant growth and stress tolerance under various environmental conditions is crucial to develop riboflavin-based interventions, paving the way for innovative practices in crop protection and stress management. Declarations Acknowledgements The author G.D. gratefully acknowledges the financial support provided by the Hellenic Foundation for Research and Innovation (H.F.R.I.) under the “2nd Call for H.F.R.I. Research Projects to support Faculty Members & Researchers” (Project Number: 02457). All authors acknowledge their host institute for infrastructure support. Authors thank Prof. P. Hatzopoulos for granting access to laboratory facilities and providing valuable support and guidance throughout this research. Author contributions G.D. conceived the research, designed the experiments and acquired funding. D.Tsitsekian. and G.D. performed most of the experiments and analyzed the data. E.K., D.Templalexis. and F.A. helped in bioinformatics and statistical analyses together with S.Rigas. G.D. prepared the initial draft and C.L.B, S.Roje and S.Rigas revised the manuscript. All authors have read and approved the manuscript. Funding This research was supported by the Hellenic Foundation for Research and Innovation (H.F.R.I.) under the “2nd Call for H.F.R.I. Research Projects to support Faculty Members & Researchers” (Project Number: 02457). Competing interests. The authors declare that they have no conflict of interest. Data availability The transcriptomic data of the seedlings treated with Riboflavin versus untreated wild-type seedlings have been deposited in the Gene Expression Omnibus database at the National Center for Biotechnology Information (NCBI) under accession number GSE261916. Ethic declarations. Not applicable. Consent to participate. Not applicable. Consent for publication . All authors have their consent to publish their work. References Bacher, A., Eberhardt, S., Fischer, M., Kis, K. & Richter, G. Biosynthesis of vitamin B2 (riboflavin). Annu. Rev. Nutr. 20 , 153–167 (2000). Sandoval, F. J., Zhang, Y. & Roje, S. 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Mol. Sci. 13 (6), 7828–7853. https://doi.org/10.3390/ijms13067828 (2012). Maruta, T. et al. An Arabidopsis FAD pyrophosphohydrolase, AtNUDX23, is involved in flavin homeostasis. Plant. Cell. Physiol. 53 (6), 1106–1116 (2012). Gerdes, S. et al. Plant B vitamin pathways and their compartmentation: a guide for the perplexed. J. Exp. Bot. 63 (15), 5379–5395 (2012). Additional Declarations No competing interests reported. Supplementary Files SupplementaryTableS1.xlsx Supplemental Table S1. Oligos used for RT-qPCR validation. SupplementaryTableS2.xlsx Supplemental Table S2. List of reports describing the effects of exogenous or foliar application of riboflavin against (a)biotic stress conditions. SupplementaryTableS3.xlsx Supplemental Table S3. The total clean reads per biological replicate and percentage of genome mapping of untreated wild-type (Col) and riboflavin treated seedlings (CR). SupplementaryTableS4.xlsx Supplemental Table S4. Annotation and expression profile of differential expressed genes (DEGs) upon riboflavin treatment. SupplementaryTableS5.xlsx Supplemental Table S5. Annotation and expression profile of differentially expressed TFs (DETFs) upon riboflavin treatment. SupplementaryTableS6.xlsx Supplemental Table S6. Annotation and expression profile of differential expressed genes encoding flavoproteins upon riboflavin treatment. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6377416","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":438549246,"identity":"942c85bf-075a-4b4a-924f-e86b1b635c22","order_by":0,"name":"Dikran Tsitsekian","email":"","orcid":"","institution":"Agricultural University of Athens","correspondingAuthor":false,"prefix":"","firstName":"Dikran","middleName":"","lastName":"Tsitsekian","suffix":""},{"id":438549247,"identity":"2810c414-7f1d-4db4-acde-250013836d51","order_by":1,"name":"Efstratios Kamargiakis","email":"","orcid":"","institution":"Agricultural University of Athens","correspondingAuthor":false,"prefix":"","firstName":"Efstratios","middleName":"","lastName":"Kamargiakis","suffix":""},{"id":438549248,"identity":"11de8982-866b-4225-a084-b6ad5b314321","order_by":2,"name":"Dimitris Templalexis","email":"","orcid":"","institution":"Agricultural University of Athens","correspondingAuthor":false,"prefix":"","firstName":"Dimitris","middleName":"","lastName":"Templalexis","suffix":""},{"id":438549250,"identity":"3badb754-ab40-4535-8895-8204b187e16c","order_by":3,"name":"Fengoula Avgeri","email":"","orcid":"","institution":"Agricultural University of Athens","correspondingAuthor":false,"prefix":"","firstName":"Fengoula","middleName":"","lastName":"Avgeri","suffix":""},{"id":438549252,"identity":"a397420d-f547-4b3e-9d20-af4415f8dcc3","order_by":4,"name":"Clayton Bailes","email":"","orcid":"","institution":"Washington State University","correspondingAuthor":false,"prefix":"","firstName":"Clayton","middleName":"","lastName":"Bailes","suffix":""},{"id":438549253,"identity":"27b1d04c-1eba-4e1d-8559-780fc5f93a04","order_by":5,"name":"Sanja Roje","email":"","orcid":"","institution":"Washington State University","correspondingAuthor":false,"prefix":"","firstName":"Sanja","middleName":"","lastName":"Roje","suffix":""},{"id":438549254,"identity":"5f747fc7-0e6b-4028-bc98-5c4f734dcfb1","order_by":6,"name":"Stamatis Rigas","email":"","orcid":"","institution":"Agricultural University of Athens","correspondingAuthor":false,"prefix":"","firstName":"Stamatis","middleName":"","lastName":"Rigas","suffix":""},{"id":438549255,"identity":"79008131-0a7b-48b4-b6f7-548948f26c15","order_by":7,"name":"Gerasimos Daras","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAq0lEQVRIiWNgGAWjYFCCBBBhQ7qWNNK1HCZBg7x78rMPP/6clzc4foD5dQUxWgzPPDOe2dt223DDmQQ2yzNEaZmRYMzA23CbccOBBDbDBuK0pH9m/PPnnP2G8w+I1CIvkWPMzMN2IHHDjQTmh0RpMeB5U8ws25acPPPGwzZG4mxpT9/M+OaPnW3f+eTDH4mz5QCcydgmQYwOBnkkg5k/EKVlFIyCUTAKRhwAAD4zN1W/hcKGAAAAAElFTkSuQmCC","orcid":"","institution":"Agricultural University of Athens","correspondingAuthor":true,"prefix":"","firstName":"Gerasimos","middleName":"","lastName":"Daras","suffix":""}],"badges":[],"createdAt":"2025-04-04 15:38:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6377416/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6377416/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80124031,"identity":"a6e7dac8-4528-4a8f-b210-3c53f3ff21da","added_by":"auto","created_at":"2025-04-08 08:05:47","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":690072,"visible":true,"origin":"","legend":"\u003cp\u003eHistochemical detection of ROS in riboflavin-treated Arabidopsis seedlings. Six-day-old Arabidopsis seedlings were treated with 0.2 mM riboflavin (CR) and then grown under continuous light conditions for 24h. Superoxide anion (O\u003csub\u003e2\u003c/sub\u003e\u003csup\u003e-\u003c/sup\u003e) and hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) production were monitored at certain time-points via nitro blue tetrazolium (NBT) and 3,3'-diaminobenzidine (DAB) staining, respectively.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6377416/v1/114ec35f3ff129fe4258e9c3.jpeg"},{"id":80124022,"identity":"c85c0d6b-15a4-4f45-bb3e-f8658d43e40d","added_by":"auto","created_at":"2025-04-08 08:05:47","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":864702,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of differentially expressed genes (DEGs) upon riboflavin treatment\u003cstrong\u003e. \u003c/strong\u003e(\u003cstrong\u003ea\u003c/strong\u003e) Correlation analysis of transcriptomic data between untreated wild-type (Col)\u003cem\u003e \u003c/em\u003eand seedlings treated with Riboflavin (CR) in triplicates. The X and Y axes represent each sample. Blue color represents the correlation coefficient (darker blue represents higher correlation, lighter blue represents lower correlations). (\u003cstrong\u003eb\u003c/strong\u003e)\u003cstrong\u003e \u003c/strong\u003eVolcano plot of DEGs between Col and CR plants. (\u003cstrong\u003ec\u003c/strong\u003e) Total number of down- and up-regulated genes upon Riboflavin treatment (CR) compared to untreated wild type plants (Col). DEGs display at least two-fold difference in expression and Padj ≤0.05. (\u003cstrong\u003ed\u003c/strong\u003e) Fold enrichment analysis of UniprotKB keyword ligands of the DEGs. Horizontal axis displays the fold enrichment, vertical axis displays the enriched term of ligand. The size of the dot represents the number of DEGs and the color of the dot represents the \u003cem\u003ep­\u003c/em\u003e-value. (\u003cstrong\u003ee\u003c/strong\u003e) KEGG pathway enrichment analysis of DEGs between untreated wild-type and seedlings treated with riboflavin. Horizontal axis displays the degree of enrichment (rich factor), whereas the vertical axis displays the enriched KEGG pathway. The size of the dot represents the number of differential expressed genes enriched in the pathway, whereas the color of the dot represents the Q value.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6377416/v1/55d9e2df7589e916040d7bb0.jpeg"},{"id":80124024,"identity":"9f9b39af-2b84-4ce8-9597-8d07ea582f75","added_by":"auto","created_at":"2025-04-08 08:05:47","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1809122,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional classification of responsive genes upon riboflavin treatment. (\u003cstrong\u003ea\u003c/strong\u003e) Gene ontology (GO) analysis and classification of upregulated genes. (\u003cstrong\u003eb\u003c/strong\u003e) Gene ontology (GO) analysis and classification of downregulated genes. Horizontal axis displays the degree of enrichment (rich factor), whereas vertical axis displays the enriched GO term. The size of the dot represents the number of differential expressed genes enriched in a GO term. The color of the dot represents the different Qvalue. Rich factor represents the number of differential genes in GO term divided by the total number of genes belonging to the GO term.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6377416/v1/5791bd1781a49d81f241e42d.jpeg"},{"id":80124023,"identity":"d7b02238-43e5-4fe2-8eec-7c67800ff71f","added_by":"auto","created_at":"2025-04-08 08:05:47","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1784851,"visible":true,"origin":"","legend":"\u003cp\u003eDifferentially expressed transcription factors (DETFs) identified upon riboflavin treatment. (\u003cstrong\u003ea\u003c/strong\u003e) Volcano plot showing distribution of the DETFs. Upregulated DETFs are colored in red, downregulated DETFs are colored in blue, both displaying two-fold change or greater with \u003cem\u003ePadj \u003c/em\u003e≤0.05. (\u003cstrong\u003eb\u003c/strong\u003e) Classification of the DETFs in TF families. Upregulated DETFs are colored in red, whereas downregulated DETFs are colored in blue. (\u003cstrong\u003ec\u003c/strong\u003e) Functional enrichment analysis of GO Biological Processes of the upregulated TFs. (\u003cstrong\u003ed\u003c/strong\u003e) Functional enrichment analysis of GO Biological Processes of the downregulated TFs. The horizontal axis displays the degree of enrichment (rich factor), whereas the vertical axis displays the enriched GO term. The size of the dot represents the number of differential expressed genes enriched in the pathway, whereas the color of the dot represents the q-value. (\u003cstrong\u003ee\u003c/strong\u003e) Selected representative DETFs with the highest or lowest fold changes.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6377416/v1/7f1cef62645d6f19f19b2c8b.jpeg"},{"id":80125171,"identity":"7c16a5b1-4070-4c83-81e9-b934ef509a69","added_by":"auto","created_at":"2025-04-08 08:13:47","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1034485,"visible":true,"origin":"","legend":"\u003cp\u003eComparative expression analysis of a subset of highly differentially expressed transcription factors (DETFs) identified upon riboflavin treatment. (\u003cstrong\u003ea\u003c/strong\u003e) RT-qPCR analysis and TPM values of upregulated DETFS. (\u003cstrong\u003eb\u003c/strong\u003e) RT-qPCR analysis and TPM values of downregulated DETFs. Values are mean ± SD, (n = 3 replicates). Asterisks indicate significant differences (t-test) between riboflavin-treated (CR) and control (Col) Arabidopsis seedlings (P ≤ 0.05) (\u003cstrong\u003ec\u003c/strong\u003e) Comparison of gene expression results derived from RT-qPCR and RNA-seq transcriptome analysis. Magenta bars represent the fold change of gene expression of seedlings treated with riboflavin (CR) relative to the control plants (Col), calculated as log\u003csub\u003e2\u003c/sub\u003e transformed ratio of FPKM values obtained by RNA-seq analysis. Green bars represent log\u003csub\u003e2\u003c/sub\u003e transformed fold change expression values obtained from RT-qPCR analysis. Positive values correlate with upregulated gene expression, negative values with downregulation of gene expression.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6377416/v1/204be9239c65addf11039ab6.jpeg"},{"id":80124028,"identity":"55bd2f81-ec8a-49d0-959d-5ec7e307bd04","added_by":"auto","created_at":"2025-04-08 08:05:47","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1161742,"visible":true,"origin":"","legend":"\u003cp\u003eCritical stress-related pathways and photosynthesis were affected by riboflavin. Cells in red and blue indicate an upstream and downstream fold change of gene expression, respectively (\u003cem\u003ePadj\u003c/em\u003e ≤ 0.05). Images were adopted from KEGG pathways visualized by Pathview using fold changes and “Node sum=mean”.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6377416/v1/b500ad7ca35f2daf853628ed.jpeg"},{"id":80124030,"identity":"ae39c286-cbd0-474b-8879-05086a441388","added_by":"auto","created_at":"2025-04-08 08:05:47","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1440387,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Riboflavin treatment in the gene expression of flavoproteins. Heatmap represents the differentially expressed (up- or downregulated) riboflavin-affected flavoproteins. Red cells indicate an upstream fold change, whereas blue cells indicate a downstream fold change of gene expression with \u003cem\u003ePadj\u003c/em\u003e ≤0.05.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6377416/v1/eddcd36a5ddc458777ab2ba6.jpeg"},{"id":80124033,"identity":"b00c7672-d66d-437b-a091-608607a8d06c","added_by":"auto","created_at":"2025-04-08 08:05:47","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":601079,"visible":true,"origin":"","legend":"\u003cp\u003eThe autoregulatory transcriptional effect of riboflavin on its own biosynthetic pathway.\u003cstrong\u003e \u003c/strong\u003eRed cells indicate an upstream fold change, and a blue cell indicates a downstream fold change of gene expression. Asterisk indicates fold change of gene expression with \u003cem\u003ePadj\u003c/em\u003e ≤0.05. This figure is based on the riboflavin biosynthesis pathway in Arabidopsis as described previously\u003csup\u003e71\u003c/sup\u003e.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6377416/v1/107b5cb2c2e84a0ce33e5e90.jpeg"},{"id":87547623,"identity":"d5c753a8-f449-46ad-a821-1e46f963aee7","added_by":"auto","created_at":"2025-07-25 05:32:43","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10551815,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6377416/v1/5d653926-ad2d-4d67-b555-41c831c62724.pdf"},{"id":80125167,"identity":"b9c3d2a7-1c20-4271-a6c4-6c17c2163a5f","added_by":"auto","created_at":"2025-04-08 08:13:47","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":22991,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Table S1. \u003c/strong\u003eOligos used for RT-qPCR validation\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SupplementaryTableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6377416/v1/7947ac81186b829a4c656005.xlsx"},{"id":80124039,"identity":"9a93d5ce-03cb-487f-8bbe-d6fa2885ff29","added_by":"auto","created_at":"2025-04-08 08:05:47","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":11964,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Table S2.\u003c/strong\u003e List of reports describing the effects of exogenous or foliar application of riboflavin against (a)biotic stress conditions.\u003c/p\u003e","description":"","filename":"SupplementaryTableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6377416/v1/c2cb36aff63466dbf1ae9cce.xlsx"},{"id":80125168,"identity":"534e89af-71b7-4665-a99b-7e51710e947a","added_by":"auto","created_at":"2025-04-08 08:13:47","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":10647,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Table S3. \u003c/strong\u003eThe total clean reads per biological replicate and percentage of genome mapping of untreated wild-type (Col) and riboflavin treated seedlings (CR).\u003c/p\u003e","description":"","filename":"SupplementaryTableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6377416/v1/9cc2a6975becdeec9970cd2a.xlsx"},{"id":80125169,"identity":"6965c59a-bb4c-48ba-8a15-350b4f2d1aa3","added_by":"auto","created_at":"2025-04-08 08:13:47","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":717752,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Table S4. \u003c/strong\u003eAnnotation and expression profile of differential expressed genes (DEGs) upon riboflavin treatment.\u003c/p\u003e","description":"","filename":"SupplementaryTableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6377416/v1/e636d194a21088a037e264d5.xlsx"},{"id":80124026,"identity":"eb2c6724-d38c-43ef-b404-866385192fc5","added_by":"auto","created_at":"2025-04-08 08:05:47","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":136578,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Table S5\u003c/strong\u003e. Annotation and expression profile of differentially expressed TFs (DETFs) upon riboflavin treatment.\u003c/p\u003e","description":"","filename":"SupplementaryTableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6377416/v1/b2d7364538cdca0d801c980f.xlsx"},{"id":80125170,"identity":"2540d241-648c-4ff1-aa27-57045bb0fe8d","added_by":"auto","created_at":"2025-04-08 08:13:47","extension":"xlsx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":155088,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplemental Table S6.\u003c/strong\u003e Annotation and expression profile of differential expressed genes encoding flavoproteins upon riboflavin treatment.\u003c/p\u003e","description":"","filename":"SupplementaryTableS6.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-6377416/v1/a63deaeec9f0f813ea8783ed.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Riboflavin treatment triggers stress-responsive gene networks for enhanced adaptation in Arabidopsis","fulltext":[{"header":"Key Message","content":"\u003cp\u003eRiboflavin induces significant transcriptional changes shifting cellular processes from growth to stress management by reprogramming the expression of transcription factors and stress-responsive genes thereby enhancing stress tolerance and plant adaptation\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eRiboflavin, also known as vitamin B2, is a water-soluble vitamin that was first identified and isolated from milk in 1879. While animals depend on exogenous riboflavin sources, plants and most microorganisms are able to synthesize riboflavin through a conserved biosynthetic pathway\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Riboflavin is critical for primary and secondary metabolism, serving as a precursor to the essential coenzymes, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Flavins are indispensable components of photosynthesis, energy generation and redox metabolism in plants, thereby contributing to plant growth, development and stress responses\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. Additionally, riboflavin regulates the corn grain filling process through coordination of the mitochondrial energy metabolism and cell cycle\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Enhancement of vitamin content in plants through genetic engineering or other breeding approaches could stimulate plant stress tolerance or improve human nutrition\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. In this context, vitamin B2 content has been enhanced in rice endosperm via metabolic engineering of enzymes from the \u003cem\u003eSaccharomyces\u003c/em\u003e riboflavin biosynthetic pathway\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eFor more than three decades, exogenous application of riboflavin has been recognized as a process to enhance pathogen resistance in plants. Formulas containing riboflavin have been demonstrated to be effective in controlling powdery mildew, crown gall, Phytopthora and other diseases in numerous crops\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Molecular analysis in Arabidopsis and tobacco showed that exogenous application of riboflavin could activate signaling pathways leading to systemic resistance by stimulating Pathogenesis-Relatedgenes, involving protein kinases and regulation by NIM1/NPR1\u003csup\u003e11\u003c/sup\u003e. In Arabidopsis, riboflavin primed defense responses against \u003cem\u003ePseudomonas\u003c/em\u003e infection and induced resistance that was associated with the expression of defense genes and induction of cellular defense mechanisms\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Application of riboflavin was able to trigger systemic acquired resistance through the accumulation of ROS and activation of hormonal signaling pathways, which in turn promoted the phenylpropanoid pathway and the accumulation of phenolic and antioxidant compounds\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. The role of riboflavin in enhancing disease resistance has been reported for various plant species mainly through the activation of host defense responses and callose deposition in stomatal cells\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e .\u003c/p\u003e \u003cp\u003eThe interplay between riboflavin, enzyme cofactors and abiotic stresses has also been well-documented\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Exogenous application of riboflavin positively affected plant growth, pigment biosynthesis and stress tolerance across various plant species\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Particularly, foliar application of riboflavin enhanced \u003cem\u003eHibiscus\u003c/em\u003e resistance to salinity stress by increasing the activity of antioxidant enzymes and the stability of the plasma membrane\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Additionally, riboflavin application modified the expression of Na\u003csup\u003e+\u003c/sup\u003e transporters to enhance ionic stress tolerance and restrict Na\u003csup\u003e+\u003c/sup\u003e accumulation in the leaf blades of rice, maintaining a favorable Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003e balance\u003csup\u003e23\u003c/sup\u003e. Foliar application of riboflavin in rice increased yield and accumulation of 2-acetyl-1-pyrroline fragrance during the heading stage\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Moreover, rice seedlings pretreated with riboflavin demonstrated higher plant biomass, lower electrolyte leakage ratio and lower levels of hydrogen peroxide\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSignificant progress has been made in understanding the physiological and biochemical effects of exogenous riboflavin application in plants. However, the genes involved in plant stress responses due to riboflavin treatment remain largely unknown. In this study, we investigated the gene expression patterns in \u003cem\u003eArabidopsis\u003c/em\u003e seedlings treated with riboflavin through transcriptome analysis. We identified changes in key genes and pathways related to stress responses. Additionally, we identified a set of differentially expressed transcription factors and examined at the transcriptional level the effect of exogenous riboflavin application on its own biosynthetic pathway. Our analysis reveals the gene networks and stress-signaling responses triggered by riboflavin treatment, providing insights into the transcriptional regulation involved in flavin metabolism.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material and growth conditions\u003c/h2\u003e \u003cp\u003e \u003cem\u003eArabidopsis thaliana\u003c/em\u003e seeds of the Col-0 ecotype were surface-sterilized and sown on Petri dishes containing 0.5x Murashige and Skoog (MS) medium (Duchefa), pH 5.7, supplemented with 1% sucrose and solidified with 0.6% Agarose (Sigma). After 24 h of stratification at 4 \u003csup\u003eo\u003c/sup\u003eC, plants were positioned to grow vertically at 22\u0026deg;C in a Fitotron (Weiss Gallenkamp) growth chamber with 100 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e light intensity under 16 h light/8 h dark photoperiod for 6 days. After 6 days seedlings were transferred to the same media composition supplemented with 0.2 mM riboflavin (Applichem) during the 8 h of the darkness cycle to avoid photodegradation of riboflavin. The concentration of 0.2 mM riboflavin was selected based on previous studies applying a range of 0.01 mM to 2.5 mM (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Petri dishes devoid of riboflavin were used as control. After 8 h of treatment with riboflavin, plants were retransferred to Petri dishes containing 0.5x MS medium and placed under a continuous light regime for 24 h. Samples were collected at 0, 4, 8, 12 and 24 h of continuous light treatment and histochemical staining for ROS detection was performed at each sampling point. For the transcriptomic analysis, plant samples were collected right after the 8 h Riboflavin treatment and were stab frozen in liquid nitrogen and stored at -80 \u003csup\u003eo\u003c/sup\u003eC until RNA extraction.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistochemical staining for ROS detection\u003c/h3\u003e\n\u003cp\u003e \u003cem\u003eIn situ\u003c/em\u003e detection of superoxide and hydrogen peroxide were performed by nitro blue tetrazolium (NBT) and 3,3\u0026rsquo; diaminobenzidine (DAB) staining respectively, as previously described\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Briefly, seedlings were collected, and transferred in NBT (Applichem) staining solution (2 mM NBT, 20 mM potassium phosphate buffer pH 6.1, 100 mM NaCl), or DAB (Sigma-Aldrich) staining solution (DAB 1 mg/mL dissolved in water with the pH adjusted to 3.8 with 1N KOH), vacuum infiltrated for 5 min and incubated for 15 min (NBT) and 2 h (DAB) at room temperature in the dark. Stained plantlets were bleached in acetic acid-glycerol-ethanol (1/1/3) (v/v/v) solution and then stored in 20% glycerol (v/v) solution until photographed with a Leica M205 FCA stereomicroscope equipped with a Leica DFC7000 T digital camera (Leica Microsystems, Wetzlar, Germany).\u003c/p\u003e\n\u003ch3\u003eExtraction of RNA and transcriptome analysis\u003c/h3\u003e\n\u003cp\u003eThe RNA preparation and RNA-seq analysis were performed as previously described\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Briefly, total RNA was extracted from \u003cem\u003eCol-0\u003c/em\u003e (wild-type) seedlings and \u003cem\u003eCol-0\u003c/em\u003e seedlings treated with 0.2 mM riboflavin using the Direct-zol RNA Miniprep kit (Zymo Research, Irvine, CA, USA) with an on-column DNase treatment according to the manufacturer\u0026rsquo;s instructions from control and PepMV-infected tomato tissue. The quantity and quality of RNA were assessed using a NanoDrop ND-1000 spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA) and agarose gel electrophoresis. RNA-seq libraries were generated using the polyA Strand-Specific Transcriptome Library method of BGI for DNBSEQ platform. Sequencing was performed on DNBSEQ platform instrument at BGI (Beijing Genomics Institute) in three biological replicates for each sample. Raw reads were filtered into clean reads and aligned to the Arabidopsis genome (GCF_000001735.4_TAIR10.1). RNA-seq data were analyzed using SOAPnuke (version 1.5.2) with parameters \u0026ldquo;-l 15 -q 0.2 -n 0.05\" and the HISAT2 pipeline (version 2.0.4) with parameters \u0026ldquo;--sensitive --no-discordant --no-mixed -I 1 -X 1000 -p 8\". For the detection of differentially expressed genes (DEGs), clean reads were mapped to the reference genome with Bowtie2, then the gene expression level was calculated using RSEM with default parameters. Statistical analysis of differential gene expression was conducted utilizing DESeq2\u003csup\u003e28\u003c/sup\u003e. A multiple-test corrected p-value of 0.05 was adjusted using the Benjamini and Hochberg\u0026rsquo;s approach, resulting in adjusted p-value (Padj). Transcripts with fold change greater than 2 and a Padj value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, identified by DESeq2, were assigned as differentially expressed. Heatmaps and volcano plots of the DEGs were constructed with Perseus software (version 1.6.8.0) using Euclidean distancing with average linkage and without any constraints in the algorithm.\u003c/p\u003e\n\u003ch3\u003eReverse-transcription qPCR analysis\u003c/h3\u003e\n\u003cp\u003eTotal RNA was isolated from plant tissues using the phenol-sodium dodecyl sulfate (SDS) extraction method as previously described\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. RNA concentrations were determined using a NanoDrop ND-1000 spectrophotometer (Thermo Fischer Scientific, Waltham, MA, USA) and verified by ethidium bromide staining on agarose gels. DNA was eliminated with RQ1 RNase-free DNase (Promega, Madison, WI, USA). Reverse transcription (RT) was performed on 1 \u0026micro;g total DNA free RNA using Invitrogen SuperScript IV VILO Master Mix (Thermo Fisher Scientific). Quantitative gene expression analysis was performed in the PikoReal Real-Time PCR System (Thermo Fisher Scientific) using SYBR Green I as the DNA-binding dye provided in SYBR Select Master Mix (Applied Biosystems, Waltham, MA, USA) and applying the following cycler conditions: 2 min at 50 \u003csup\u003eo\u003c/sup\u003eC, 2 min at 95 \u003csup\u003eo\u003c/sup\u003eC, followed by 40 cycles of 15 s at 95 \u003csup\u003eo\u003c/sup\u003eC, 1 min at 60 \u003csup\u003eo\u003c/sup\u003eC. All quantitative PCR reactions were performed as triplicates of three biological repeats. At the end of each reaction, the cycle threshold (Ct) was automatically set up at the level that reflected the best kinetic PCR parameters by the PikoReal Software 2.1 and melting curve analysis was performed to monitor primer specificity. Τhe specificity of the amplification was verified on agarose gel. Negative controls included samples without a template and those without prior reverse transcription. The primers and amplicon length per gene are listed in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e. Quantification of gene expression was calculated as expression of the gene of interest relative to \u003cem\u003eglyceraldehyde-3-phosphate dehydrogenase\u003c/em\u003e (\u003cem\u003eGAPDH\u003c/em\u003e) and \u003cem\u003epolyubiquitin 10\u003c/em\u003e (\u003cem\u003eUBQ10\u003c/em\u003e) gene expression based on the 2\u003csup\u003e\u0026minus;ΔCt\u003c/sup\u003e method\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. The fold change of transcripts between Riboflavin treated (CR) and untreated (Col) samples was calculated with the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method as previously described\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, using the untreated (Col) as the normalizing samples.\u003c/p\u003e \u003cp\u003e \u003cb\u003eBioinformatics resources for\u003c/b\u003e \u003cb\u003ein silico\u003c/b\u003e \u003cb\u003eanalysis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFunctional enrichment analysis of the differentially expressed genes was performed as previously described\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were performed utilizing the Database for Annotation, Visualization, and Integrated Discovery (DAVID)\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. GO analysis was conducted to elucidate significant genetic regulatory networks by organizing genes into hierarchical categories based on Biological Process (BP), Cellular Component (CC), and Molecular Function (MF). Functional annotation to identify key biological pathways was carried out using KEGG. The enriched KEGG pathways were visualized with Pathview as previously described\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, a toolset designed for pathway-based data integration and visualization, which maps and renders diverse biological data onto relevant pathway graphs from KEGG and related databases\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eExogenous riboflavin treatment results in alterations to\u003c/b\u003e \u003cb\u003eArabidopsis\u003c/b\u003e \u003cb\u003egene expression\u003c/b\u003e\u003c/p\u003e \u003cp\u003eExtensive research has demonstrated the beneficial effects of exogenous application of riboflavin on plants, highlighting the protective role against abiotic and biotic stresses, as well as the ability to reduce post-harvest decay (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). To elucidate the transcriptional responses to exogenous riboflavin application, we transferred 7-day-old Arabidopsis seedlings to a growth medium supplemented with riboflavin in darkness. Riboflavin pretreatment has been shown to significantly mitigate oxidative stress by enhancing antioxidant enzyme activity and reducing lipid peroxidation\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Hence, we tracked the production of ROS, specifically superoxide anion and hydrogen peroxide, over a 24-hour period under continuous light conditions using nitro blue tetrazolium (NBT) and 3,3\u0026prime;-diaminobenzidine (DAB) staining. Seedlings treated with riboflavin (CR) displayed less ROS accumulation between 4 and 12 hours after treatment compared to untreated wild-type plants (Col) aligning with previous studies\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince transcriptional responses to exogenous stimuli occur rapidly, we opted to collect samples for RNA sequencing (RNA-seq) after 8 hours of riboflavin treatment to capture early changes in gene expression. RNA-seq analysis resulted in an average of 45 M clean reads and an average mapping ratio of 98.32% (Supplementary Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). Correlation analysis showed a high consistency among the replicates, which clustered together, while the gene expression profile of CR differed significantly from that of the control Col (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The results were highly reproducible within each RNA-seq sample revealing a deep and satisfactory representation of the Arabidopsis transcriptome. The analysis revealed 3,780 DEGs of which 1711 genes were up-regulated and 2069 genes were down-regulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c; Supplementary Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). Interestingly, fold enrichment analysis of UniprotKB keyword ligands classification of the DEGs mainly revealed the terms \u0026ldquo;Chlorophyll\u0026rdquo;, \u0026ldquo;Pyridoxal phosphate\u0026rdquo;, \u0026ldquo;FAD\u0026rdquo; and \u0026ldquo;Flavoprotein\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Pathway enrichment analysis of the DEGs using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database revealed that \u0026ldquo;Photosynthesis\u0026rdquo;, \u0026ldquo;Glutathione metabolism\u0026rdquo;, \u0026ldquo;Fatty acid metabolism\u0026rdquo;, \u0026ldquo;Glycine, serine and threonine metabolism\u0026rdquo;, \u0026ldquo;Glucosinolate biosynthesis\u0026rdquo; and \u0026ldquo;Plant hormone signal transduction\u0026rdquo; were included as significant prominent enriched terms (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Based on these observations, it is reasonable to suggest that the exogenous application of riboflavin resulted in a dynamic and coordinated reprogramming of the Arabidopsis transcriptome by altering the expression of genes involved in specific biological processes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eRiboflavin modified the expression of specific gene categories involved in various defense mechanisms\u003c/h2\u003e \u003cp\u003eA Gene Ontology (GO) functional enrichment analysis was performed on the up- and down-regulated DEGs to highlight the main functional categories that were affected by riboflavin. GO consists of three ontologies describing the molecular function (MF), cellular component (CC) and biological process (BP) of the transcripts (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Significant enrichment of the upregulated genes in BP was evident mainly for the terms \u0026ldquo;Cellular response to hypoxia\u0026rdquo;, \u0026ldquo;response to water deprivation\u0026rdquo;, \u0026ldquo;glutathione metabolic process\u0026rdquo;, responses to \u0026ldquo;abscisic acid\u0026rdquo;, \u0026ldquo;salt stress\u0026rdquo;, \u0026ldquo;heat\u0026rdquo;, \u0026ldquo;hydrogen peroxide\u0026rdquo;, \u0026ldquo;karrikin\u0026rdquo;, \u0026ldquo;wounding\u0026rdquo; and \u0026ldquo;to other organisms\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Molecular Function classification of the up-regulated genes primary involved \u0026ldquo;oxidoreductase activity\u0026rdquo;, \u0026ldquo;glutathione transferase activity\u0026rdquo; and \u0026ldquo;FAD binding\u0026rdquo;. Concerning the cellular component, the prime category genes were associated with \u0026ldquo;membrane\u0026rdquo;, \u0026ldquo;cytoplasm\u0026rdquo;, \u0026ldquo;chloroplast\u0026rdquo; and \u0026ldquo;mitochondrial membranes\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). Taken together, these findings strongly suggest that application of riboflavin on Arabidopsis seedlings primarily stimulates the expression of genes associated with stress responses within various defense mechanisms consistent with data of previous studies demonstrating the protective role of riboflavin against biotic and abiotic stresses\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. On the other hand, functional enrichment analysis for the BPs of the down-regulated genes revealed critical terms, including \u0026ldquo;photosynthesis\u0026rdquo;, \u0026ldquo;cell division and cycle\u0026rdquo;, \u0026ldquo;response to auxin\u0026rdquo;, \u0026ldquo;response to light stimulus\u0026rdquo;, \u0026ldquo;cell wall organization\u0026rdquo; and \u0026ldquo;regulation of growth\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Regarding MF analysis of the down-regulated genes, those exhibiting the highest abundance were \u0026ldquo;microtubule binding\u0026rdquo;, \u0026ldquo;microtubule motor activity\u0026rdquo; and \u0026ldquo;cyclin-dependent protein serine/threonine kinases\u0026rdquo;. In terms of CC analysis, the down-regulated genes were annotated to \u0026ldquo;chloroplast\u0026rdquo;, \u0026ldquo;apoplast\u0026rdquo;, \u0026ldquo;cell wall\u0026rdquo; and \u0026ldquo;microtubule\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb). Remarkably, the functional classification analysis of up- and down-regulated genes revealed that the application of riboflavin affects the expression of specific gene categories across all ontologies, indicating a distinct pattern of differential gene regulation for the induction or repression of these genes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRiboflavin triggers the reprogramming of stress response transcriptional controllers\u003c/h3\u003e\n\u003cp\u003eTranscription factors (TFs) are central regulatory hubs of the organismal transcriptome mediating responses to external stimuli through a perplexed network of signaling cascades. We therefore conducted a comprehensive analysis of RNA-seq data to identify differentially expresses TFs (DETFs) with significant differential expression. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea; Supplementary Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e). The analysis revealed that the majority of upregulated TFs were categorized in the \u003cem\u003eNAC\u003c/em\u003e, \u003cem\u003eERF\u003c/em\u003e, \u003cem\u003eMYC\u003c/em\u003e, and \u003cem\u003ebHLH\u003c/em\u003e families. Conversely, the downregulated TFs predominantly belonged to the \u003cem\u003ebHLH\u003c/em\u003e, \u003cem\u003eERF\u003c/em\u003e, \u003cem\u003eMYB\u003c/em\u003e, and \u003cem\u003eB3\u003c/em\u003e families, with only a small proportion falling into the NAC category (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). This distinct categorization suggests divergent regulatory pathways and signaling mechanisms between upregulated and downregulated TFs in response to riboflavin treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGene Ontology analysis of the Biological Process category of the upregulated transcription factors identified significant enrichment in processes related to stress responses, whereas the GO analysis concerning the downregulated transcription factors identified terms associated with either plant growth or control of cellular processes. (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This pattern indicates a shift in resource allocation from growth to stress management, reflecting a strategic prioritization of survival under possible adverse conditions. Further classification of the DETFs resulted in a list of the top upregulated and downregulated TFs, using a threshold of the gene expression value TPM (Transcripts per Million)\u0026thinsp;\u0026gt;\u0026thinsp;1.00 as an additional selection criterion (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ee). The upregulated TFs included \u003cem\u003eDREB19\u003c/em\u003e, \u003cem\u003eDREB2C\u003c/em\u003e, \u003cem\u003eERF71\u003c/em\u003e, \u003cem\u003eABR1\u003c/em\u003e, \u003cem\u003eMYB112\u003c/em\u003e and \u003cem\u003eAT4G28140\u003c/em\u003e which are associated with responses to stresses. On the other hand, the list of downregulated TFs included \u003cem\u003eDDF2\u003c/em\u003e and \u003cem\u003ePRE5\u003c/em\u003e, which are involved in the response to gibberellic acid, as well as \u003cem\u003eAT1G33760\u003c/em\u003e, which is related to the ethylene-activated signaling pathway. Furthermore, transcription factor \u003cem\u003ePIL1\u003c/em\u003e, associated with shade avoidance together with the red/far-red light signaling pathway. \u003cem\u003eMYB29\u003c/em\u003e and \u003cem\u003eDDF1\u003c/em\u003e transcription factors, which respond to various biotic and abiotic stresses, are also included in this list.\u003c/p\u003e \u003cp\u003eTo further validate the accuracy and reproducibility of the transcriptome analysis, we performed reverse transcription quantitative polymerase chain reaction (RT-qPCR) on a subset of the DETFs identified upon riboflavin treatment. Notably, the expression pattern of both upregulated and downregulated DETFs, as assessed by RT-qPCR, was almost identical between the two experimental approaches (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea-b). Additionally, a direct comparison of fold change values using a log\u003csub\u003e2\u003c/sub\u003e scale, demonstrated a strong correlation between the two methodologies, with RNA-seq-derived expression levels aligning well with RT-qPCR measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). These results collectively validate the reliability of the RNA-seq analysis and indicate a strong and satisfactory correlation of RT-qPCR data with the outcome of transcriptome analysis, confirming the pattern of DETFs identified by RNA-seq analysis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eRiboflavin-induced transcriptional modulations in stress and photosynthesis\u003c/h3\u003e\n\u003cp\u003eTo comprehend in depth the way Arabidopsis plants responded to exogenous application of riboflavin, we annotated the DEGs to the KEGG PATHWAY database (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Certain genes of various stress responses were annotated, such as genes of the abscisic acid (ABA) pathway, the plant defensin gene \u003cem\u003ePDF1.2\u003c/em\u003e, the wounding response gene \u003cem\u003eVSP2\u003c/em\u003e and the \u003cem\u003ePAD3\u003c/em\u003e gene responsible for camaxelin synthesis, the characteristic phytoalexin of \u003cem\u003eArabidopsis\u003c/em\u003e, which is induced by a great variety of plant pathogens\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Additionally, the peroxisomal catalase gene \u003cem\u003eCAT2\u003c/em\u003e which codes for an important hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e)-scavenging enzyme is upregulated, whereas the \u003cem\u003eFSD3\u003c/em\u003e (\u003cem\u003eSOD\u003c/em\u003e) gene encoding for the iron superoxide dismutaseshows lower expression compared to the control plants. Genes related to pathogen attack were also upregulated including the transcription factors EFR, WRKY22/25/33 and WRKY29 characterized for their important role in plant immunity\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e(Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsidering that glutathione constitutes a major component of the antioxidant system, we focused on key genes of its metabolism. The expression of GLUTATHIONE PEROXIDASE 6 (GPX6) that convert reduced glutathione (GSH) to oxidized glutathione (GSSG) is positively regulated upon riboflavin treatment and another important peroxide reductase, the 1-Cys Prx (PER1) is also upregulated. The enzymes 6-phosphogluconate dehydrogenase (PGD) and glucose-6-phosphate dehydrogenase (G6PDH) are involved in the conversion of Glc-6-P to ribulose-5-phosphate (Ru-5-P), yielding NADPH and to this extent contribute to glutathione-based redox homeostasis (GSH/GSSG ratio). Under riboflavin treatment PGD3, G6PD2 and G6PD3 (GSTF/GSTU) are upregulated showing a transcriptional demand for NADPH production.\u003c/p\u003e \u003cp\u003eIntriguingly, riboflavin triggered the expression of genes involved in the catabolism of phytol which is a product of chlorophyll degradation. The genes \u003cem\u003eHPCL\u003c/em\u003e and \u003cem\u003ePAHX\u003c/em\u003e (\u003cem\u003ePHYH\u003c/em\u003e) that encode for phytanoyl-CoA hydroxylase and 2-hydroxy-phytanoyl-CoA lyase, respectively, show higher expression level compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Additionally, the gene \u003cem\u003eKAT2/PKT3\u003c/em\u003e (\u003cem\u003eΑCAA1\u003c/em\u003e), which encodes for the enzyme 3-ketoacyl-CoA thiolase-2 and catalyzes \u003cem\u003eβ\u003c/em\u003e-oxidation of fatty acids, is upregulated. KAT2/PKT3 positively regulates ABA signaling and has been shown to be important for ROS production\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Regarding unsaturated fatty acid \u003cem\u003eβ\u003c/em\u003e-oxidation, the gene encoding for the peroxisomal short-chain dehydrogenase-reductase B (SDRB/PDCR) displays elevated expression levels, while on the contrary the genes encoding for long-chain acyl-CoA synthetases 2 and 9 (LACS2/9, ACSL) are downregulated.\u003c/p\u003e \u003cp\u003eFurther, the expression of genes of the oxidative phosphorylation (OXPHOS) system was altered including NADH dehydrogenases, Cytochrome c oxidases and reductases indicating a potential shift in cellular energy metabolism and redox balance in response to riboflavin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Regulating protein synthesis and post-translational modifications enables cells to quickly respond to environmental signals. It is not surprising that genes involved in the protein processing pathway of the endoplasmic reticulum (ER) such as SEC61, SC13/31, BIP and genes involved in the ER-associated degradation and Ubuiquitin ligase complex were upregulated implicating a biological need for protein recycling upon Riboflavin application (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile exogenous application of riboflavin activated the expression of various genes involved in antioxidant mechanisms and adaptation to abiotic and biotic stresses, the process of photosynthesis was downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In particular, the majority of the components of the light-harvesting chlorophyll protein complexes, transferring the energy of photons to the attached complexes of Photosystem II (PSII) and Photosystem I (PSI), respectively, showed reduced expression. Likewise, a notable effect was evident on the expression of genes encoding components of the photosynthetic apparatus. Especially, components of PSII and PSI, cytochrome b6/f complex and F-type ATPase were downregulated potentially activating retrograde signaling and transcriptional reprogramming. Further, exogenous riboflavin application led to the down-regulation of the genes encoding for Brassinosteroid signaling Kinases 2 and 5 (\u003cem\u003eBSK2\u003c/em\u003e and \u003cem\u003eBSK5\u003c/em\u003e) and the downstream transcription factors \u003cem\u003eBES1\u003c/em\u003e and \u003cem\u003eBES1-Homolog 1\u003c/em\u003e (\u003cem\u003eBZR1/2\u003c/em\u003e), which regulate the expression of brassinosteroid responsive genes and ultimately affect plant growth, development, and stress adaptation\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Overall, these data suggest that exogenous riboflavin application initiates a profound reprogramming of transcriptional stress responses and a coordinated alteration in photosynthetic machinery, reflecting a strategic shift in resource allocation that may enhance survival and adaptation under stress conditions.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExogenous riboflavin alters the flavoproteome at the transcriptional level\u003c/h2\u003e \u003cp\u003eFlavoproteins constitute a highly diverse group of proteins, primarily enzymes, which incorporate (FMN) and/or (FAD) as cofactors, and are capable of participating in a broad spectrum of physiological reactions mostly catalyzing redox reactions\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. The list of Arabidopsis flavoproteome includes 249 genes encoding potential FAD/FMN-binding proteins of which 211 and 32 proteins exclusively bind FAD and FMN, respectively\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Interestingly, the exogenous application of riboflavin affected the expression of genes encoding flavoproteins, with 70 out of 249 genes in the flavoproteome group showing differential expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e; Supplementary Table \u003cspan refid=\"MOESM6\" class=\"InternalRef\"\u003eS6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eApproximately two-thirds of these genes exhibited increased expression. Notably, a large group of FAD-binding Berberine genes, or Berberine bridge enzyme-like (BBE) genes, including 11 genes, were predominantly upregulated, with the exception of two genes that showed a decreased expression pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). In plants, BBE-like enzymes are implicated in a variety of physiological processes, including defense mechanisms and secondary metabolism\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. These enzymes, characterized by their berberine bridge enzyme activity, mainly catalyze the oxidation of carbohydrates at the anomeric center to the appropriate lactones playing a key role as oligosaccharide oxidases and reducing the activity of olygogalacturonanes\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Additionally, components of the alternative electron transport pathway, namely the mitochondrial alternative NAD(P)H dehydrogenases (NDA1-2 and NDB2-4) were upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Alternative NAD(P)H dehydrogenases and alternative oxidase (AOX) are upregulated in response to a wide range of environmental and chemical stresses to reduce ROS production in mitochondria\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. Other upregulated genes encoding flavoenzymes included various FAD and FMN-linked oxidoreductases, acyl-CoA oxidases, the absicic aldehyde oxidase 3, the L-apsarate oxidase and the monooxygenase 1 gene. Particularly, the 12-oxophytodienoate reductases 1 and 2 (OPR1/2) that belong to the class of the FMN-dependent oxidoreductases, showed increased stimulation of expression upon riboflavin treatment. On the contrary, genes encoding ferric reduction oxidases or members of the YUCCA flavin monooxygenases family that play a key role in auxin biosynthesis, were downregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Overall, these data suggest that exogenous riboflavin application acts as a modulator of cellular redox homeostasis, potentially by influencing the oxidative stress response pathways through the transcriptional regulation of flavoenzymes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eRiboflavin regulates the expression of genes in its own biosynthetic pathway\u003c/h2\u003e \u003cp\u003eTo determine the regulatory effect of exogenous riboflavin on the expression of the genes involved in the riboflavin biosynthetic pathway, we mapped the expression pattern of genes involved in this pathway. The expression of most pathway genes was downregulated or remained unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). On the contrary, the \u003cem\u003ePYRD\u003c/em\u003e and \u003cem\u003ePYRP2\u003c/em\u003e genes were upregulated. The \u003cem\u003ePYRD\u003c/em\u003e gene encodes for a monofunctional pyrimidine deaminase and the \u003cem\u003ePYRP2\u003c/em\u003e gene is responsible for the dephosphorylation of the intermediate 5-amino-6-ribitylamino-2,4(1\u003cem\u003eH\u003c/em\u003e,3\u003cem\u003eH\u003c/em\u003e) pyrimidinedione 5\u0026rsquo;-phosphate in plastids\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. In addition, the expression of the \u003cem\u003eFMN/FHY\u003c/em\u003e gene, which is responsible for converting riboflavin to flavin mononucleotide (FMN) in the cytosol\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, was increased (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). This upregulation profile potentially emanates from an increase in riboflavin concentration that in turn stimulates conversion of the excess riboflavin into FMN within the cell. In line with this hypothesis, the gene \u003cem\u003eFHY1/PYRP1/At1g79790\u003c/em\u003e, which encodes an FMN hydrolase located in the chloroplast\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e, was slightly downregulated contrary to \u003cem\u003eFMN/FHY\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These results support the notion that exogenous riboflavin predominantly downregulates the expression of genes involved in its own synthesis, except for the \u003cem\u003ePYRD\u003c/em\u003e, \u003cem\u003ePYRP2\u003c/em\u003e and especially the \u003cem\u003eFMN/FHY\u003c/em\u003e gene, which is induced to convert riboflavin to FMN. In addition, the decreased expression of the FMN hydrolase highlights a possible endogenous regulatory mechanism that maintains a balance between riboflavin synthesis and conversion, in accordance with the cellular needs\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eNumerous studies have demonstrated that riboflavin treatment enhances plant tolerance to both biotic and abiotic stresses across various species, including model plants like Arabidopsis and economically important crops such as rice, grapevine, soybean, sugar beet, and tobacco\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Riboflavin application has been shown to enhance abiotic stress tolerance and prime plant immune responses, leading to reduced disease severity against a wide range of pathogens (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These findings highlight riboflavin as a potent modulator of plant immunity, reinforcing its potential role as a sustainable strategy for crop protection.\u003c/p\u003e \u003cp\u003eWhile significant progress in understanding the physiological effects of exogenous riboflavin application in plants has been made, a critical gap remains regarding riboflavin effect on regulation of gene expression. To gain insights into the gene expression profiles underlying this enhanced tolerance, we analyzed the transcriptional profiles of plants grown in riboflavin-supplemented media. The application of exogenous riboflavin led to significant alterations in the gene expression profile of the treated seedlings, suggesting a possible role for riboflavin as a potent direct or indirect regulator of plant stress responses.\u003c/p\u003e \u003cp\u003eThe GO enrichment analysis of the DEGs demonstrated that the exogenous application of riboflavin results in induction of stress response mechanisms, as evidenced by the upregulation of genes associated with defense and stress adaptation. These results are in line with previous reports, which emphasize the protective role of riboflavin against various stresses, either abiotic or biotic\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. The molecular function (MF) analysis revealed that products of the upregulated genes predominantly exhibit functions essential for maintaining cellular redox homeostasis and detoxification of reactive oxygen species, thereby supporting the plant's defense mechanisms.\u003c/p\u003e \u003cp\u003eConversely, the functional enrichment analysis of the downregulated genes revealed suppression of pathways linked to plant growth and development, indicating a downregulation of cellular processes critical for growth and cell proliferation. Additionally, the cellular components associated with these genes, including the \"chloroplast\", \"apoplast\", \"cell wall\" and \"microtubule\", further suggest a strategic reallocation of resources away from growth towards stress management. This shift in expression patterns reflects a trade-off between growth and stress responses, further supporting the idea that riboflavin uptake leads to activation of defense mechanisms and their prioritization over growth-related processes to enhance plant resilience.\u003c/p\u003e \u003cp\u003eA comprehensive analysis of differentially expressed transcription factors revealed that the majority of upregulated TFs belong to the \u003cem\u003eNAC\u003c/em\u003e, \u003cem\u003eERF\u003c/em\u003e, \u003cem\u003eMYC\u003c/em\u003e, and \u003cem\u003ebHLH\u003c/em\u003e families, while the downregulated TFs are predominantly members of the \u003cem\u003ebHLH\u003c/em\u003e, \u003cem\u003eERF\u003c/em\u003e, \u003cem\u003eMYB\u003c/em\u003e, and \u003cem\u003eB3\u003c/em\u003e families, suggesting the activation of specific regulatory networks involved in stress responses and adaptation. GO analysis of upregulated TFs indicated significant enrichment in stress-related processes, highlighting the role of riboflavin in enhancing stress adaptation and protective mechanisms. Conversely, downregulated TFs were associated with growth and cellular processes. This suggests a strategic reallocation of resources from plant growth to stress management, reflecting a prioritization of survival over growth under stress conditions.\u003c/p\u003e \u003cp\u003eA recent study demonstrated that the transcription factor \u003cem\u003eAtDREB2G\u003c/em\u003e is a novel regulator of riboflavin biosynthesis under low-temperature stress and abscisic acid treatment\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eAtDREB2G\u003c/em\u003e was upregulated after FMN treatment and the \u003cem\u003edreb2g\u003c/em\u003e mutants exhibited reduced flavin levels and decreased expression of riboflavin biosynthetic genes compared to wild-type plants. Conversely, conditional overexpression of \u003cem\u003eAtDREB2G\u003c/em\u003e led to an increase in the expression of riboflavin biosynthesis genes and elevated flavin levels. Interestingly, in our analysis, \u003cem\u003eDREB19\u003c/em\u003e and \u003cem\u003eDREB2C\u003c/em\u003e were also identified among the top hits of transcription factors highly expressed upon riboflavin treatment. These factors share a relatively high degree of homology with \u003cem\u003eAtDREB2G\u003c/em\u003e, belonging to the same group and clade in the phylogenetic tree of \u003cem\u003eDREB\u003c/em\u003e transcription factors\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, further supporting the involvement of this specific group of \u003cem\u003eDREB\u003c/em\u003e transcription factors in the regulation of riboflavin biosynthesis.\u003c/p\u003e \u003cp\u003eThe KEGG analysis provides further insights into specific pathways affected by riboflavin treatment. Notably, the upregulation of genes of the ABA signaling pathway, including the plant defensin gene, the wounding response gene and the phytoalexin synthesis gene emphasizes the enhanced defense capabilities against biotic stressors. Further, the induction of transcription factors such as \u003cem\u003eEFR\u003c/em\u003e, \u003cem\u003eWRKY22/25/33\u003c/em\u003e, and \u003cem\u003eWRKY29\u003c/em\u003e highlights a role in plant immunity, as these factors are crucial for pathogen recognition and defense signaling\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Specifically, Arabidopsis \u003cem\u003eWRKY29\u003c/em\u003e has been shown to act downstream of \u003cem\u003eFlagellin Sensing 2\u003c/em\u003e (\u003cem\u003eFLS2\u003c/em\u003e), where it plays a critical role in mediating resistance against bacterial and fungal pathogens\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Additionally, \u003cem\u003eWRKY33\u003c/em\u003e positively regulates the defense against necrotrophic fungi by regulating camalexin and ethylene biosynthesis\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e,\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn plant mitochondria, oxidative phosphorylation (OXPHOS) is a major source of ROS, especially under stress conditions, where disruption of the electron transport increases ROS production. Elevated ROS levels cause oxidative injury, but ROS can also act as signaling molecules to activate stress response pathways\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Riboflavin treatment resulted in an altered expression of genes associated with the OXPHOS system. This alteration indicates a potential reprogramming of energy metabolism, possibly to meet the increased energy demands associated with stress responses and ROS accumulation. Additionally, the upregulation of genes involved in protein processing within the endoplasmic reticulum (ER)suggests an enhanced capacity for protein recycling and post-translational modifications, further supporting the plant's adaptability to stress. The SEC61 complex, involved in protein translocation across the ER membrane, and the SEC13/31 complex, the key player in ER-Golgi trafficking, are critical for maintaining ER homeostasis during stress in plants\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. Under stress conditions, Binding Immunoglobulin Protein (BiP) acts as a chaperone to assist in protein folding and alleviate ER stress, linking the activity of SEC61 and SEC13/31 with the unfolded protein response (UPR)\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMoreover, the effect of riboflavin uptake on the control of the antioxidant mechanisms is evident due to the upregulation of genes like \u003cem\u003eCAT2\u003c/em\u003e, which encodes for a peroxisomal catalase, a key enzyme in hydrogen peroxide detoxification. The modulation of the glutathione metabolism pathway further emphasizes the central role of riboflavin in maintaining cellular redox balance and enhancing oxidative stress tolerance. Interestingly, riboflavin treatment also influenced fatty acid metabolism and brassinosteroid signaling. The downregulation of genes encoding for brassinosteroid signaling kinases suggests a suppression of growth-promoting pathways, reinforcing the plant's shift towards stress response. The upregulation of genes involved in phytol catabolism and \u003cem\u003eβ\u003c/em\u003e-oxidationindicates an enhanced turnover of chlorophyll and fatty acids, potentially providing additional substrates for energy production and stress adaptation. Notably, the downregulation of components related to photosynthesispoints to a deliberate downshift in photosynthetic activity. This suppression may act as a protective mechanism, conserving energy and resources for more immediate stress responses, thereby preventing potential damage from excess light or oxidative stress. This observation is consistent with other reports supporting that photosynthesis is frequently and significantly impacted in many plant systems under stress conditions\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe analysis of transcriptional changes regarding the flavoproteome upon riboflavin application, demonstrates a substantial modulation of genes encoding flavoproteins. The genes encoding for BBE proteins, which are involved in carbohydrate oxidation and secondary metabolism, were prominently featured among the upregulated genes. BBEs play critical roles in plant defense and secondary metabolism, supporting the notion that riboflavin treatment may enhance defensive and metabolic responses\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. Additionally, mitochondrial alternative NAD(P)H dehydrogenases, which are known to mitigate ROS production, were upregulated. This suggests that riboflavin treatment may alleviate oxidative stress by modulating mitochondrial electron transport and ROS management. Additional upregulated flavoproteins include various FAD and FMN-linked oxidoreductases, acyl-CoA oxidases, and the abscisic aldehyde oxidase 3. These enzymes are integral to redox reactions and stress responses. Particularly, the 12-oxophytodienoate reductases, which are inducible by environmental stress, were upregulated, indicating a potential enhancement in stress-responsive metabolic pathways\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. On the contrary, downregulation was observed in genes encoding ferric reduction oxidases and YUCCA flavin monooxygenases, which are involved in auxin biosynthesis. This downregulation may reflect a shift in metabolic priorities, possibly towards stress management at the expense of plant growth-related processes.\u003c/p\u003e \u003cp\u003eExogenous flavin application has been documented to alter the intracellular flavin levels, which can either positively or negatively impact the expression of genes involved in flavin biosynthesis\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. This suggests a potential negative feedback mechanism in response to elevated flavin levels within plant cells\u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Additionally, exogenous riboflavin application has been shown to enhance salinity tolerance by stimulating riboflavin biosynthesis in rice seedlings under salinity stress\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. Consistent with these findings, we observed a slight downregulation of most genes related to riboflavin synthesis. Nevertheless, the \u003cem\u003eFMN/FHY\u003c/em\u003e gene, which encodes a bifunctional enzyme responsible for the hydrolysis of FMN to riboflavin and the phosphorylation of riboflavin to FMN\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e, exhibited increased expression. This increase suggests a cellular response to convert excess riboflavin into FMN, potentially to regulate riboflavin levels and maintain homeostasis. In contrast, the gene encoding the chloroplast \u003cem\u003eFMN hydrolase\u003c/em\u003e was downregulated. This decrease may indicate a feedback mechanism where elevated riboflavin levels suppress its own synthesis or conversion processes.\u003c/p\u003e \u003cp\u003eIn conclusion, our findings provide a comprehensive overview of transcriptional reprogramming of Arabidopsis genes due to riboflavin exogenous application. Riboflavin has been shown to inhibit post-harvest decay and trigger resistance mechanisms against pathogens and abiotic stresses by priming defense responses. Our study provides compelling evidence that exogenous application of riboflavin stimulates a dynamic reprogramming of the Arabidopsis transcriptome, promoting stress adaptation through targeted changes in gene expression across a range of biological pathways. Riboflavin application facilitates key metabolic shifts essential for enhancing stress resistance, further strengthening plant\u0026rsquo;s capacity to cope with oxidative stress and maintain metabolic integrity under adverse conditions. The differentially expressed genes identified in this study could serve as potential targets for biotechnological applications aiming to at improve plant responses to biotic and abiotic stress factors, and improve fruit resistance to decay. Future studies may elucidate the precise molecular mechanisms and signaling networks through which riboflavin exerts these protective effects. Investigating the long-term effects of riboflavin on plant growth and stress tolerance under various environmental conditions is crucial to develop riboflavin-based interventions, paving the way for innovative practices in crop protection and stress management.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author G.D. gratefully acknowledges the financial support provided by the Hellenic Foundation for Research and Innovation (H.F.R.I.) under the \u0026ldquo;2nd Call for H.F.R.I. Research Projects to support Faculty Members \u0026amp; Researchers\u0026rdquo; (Project Number: 02457). All authors acknowledge their host institute for infrastructure support. Authors thank Prof. P. Hatzopoulos for granting access to laboratory facilities and providing valuable support and guidance throughout this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eG.D. conceived the research, designed the experiments and acquired funding. D.Tsitsekian. and G.D. performed most of the experiments and analyzed the data. E.K., D.Templalexis. and F.A. helped in bioinformatics and statistical analyses together with S.Rigas. G.D. prepared the initial draft and C.L.B, S.Roje and S.Rigas revised the manuscript. All authors have read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Hellenic Foundation for Research and Innovation (H.F.R.I.) under the \u0026ldquo;2nd Call for H.F.R.I. Research Projects to support Faculty Members \u0026amp; Researchers\u0026rdquo; (Project Number: 02457).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests.\u003c/strong\u003e The authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe transcriptomic data of the seedlings treated with Riboflavin versus untreated wild-type seedlings have been deposited in the Gene Expression Omnibus database at the National Center for Biotechnology Information (NCBI) under accession number GSE261916.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthic declarations.\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate.\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e. \u0026nbsp;All authors have their consent to publish their work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBacher, A., Eberhardt, S., Fischer, M., Kis, K. \u0026amp; Richter, G. 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Bot.\u003c/em\u003e \u003cb\u003e63\u003c/b\u003e (15), 5379\u0026ndash;5395 (2012).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Riboflavin, Vitamin B2, flavins, transcription factors, gene expression, stress response, plant resilience","lastPublishedDoi":"10.21203/rs.3.rs-6377416/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6377416/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRiboflavin is the precursor of the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) which are vital coenzymes to a wide array of plant metabolic processes. While the exogenous application of riboflavin has been well-documented to enhance plant stress tolerance, the molecular mechanisms underlying this protective effect remain largely unknown. Here, we present a comprehensive transcriptomic analysis of riboflavin-treated Arabidopsis seedlings, revealing significant changes in gene expression related to stress responses, signaling transduction and secondary metabolism. Riboflavin treatment altered the expression of genes within specific cellular functional categories, supporting the role of riboflavin in regulating plant metabolism and enhancing stress adaptation. The transcriptional changes indicate a shift from growth to stress management, potentially downregulating photosynthesis to preserve energy for immediate stress responses and protect against damage from excess light or oxidative stress. Further, we identified a feedback mechanism where elevated riboflavin levels regulate the expression of genes of its own biosynthetic pathway, controlling both its synthesis and chemical conversion processes. Our study provides novel and valuable insights into the gene expression mechanisms underlying riboflavin-mediated stress tolerance and highlights a potential application of exogenous riboflavin as a strategy for improving crop plasticity and adaptation in the face of environmental challenges.\u003c/p\u003e","manuscriptTitle":"Riboflavin treatment triggers stress-responsive gene networks for enhanced adaptation in Arabidopsis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-08 08:05:42","doi":"10.21203/rs.3.rs-6377416/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1c19c78d-fb16-48f7-b04c-769893eb38ce","owner":[],"postedDate":"April 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46701920,"name":"Biological sciences/Plant sciences/Plant genetics"},{"id":46701921,"name":"Biological sciences/Plant sciences/Plant molecular biology"},{"id":46701922,"name":"Biological sciences/Plant sciences/Plant stress responses"}],"tags":[],"updatedAt":"2025-07-25T05:23:44+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-08 08:05:42","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6377416","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6377416","identity":"rs-6377416","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}