Muti-omics revealed the global response of Hordeum vulgare to different wavelength of UV exposure

Muti-omics revealed the global response of Hordeum vulgare to different wavelength of UV exposure | 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 Research Article Muti-omics revealed the global response of Hordeum vulgare to different wavelength of UV exposure Zhihua Hao, Xiaojun Yang, Ciren Deji, Yifan Zhang, Xuelian Wu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5763885/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 Background UV seriously affected Hordeum vulgare growth, which limited the its development and production in the plateau region. However, the detailed response mechanisms of Hordeum vulgare to UV exposure remain unclear. Results We found that UVA, UVAB and UVB reprogrammed the proteome pattern of H. vulgare . UVA mainly affected the expression of proteins involved in cell cycle and DNA repair, while UVB and UVAB mainly activated proteins involved in anthocyanin biosynthesis and antioxidant systems, leading to accumulation of anthocyanin in H. vulgare . Then, we constructed the network for regulating UV-triggered anthocyanin biosynthesis in H. vulgare using proteome profiles. Importantly, we identified that NAC transcription together with numerous kinases were responsible for regulating UVAB/UVB-triggered anthocyanin biosynthesis in H. vulgare . Of note, NAC1 directly bound on the promoters of CAD1 and CHS to trigger their expression, then activated anthocyanin biosynthesis in H. vulgare . Conclusions Our research gain insight into the global responses of H. vulgare to UV exposures, then provided guidance for protecting plant from UV stress in plateau. Hordeum vulgare Multi-omics UV exposure Regulation network Transcription factor Anthocyanin biosynthesis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Plants are often challenged by numerous adverse abiotic stresses, including drought, high salinity, and especially UV exposure (i.e., UVA and UVB) [ 1 ]. With the increasingly prominent global environmental changes, the increasing trend of ultraviolet radiation is becoming more and more obvious. Studies have shown that the ultraviolet radiation on the Earth's surface has increased by 6–14% and will continue to increase in the future [ 2 ]. UV-B is the ionizing radiation with a wavelength of 280 to 315 nm that can penetrate the ozone layer and is a part of the solar radiation. In recent years, with the decrease of the ozone layer, UV-B radiation reaching the Earth's surface has increased [ 3 , 4 ]. High doses of UV-B damage amino acid residues, leading to protein and enzyme inactivation of amino acids in synthesis [ 5 , 6 ]. Studies have shown that UV-B radiation can significantly inhibit the photosynthetic rate of plants, hinder plant growth and development, and lead to a decrease in crop yield [ 7 – 10 ]. Highland barley ( Hordeum vulgare L.) is a kind of Chinese general name for naked barley in the Tibetan plateau area, and always challenged by strong UV challenge in production region. Typically, highland barley has the advantages of drought resistance, cold resistance, UV resistance and humidity resistance, which lead to it adapted to plant in a range of Tibetan plateau area. Thus, it has been the most important food crop for farmers and herdsmen in Tibetan areas. At present, China's highland barley planting area is about 333,300 hm 2 , 95% of which is distributed in the Qinghai-Tibet Plateau, while Tibet's highland barley planting area is the largest, highest yield, and most widely distributed, its planting area is about 240,000 hm 2 , accounting for 72% of the major planting area. Moreover, the proportion of grain crop planting area and output in the whole region has always remained above 70%. Thus, understanding the detailed responses of plants to UV exposure is of great significance for their development and cultivation. In order to cope with the harsh living environment, plants growing on the Qinghai-Tibet Plateau show different adaptive traits, such as the shape and color of roots, stems and leaves, the interaction between different plants, and the change of reproduction mode [ 11 , 12 ]. Typically, plant altered the expression levels of soluble sugar, superoxide dismutase, peroxidase, etc., in the leaf tissues to strong ultraviolet radiation in the Qinghai-Tibet Plateau, and the physiological characteristics of plants are transformed to protect plants from UV exposure [ 11 ]. In addition, wild barley on the plateau mainly changed genes related to DNA damage repair, nitrogen metabolism, cell membrane stability regulation, UV radiation resistance and other functions, so as to adapt to excessive UV radiation on the Qinghai-Tibet Plateau [ 11 ]. It has shown that UV-B exerted significant effect on gene expression related to the biosynthesis of plant secondary metabolites [ 12 , 13 ], in which increased accumulation of secondary metabolites in plants [ 14 ]. These secondary metabolites include terpenoids [ 15 ], alkaloids, and phenols [ 16 ].and secondary nitrogen-containing compound [ 17 ]. Flavonoids also exerted many important physiological effects on plants, especially protecting plants from biotic and abiotic stresses [ 18 ]. Recently, it was found that UV radiation significantly affected the compositions of flavonoids and anthocyanins in plant [ 19 , 20 ]. Park et al. (2008) [ 21 ] found that the synthesis of plant flavonoids and anthocyanins is related to many factors, typically their synthetic genes are affected by UV-B radiation [ 21 ]. In detail, flavonoids can accumulate in some special parts of plant cells, such as cell walls, vacuoles, chloroplasts, and glands [ 21 ]. Additionally, the contents of flavonoids and anthocyanins in C. mongolica and Cymbopogon citratus were significantly increased after UV-B stress [ 22 – 24 ]. Anthocyanins are distributed in plant epidermal cells, while numerous structural genes and transcription factors play an important role in the synthesis of anthocyanins and flavonoids [ 25 ]. Ohnishi et al. (1992) [ 26 ] found that anthocyanin could inhibit DNA damage in Centaurea cyanus under the condition of UV-B increase, thus protecting the normal growth and development of plants under UVB condition. However, the detailed metabolic compositions in Hordeum vulgare under different wavelength of UV exposures remained unclear. Hordeum vulgare , which is widely distributed in habitat, is always exposed under UV challenges, however its detailed responses to UV exposure remain unclear. In this study, we deployed 4-D proteome and anthocyanins metabolomics to investigate the responses of Hordeum vulgare to different wavelength and composition of UV exposures, including UVA, UVAB and UVB. It was found that UVA, UVAB, and UVB exposures caused different responses in H. vulgare at proteome levels. Typically, H. vulgare reprogrammed anthocyanins biosynthesis to accumulated more related metabolites in response to UVAB and UVB exposures. And NAC1 was responsible for activating anthocyanin biosynthesis of H. vulgare in response to UV exposure. Overall, our research depicted global proteome and anthocyanins landscape of H. vulgare under different wavelength of UV exposures, which contributed to the cultivation and management of H. vulgare from UV stress, especially in plateau. Materials and Methods Sample preparation Here, Longzi black highland barley, a variety of black highland barley rich in anthocyanins, was planted in pots with vegetative soil as the substrate. Totally, 120–130 seedlings were planted in each pot at Xizang Academy of Agricultural and Animal Husbandry Sciences experimental fields. Plants were grown in a greenhouse at a temperature of 20°C/day and 15°C/night, and watered every 5 days. The highland barley was subjected to ultraviolet stress at the earing stage. Each treatment was divided into experimental group and control group. Typically, three kinds of UV stress treatments were set up and compared with P normal sunlight exposure, treatment 1 was PA (sunlight P + UVA 400-320nm), treatment 2 was PB (sunlight P + UVB 320-280nm), treatment 3 was PAB (sunshine P + UVA + UVB) mixed irradiation, treatment for 8 hours a day, treatment for 8 days, control for outdoor planting, three parallel treatments, a total of 36 samples. The information of UV-B light is 5.5 W cm − 2 , equivalent to 12 µmol m − 2 s − 1 (measured by a UV-297 UV-B Light Meter, HANDY) provided by Philips TL20W/01RS narrowband UV-B tubes11. And the information of UV-A light is 5.5 W m − 2 , PPFD = 12 µmol·m − 2 s − 1 , as measured by a portable digital radiometer for UV (UV-340A, Lutron, Taiwan). Protein extraction and digestion To investigate the responses of Hordeum vulgare to UVA, UVAB and UVB exposure, three biological replicates of H. vulgare were collected from all experimental groups (UVA, UVAB and UVB) for proteome analysis. Sample analysis and protein extraction were performed using SDT (4% SDS, 100 mM Tris-HCl, 1 mM DTT, pH 7.6) buffer and quantified with the BCA Protein Assay Kit (BioRad, Hercules, CA, USA). Then, the protein was digested by trypsin and desalted on C18 cartridges (Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 mL, Sigma, Burlington, MA, USA), concentrated via vacuum centrifugation and reconstituted in 40 µL of 0.1% (v/v) formic acid. Filter-aided sample preparation (FASP digestion) procedure: 200 µg of protein for each sample was incorporated into 30 µL SDT buffer (4% SDS, 100 mM DTT, 150 mM Tris-HCl pH 8.0). The detergent, DTT and other low-molecular-weight components were removed using UA buffer (8 M urea, 150 mM Tris-HCl pH 8.0) via repeated ultrafiltration (Microcon units, 10 kD). Then, 100 µL iodoacetamide (100 mM IAA in UA buffer) was added to block reduced cysteine residues and the samples were incubated for 30 min in darkness. The filters were washed with 100 µL UA buffer three times and then 100 µL 25 mM NH 4 HCO 3 buffer twice. Finally, the protein suspensions were digested with 4 µg trypsin (Promega) in 40 µL 25 mM NH 4 HCO 3 buffer overnight at 37°C, and the resulting peptides were collected as a filtrate. The peptides of each sample were desalted on C18 cartridges (Empore™ SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 mL, Sigma, Burlington, MA, USA), concentrated via vacuum centrifugation and reconstituted in 40 µL of 0.1% (v/v) formic acid. The peptide content was estimated based on the UV light at 280 nm using an extinction coefficient of 1.1 of 0.1% (g/L) solution that was calculated based on the frequency of tryptophan and tyrosine in vertebrate proteins according to Cheng et al. (2020)[ 27 ]. LC-MS/MS detection for proteome analysis For each sample, 200 ng of total peptides were separated and analyzed with a nano­UPLC (Evosep one) coupled to a timsTOF Pro2 instrument (Bruker) with a nano­electrospray ion source. Separation was performed using a reversed­phase column (PePSep C18, 1.9 µ, 150 µ × 15 cm, Bruker, Germany). Mobile phases were H 2 O with 0.1% FA (phase A) and CAN with 0.1% FA (phase B). Separation of sample was executed with a 44 min gradient. The mass spectrometer adopts DDA PaSEF mode for DDA data acquisition, and the scanning range is from 100 to 1700 m/z for MS1. During PASEF MS/MS scanning, the impact energy increases linearly with ion mobility, from 20 eV (1/K 0 = 0.6 Vs/cm 2 ) to 59 eV (1/K 0 = 1.6 Vs/cm 2 ). SpectroMine database search Vendor’s raw MS files were processed using SpectroMine software (4.2.230428.52329) and the built­in Pulsar search engine. MS spectra lists were searched against their species­level UniProt FASTA databases (uniprotkb_Hordeum vulgare_id_4513_2024_05_17), Carbamidomethyl [C] as a fixed modification, Oxidation (M) and Acetyl (Protein N­term) as variable modifications. Trypsin was used as proteases. A maximum of 2 missed cleavage (s) was allowed. The false discovery rate (FDR) was set to 0.01 for both PSM and peptide levels. Peptide identification was performed with an initial precursor mass deviation of up to 20 ppm and a fragment mass deviation of 20 ppm. All the other parameters were reserved as default. Bioinformatic Analysis CELLO v2.5 ( http://cello.life.nctu.edu.tw/ accessed on 31 July 2022), a multi-class SVM classification system, was used to predict protein subcellular localization. The protein sequences of the selected differentially expressed proteins were locally searched using the NCBI BLAST + client software (ncbi-blast-2.2.28+-win32.exe) and InterProScan-5.25-64.0 to find homologue sequences, then, gene ontology (GO) terms were mapped and sequences were annotated using the software program Blast2GO 6.0. The GO annotation results were plotted by R scripts. The BLAST2GO 6.0 software was used to perform functional annotation following Ye et al. (2006) [ 28 ]. The DEPs of each comparison were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database ( http://www.genome.jp/kegg/ accessed on 31 July 2022) [ 29 ]. GO and KEGG pathway enrichment analyses were performed by clusterprofiler 2.0. Only functional categories and pathways with p-values under a threshold of 0.05 were considered significant. Proteins with P-value 1 was set as the threshold for significantly differential expression (Table S4 ). WGCNA was performed using R package WGCNA following Du et al. (2023) [ 30 ]. The detailed information of proteome data were listed in Table S4 . Anthocyanin preparation and extraction Each experimental group contained three biological replicates to ensure the validity of altitude-induced metabolic changes in H. vulgare . Totally, 20 mg of each H. vulgare sample from CK, UVA, UVB and UVAB groups was weighted to an EP tube after grinding with liquid nitrogen, and 1000 µL extract solution (methanol: water = 3: 1, with isotopically-labelled internal standard mixture) was added. The sample was freeze-dried, ground into powder (30 Hz, 1.5 min), and stored at -80°C until needed. 50 mg powder was weighted and extracted with 0.5 mL methanol/water/hydrochloric acid (500:500:1, V/V/V). Then the extract was vortexed for 5 min and ultrasound for 5 min and centrifuged at 12, 000 g under 4°C for 3 min. The residue was re-extracted by repeating the above steps again under the same conditions. The supernatants were collected, and filtrated through a membrane filter (0.22 µm, Anpel) before LC-MS/MS analysis. All of the standards were purchased from isoReag (Shanghai, China). Formic acid was purchased from Sigma-Aldrich (St Louis, MO, USA). Hydrochloric acid was bought from Xinyang ChemicalReagent (China). The stock solutions of standards were prepared at the concentration of 1 mg/mL in 50% MeOH. All stock solutions were stored at -20°C. The stock solutions were diluted with 50% MeOH to working solutions before analysis. LC–MS/MS Analysis for anthocyanin determination HPLC-grade methanol (MeOH) was purchased from Merck (Darmstadt, Germany). MilliQ water (Millipore, Bradford, USA) was used in all experiments. The sample extracts were analyzed using an UPLC-ESI-MS/MS system (UPLC, ExionLC™ AD, https://sciex.com.cn/ , MS, Applied Biosystems 6500 Triple Quadrupole, https://sciex.com.cn/ ). The analytical conditions were as follows, UPLC: column, WatersACQUITY BEH C18 (1.7 µm, 2.1 mm*100 mm), solvent system, water (0.1% formic acid): methanol (0.1% formic acid), gradient program, 95:5 V/V at 0 min, 50:50 V/V at 6 min, 5:95 V/V at 12 min, hold for 2 min, 95:5 V/V at 14 min, hold for 2 min, flow rate, 0.35 mL/min, temperature, 40°C, injection volume, 2 µL. Linear ion trap (LIT) and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (QTRAP), QTRAP® 6500 + LC-MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in positive ion mode and controlled by Analyst 1.6.3 software (Sciex). The ESI source operation parameters were as follows: ion source, ESI+, source temperature 550°C, ion spray voltage (IS) 5500 V, curtain gas (CUR) was set at 35 psi, respectively. Anthocyanins were analyzed using scheduled multiple reaction monitoring (MRM). Data acquisitions were performed using Analyst 1.6.3 software (Sciex). Multiquant 3.0.3 software (Sciex) was used to quantify all metabolites. Mass spectrometer parameters including the declustering potentials (DP) and collision energies (CE) for individual MRM transitions were done with further DP and CE optimization. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period. Data Preprocessing and Annotation The raw data of all samples were converted to the mzXML format using ProteoWizard and processed with an in-house program, which was developed using R and based on XCMS, for peak detection, extraction, alignment, and integration. Then an in-house MS2 database (BiotreeDB) was applied in metabolite annotation. The cutoff for annotation was set at 0.3. The data variation was performed by R packages XCMS software [ 31 ]. The Principal Component Analysis (PCA) was performed using Metaboanalyst 3.0 based on the normalized peak area of the metabolites in each sample. Significantly regulated metabolites between groups were determined by absolute Log1.5FC (fold change). Therapy FDR 1.5 were used to identify significantly differential metabolites. The HCA (hierarchical cluster analysis) results of samples and metabolites were presented as heatmaps with dendrograms. HCA was carried out by R package pheatmap. For HCA, normalized signal intensities of metabolites (unit variance scaling) are visualized as a color spectrum. The detailed information of anthocyanins were listed in Table S5 . LUC assay The LUC assay was performed according to Du et al. (2024a). The promoter sequence of CAD and CHS were cloned into the reporter effector pGreenII LUC0800 (pLUC-Pro) and the coding sequence of NAC1 was fused into pGreenII 62SK (pSK-NAC1). The Agrobacterium strains (GV3101) were incubated overnight and resuspended in infiltration buffer (20 mM MES, 0.1 mM acetosyringone, and 10 mM MgCl 2 ) and final diluted to the concentration of OD 600 = 0.6–0.8. Suspensions of pLUC-Pro and pSK-NAC1 were mixed in equal volumes and infiltrated into Nicotiana benthamiana leaves. After 48 h, the transformed leaves were sprayed with 0.1 M luciferin solution and kept in the dark for 7 min. A low light-cooled CCD imaging apparatus (Lumazone Pylon 2048 B, Princeton Instruments) was used to capture and analyze the acquired LUC images. Transient overexpression assay The open reading frames (ORF) of NAC1 were cloned using a primer pair containing EcoRI and BamHI sites and ligated to pBIN-eGFP. Then, the positive vector was confirmed using sequencing, and transduced into Agrobacterium tumefaciens LBA4404. GFP controls, pBIN-NAC1 was transferred into Nicotiana benthamiana using A. tumefaciens -mediated method. Four days after infiltration, the representative fruit was collected for further experiments. Results UVA, UVAB and UVB altered the proteome patterns of H. vulgare . To investigate the responses of H. vulgare to UVB, UVA and UVA + UVB (UVAB) stresses, three biological replicates for H. vulgare from CK, UVB, UVA and UVAB treatments were collected for proteome sequencing. Totally, 3949 proteins were identified in H. vulgare . Then, we performed principal component analysis (PCA) on all proteome profiles to investigate the proteomic pattern of H. vulgare following UVB, UVA and UVAB exposures (Fig. 1 A). PCA plot was constructed using principal component 1 (PC1) and PC2, which explained 47.8% and 8.1% variations between all samples (Fig. 1 A). The PCA plot showed that the samples from CK clearly distributed at a single region and separated with UVB, UVA and UVAB (Fig. 1 A), suggesting that all UVB, UVA and UVAB treatments dramatically altered the proteome pattern of H. vulgare . Typically, the differences between experimental groups were mainly represented by PC1, while PC2 mainly explained the differences among biological replicates from the same treatment (Fig. 1 A). Additionally, UVB samples also clearly separated with UVA, UVAB and CK, and the differences between UVB and CK were more significant than that between UVAB/UVA vs. CK (Fig. 1 A), suggesting that H. vulgare are more responsive to UVB. Meanwhile, H. vulgare exhibited similar responses to UVA and UVB, and the effect of UVA on plant protein expression was minimal that UVA samples were closet to CK (Fig. 1 A). Then, to investigate the detailed changes in proteome of H. vulgare following UVA, UVB and UVAB exposures, we analyzed the differentially expressed proteins (DEPs) of all 3949 identified proteins in UVA vs. CK, UVB vs. CK and UVAB vs. CK comparisons based on P 1.5 (Fig. 1 B). Totally, 73 DEPs were identified in UVA vs. CK, with 49 downregulated DEPs and 24 upregulated DEPs (Fig. 1 B). Additionally, we identified 272 DEPs (228 down- and 44 up-regulated DEPs) in UVAB vs. CK and 678 DEPs (477 down- and 201 up-regulated DEPs) in UVB vs. CK comparison (Fig. 1 B). Typically, most DEPs in H. vulgare associated with UVA, UVB and UVAB exposures were mainly located in nucleus, cytoplasm, secreted and chloroplast (Fig. 1 C). It has been well documented that chloroplast involved in responses of plants to UV by regulating reactive oxygen homeostasis caused by UV stress [ 32 ]. Moreover, COG/KOG enrichment showed that most of proteins among the DEPs from these 3 pairwise comparisons mainly involved in RNA processing and modification, Energy production and conversion, Translation, ribosomal structure and biogenesis, Posttranslational modification, protein turnover, chaperones, General function prediction only and Intracellular trafficking, secretion, and vesicular transport (Figure S1 ). Thus, these results suggested UVA, UVAB and especially UVB could alter the proteome pattern of H. vulgare , especially leading changes in levels of proteins located in nucleus, cytoplasm, secreted and chloroplast. Different wavelengths of UV cause changes in expression of different functional proteins in H. vulgare . Plants make different responses to different wavelength of light [ 33 ]. Here, we investigated the function of DEPs in H. vulgare following UVA, UVB and UVAB exposures using gene ontology (GO) enrichment analysis. For UVA vs. CK comparison, 90 GO terms were significantly enriched against all DEPs (Fig. 2 A, Table S1 ). Typically, these DEPs mainly functioned in protein storage vacuole membrane, vacuolar proton-transporting V-type ATPase, plant-type vacuole membrane and post-spliceosomal complex, with thiol-dependent ubiquitin-specific protease activity, ubiquitinyl hydrolase activity, receptor binding, cysteine-type peptidase activity, hydrolase activity, signaling receptor activity, cytokine activity, vascular endothelial growth factor receptor binding, molecular transducer activity, ATPase activity, monocarboxylic acid binding, inositol monophosphate 3-phosphatase activity, ion transmembrane transporter activity and peptidase activity (Fig. 2 A, Table S1 ). These DEPs associated with UVA mainly involved in regulation of protein complex disassembly, DNA replication checkpoint, DNA damage response, single organism reproductive process, negative regulation of G2/M transition of mitotic cell cycle, DNA integrity checkpoint, glyoxal metabolic process and cell communication (Fig. 2 A, Table S1 ). These results showed that UVA mainly affected proteins involved in DNA replication and cell cycle, which then may cause damage to cell growth in plants. For UVAB vs. CK comparison, DEPs mainly functioned in ESCRT complex, organelle membrane, endosome, ESCRT I complex and cis-Golgi network in plant cell (Fig. 2 B, Table S2 ). For molecular function, 21 terms were significantly enriched, including protein disulfide oxidoreductase activity, peptidase inhibitor activity, glutathione transferase activity, peroxiredoxin activity, oxidoreductase activity, lipid binding, transferase activity and thioredoxin peroxidase activity (Fig. 2 B, Table S2 ). And these DEPs associated with UVAB mainly involved in 46 biological progresses, especially lipid transport, response to stimulus, endosomal transport, vacuolar transport, positive regulation of macromolecule metabolic process, cell surface receptor signaling pathway, glutathione metabolic process, cell redox homeostasis, response to abscisic acid, glutamate catabolic process and seed development (Fig. 2 B, Table S2 ). Of note, numerous processes relevant to oxidant responses were identified in UVAB vs. CK comparison, including glutathione metabolic process, cell redox homeostasis, response to abscisic acid, glutamate catabolic process, while it also affected proteins relevant to seed development (Fig. 2 B, Table S2 ). It has been found that UV exposure always accompanied by oxidant stress and disordered oxygen-radical homeostasis, leading to damage to plant cell and growth [ 34 ]. Thus, these results suggested that UVAB may cause oxidant stress in plant cell to affect seed development. Subsequently, we analyzed the function of DEPs from UVB vs. CK comparison. The GO results showed that these DEPs mainly distributed in respiratory chain complex, mitochondrial membrane, oxidoreductase complex, mitochondrion, plasma membrane, NADH dehydrogenase complex, chromatin, organellar ribosome, nucleosome and protein-DNA complex (Fig. 2 C, Table S3 ). Typically, these DEPs with functions of voltage-gated channel activity, gated channel activity, transcription corepressor activity, DNA binding, kinase regulator activity, phospholipid transporter activity, sphingolipid binding, ceramide binding and ATPase activity, mainly involved in protein-DNA complex assembly, nucleosome assembly, chromatin assembly, DNA packaging and anthocyanin-containing compound biosynthetic process (Fig. 2 C, Table S3 ). Anthocyanin biosynthesis was the well-documented processes in plants in response to UV stress, anthocyanin could protect plants from oxidant damage caused by UV exposure [ 35 ]. Thus, these results suggested that plant could trigger the anthocyanin biosynthesis in response to UVB exposure. Changes in anthocyanin biosynthesis were the main responses in H. vulgare to UVAB and UVB exposures. Here, we further performed KEGG pathway enrichment analysis on DEPs from three pairwise comparisons (Fig. 3 A). For DEPs from UVA vs. CK, KEGG results showed that these DEPs mainly involved in Phagosome, Vitamin B6 metabolism, Diterpenoid biosynthesis, Oxidative phosphorylation, Phosphatidylinositol signaling system, Phenylalanine metabolism, Plant hormone signal transduction, Tyrosine metabolism, Protein export and Metabolic pathways (Fig. 3 A). In parallel, DEPs from UVAB vs. CK mainly functioned in Glutathione metabolism, Phagosome, Protein processing in endoplasmic reticulum, Valine, leucine and isoleucine biosynthesis, Porphyrin metabolism, Phenylpropanoid biosynthesis, D-Amino acid metabolism, Monoterpenoid biosynthesis, Phenylalanine metabolism, Butanoate metabolism, Thiamine metabolism, Plant hormone signal transduction and 2-Oxocarboxylic acid metabolism (Fig. 3 A). In addition, KEGG pathway enrichment analysis on DEPs from UVB vs. CK showed that 8 biosynthesis pathways were significantly enriched, including Oxidative phosphorylation, Plant hormone signal transduction, Anthocyanin biosynthesis, Basal transcription factors, Diterpenoid biosynthesis, Phenylpropanoid biosynthesis, Flavonoid biosynthesis and MAPK signaling pathway (Fig. 3 A). Typically, we noted the significant enrichment of anthocyanin and flavonoid biosynthesis in UVB vs. CK (Fig. 3 B), reaching a consensus with GO results that UVB could trigger the proteome pattern of flavonoid and anthocyanin biosynthesis in H. vulgare (Fig. 3 A). Additionally, UVA vs. CK, UVAB vs. CK and UVB vs. CK shared 25 pathways, such as Ribosome, Phagosome, Plant hormone signal transduction, Starch and sucrose metabolism, Biosynthesis of amino acids, Glycolysis / Gluconeogenesis, Protein export, Proteasome, Diterpenoid biosynthesis, Tyrosine metabolism, MAPK signaling pathway and Oxidative phosphorylation (Fig. 3 B). Of note, there are 45 pathways were specifically enriched under UVB exposure, especially DNA replication, Glycerophospholipid metabolism, Basal transcription factors, Photosynthesis, Fatty acid degradation, alpha-Linolenic acid metabolism, Aminoacyl-tRNA biosynthesis, Anthocyanin biosynthesis, Fatty acid metabolism, Flavonoid biosynthesis, ABC transporters and Sphingolipid metabolism (Fig. 3 B). Considering the effects of importance of anthocyanin synthesis and antioxidant system in plant in response to UV exposure, we further analyzed the expression of related proteins in H. vulgare following UVB, UVAB and UVA exposures. The expression heat map of proteins relevant to antioxidant system showed that the expression levels of most related proteins were higher in H. vulgare under UVAB and especially UVB exposures, compared to UVA and CK (Fig. 3 C). Meanwhile, the proteins involved in anthocyanin synthesis were also triggered to highly expressed in H. vulgare by UVAB and especially UVB exposures, but not UVA (Fig. 3 C). Taken together, we proposed that different wavelength of UV could trigger different responses in plants, typically anthocyanin biosynthesis and antioxidant reaction was the main responses in H. vulgare to UVB exposure. UVAB and UVB triggered accumulation of anthocyanins in H. vulgare . Further, we compared the anthocyanin-related metabolic profiles of H. vulgare following UVA, UVAB and UVB exposures. To identify the overview of changes in anthocyanin composition of H. vulgare following UVA, UVAB and UVB exposures, we performed PCA analysis on all metabolic profiles of H. vulgare (Fig. 4 A). The PCA plots constructed by PC 1 and 2 showed significant variations in anthocyanin composition among all groups, while PC1 and PC2 explained 33.2% and 15.4% variations associated with experimental treatments (Fig. 4 A). Typically, PC1 mainly explained the variations in anthocyanins compositions of H. vulgare caused by UVAB and UVB exposures (Fig. 4 A), supporting the involvement of anthocyanins synthesis of H. vulgare in response to UVAB and UVB exposures. Totally, 28 anthocyanins were identified in H. vulgare (Fig. 4 B, Figure S2 ). Typically, the contents of most of anthocyanins were higher in H. vulgare under UVB exposure in comparison with UVA and CK, including Procyanidin B2, Cyanidin-3-O-sambubioside, Peonidin-3-O-glucoside, Cyanidin-3-O-glucoside, Pelargonidin-3-O-glucoside, Procyanidin B1, Peonidin-3-O-sophoroside, Petunidin-3-O-glucoside, Cyanidin-3-(malonyl)glucoside-5-rhamnoside, Delphinidin-3-O-glucoside, Cyanidin-3-O-sophoroside, Cyanidin-3-O-(6-O-malonyl-beta-D-glucoside), Cyanidin-3,5-O-diglucoside, Cyanidin-3-O-xyloside and Peonidin (Fig. 4 B). Of note, the contents of all anthocyanins in H. vulgare under UVAB exposure was higher than that in CK and UVA, while UVA did not induce increases of anthocyanins in H. vulgare (Fig. 4 B). Thus, we concluded that anthocyanins accumulation was the main response in H. vulgare to UVAB and UVB exposures. Active regulation network involved in regulating anthocyanin synthesis in H. vulgare in response to UVB and UVAB exposures. To identify the hub proteins involved in anthocyanin synthesis in H. vulgare in response to UVB and UVAB exposures, we analyzed the correlation between levels of proteins and anthocyanins using Pearson algorithm. The results showed that proteins represented by Q40069, F2D3Q9, F2E8A9, M0WRX0, F2E7V8, F2CXT3 and F2E8K5 were highly correlated with the anthocyanin level in H. vulgare (Fig. 5 A), implying their involvements in synthesizing anthocyanin in H. vulgare . Considering the involvement of anthocyanin synthesis H. vulgare in response to UVB and UVAB exposures, we further constructed the co-expression network of proteins (Q40069, F2D3Q9, F2E8A9, M0WRX0, F2E7V8, F2CXT3 and F2E8K5) relevant to anthocyanin synthesis using WGCNA based on all proteome profiles of H. vulgare (Fig. 5 B-D). The expression data of all transcription factors and kinases of H. vulgare were selected via Nr annotation for WGCNA construction. We set the soft threshold to 26 ( R 2 = 0.85) to construct a scale-free network (Figure S3 A). Then, 9 modules were identified by hierarchical clustering and the dynamic branch cutting, each module was assigned a unique color as an identifier (Fig. 5 B, C, Figure S3 B). Then, the modules highly correlated with the related traits were filtered out for further construction of regulation network of anthocyanin synthesis in H. vulgare (Fig. 5 C). Based on the WGCNA results, we identified that MEbrown, MEyellow and MEturquoise were highly related to proteins relevant to anthocyanin synthesis (Fig. 5 C). Among proteins from these modules, we constructed the co-expression regulation networks for anthocyanin synthesis in H. vulgare (Fig. 5 D). Totally, we identified that 4 transcription factors and 34 kinases were responsible for positively regulating anthocyanin synthesis in H. vulgare in response to UVB and UVAB exposures (P < 0.05, Fig. 5 D). As shown in Fig. 5 D, A0A8I6WJ93, A0A8I6WLA6, A0A8I7B6M6 and especially F6IAY3 (NAC transcription factor) were the hub transcription factors involved in the regulation network of anthocyanin synthesis in H. vulgare , and it was positively correlated with the expression of anthocyanin-related proteins (Fig. 5 D). Of note, most of these regulators were upregulated to higher expression levels in H. vulgare by UVAB and UVB exposures, supporting their involvements in regulating responses in H. vulgare to UVAB and UVB exposures (Fig. 5 D). Further RT-qPCR assay confirmed that transcription factors including A0A8I6WJ93, A0A8I6WLA6, A0A8I7B6M6 and especially F6IAY3 were activated to upregulate in H. vulgare by UVAB and UVB exposures, but not UVA (Fig. 5 E). Of note, LUC assay confirmed that NAC1 could directly bind on the promoters of CAD1 and CHS to trigger their expression (Fig. 5 F). Further transient overexpression assay showed that NAC1 overexpression activated expression of CHS , CAD and ANS in plants, supporting its function in activating anthocyanin biosynthesis in H. vulgare (Fig. 5 G). Taken together, these results suggested that the UVAB/UVB-reprogrammed anthocyanin synthesis in H. vulgare was regulated by a complex network. Discussion In the plateau region, especially Qinghai-Tibet Plateau, UV stress has become a nonnegligible obstacle to the development of crops. Thus, it is of great practical significance to investigate the detailed plant responses to UV exposures for guiding the agriculture development in these regions. Presently, we found that UVA, UVAB and UVB reprogrammed the proteome pattern of H. vulgare . In detail, UVA mainly affected the expression of proteins involved in cell cycle and DNA repair, while UVB and UVAB mainly altered proteins involved in anthocyanin biosynthesis and antioxidant systems. Typically, all identified proteins involved in anthocyanin biosynthesis were activated to upregulate in H. vulgare by UVB and UVAB exposures. Further anthocyanin profiles showed that UVB and UVAB caused accumulation of anthocyanin in H. vulgare , which may protect H. vulgare from UV-associated oxidant damage to plant cell. Then, we constructed the network for regulating UV-triggered anthocyanin biosynthesis in H. vulgare using proteome profiles. Importantly, we identified NAC transcription together with numerous kinases were responsible for regulating UVAB/UVB-triggered anthocyanin biosynthesis in H. vulgare . Of note, NAC1 directly bound on the promoters of CAD1 and CHS to trigger their expression, then activated anthocyanin biosynthesis in H. vulgare . Overall, our research depicted the detailed global responses of H. vulgare to UV challenge, which contributed to our understanding of adaptive mechanisms of plants in plateau. With the destruction of the ozone layer, the research on ultraviolet radiation has attracted increasing attention. Ultraviolet-B radiation is a light wave with a wavelength between 280 and 320 nm, and its high energy can directly lead to DNA strand breakage and blocked cross-linked replication between DNA strand and DNA strand or protein [ 36 , 37 ]. Meanwhile, UV exposure also caused the disruption and structural changes of polypeptide chains in protein molecules in plants and changes in multiple metabolic activities of organisms [ 38 ]. Similarly, we found that the UVA could affected the expression of proteins involved in cell cycle and DNA replications. In addition, UV-B can also trigger the accumulation of reactive oxygen species, resulting in lipid peroxidation of biofilm [ 39 ], then change the fluidity and permeability of the membrane and affecting a variety of physiological activities of organisms [ 40 ]. UV-B radiation can directly or indirectly affect the synthesis, accumulation and metabolism of primary metabolism and secondary metabolites in plants [ 11 ]. Of note, combining with metabolomics, genomics and proteomics provides theoretical basis and technical support for the application of UV-B radiation in agricultural production and agricultural product processing. Since UV-B irradiation can induce oxidative stress in plants, the biosynthesis pathway of antioxidant active substances such as polyphenols and flavonoids will be activated, so as to rapidly synthesize a large number of antioxidant active substances to effectively repair the body damage caused by oxidative stress [ 41 , 42 ]. Polyphenols are a class of compounds formed by the substitution of polyhydroxyl groups on the basic skeleton of phenol. They are important bioactive substances in human diet and secondary metabolites closely related to plant defense mechanisms [ 43 ]. A large number of studies have shown that that UVB treatment can significantly increase flavonoid and anthocyanin content in plant tissues [ 44 – 46 ]. Here, we also found that UV-B is an important factor in inducing the biosynthesis of plant anthocyanins, while UVA + UVB also could promote the anthocyanin accumulation in H. vulgare . Anthocyanins and flavonoid metabolites have efficient scavenging function of reactive oxygen species [ 42 ]. Typically, both low and high doses of UV-B radiation lead to an increase in ROS in the plant [ 47 ]. Therefore, the accumulation of anthocyanin metabolites may be an important way for plants to resist strong UV stress at high altitude. Because anthocyanins and flavonoids metabolites not only help plants resist abiotic and biological stresses, they are also important nutrients in crops [ 48 ]. Therefore, the rational use of ultraviolet rays can be used as an important means of metabolic regulation in agricultural production, so as to improve crop yield and carry out targeted regulation and accumulation of target products. It was found that phenylpropanoid-derived flavonoid and anthocyanin biosynthesis was mainly regulated by MYB transcription factors to perform related function in plant [ 30 ]. Of note, we identified NAC transcription factor as regulator for mediating UV-associated anthocyanin biosynthesis via constructing regulation network of anthocyanin biosynthesis in H. vulgare . And the detailed regulation mechanism of NAC in mediating UV-associated anthocyanin biosynthesis in H. vulgare will be the focus in our future work. Overall, the study on the effects of UV-B on the main nutrients and bioactive substances in plants is of great significance for guiding agricultural production, especially for the directed regulation of specific nutrients and bioactive substances in agricultural products. Conclusion In the present study, we deciphered the global responses of H. vulgare to UV challenge via integrating metabolome and proteome. we found that UVA, UVAB and UVB reprogrammed the proteome pattern of H. vulgare . UVA mainly affected the expression of proteins involved in cell cycle and DNA repair. In addition, UVB and UVAB mainly activated proteins involved in anthocyanin biosynthesis and antioxidant systems, leading to accumulation of anthocyanin in H. vulgare to protect H. vulgare from UV-associated oxidant damage. Of note, we identified that NAC transcription together with numerous kinases were responsible for regulating UVAB/UVB-triggered anthocyanin biosynthesis in H. vulgare. Thus, the present study gain insight into the adaptive mechanisms of plants in plateau. Declarations Author Contributions : Z.H.: writing-original draft, writing-review and editing. T.Z.: data curation, visualization, methodology. X.Y. and C.DJ: methodology, software. Y.Z.: formal analysis, validation, conceptualization. X.W. and C.D: supervision, funding acquisition. Y.L. and T.Z.: resources, supervision, funding acquisition. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by Natural Science Funds of Xizang (XZ202101ZR0073G). Ethics approval and consent to participate The collection of plant materials used in our study complied with permission of related institutions, and complied with national or international guidelines and legislation. The experiments did not involve endangered or protected species. Consent for publication Not applicable. Institutional Review Board Statement : Not applicable. Informed Consent Statement : Not applicable. Data Availability Statement : The data reported in this paper have been deposited in the OMIX, China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (https://ngdc.cncb.ac.cn/omix: accession no.OMIX009895). Conflicts of Interest : The authors declare no conflicts of interest. References Huang R, Gao HY, Liu J, Li XP. WRKY transcription factors in moso bamboo that are responsive to abiotic stresses. J Plant Biochem Biotechnol. 2022;31:107–14. Kataria S, Jajoo A, Guruprasad KN. 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Supplementary Files TableS1.xlsx TableS2.xlsx TableS3.xlsx TableS4.xlsx TableS5.xlsx Supplementalfigures.docx 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-5763885","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":445903997,"identity":"a3cc6d9b-ffb9-41cb-b2b5-4ddf579a8ddd","order_by":0,"name":"Zhihua Hao","email":"","orcid":"","institution":"Xizang Academy of Agricultural and Animal Husbandry Sciences","correspondingAuthor":false,"prefix":"","firstName":"Zhihua","middleName":"","lastName":"Hao","suffix":""},{"id":445904000,"identity":"b7c8cc86-ab50-40db-874d-c1f77b68867f","order_by":1,"name":"Xiaojun Yang","email":"","orcid":"","institution":"Xizang Academy of Agricultural and Animal Husbandry Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xiaojun","middleName":"","lastName":"Yang","suffix":""},{"id":445904004,"identity":"7e03704d-e371-4a6a-b0e8-20dc20cb36da","order_by":2,"name":"Ciren Deji","email":"","orcid":"","institution":"Xizang Academy of Agricultural and Animal Husbandry Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ciren","middleName":"","lastName":"Deji","suffix":""},{"id":445904010,"identity":"575d5dba-e52d-44a2-9044-19a611840ccc","order_by":3,"name":"Yifan Zhang","email":"","orcid":"","institution":"Xizang Academy of Agricultural and Animal Husbandry Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yifan","middleName":"","lastName":"Zhang","suffix":""},{"id":445904012,"identity":"c66e952d-aa6b-4410-8b2d-06bec80d2090","order_by":4,"name":"Xuelian Wu","email":"","orcid":"","institution":"Xizang Academy of Agricultural and Animal Husbandry Sciences","correspondingAuthor":false,"prefix":"","firstName":"Xuelian","middleName":"","lastName":"Wu","suffix":""},{"id":445904014,"identity":"32d77fd6-9973-474d-98a7-0eb255d178ad","order_by":5,"name":"Ying Li","email":"","orcid":"","institution":"Xizang Academy of Agricultural and Animal Husbandry Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ying","middleName":"","lastName":"Li","suffix":""},{"id":445904016,"identity":"05c637db-732b-455d-8679-c42a9228598d","order_by":6,"name":"Ci Dun","email":"","orcid":"","institution":"Xizang Academy of Agricultural and Animal Husbandry Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ci","middleName":"","lastName":"Dun","suffix":""},{"id":445904018,"identity":"20f7c152-5cb6-4796-8188-d19ae3aed822","order_by":7,"name":"Tangwei Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBElEQVRIiWNgGAWjYDACCST2gQQeGx5+/gZitCRAtXyQSZORnHGABC2MM2wO2xg0JODXIT+7+dnDrz/s8uQjkg8e5sk5z2PAcIDxw8cc3FoY5xwzN5ZJSC42vJGWcJjnzG0ec+YGZsmZ23BrYZZIMJOWSGBO3Dg7x+Awb89tHsuGA2zMvHi0sEmkfwNqqQdqyf9wmPffOR6DAwn4tfBI5JhJfkg4nDhfOofh4AyeA4S1SEjklEkzpB1P3CD/zODAB55kHskZB5vx+kV+Rvo2yR821Ynzew4//pDAY2fPz9988MNHPFrAQcADJAwOwPmMDfjVg5T8AFlHWN0oGAWjYBSMVAAAXBxU+stz4E0AAAAASUVORK5CYII=","orcid":"","institution":"Xizang Academy of Agricultural and Animal Husbandry Sciences","correspondingAuthor":true,"prefix":"","firstName":"Tangwei","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2025-01-04 13:53:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5763885/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5763885/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81239304,"identity":"9de43a40-8148-43b9-9a72-3333ded4765c","added_by":"auto","created_at":"2025-04-23 21:09:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":490435,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUVA, UVB and UVAB altered the global proteome pattern of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. vulgare\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA, D: \u003c/strong\u003ePCA scores plot of the samples showing distinct separation between UVA, CK, UVB and UVAB at proteome levels. The ellipse represents the 95% confidence interval. \u003cstrong\u003eB:\u003c/strong\u003e Volcano plot shows the differential proteins (DEPs) with log1.5fold changed ≥ 1.0 and P ≤ 0.05 in UVA vs. CK, UVAB vs. CK and UVB vs. CK pairwise comparisons. The upregulated proteins are shown in red, while the downregulated ones are shown in blue. \u003cstrong\u003eC:\u003c/strong\u003e Subcellular location analysis on DEPs of \u003cem\u003eH. vulgare\u003c/em\u003e from each comparison.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-5763885/v1/dadfc73fe87e1ff1e232fc22.png"},{"id":81238496,"identity":"921535b4-33e8-4f4a-bd22-6ccddcf9b157","added_by":"auto","created_at":"2025-04-23 20:53:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":520274,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUVA, UVAB and UVB altered the expressions of proteins of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eH. vulgare\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e with different function.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-\u003cstrong\u003eC: \u003c/strong\u003eGene ontology (GO) enrichment analysis on DEPs from UVA vs. CK (A), UVAB vs. CK (B) and UVB vs. CK (C) pairwise comparisons. The various color levels displayed different levels of significance of metabolic pathways from low (green) to high (red). The terms of cellular component, molecular function and biological progress are shown in green, blue and orange, respectively.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-5763885/v1/4664f64a8cdb1bd5b345b051.png"},{"id":81238854,"identity":"6a2730e7-41c5-415e-afcb-fb9d0dee74a2","added_by":"auto","created_at":"2025-04-23 21:01:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":472615,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUVB and UVAB triggered the expression of proteins involved in anthocyanin synthesis and antioxidant systems in\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e H. vulgare\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003eScatter plot of the most enriched KEGG pathways of all DEPs from UVA vs. CK, UVAB vs. CK and UVB vs. CK pairwise comparisons. The size and color of each plots represented the protein number and significance of each related pathways. The x-axis represents the rich factor of each pathway. Rich factor was generated by summation of importance measures of matched proteins to all proteins present within the pathway. \u003cstrong\u003eB: \u003c/strong\u003eVenn diagram displays the overlap of pathways among UVA vs. CK, UVAB vs. CK and UVB vs. CK pairwise comparisons.\u003cstrong\u003e C: \u003c/strong\u003eHeat maps of the relative expression abundances of proteins\u003cem\u003e \u003c/em\u003ein \u003cem\u003eH. vulgare\u003c/em\u003efollowing UVA, UVAB and UVB exposures. The scale bar showed the mean values of proteins in each group.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-5763885/v1/c8366c14764ec2ef3694eb82.png"},{"id":81238495,"identity":"9be4057f-8567-4852-93a3-d43e1752c63e","added_by":"auto","created_at":"2025-04-23 20:53:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":225937,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMT activated the regulation network for zeatin synthesis in ENG to enhance its cold tolerance.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e PCA plots displays the separation of all treatments based on anthocyanin composition and stability of replicates with the same treatment. Each circle represents a sample, the circles labeled by same color were the samples with the same treatment. PCA plots are constructed using top 2 principal component. The ellipse represents the 95% confidence interval. \u003cstrong\u003eB: \u003c/strong\u003eHeatmap displays the levels of related anthocyanins in \u003cem\u003eH. vulgare\u003c/em\u003e following UVA, UVAB and UVB exposures. The high and low expression levels of metabolites in each biological replicate are shown in blue and yellow, respectively.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-5763885/v1/3aa6687a6c1788a3cacf362a.png"},{"id":81238533,"identity":"4a0fb612-e675-415c-86cf-8fd7fc53a6a3","added_by":"auto","created_at":"2025-04-23 20:53:57","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":765023,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComplex regulation network involved in mediating the anthocyanin responses in\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e H. vulgare\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e to UVB exposure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA: \u003c/strong\u003eCorrelation analysis between proteins responsible for anthocyanin synthesis and anthocyanins in \u003cem\u003eH. vulgare\u003c/em\u003efollowing UVA, UVAB and UVB exposures. \u003cstrong\u003eB: \u003c/strong\u003eAssociation between modules and experiment trait. The darker the module color, the more significant their relationship. \u003cstrong\u003eC:\u003c/strong\u003e Heatmap displaying the Module-trait relationships. Each row represents a module eigengene, and column represents a trait. Each cell contains the corresponding correlation and p value. The table is color-coded by correlation based on the color legend. \u003cstrong\u003eD:\u003c/strong\u003eThe weight network of the significant genes involved in the module labeled by MEblack. The color and the size of the circle represented the weight value of each protein in the network. The circle sizes displayed the degree value of corresponding genes in the network, which showed the gene importance. \u003cstrong\u003eE: \u003c/strong\u003eRT-qPCR detects the expression levels of genes encoding hub transcription factors in \u003cem\u003eH. vulgare\u003c/em\u003e following UVA, UVAB and UVB exposures, “Different letters represented significance based on P \u0026lt; 0.01 from Student’s T-test”. \u003cstrong\u003eF: \u003c/strong\u003eLUC report assay detects the binding affinity of NAC1 on promoters of genes encoding anthocyanin biosynthesis. \u003cstrong\u003eG: \u003c/strong\u003eRT-qPCR determines the expression levels of genes involved in anthocyanin biosynthesis in plant overexpressing\u003cem\u003e NAC1\u003c/em\u003e. GFP controls, pBIN-NAC1 was transferred into \u003cem\u003eN\u003c/em\u003e.\u003cem\u003ebenthamiana\u003c/em\u003e using \u003cem\u003eA. tumefaciens\u003c/em\u003e-mediated method.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-5763885/v1/03a9dca4c587bc5156ffdea1.png"},{"id":97326435,"identity":"dd46c860-d068-4549-bbd1-5b3b6576b206","added_by":"auto","created_at":"2025-12-03 08:40:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3143706,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5763885/v1/4ef19623-044e-4535-8d2f-af1cbae44d87.pdf"},{"id":81239569,"identity":"e41fb968-13d6-4f81-ab24-c37a7700f59b","added_by":"auto","created_at":"2025-04-23 21:17:56","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":70872,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5763885/v1/deface9b6259cf0c1bce1db1.xlsx"},{"id":81239302,"identity":"fc3bdda6-b474-4d33-9d1d-5ca07b427877","added_by":"auto","created_at":"2025-04-23 21:09:56","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":116870,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5763885/v1/0954505836c49ca6d9d867ea.xlsx"},{"id":81238847,"identity":"c5681220-0a7f-482c-ae2c-dfd0453fd9cf","added_by":"auto","created_at":"2025-04-23 21:01:56","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":199297,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5763885/v1/7f32ee6378fd08d5516ebfec.xlsx"},{"id":81239309,"identity":"e5746326-d859-43f4-ad35-101c782925e8","added_by":"auto","created_at":"2025-04-23 21:09:57","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":1132434,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5763885/v1/ca2cd1ac5767814ca45e801e.xlsx"},{"id":81238864,"identity":"c6b32d55-5f58-42d2-98e7-60749d8b8d0a","added_by":"auto","created_at":"2025-04-23 21:01:57","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":29823,"visible":true,"origin":"","legend":"","description":"","filename":"TableS5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-5763885/v1/dabec315177694e0b433fdd7.xlsx"},{"id":81238505,"identity":"80a40385-9a1f-48d8-82bf-c67f195af8eb","added_by":"auto","created_at":"2025-04-23 20:53:56","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":389589,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementalfigures.docx","url":"https://assets-eu.researchsquare.com/files/rs-5763885/v1/cd0d4447a02bf8e4816f0f02.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Muti-omics revealed the global response of Hordeum vulgare to different wavelength of UV exposure","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlants are often challenged by numerous adverse abiotic stresses, including drought, high salinity, and especially UV exposure (i.e., UVA and UVB) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. With the increasingly prominent global environmental changes, the increasing trend of ultraviolet radiation is becoming more and more obvious. Studies have shown that the ultraviolet radiation on the Earth's surface has increased by 6\u0026ndash;14% and will continue to increase in the future [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. UV-B is the ionizing radiation with a wavelength of 280 to 315 nm that can penetrate the ozone layer and is a part of the solar radiation. In recent years, with the decrease of the ozone layer, UV-B radiation reaching the Earth's surface has increased [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. High doses of UV-B damage amino acid residues, leading to protein and enzyme inactivation of amino acids in synthesis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Studies have shown that UV-B radiation can significantly inhibit the photosynthetic rate of plants, hinder plant growth and development, and lead to a decrease in crop yield [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Highland barley (\u003cem\u003eHordeum vulgare\u003c/em\u003e L.) is a kind of Chinese general name for naked barley in the Tibetan plateau area, and always challenged by strong UV challenge in production region. Typically, highland barley has the advantages of drought resistance, cold resistance, UV resistance and humidity resistance, which lead to it adapted to plant in a range of Tibetan plateau area. Thus, it has been the most important food crop for farmers and herdsmen in Tibetan areas. At present, China's highland barley planting area is about 333,300 hm\u003csup\u003e2\u003c/sup\u003e, 95% of which is distributed in the Qinghai-Tibet Plateau, while Tibet's highland barley planting area is the largest, highest yield, and most widely distributed, its planting area is about 240,000 hm\u003csup\u003e2\u003c/sup\u003e, accounting for 72% of the major planting area. Moreover, the proportion of grain crop planting area and output in the whole region has always remained above 70%. Thus, understanding the detailed responses of plants to UV exposure is of great significance for their development and cultivation.\u003c/p\u003e \u003cp\u003eIn order to cope with the harsh living environment, plants growing on the Qinghai-Tibet Plateau show different adaptive traits, such as the shape and color of roots, stems and leaves, the interaction between different plants, and the change of reproduction mode [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Typically, plant altered the expression levels of soluble sugar, superoxide dismutase, peroxidase, etc., in the leaf tissues to strong ultraviolet radiation in the Qinghai-Tibet Plateau, and the physiological characteristics of plants are transformed to protect plants from UV exposure [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition, wild barley on the plateau mainly changed genes related to DNA damage repair, nitrogen metabolism, cell membrane stability regulation, UV radiation resistance and other functions, so as to adapt to excessive UV radiation on the Qinghai-Tibet Plateau [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It has shown that UV-B exerted significant effect on gene expression related to the biosynthesis of plant secondary metabolites [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], in which increased accumulation of secondary metabolites in plants [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These secondary metabolites include terpenoids [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], alkaloids, and phenols [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].and secondary nitrogen-containing compound [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Flavonoids also exerted many important physiological effects on plants, especially protecting plants from biotic and abiotic stresses [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Recently, it was found that UV radiation significantly affected the compositions of flavonoids and anthocyanins in plant [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Park et al. (2008) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] found that the synthesis of plant flavonoids and anthocyanins is related to many factors, typically their synthetic genes are affected by UV-B radiation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In detail, flavonoids can accumulate in some special parts of plant cells, such as cell walls, vacuoles, chloroplasts, and glands [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Additionally, the contents of flavonoids and anthocyanins in \u003cem\u003eC. mongolica\u003c/em\u003e and \u003cem\u003eCymbopogon citratus\u003c/em\u003e were significantly increased after UV-B stress [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Anthocyanins are distributed in plant epidermal cells, while numerous structural genes and transcription factors play an important role in the synthesis of anthocyanins and flavonoids [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Ohnishi et al. (1992) [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] found that anthocyanin could inhibit DNA damage in \u003cem\u003eCentaurea cyanus\u003c/em\u003e under the condition of UV-B increase, thus protecting the normal growth and development of plants under UVB condition. However, the detailed metabolic compositions in Hordeum vulgare under different wavelength of UV exposures remained unclear.\u003c/p\u003e \u003cp\u003e \u003cem\u003eHordeum vulgare\u003c/em\u003e, which is widely distributed in habitat, is always exposed under UV challenges, however its detailed responses to UV exposure remain unclear. In this study, we deployed 4-D proteome and anthocyanins metabolomics to investigate the responses of \u003cem\u003eHordeum vulgare\u003c/em\u003e to different wavelength and composition of UV exposures, including UVA, UVAB and UVB. It was found that UVA, UVAB, and UVB exposures caused different responses in \u003cem\u003eH. vulgare\u003c/em\u003e at proteome levels. Typically, \u003cem\u003eH. vulgare\u003c/em\u003e reprogrammed anthocyanins biosynthesis to accumulated more related metabolites in response to UVAB and UVB exposures. And NAC1 was responsible for activating anthocyanin biosynthesis of \u003cem\u003eH. vulgare\u003c/em\u003e in response to UV exposure. Overall, our research depicted global proteome and anthocyanins landscape of \u003cem\u003eH. vulgare\u003c/em\u003e under different wavelength of UV exposures, which contributed to the cultivation and management of \u003cem\u003eH. vulgare\u003c/em\u003e from UV stress, especially in plateau.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample preparation\u003c/h2\u003e \u003cp\u003eHere, Longzi black highland barley, a variety of black highland barley rich in anthocyanins, was planted in pots with vegetative soil as the substrate. Totally, 120\u0026ndash;130 seedlings were planted in each pot at Xizang Academy of Agricultural and Animal Husbandry Sciences experimental fields. Plants were grown in a greenhouse at a temperature of 20\u0026deg;C/day and 15\u0026deg;C/night, and watered every 5 days. The highland barley was subjected to ultraviolet stress at the earing stage. Each treatment was divided into experimental group and control group. Typically, three kinds of UV stress treatments were set up and compared with P normal sunlight exposure, treatment 1 was PA (sunlight P\u0026thinsp;+\u0026thinsp;UVA 400-320nm), treatment 2 was PB (sunlight P\u0026thinsp;+\u0026thinsp;UVB 320-280nm), treatment 3 was PAB (sunshine P\u0026thinsp;+\u0026thinsp;UVA\u0026thinsp;+\u0026thinsp;UVB) mixed irradiation, treatment for 8 hours a day, treatment for 8 days, control for outdoor planting, three parallel treatments, a total of 36 samples. The information of UV-B light is 5.5 W cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, equivalent to 12 \u0026micro;mol m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (measured by a UV-297 UV-B Light Meter, HANDY) provided by Philips TL20W/01RS narrowband UV-B tubes11. And the information of UV-A light is 5.5 W m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e, PPFD\u0026thinsp;=\u0026thinsp;12 \u0026micro;mol\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, as measured by a portable digital radiometer for UV (UV-340A, Lutron, Taiwan).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProtein extraction and digestion\u003c/h3\u003e\n\u003cp\u003eTo investigate the responses of \u003cem\u003eHordeum vulgare\u003c/em\u003e to UVA, UVAB and UVB exposure, three biological replicates of \u003cem\u003eH. vulgare\u003c/em\u003e were collected from all experimental groups (UVA, UVAB and UVB) for proteome analysis. Sample analysis and protein extraction were performed using SDT (4% SDS, 100 mM Tris-HCl, 1 mM DTT, pH 7.6) buffer and quantified with the BCA Protein Assay Kit (BioRad, Hercules, CA, USA). Then, the protein was digested by trypsin and desalted on C18 cartridges (Empore\u0026trade; SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 mL, Sigma, Burlington, MA, USA), concentrated via vacuum centrifugation and reconstituted in 40 \u0026micro;L of 0.1% (v/v) formic acid. Filter-aided sample preparation (FASP digestion) procedure: 200 \u0026micro;g of protein for each sample was incorporated into 30 \u0026micro;L SDT buffer (4% SDS, 100 mM DTT, 150 mM Tris-HCl pH 8.0). The detergent, DTT and other low-molecular-weight components were removed using UA buffer (8 M urea, 150 mM Tris-HCl pH 8.0) via repeated ultrafiltration (Microcon units, 10 kD). Then, 100 \u0026micro;L iodoacetamide (100 mM IAA in UA buffer) was added to block reduced cysteine residues and the samples were incubated for 30 min in darkness. The filters were washed with 100 \u0026micro;L UA buffer three times and then 100 \u0026micro;L 25 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e buffer twice. Finally, the protein suspensions were digested with 4 \u0026micro;g trypsin (Promega) in 40 \u0026micro;L 25 mM NH\u003csub\u003e4\u003c/sub\u003eHCO\u003csub\u003e3\u003c/sub\u003e buffer overnight at 37\u0026deg;C, and the resulting peptides were collected as a filtrate. The peptides of each sample were desalted on C18 cartridges (Empore\u0026trade; SPE Cartridges C18 (standard density), bed I.D. 7 mm, volume 3 mL, Sigma, Burlington, MA, USA), concentrated via vacuum centrifugation and reconstituted in 40 \u0026micro;L of 0.1% (v/v) formic acid. The peptide content was estimated based on the UV light at 280 nm using an extinction coefficient of 1.1 of 0.1% (g/L) solution that was calculated based on the frequency of tryptophan and tyrosine in vertebrate proteins according to Cheng et al. (2020)[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e\n\u003ch3\u003eLC-MS/MS detection for proteome analysis\u003c/h3\u003e\n\u003cp\u003eFor each sample, 200 ng of total peptides were separated and analyzed with a nano\u0026shy;UPLC (Evosep one) coupled to a timsTOF Pro2 instrument (Bruker) with a nano\u0026shy;electrospray ion source. Separation was performed using a reversed\u0026shy;phase column (PePSep C18, 1.9 \u0026micro;, 150 \u0026micro;\u0026thinsp;\u0026times;\u0026thinsp;15 cm, Bruker, Germany). Mobile phases were H\u003csub\u003e2\u003c/sub\u003eO with 0.1% FA (phase A) and CAN with 0.1% FA (phase B). Separation of sample was executed with a 44 min gradient. The mass spectrometer adopts DDA PaSEF mode for DDA data acquisition, and the scanning range is from 100 to 1700 m/z for MS1. During PASEF MS/MS scanning, the impact energy increases linearly with ion mobility, from 20 eV (1/K\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.6 Vs/cm\u003csup\u003e2\u003c/sup\u003e) to 59 eV (1/K\u003csub\u003e0\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.6 Vs/cm\u003csup\u003e2\u003c/sup\u003e).\u003c/p\u003e\n\u003ch3\u003eSpectroMine database search\u003c/h3\u003e\n\u003cp\u003eVendor\u0026rsquo;s raw MS files were processed using SpectroMine software (4.2.230428.52329) and the built\u0026shy;in Pulsar search engine. MS spectra lists were searched against their species\u0026shy;level UniProt FASTA databases (uniprotkb_Hordeum vulgare_id_4513_2024_05_17), Carbamidomethyl [C] as a fixed modification, Oxidation (M) and Acetyl (Protein N\u0026shy;term) as variable modifications. Trypsin was used as proteases. A maximum of 2 missed cleavage (s) was allowed. The false discovery rate (FDR) was set to 0.01 for both PSM and peptide levels. Peptide identification was performed with an initial precursor mass deviation of up to 20 ppm and a fragment mass deviation of 20 ppm. All the other parameters were reserved as default.\u003c/p\u003e\n\u003ch3\u003eBioinformatic Analysis\u003c/h3\u003e\n\u003cp\u003eCELLO v2.5 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://cello.life.nctu.edu.tw/\u003c/span\u003e\u003cspan address=\"http://cello.life.nctu.edu.tw/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e accessed on 31 July 2022), a multi-class SVM classification system, was used to predict protein subcellular localization. The protein sequences of the selected differentially expressed proteins were locally searched using the NCBI BLAST\u0026thinsp;+\u0026thinsp;client software (ncbi-blast-2.2.28+-win32.exe) and InterProScan-5.25-64.0 to find homologue sequences, then, gene ontology (GO) terms were mapped and sequences were annotated using the software program Blast2GO 6.0. The GO annotation results were plotted by R scripts. The BLAST2GO 6.0 software was used to perform functional annotation following Ye et al. (2006) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The DEPs of each comparison were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.genome.jp/kegg/\u003c/span\u003e\u003cspan address=\"http://www.genome.jp/kegg/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e accessed on 31 July 2022) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. GO and KEGG pathway enrichment analyses were performed by clusterprofiler 2.0. Only functional categories and pathways with p-values under a threshold of 0.05 were considered significant. Proteins with P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 \u0026amp; |log\u003csub\u003e2\u003c/sub\u003e(Foldchange) |\u0026gt;1 was set as the threshold for significantly differential expression (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e). WGCNA was performed using R package WGCNA following Du et al. (2023) [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The detailed information of proteome data were listed in Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eAnthocyanin preparation and extraction\u003c/h2\u003e \u003cp\u003eEach experimental group contained three biological replicates to ensure the validity of altitude-induced metabolic changes in \u003cem\u003eH. vulgare\u003c/em\u003e. Totally, 20 mg of each \u003cem\u003eH. vulgare\u003c/em\u003e sample from CK, UVA, UVB and UVAB groups was weighted to an EP tube after grinding with liquid nitrogen, and 1000 \u0026micro;L extract solution (methanol: water\u0026thinsp;=\u0026thinsp;3: 1, with isotopically-labelled internal standard mixture) was added. The sample was freeze-dried, ground into powder (30 Hz, 1.5 min), and stored at -80\u0026deg;C until needed. 50 mg powder was weighted and extracted with 0.5 mL methanol/water/hydrochloric acid (500:500:1, V/V/V). Then the extract was vortexed for 5 min and ultrasound for 5 min and centrifuged at 12, 000 g under 4\u0026deg;C for 3 min. The residue was re-extracted by repeating the above steps again under the same conditions. The supernatants were collected, and filtrated through a membrane filter (0.22 \u0026micro;m, Anpel) before LC-MS/MS analysis. All of the standards were purchased from isoReag (Shanghai, China). Formic acid was purchased from Sigma-Aldrich (St Louis, MO, USA). Hydrochloric acid was bought from Xinyang ChemicalReagent (China). The stock solutions of standards were prepared at the concentration of 1 mg/mL in 50% MeOH. All stock solutions were stored at -20\u0026deg;C. The stock solutions were diluted with 50% MeOH to working solutions before analysis.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLC–MS/MS Analysis for anthocyanin determination\u003c/h3\u003e\n\u003cp\u003eHPLC-grade methanol (MeOH) was purchased from Merck (Darmstadt, Germany). MilliQ water (Millipore, Bradford, USA) was used in all experiments. The sample extracts were analyzed using an UPLC-ESI-MS/MS system (UPLC, ExionLC\u0026trade; AD, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sciex.com.cn/\u003c/span\u003e\u003cspan address=\"https://sciex.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e, MS, Applied Biosystems 6500 Triple Quadrupole, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://sciex.com.cn/\u003c/span\u003e\u003cspan address=\"https://sciex.com.cn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The analytical conditions were as follows, UPLC: column, WatersACQUITY BEH C18 (1.7 \u0026micro;m, 2.1 mm*100 mm), solvent system, water (0.1% formic acid): methanol\u0026ensp;(0.1% formic acid), gradient program, 95:5 V/V at 0 min, 50:50 V/V at 6 min, 5:95 V/V at 12 min, hold for 2 min, 95:5 V/V at 14 min, hold for 2 min, flow rate, 0.35 mL/min, temperature, 40\u0026deg;C, injection volume, 2 \u0026micro;L.\u003c/p\u003e \u003cp\u003eLinear ion trap (LIT) and triple quadrupole (QQQ) scans were acquired on a triple quadrupole-linear ion trap mass spectrometer (QTRAP), QTRAP\u0026reg; 6500\u0026thinsp;+\u0026thinsp;LC-MS/MS System, equipped with an ESI Turbo Ion-Spray interface, operating in positive ion mode and controlled by Analyst 1.6.3 software (Sciex). The ESI source operation parameters were as follows: ion source, ESI+, source temperature 550\u0026deg;C, ion spray voltage (IS) 5500 V, curtain gas (CUR) was set at 35 psi, respectively. Anthocyanins were analyzed using scheduled multiple reaction monitoring (MRM). Data acquisitions were performed using Analyst 1.6.3 software (Sciex). Multiquant 3.0.3 software (Sciex) was used to quantify all metabolites. Mass spectrometer parameters including the declustering potentials (DP) and collision energies (CE) for individual MRM transitions were done with further DP and CE optimization. A specific set of MRM transitions were monitored for each period according to the metabolites eluted within this period.\u003c/p\u003e\n\u003ch3\u003eData Preprocessing and Annotation\u003c/h3\u003e\n\u003cp\u003eThe raw data of all samples were converted to the mzXML format using ProteoWizard and processed with an in-house program, which was developed using R and based on XCMS, for peak detection, extraction, alignment, and integration. Then an in-house MS2 database (BiotreeDB) was applied in metabolite annotation. The cutoff for annotation was set at 0.3. The data variation was performed by R packages XCMS software [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The Principal Component Analysis (PCA) was performed using Metaboanalyst 3.0 based on the normalized peak area of the metabolites in each sample. Significantly regulated metabolites between groups were determined by absolute Log1.5FC (fold change). Therapy FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and fold change\u0026thinsp;\u0026gt;\u0026thinsp;1.5 were used to identify significantly differential metabolites. The HCA (hierarchical cluster analysis) results of samples and metabolites were presented as heatmaps with dendrograms. HCA was carried out by R package pheatmap. For HCA, normalized signal intensities of metabolites (unit variance scaling) are visualized as a color spectrum. The detailed information of anthocyanins were listed in Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eLUC assay\u003c/h2\u003e \u003cp\u003eThe LUC assay was performed according to Du et al. (2024a). The promoter sequence of \u003cem\u003eCAD\u003c/em\u003e and \u003cem\u003eCHS\u003c/em\u003e were cloned into the reporter effector pGreenII LUC0800 (pLUC-Pro) and the coding sequence of \u003cem\u003eNAC1\u003c/em\u003e was fused into pGreenII 62SK (pSK-NAC1). The \u003cem\u003eAgrobacterium\u003c/em\u003e strains (GV3101) were incubated overnight and resuspended in infiltration buffer (20 mM MES, 0.1 mM acetosyringone, and 10 mM MgCl\u003csub\u003e2\u003c/sub\u003e) and final diluted to the concentration of OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.6\u0026ndash;0.8. Suspensions of pLUC-Pro and pSK-NAC1 were mixed in equal volumes and infiltrated into \u003cem\u003eNicotiana benthamiana\u003c/em\u003e leaves. After 48 h, the transformed leaves were sprayed with 0.1 M luciferin solution and kept in the dark for 7 min. A low light-cooled CCD imaging apparatus (Lumazone Pylon 2048 B, Princeton Instruments) was used to capture and analyze the acquired LUC images.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTransient overexpression assay\u003c/h2\u003e \u003cp\u003eThe open reading frames (ORF) of \u003cem\u003eNAC1\u003c/em\u003e were cloned using a primer pair containing EcoRI and BamHI sites and ligated to pBIN-eGFP. Then, the positive vector was confirmed using sequencing, and transduced into \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e LBA4404. GFP controls, pBIN-NAC1 was transferred into \u003cem\u003eNicotiana benthamiana\u003c/em\u003e using \u003cem\u003eA. tumefaciens\u003c/em\u003e-mediated method. Four days after infiltration, the representative fruit was collected for further experiments.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eUVA, UVAB and UVB altered the proteome patterns of\u003c/strong\u003e \u003cstrong\u003eH. vulgare\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eTo investigate the responses of \u003cem\u003eH. vulgare\u003c/em\u003e to UVB, UVA and UVA\u0026thinsp;+\u0026thinsp;UVB (UVAB) stresses, three biological replicates for \u003cem\u003eH. vulgare\u003c/em\u003e from CK, UVB, UVA and UVAB treatments were collected for proteome sequencing. Totally, 3949 proteins were identified in \u003cem\u003eH. vulgare\u003c/em\u003e. Then, we performed principal component analysis (PCA) on all proteome profiles to investigate the proteomic pattern of \u003cem\u003eH. vulgare\u003c/em\u003e following UVB, UVA and UVAB exposures (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). PCA plot was constructed using principal component 1 (PC1) and PC2, which explained 47.8% and 8.1% variations between all samples (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). The PCA plot showed that the samples from CK clearly distributed at a single region and separated with UVB, UVA and UVAB (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), suggesting that all UVB, UVA and UVAB treatments dramatically altered the proteome pattern of \u003cem\u003eH. vulgare\u003c/em\u003e. Typically, the differences between experimental groups were mainly represented by PC1, while PC2 mainly explained the differences among biological replicates from the same treatment (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA). Additionally, UVB samples also clearly separated with UVA, UVAB and CK, and the differences between UVB and CK were more significant than that between UVAB/UVA vs. CK (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA), suggesting that \u003cem\u003eH. vulgare\u003c/em\u003e are more responsive to UVB. Meanwhile, \u003cem\u003eH. vulgare\u003c/em\u003e exhibited similar responses to UVA and UVB, and the effect of UVA on plant protein expression was minimal that UVA samples were closet to CK (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e\n\u003cp\u003eThen, to investigate the detailed changes in proteome of \u003cem\u003eH. vulgare\u003c/em\u003e following UVA, UVB and UVAB exposures, we analyzed the differentially expressed proteins (DEPs) of all 3949 identified proteins in UVA vs. CK, UVB vs. CK and UVAB vs. CK comparisons based on P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and Foldchange\u0026thinsp;\u0026gt;\u0026thinsp;1.5 (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Totally, 73 DEPs were identified in UVA vs. CK, with 49 downregulated DEPs and 24 upregulated DEPs (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Additionally, we identified 272 DEPs (228 down- and 44 up-regulated DEPs) in UVAB vs. CK and 678 DEPs (477 down- and 201 up-regulated DEPs) in UVB vs. CK comparison (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). Typically, most DEPs in \u003cem\u003eH. vulgare\u003c/em\u003e associated with UVA, UVB and UVAB exposures were mainly located in nucleus, cytoplasm, secreted and chloroplast (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). It has been well documented that chloroplast involved in responses of plants to UV by regulating reactive oxygen homeostasis caused by UV stress [\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. Moreover, COG/KOG enrichment showed that most of proteins among the DEPs from these 3 pairwise comparisons mainly involved in RNA processing and modification, Energy production and conversion, Translation, ribosomal structure and biogenesis, Posttranslational modification, protein turnover, chaperones, General function prediction only and Intracellular trafficking, secretion, and vesicular transport (Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Thus, these results suggested UVA, UVAB and especially UVB could alter the proteome pattern of \u003cem\u003eH. vulgare\u003c/em\u003e, especially leading changes in levels of proteins located in nucleus, cytoplasm, secreted and chloroplast.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDifferent wavelengths of UV cause changes in expression of different functional proteins in\u003c/strong\u003e \u003cstrong\u003eH. vulgare\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003ePlants make different responses to different wavelength of light [\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e]. Here, we investigated the function of DEPs in \u003cem\u003eH. vulgare\u003c/em\u003e following UVA, UVB and UVAB exposures using gene ontology (GO) enrichment analysis. For UVA vs. CK comparison, 90 GO terms were significantly enriched against all DEPs (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). Typically, these DEPs mainly functioned in protein storage vacuole membrane, vacuolar proton-transporting V-type ATPase, plant-type vacuole membrane and post-spliceosomal complex, with thiol-dependent ubiquitin-specific protease activity, ubiquitinyl hydrolase activity, receptor binding, cysteine-type peptidase activity, hydrolase activity, signaling receptor activity, cytokine activity, vascular endothelial growth factor receptor binding, molecular transducer activity, ATPase activity, monocarboxylic acid binding, inositol monophosphate 3-phosphatase activity, ion transmembrane transporter activity and peptidase activity (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). These DEPs associated with UVA mainly involved in regulation of protein complex disassembly, DNA replication checkpoint, DNA damage response, single organism reproductive process, negative regulation of G2/M transition of mitotic cell cycle, DNA integrity checkpoint, glyoxal metabolic process and cell communication (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA, Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e). These results showed that UVA mainly affected proteins involved in DNA replication and cell cycle, which then may cause damage to cell growth in plants.\u003c/p\u003e\n\u003cp\u003eFor UVAB vs. CK comparison, DEPs mainly functioned in ESCRT complex, organelle membrane, endosome, ESCRT I complex and cis-Golgi network in plant cell (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). For molecular function, 21 terms were significantly enriched, including protein disulfide oxidoreductase activity, peptidase inhibitor activity, glutathione transferase activity, peroxiredoxin activity, oxidoreductase activity, lipid binding, transferase activity and thioredoxin peroxidase activity (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). And these DEPs associated with UVAB mainly involved in 46 biological progresses, especially lipid transport, response to stimulus, endosomal transport, vacuolar transport, positive regulation of macromolecule metabolic process, cell surface receptor signaling pathway, glutathione metabolic process, cell redox homeostasis, response to abscisic acid, glutamate catabolic process and seed development (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). Of note, numerous processes relevant to oxidant responses were identified in UVAB vs. CK comparison, including glutathione metabolic process, cell redox homeostasis, response to abscisic acid, glutamate catabolic process, while it also affected proteins relevant to seed development (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB, Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). It has been found that UV exposure always accompanied by oxidant stress and disordered oxygen-radical homeostasis, leading to damage to plant cell and growth [\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e]. Thus, these results suggested that UVAB may cause oxidant stress in plant cell to affect seed development.\u003c/p\u003e\n\u003cp\u003eSubsequently, we analyzed the function of DEPs from UVB vs. CK comparison. The GO results showed that these DEPs mainly distributed in respiratory chain complex, mitochondrial membrane, oxidoreductase complex, mitochondrion, plasma membrane, NADH dehydrogenase complex, chromatin, organellar ribosome, nucleosome and protein-DNA complex (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC, Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e). Typically, these DEPs with functions of voltage-gated channel activity, gated channel activity, transcription corepressor activity, DNA binding, kinase regulator activity, phospholipid transporter activity, sphingolipid binding, ceramide binding and ATPase activity, mainly involved in protein-DNA complex assembly, nucleosome assembly, chromatin assembly, DNA packaging and anthocyanin-containing compound biosynthetic process (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC, Table \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003e). Anthocyanin biosynthesis was the well-documented processes in plants in response to UV stress, anthocyanin could protect plants from oxidant damage caused by UV exposure [\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e]. Thus, these results suggested that plant could trigger the anthocyanin biosynthesis in response to UVB exposure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eChanges in anthocyanin biosynthesis were the main responses in\u003c/strong\u003e \u003cstrong\u003eH. vulgare\u003c/strong\u003e \u003cstrong\u003eto UVAB and UVB exposures.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHere, we further performed KEGG pathway enrichment analysis on DEPs from three pairwise comparisons (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). For DEPs from UVA vs. CK, KEGG results showed that these DEPs mainly involved in Phagosome, Vitamin B6 metabolism, Diterpenoid biosynthesis, Oxidative phosphorylation, Phosphatidylinositol signaling system, Phenylalanine metabolism, Plant hormone signal transduction, Tyrosine metabolism, Protein export and Metabolic pathways (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). In parallel, DEPs from UVAB vs. CK mainly functioned in Glutathione metabolism, Phagosome, Protein processing in endoplasmic reticulum, Valine, leucine and isoleucine biosynthesis, Porphyrin metabolism, Phenylpropanoid biosynthesis, D-Amino acid metabolism, Monoterpenoid biosynthesis, Phenylalanine metabolism, Butanoate metabolism, Thiamine metabolism, Plant hormone signal transduction and 2-Oxocarboxylic acid metabolism (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). In addition, KEGG pathway enrichment analysis on DEPs from UVB vs. CK showed that 8 biosynthesis pathways were significantly enriched, including Oxidative phosphorylation, Plant hormone signal transduction, Anthocyanin biosynthesis, Basal transcription factors, Diterpenoid biosynthesis, Phenylpropanoid biosynthesis, Flavonoid biosynthesis and MAPK signaling pathway (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Typically, we noted the significant enrichment of anthocyanin and flavonoid biosynthesis in UVB vs. CK (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB), reaching a consensus with GO results that UVB could trigger the proteome pattern of flavonoid and anthocyanin biosynthesis in \u003cem\u003eH. vulgare\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA). Additionally, UVA vs. CK, UVAB vs. CK and UVB vs. CK shared 25 pathways, such as Ribosome, Phagosome, Plant hormone signal transduction, Starch and sucrose metabolism, Biosynthesis of amino acids, Glycolysis / Gluconeogenesis, Protein export, Proteasome, Diterpenoid biosynthesis, Tyrosine metabolism, MAPK signaling pathway and Oxidative phosphorylation (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). Of note, there are 45 pathways were specifically enriched under UVB exposure, especially DNA replication, Glycerophospholipid metabolism, Basal transcription factors, Photosynthesis, Fatty acid degradation, alpha-Linolenic acid metabolism, Aminoacyl-tRNA biosynthesis, Anthocyanin biosynthesis, Fatty acid metabolism, Flavonoid biosynthesis, ABC transporters and Sphingolipid metabolism (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e\n\u003cp\u003eConsidering the effects of importance of anthocyanin synthesis and antioxidant system in plant in response to UV exposure, we further analyzed the expression of related proteins in \u003cem\u003eH. vulgare\u003c/em\u003e following UVB, UVAB and UVA exposures. The expression heat map of proteins relevant to antioxidant system showed that the expression levels of most related proteins were higher in \u003cem\u003eH. vulgare\u003c/em\u003e under UVAB and especially UVB exposures, compared to UVA and CK (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). Meanwhile, the proteins involved in anthocyanin synthesis were also triggered to highly expressed in \u003cem\u003eH. vulgare\u003c/em\u003e by UVAB and especially UVB exposures, but not UVA (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). Taken together, we proposed that different wavelength of UV could trigger different responses in plants, typically anthocyanin biosynthesis and antioxidant reaction was the main responses in \u003cem\u003eH. vulgare\u003c/em\u003e to UVB exposure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUVAB and UVB triggered accumulation of anthocyanins in\u003c/strong\u003e \u003cstrong\u003eH. vulgare\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eFurther, we compared the anthocyanin-related metabolic profiles of \u003cem\u003eH. vulgare\u003c/em\u003e following UVA, UVAB and UVB exposures. To identify the overview of changes in anthocyanin composition of \u003cem\u003eH. vulgare\u003c/em\u003e following UVA, UVAB and UVB exposures, we performed PCA analysis on all metabolic profiles of \u003cem\u003eH. vulgare\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). The PCA plots constructed by PC 1 and 2 showed significant variations in anthocyanin composition among all groups, while PC1 and PC2 explained 33.2% and 15.4% variations associated with experimental treatments (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA). Typically, PC1 mainly explained the variations in anthocyanins compositions of \u003cem\u003eH. vulgare\u003c/em\u003e caused by UVAB and UVB exposures (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA), supporting the involvement of anthocyanins synthesis of \u003cem\u003eH. vulgare\u003c/em\u003e in response to UVAB and UVB exposures. Totally, 28 anthocyanins were identified in \u003cem\u003eH. vulgare\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB, Figure \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e). Typically, the contents of most of anthocyanins were higher in \u003cem\u003eH. vulgare\u003c/em\u003e under UVB exposure in comparison with UVA and CK, including Procyanidin B2, Cyanidin-3-O-sambubioside, Peonidin-3-O-glucoside, Cyanidin-3-O-glucoside, Pelargonidin-3-O-glucoside, Procyanidin B1, Peonidin-3-O-sophoroside, Petunidin-3-O-glucoside, Cyanidin-3-(malonyl)glucoside-5-rhamnoside, Delphinidin-3-O-glucoside, Cyanidin-3-O-sophoroside, Cyanidin-3-O-(6-O-malonyl-beta-D-glucoside), Cyanidin-3,5-O-diglucoside, Cyanidin-3-O-xyloside and Peonidin (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Of note, the contents of all anthocyanins in \u003cem\u003eH. vulgare\u003c/em\u003e under UVAB exposure was higher than that in CK and UVA, while UVA did not induce increases of anthocyanins in \u003cem\u003eH. vulgare\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB). Thus, we concluded that anthocyanins accumulation was the main response in \u003cem\u003eH. vulgare\u003c/em\u003e to UVAB and UVB exposures.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eActive regulation network involved in regulating anthocyanin synthesis in\u003c/strong\u003e \u003cstrong\u003eH. vulgare\u003c/strong\u003e \u003cstrong\u003ein response to UVB and UVAB exposures.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo identify the hub proteins involved in anthocyanin synthesis in \u003cem\u003eH. vulgare\u003c/em\u003e in response to UVB and UVAB exposures, we analyzed the correlation between levels of proteins and anthocyanins using Pearson algorithm. The results showed that proteins represented by Q40069, F2D3Q9, F2E8A9, M0WRX0, F2E7V8, F2CXT3 and F2E8K5 were highly correlated with the anthocyanin level in \u003cem\u003eH. vulgare\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA), implying their involvements in synthesizing anthocyanin in \u003cem\u003eH. vulgare\u003c/em\u003e. Considering the involvement of anthocyanin synthesis \u003cem\u003eH. vulgare\u003c/em\u003e in response to UVB and UVAB exposures, we further constructed the co-expression network of proteins (Q40069, F2D3Q9, F2E8A9, M0WRX0, F2E7V8, F2CXT3 and F2E8K5) relevant to anthocyanin synthesis using WGCNA based on all proteome profiles of \u003cem\u003eH. vulgare\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB-D). The expression data of all transcription factors and kinases of \u003cem\u003eH. vulgare\u003c/em\u003e were selected via Nr annotation for WGCNA construction. We set the soft threshold to 26 (\u003cem\u003eR\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.85) to construct a scale-free network (Figure \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003eA). Then, 9 modules were identified by hierarchical clustering and the dynamic branch cutting, each module was assigned a unique color as an identifier (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB, C, Figure \u003cspan class=\"InternalRef\"\u003eS3\u003c/span\u003eB). Then, the modules highly correlated with the related traits were filtered out for further construction of regulation network of anthocyanin synthesis in \u003cem\u003eH. vulgare\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). Based on the WGCNA results, we identified that MEbrown, MEyellow and MEturquoise were highly related to proteins relevant to anthocyanin synthesis (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). Among proteins from these modules, we constructed the co-expression regulation networks for anthocyanin synthesis in \u003cem\u003eH. vulgare\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). Totally, we identified that 4 transcription factors and 34 kinases were responsible for positively regulating anthocyanin synthesis in \u003cem\u003eH. vulgare\u003c/em\u003e in response to UVB and UVAB exposures (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). As shown in Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD, A0A8I6WJ93, A0A8I6WLA6, A0A8I7B6M6 and especially F6IAY3 (NAC transcription factor) were the hub transcription factors involved in the regulation network of anthocyanin synthesis in \u003cem\u003eH. vulgare\u003c/em\u003e, and it was positively correlated with the expression of anthocyanin-related proteins (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). Of note, most of these regulators were upregulated to higher expression levels in \u003cem\u003eH. vulgare\u003c/em\u003e by UVAB and UVB exposures, supporting their involvements in regulating responses in \u003cem\u003eH. vulgare\u003c/em\u003e to UVAB and UVB exposures (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). Further RT-qPCR assay confirmed that transcription factors including A0A8I6WJ93, A0A8I6WLA6, A0A8I7B6M6 and especially F6IAY3 were activated to upregulate in \u003cem\u003eH. vulgare\u003c/em\u003e by UVAB and UVB exposures, but not UVA (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE). Of note, LUC assay confirmed that NAC1 could directly bind on the promoters of \u003cem\u003eCAD1\u003c/em\u003e and \u003cem\u003eCHS\u003c/em\u003e to trigger their expression (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eF). Further transient overexpression assay showed that \u003cem\u003eNAC1\u003c/em\u003e overexpression activated expression of \u003cem\u003eCHS\u003c/em\u003e, \u003cem\u003eCAD\u003c/em\u003e and \u003cem\u003eANS\u003c/em\u003e in plants, supporting its function in activating anthocyanin biosynthesis in \u003cem\u003eH. vulgare\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG). Taken together, these results suggested that the UVAB/UVB-reprogrammed anthocyanin synthesis in \u003cem\u003eH. vulgare\u003c/em\u003e was regulated by a complex network.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the plateau region, especially Qinghai-Tibet Plateau, UV stress has become a nonnegligible obstacle to the development of crops. Thus, it is of great practical significance to investigate the detailed plant responses to UV exposures for guiding the agriculture development in these regions. Presently, we found that UVA, UVAB and UVB reprogrammed the proteome pattern of \u003cem\u003eH. vulgare\u003c/em\u003e. In detail, UVA mainly affected the expression of proteins involved in cell cycle and DNA repair, while UVB and UVAB mainly altered proteins involved in anthocyanin biosynthesis and antioxidant systems. Typically, all identified proteins involved in anthocyanin biosynthesis were activated to upregulate in \u003cem\u003eH. vulgare\u003c/em\u003e by UVB and UVAB exposures. Further anthocyanin profiles showed that UVB and UVAB caused accumulation of anthocyanin in \u003cem\u003eH. vulgare\u003c/em\u003e, which may protect \u003cem\u003eH. vulgare\u003c/em\u003e from UV-associated oxidant damage to plant cell. Then, we constructed the network for regulating UV-triggered anthocyanin biosynthesis in \u003cem\u003eH. vulgare\u003c/em\u003e using proteome profiles. Importantly, we identified NAC transcription together with numerous kinases were responsible for regulating UVAB/UVB-triggered anthocyanin biosynthesis in \u003cem\u003eH. vulgare\u003c/em\u003e. Of note, NAC1 directly bound on the promoters of \u003cem\u003eCAD1\u003c/em\u003e and \u003cem\u003eCHS\u003c/em\u003e to trigger their expression, then activated anthocyanin biosynthesis in \u003cem\u003eH. vulgare\u003c/em\u003e. Overall, our research depicted the detailed global responses of \u003cem\u003eH. vulgare\u003c/em\u003e to UV challenge, which contributed to our understanding of adaptive mechanisms of plants in plateau.\u003c/p\u003e \u003cp\u003eWith the destruction of the ozone layer, the research on ultraviolet radiation has attracted increasing attention. Ultraviolet-B radiation is a light wave with a wavelength between 280 and 320 nm, and its high energy can directly lead to DNA strand breakage and blocked cross-linked replication between DNA strand and DNA strand or protein [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Meanwhile, UV exposure also caused the disruption and structural changes of polypeptide chains in protein molecules in plants and changes in multiple metabolic activities of organisms [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Similarly, we found that the UVA could affected the expression of proteins involved in cell cycle and DNA replications. In addition, UV-B can also trigger the accumulation of reactive oxygen species, resulting in lipid peroxidation of biofilm [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], then change the fluidity and permeability of the membrane and affecting a variety of physiological activities of organisms [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. UV-B radiation can directly or indirectly affect the synthesis, accumulation and metabolism of primary metabolism and secondary metabolites in plants [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Of note, combining with metabolomics, genomics and proteomics provides theoretical basis and technical support for the application of UV-B radiation in agricultural production and agricultural product processing. Since UV-B irradiation can induce oxidative stress in plants, the biosynthesis pathway of antioxidant active substances such as polyphenols and flavonoids will be activated, so as to rapidly synthesize a large number of antioxidant active substances to effectively repair the body damage caused by oxidative stress [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Polyphenols are a class of compounds formed by the substitution of polyhydroxyl groups on the basic skeleton of phenol. They are important bioactive substances in human diet and secondary metabolites closely related to plant defense mechanisms [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. A large number of studies have shown that that UVB treatment can significantly increase flavonoid and anthocyanin content in plant tissues [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Here, we also found that UV-B is an important factor in inducing the biosynthesis of plant anthocyanins, while UVA\u0026thinsp;+\u0026thinsp;UVB also could promote the anthocyanin accumulation in \u003cem\u003eH. vulgare\u003c/em\u003e. Anthocyanins and flavonoid metabolites have efficient scavenging function of reactive oxygen species [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Typically, both low and high doses of UV-B radiation lead to an increase in ROS in the plant [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Therefore, the accumulation of anthocyanin metabolites may be an important way for plants to resist strong UV stress at high altitude.\u003c/p\u003e \u003cp\u003eBecause anthocyanins and flavonoids metabolites not only help plants resist abiotic and biological stresses, they are also important nutrients in crops [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Therefore, the rational use of ultraviolet rays can be used as an important means of metabolic regulation in agricultural production, so as to improve crop yield and carry out targeted regulation and accumulation of target products. It was found that phenylpropanoid-derived flavonoid and anthocyanin biosynthesis was mainly regulated by MYB transcription factors to perform related function in plant [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Of note, we identified NAC transcription factor as regulator for mediating UV-associated anthocyanin biosynthesis via constructing regulation network of anthocyanin biosynthesis in \u003cem\u003eH. vulgare\u003c/em\u003e. And the detailed regulation mechanism of NAC in mediating UV-associated anthocyanin biosynthesis in \u003cem\u003eH. vulgare\u003c/em\u003e will be the focus in our future work. Overall, the study on the effects of UV-B on the main nutrients and bioactive substances in plants is of great significance for guiding agricultural production, especially for the directed regulation of specific nutrients and bioactive substances in agricultural products.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn the present study, we deciphered the global responses of \u003cem\u003eH. vulgare\u003c/em\u003e to UV challenge via integrating metabolome and proteome. we found that UVA, UVAB and UVB reprogrammed the proteome pattern of \u003cem\u003eH. vulgare\u003c/em\u003e. UVA mainly affected the expression of proteins involved in cell cycle and DNA repair. In addition, UVB and UVAB mainly activated proteins involved in anthocyanin biosynthesis and antioxidant systems, leading to accumulation of anthocyanin in \u003cem\u003eH. vulgare\u003c/em\u003e to protect \u003cem\u003eH. vulgare\u003c/em\u003e from UV-associated oxidant damage. Of note, we identified that NAC transcription together with numerous kinases were responsible for regulating UVAB/UVB-triggered anthocyanin biosynthesis in \u003cem\u003eH. vulgare.\u003c/em\u003e Thus, the present study gain insight into the adaptive mechanisms of plants in plateau.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e: Z.H.: writing-original draft, writing-review and editing. T.Z.: data curation, visualization, methodology. X.Y. and C.DJ: methodology, software. Y.Z.: formal analysis, validation, conceptualization. X.W. and C.D: supervision, funding acquisition. Y.L. and T.Z.: resources, supervision, funding acquisition. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work was supported by Natural Science Funds of Xizang (XZ202101ZR0073G).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe collection of plant materials used in our study complied with permission\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eof related institutions, and complied with national or international guidelines\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eand legislation. The experiments did not involve endangered or protected\u0026nbsp;\u003c/p\u003e\n\u003cp\u003especies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement\u003c/strong\u003e: Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement\u003c/strong\u003e: Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e: The data reported in this paper have been deposited in the OMIX, China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (https://ngdc.cncb.ac.cn/omix: accession no.OMIX009895).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e: The authors declare no conflicts of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHuang R, Gao HY, Liu J, Li XP. 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Rocz Panstw Zakl Hig. 2014;65:79\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"Hordeum vulgare, Multi-omics, UV exposure, Regulation network, Transcription factor, Anthocyanin biosynthesis","lastPublishedDoi":"10.21203/rs.3.rs-5763885/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5763885/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eUV seriously affected \u003cem\u003eHordeum vulgare\u003c/em\u003e growth, which limited the its development and production in the plateau region. However, the detailed response mechanisms of \u003cem\u003eHordeum vulgare\u003c/em\u003e to UV exposure remain unclear.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe found that UVA, UVAB and UVB reprogrammed the proteome pattern of \u003cem\u003eH. vulgare\u003c/em\u003e. UVA mainly affected the expression of proteins involved in cell cycle and DNA repair, while UVB and UVAB mainly activated proteins involved in anthocyanin biosynthesis and antioxidant systems, leading to accumulation of anthocyanin in \u003cem\u003eH. vulgare\u003c/em\u003e. Then, we constructed the network for regulating UV-triggered anthocyanin biosynthesis in \u003cem\u003eH. vulgare\u003c/em\u003e using proteome profiles. Importantly, we identified that NAC transcription together with numerous kinases were responsible for regulating UVAB/UVB-triggered anthocyanin biosynthesis in \u003cem\u003eH. vulgare\u003c/em\u003e. Of note, NAC1 directly bound on the promoters of \u003cem\u003eCAD1\u003c/em\u003e and \u003cem\u003eCHS\u003c/em\u003e to trigger their expression, then activated anthocyanin biosynthesis in \u003cem\u003eH. vulgare\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eOur research gain insight into the global responses of \u003cem\u003eH. vulgare\u003c/em\u003e to UV exposures, then provided guidance for protecting plant from UV stress in plateau.\u003c/p\u003e","manuscriptTitle":"Muti-omics revealed the global response of Hordeum vulgare to different wavelength of UV exposure","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-23 20:53:51","doi":"10.21203/rs.3.rs-5763885/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":"109aab16-6c09-4f00-9a0a-28c3de1956ca","owner":[],"postedDate":"April 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-03T08:39:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-23 20:53:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5763885","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5763885","identity":"rs-5763885","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}