Genome-Wide Association Study to Identify Marker-Trait Associations for Seed Color in Colored Wheat (Triticum aestivum L.).

This study conducted phenotypic evaluations on a wheat F3 population derived from 155 F2 plants. Traits related to seed color, including chlorophyll a, chlorophyll b, carotenoid, anthocyanin, L*, a*, and b*, were assessed, revealing highly significant correlations among various traits. Genotyping using 81,587 SNP markers resulted in 3969 high-quality markers, revealing a genome-wide distribution with varying densities across chromosomes. A genome-wide association study using fixed and random model circulating probability unification (FarmCPU) and Bayesian-information and linkage-disequilibrium iteratively nested keyway (BLINK) identified 11 significant marker-trait associations (MTAs) associated with L*, a*, and b*, and chromosomal distribution patterns revealed predominant locations on chromosomes 2A, 2B, and 4B. A comprehensive annotation uncovered 69 genes within the genomic vicinity of each MTA, providing potential functional insights. Gene expression analysis during seed development identified greater than 2-fold increases or decreases in expression in colored wheat for 16 of 69 genes. Among these, eight genes, including transcription factors and genes related to flavonoid and ubiquitination pathways, exhibited distinct expression patterns during seed development, providing further approaches for exploring seed coloration. This comprehensive exploration expands our understanding of the genetic basis of seed color and paves the way for informed discussions on the molecular intricacies contributing to this phenotypic trait.

Molecular characterization of wheat floret development-related F-box protein (TaF-box2): Possible involvement in regulation of Arabidopsis flowering.

In wheat (Triticum aestivum L.), the floret development stage is an important step in determining grain yield per spike; however, the molecular mechanisms underlying floret development remain unclear. In this study, we elucidated the role of TaF-box2, a member of the F-box-containing E3 ubiquitin protein ligases, which is involved in floret development and anthesis of wheat. TaF-box2 was transiently expressed in the plasma membrane and cytoplasm of both tobacco and wheat. We also found that the SCFF-box2 (Skp1-Cul1-Rbx1-TaF-box2) ubiquitin ligase complex mediated self-ubiquitination activity. Transgenic Arabidopsis plants that constitutively overexpressed TaF-box2 showed markedly greater hypocotyl and root length than wild-type plants, and produced early flowering phenotypes. Flowering-related genes were significantly upregulated in TaF-box2-overexpressing Arabidopsis plants. Further protein interaction analyses such as yeast two-hybrid, in vitro pull-down, and bimolecular fluorescence complementation assays confirmed that TaF-box2 physically interacted with TaCYCL1 (Triticum aestivum cyclin-L1-1). Ubiquitination and degradation assays demonstrated that TaCYCL1 was ubiquitinated by SCFF-box2 and degraded through the 26S proteasome complex. The physiological functions of the TaF-box2 protein remain unclear; however, we discuss several potential routes of involvement in various physiological mechanisms which counteract flowering in transgenic Arabidopsis plants.

Genome Wide Analysis of U-Box E3 Ubiquitin Ligases in Wheat (Triticum aestivum L.).

U-box E3 ligase genes play specific roles in protein degradation by post-translational modification in plant signaling pathways, developmental stages, and stress responses; however, little is known about U-box E3 genes in wheat. We identified 213 U-box E3 genes in wheat based on U-box and other functional domains in their genome sequences. The U-box E3 genes were distributed among 21 chromosomes and most showed high sequence homology with homoeologous U-box E3 genes. Synteny analysis of wheat U-box E3 genes was conducted with other plant species such as Brachypodium distachyon, barley, rice, Triricum uratu, and Aegilops tauschii. A total of 209 RNA-seq samples representing 22 tissue types, from grain, root, leaf, and spike samples across multiple time points, were analyzed for clustering of U-box E3 gene expression during developmental stages, and the genes responded differently in various tissues and developmental stages. In addition, expression analysis of U-box E3 genes under abiotic stress, including drought, heat, and both heat and drought, and cold conditions, was conducted to provide information on U-box E3 gene expression under specific stress conditions. This analysis of U-box E3 genes could provide valuable information to elucidate biological functions for a better understanding of U-box E3 genes in wheat.

Isolation and characterization of kelch repeat-containing F-box proteins from colored wheat.

F-box proteins play important roles in the regulation of various developmental processes in plants. Approximately 1796 F-box genes have been identified in the wheat genome, but details of their functions remain unknown. Moreover, not much was known about the roles of kelch repeat domain-containing F-box genes (TaKFBs) in wheat. In the present study, we isolated five TaKFBs to investigate the roles of KFBs at different stages of colored wheat grain development. The cDNAs encoding TaKFB1, TaKFB2, TaKFB3, TaKFB4, and TaKFB5 contained 363, 449, 353, 382, and 456 bp open reading frames, respectively. All deduced TaKFBs contained an F-box domain (IPR001810) and a kelch repeat type 1 domain (IPR006652), except TaKFB2. Expression of TaKFBs was elevated during the pigmentation stages of grain development. To clarify how TaKFB and SKP interact in wheat, we investigated whether five TaKFB proteins showed specificity for six SKP proteins using a yeast two-hybrid (Y2H) assay. An Y2H screen was performed to search for proteins capable of binding the TaKFBs and interaction was identified between TaKFB1 and aquaporin PIP1. To examine the subcellular localization of TaKFBs, we transiently expressed TaKFB-green fluorescent protein (GFP) fusions in tobacco leaves; the TaKFB-GFP fusions were detected in the nucleus and the cytoplasm. Y2H and bimolecular fluorescence complementation (BiFC) assays revealed that TaKFB1 specifically interacts with aquaporin PIP1. These results will provide useful information for further functional studies on wheat F-box proteins and their possible roles.

Overexpression of rice jacalin-related mannose-binding lectin (OsJAC1) enhances resistance to ionizing radiation in Arabidopsis.

BACKGROUND: Jacalin-related lectins in plants are important in defense signaling and regulate growth, development, and response to abiotic stress. We characterized the function of a rice mannose-binding jacalin-related lectin (OsJAC1) in the response to DNA damage from gamma radiation. RESULTS: Time- and dose-dependent changes of OsJAC1 expression in rice were detected in response to gamma radiation. To identify OsJAC1 function, OsJAC1-overexpressing transgenic Arabidopsis plants were generated. Interestingly, OsJAC1 overexpression conferred hyper-resistance to gamma radiation in these plants. Using comparative transcriptome analysis, genes related to pathogen defense were identified among 22 differentially expressed genes in OsJAC1-overexpressing Arabidopsis lines following gamma irradiation. Furthermore, expression profiles of genes associated with the plant response to DNA damage were determined in these transgenic lines, revealing expression changes of important DNA damage checkpoint and perception regulatory components, namely MCMs, RPA, ATM, and MRE11. CONCLUSIONS: OsJAC1 overexpression may confer hyper-resistance to gamma radiation via activation of DNA damage perception and DNA damage checkpoints in Arabidopsis, implicating OsJAC1 as a key player in DNA damage response in plants. This study is the first report of a role for mannose-binding jacalin-related lectin in DNA damage.

Genome-wide transcript analysis of inflorescence development in wheat.

The process of inflorescence development is directly related to yield components that determine the final grain yield in most cereal crops. Here, microarray analysis was conducted for four different developmental stages of inflorescence to identify genes expressed specifically during inflorescence development. To select inflorescence-specific expressed genes, we conducted meta-analysis using 1245 Affymetrix GeneChip array sets obtained from various development stages, organs, and tissues of members of Poaceae. The early stage of inflorescence development was accompanied by a significant upregulation of a large number of cell differentiation genes, such as those associated with the cell cycle, cell division, DNA repair, and DNA synthesis. Moreover, key regulatory genes, including the MADS-box gene, KNOTTED-1-like homeobox genes, GROWTH-REGULATING FACTOR 1 gene, and the histone methyltransferase gene, were highly expressed in the early inflorescence development stage. In contrast, fewer genes were expressed in the later stage of inflorescence development, and played roles in hormone biosynthesis and meiosis-associated genes. Our work provides novel information regarding the gene regulatory network of cell division, key genes involved in the differentiation of inflorescence in wheat, and regulation mechanism of inflorescence development that are crucial stages for determining final grain number per spike and the yield potential of wheat.

Characterization of novel mutants of hexaploid wheat (Triticum aestivum L.) with various depths of purple grain color and antioxidant capacity.

BACKGROUND: Wheat grain is recognized as a rich source of nutrients, including proteins, vitamins, minerals, fibers and antioxidants. In recent years, the focus of wheat breeding has been to increase the content of bioactive compounds to improve human health and prevent diseases. RESULTS: Five novel wheat mutant lines with variable seed color were developed using gamma irradiation of hexaploid wheat inbred line K4191 (purple seed color). The total anthocyanin contents of three mutant lines (L47, L167 and L925) were significantly higher than those of wild-type lines, including K4191 and 'Keumkang' (white seed color). L925 showed the highest total anthocyanin content, and cyanidin-3-glucoside was presented as the most predominant anthocyanin. Compared with 'Keumkang', the expression of anthocyanin biosynthesis genes was significantly up-regulated in purple seed mutant lines. The highest antioxidant activity was observed in L925 extracts. The expression of a few antioxidant-related genes and total anthocyanin content were positively correlated with antioxidant capacity. These data suggest that anthocyanins and phenolic compounds in wheat grains contribute to the antioxidant potential. CONCLUSION: Purple grain color is associated with higher anthocyanin accumulation and antioxidant capacity in wheat. Wheat mutants developed in this study may serve as a valuable source of antioxidants. © 2018 Society of Chemical Industry.

Selection of mutants with high linolenic acid contents and characterization of fatty acid desaturase 2 and 3 genes during seed development in soybean (Glycine max).

BACKGROUND: Soybean seeds contain 18-24% lipids, which are made up of 85% polyunsaturated fatty acids. Two of these (linoleic and linolenic acids) comprise essential fatty acids that are not synthesized in humans and animals. Linolenic acid plays a vital role in the maintenance of brain function and is a source of docosahexaenoic acid for retinal and nerve tissue, with its physiological functions being a focus of attention. RESULTS: We developed mutant soybean populations via gamma irradiation of Korean cultivars Danbaek and Daepung and evaluated the linolenic acid content of 78 and 154 M9 mutant progenies. We selected the four mutant lines with the highest linolenic acid contents based on 2 years of investigation of fatty acids. The selected mutant lines had linolenic acid contents that were 33.9% to 67.7% higher than those of the original cultivars and exhibited increased fatty acid desaturase (FAD) gene expression levels during seed development. We also identified nucleotide polymorphisms of FAD genes in the four mutant lines. CONCLUSION: The present study found that linolenic acid content is related to significantly increased expression levels of the FAD3C and FAD3D genes in the endoplasmic reticulum, which was uncovered by radiation mutation breeding of soybean. © 2019 Society of Chemical Industry.

Comparison of radiosensitivity response to acute and chronic gamma irradiation in colored wheat.

We aimed to investigate the biological responses induced by acute and chronic gamma irradiation in colored wheat seeds rich in natural antioxidants. After acute and chronic irradiation, the phenotypic effects on plant growth, germination rate, seedling height, and root length were examined, and the biochemical changes were investigated by analyzing the expression of antioxidant enzyme-related genes, antioxidant enzyme activities, and total antioxidant capacity. High dosages of chronic radiation reduced plant growth compared with the controls. Electron spin resonance measurement and 2,2-diphenyl-1-picrylhydrazyl activity analysis showed lower amount of free radicals in colored wheat seeds on chronic irradiation with low dosage of gamma rays compared to seeds subjected to acute irradiation. Expression levels of anthocyanin biosynthesis genes, antioxidant-related genes, and antioxidant enzyme activity in seeds and young leaves of seedling showed diverse effects in response to different dosages and types of gamma irradiation. This suggests that phenotype is affected by the dosage and type of gamma radiation, and the phytochemicals in colored wheat seeds involved in antioxidant activity to scavenge free radicals respond differently to irradiation types. This provides evidence that acute and chronic exposure to radiation have different effects on seeds and young leaves after germination.

Characterization of 4 TaGAST genes during spike development and seed germination and their response to exogenous phytohormones in common wheat.

Gibberellic acid (GA) is involved in the regulation of plant growth and development. We defined GA-stimulated transcript (GAST) gene family and characterized its four members (TaGAST1, 2, 3, and 4) in wheat spikes. Triticum aestivum whole spikes were collected at ten developmental stages and dehulled spikelets were obtained at various days after flowering. Expression of TaGAST1, 2, 3, and 4 was analyzed using RT-PCR at inflorescence development stages, in different tissues, and after phytohormones application. To identify proteins interacting with TaGAST1, yeast two-hybridization was performed and BiFC analysis was used for verification. TaGAST1 was expressed at the inflorescence stage and only expressed in seedlings under abscisic acid (ABA) treatment after phytohormone treatment. TaGAST2 and TaGAST3 showed moderate expression in the spike, vigorous transcript accumulation in the seedling, and up-regulation by exogenous GA in early germination stages. TaGAST4 was predominantly expressed in the seedling. Wheat cyclophilin A-1 (TaCypA1), identified as a TaGAST1-interacting protein, showed opposite expression pattern in the developing spike to TaGAST1. TaCypA1 transcript was slightly up-regulated by GA, slightly down-regulated by paclobutrazol, and was maintained after ABA treatment. The interaction of TaGAST1 with TaCypA1 is targeted to the plasma membrane. TaGAST1 was specifically expressed in the wheat spike and was stimulated by exogenous GA treatment. TaGAST2 and TaGAST3 expression in germinating seeds and seedlings was higher than that in the spike stage. TaGAST4 was not expressed in all developmental stages. TaGAST1 and TaCypA1 might be expressed antagonistically during wheat spike development.

Role of wheat trHb in nitric oxide scavenging.

We examined the role of wheat truncated-hemoglobin (TatrHb) in nitric oxide (NO) scavenging in transgenic Arabidopsis plants by assessing the response to an NO donor/scavenger and salt stress. The degree of increase in Na(+) and decrease in K(+) levels in the transgenic plants were more than those in the wild-type plants, and the ratio of Na(+) to K(+) increased in the transgenic plants under salt stress. Endogenous NO increased dramatically in the salt-treated wild-type plants but not in the transgenic plants. Additionally, the maximum photosystem II quantum ratio of variable to maximum fluorescence (Fv/Fm) in transgenic plants decreased more significantly than that in the wild-type plants, indicating that the transgenic plants suffered more severe photosynthetic damage because of salt stress than that by the wild type. Similar results were observed in germination experiments by using Murashige and Skoog media containing 100 mM sodium chloride. The Fv/Fm decreased in the leaves of salt-treated transgenic plants, indicating that transgenic seeds were more sensitive to salt stress than that by the wild-type seeds. In addition, the negative effect on seed germination was more severe in transgenic plants than in the wild types under NaCl treatment conditions. The results support the hypothesis that plant trHb shares NO scavenging functions and characteristics with bacterial trHb.

Identification and transcriptomic profiling of salinity stress response genes in colored wheat mutant.

Background: Salinity is a major abiotic stress that prevents normal plant growth and development, ultimately reducing crop productivity. This study investigated the effects of salinity stress on two wheat lines: PL1 (wild type) and PL6 (mutant line generated through gamma irradiation of PL1). Results: The salinity treatment was carried out with a solution consisting of a total volume of 200 mL containing 150 mM NaCl. Salinity stress negatively impacted germination and plant growth in both lines, but PL6 exhibited higher tolerance. PL6 showed lower Na+ accumulation and higher K+ levels, indicating better ion homeostasis. Genome-wide transcriptomic analysis revealed distinct gene expression patterns between PL1 and PL6 under salt stress, resulting in notable phenotypic differences. Gene ontology analysis revealed positive correlations between salt stress and defense response, glutathione metabolism, peroxidase activity, and reactive oxygen species metabolic processes, highlighting the importance of antioxidant activities in salt tolerance. Additionally, hormone-related genes, transcription factors, and protein kinases showed differential expression, suggesting their roles in the differential salt stress response. Enrichment of pathways related to flavonoid biosynthesis and secondary metabolite biosynthesis in PL6 may contribute to its enhanced antioxidant activities. Furthermore, differentially expressed genes associated with the circadian clock system, cytoskeleton organization, and cell wall organization shed light on the plant's response to salt stress. Conclusions: Understanding these mechanisms is crucial for developing stress-tolerant crop varieties, improving agricultural practices, and breeding salt-resistant crops to enhance global food production and address food security challenges.

Unveiling differential expression profiles of the wheat DOG1 gene family and functional analysis of the association between TaDOG1-1 and heat stress tolerance in transgenic Arabidopsis.

High temperatures can significantly impact wheat growth and grain yields during the grain-filling stage. In this study, we identified genes that respond to high-temperature stress during the grain-filling stage. We also identified and characterized 24 novel genes of the DOG1 gene family in hexaploid wheat. Motif analysis and conserved domain search revealed substantial similarities among TaDOG1 family members. Phylogenetic analysis demonstrated the evolutionary conservation of the TaDOG1 family across various plant species. Tissue-specific expression profiling indicated consistent patterns, with TaDOG1 genes predominantly expressed in stem tissues. Only TaDOG1-1 exhibited enhanced expression, particularly during hard dough and ripening stages. TaDOG1-1 and TaDOG1-7 exhibited increased expression under heat stress during the grain-filling stage, indicating their heat-responsive nature. Cis-element analysis revealed potential regulatory motifs, suggesting the involvement of TaDOG1-1 and TaDOG1-7 in stress tolerance mechanisms. Yeast two-hybrid screening revealed interacting proteins, including stress-responsive and grain development-associated proteins. To understand the biological function, we overexpressed TaDOG1-1 in Arabidopsis plants and observed enhanced thermotolerance under basal heat stress. Under heat stress, the transgenic plants exhibited increased biomass and elevated expression levels of heat-responsive genes. Furthermore, TaDOG1-1-overexpressing plants showed improved survival rates under soil heat stress, along with a greater accumulation of antioxidant enzymes in leaves. In this study, the identification and functions of the DOG1 gene family provide valuable insights for developing genetic engineering strategies aimed at improving wheat yield under high-temperature stress.

SKP1-like-related genes interact with various F-box proteins and may form SCF complexes with Cullin-F-box proteins in wheat.

S-phase kinase-associated protein 1 (SKP1), a core component of the SKP1-Cullin-F-box (SCF) E3 ubiquitin ligase complex, functions as an adaptor protein, connecting cullin and F-box proteins. SKP1 plays crucial roles in cell-cycle progression, transcriptional regulation, flower formation, signal transduction, and many other cellular processes. SKP1-like genes have been largely unstudied in wheat. Here, we isolated six wheat SKP1-like (TaSKP) genes from common wheat (Triticum aestivum) and analyzed the expression patterns of these six genes using reverse transcription-polymerase chain reaction (RT-PCR). Based on gene expression patterns, we divided the genes into two groups. Our data demonstrated that green fluorescent protein-tagged TaSKP proteins were targeted to the plasma membrane or cytoplasm in plant cells. In a yeast two-hybrid system, all TaSKP proteins interacted with TaCFBD, TaSKP1, and TaSKP5, while TaSKP6 interacted with RA and RLK. A BiFC assay suggested that specific combinations of TaSKP and F-box proteins may influence localization patterns in plant cells. TaSKP1, TaSKP5, and TaSKP6 interacted with TaCullin, while TaSKP2, TaSKP3, and TaSKP4 were not found to interact with TaCullin in the yeast two-hybrid system. This evidence indicated that some TaSKP proteins may have the ability to form SCF complexes. Taken together, these data suggested that TaSKP1, TaSKP5, and TaSKP6 proteins may act as a bridge between various F-box proteins and cullin proteins and that TaSKP genes may be involved in various growth and flower development processes.

Comparison of Policosanol Profiles of the Sprouts of Wheat Mutant Lines and the Effect of Differential LED Lights on Selected Lines.

Policosanols (PCs) are long-chain linear aliphatic alcohols that are present in the primary leaves of cereal crops, such as barley and wheat, sugar cane wax, and beeswax. PCs have been used as a nutraceutical for improving hyperlipidemia and hypercholesterolemia. However, the PC content in mutant wheat lines has not been investigated. To select highly functional wheat sprouts with a high content of PCs in wheat mutant lines developed via gamma-irradiated mutation breeding, we cultivated the sprouts of wheat mutant lines in a growth chamber with white LED light (6000 K) and analyzed the PC content in these samples using GC-MS. We studied the PC content in 91 wheat sprout samples: the original variety (Woori-mil × D-7; WS01), commercially available cv. Geumgang (WS87) and cv. Cheongwoo (WS91), and mutant lines (WS02-WS86 and WS88-WS90) developed from WS01 and WS87. Compared to WS01, 18 mutant lines exhibited a high total PC content (506.08-873.24 mg/100 g dry weight). Among them, the top 10 mutant lines were evaluated for their PC production after cultivating under blue (440 nm), green (520 nm), and red (660 nm) LED light irradiation; however, these colored LED lights reduced the total PC production by 35.8-49.7%, suggesting that the cultivation with white LED lights was more efficient in promoting PCs' yield, compared to different LED lights. Therefore, our findings show the potential of radiation-bred wheat varieties as functional foods against hyperlipidemia and obesity and the optimal light conditions for high PC production.

Phytochemical profile and anti-inflammatory activity of the hull of γ-irradiated wheat mutant lines (Triticum aestivum L.).

Wheat (Triticum aestivum Linn.; Poaceae) is the second most cultivated food crop among all global cereal crop production. The high carbohydrate content of its grains provides energy, multiple nutrients, and dietary fiber. After threshing, a substantial amount of wheat hull is produced, which serves as the non-food component of wheat. For the valorization of these by-products as a new resource from which functional components can be extracted, the hull from the seeds of cultivated wheat mutant lines bred after γ-irradiation were collected. Untargeted metabolite analysis of the hull of the original cultivar (a crossbreeding cultivar., Woori-mil × D-7) and its 983 mutant lines were conducted using ultra-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry technique. A total of 55 molecules were tentatively identified, including 21 compounds found in the Triticum species for the first time and 13 compounds not previously described. Among them, seven flavonolignans with a diastereomeric structure, isolated as a single compound from the hull of T. aestivum in our previous study, were used as the standards in the metabolite analysis. The differences in their collision cross-section values were shown to contribute to the clear distinction between tricine-lignan stereoisomers. To select functionally active agents with anti-inflammatory activity among the identified compounds, the wheat hull samples were evaluated for their inhibitory effect on nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 cells. As a result of multivariate analysis based on the results of chemical and biological profiles of the wheat hull samples, 10 metabolites were identified as key markers, contributing to the distinction between active and inactive mutant lines. Considering that one of the four key markers attributed to anti-inflammatory activity has been identified to be a flavonolignan, the wheat hull could be a valuable source of diverse tricin-lignan type compounds and used as a natural health-promoting product in food supplements.

Wheat F-box protein recruits proteins and regulates their abundance during wheat spike development.

F-box proteins, components of the Skp1-Cullin1-F-box (SCF) protein E3 ubiquitin ligase complex, serve as the variable component responsible for substrate recognition and recruitment in SCF-mediated proteolysis. F-box proteins interact with Skp1 through the F-box motif and with ubiquitination substrates through C-terminal protein interaction domains. F-box proteins regulate plant development, various hormonal signal transduction processes, circadian rhythm, and cell cycle control. We isolated an F-box protein gene from wheat spikes at the onset of flowering. The Triticum aestivum cyclin F-box domain (TaCFBD) gene showed elevated expression levels during early inflorescence development and under cold stress treatment. TaCFBD green fluorescent protein signals were localized in the cytoplasm and plasma membrane. We used yeast two-hybrid screening to identify proteins that potentially interact with TaCFBD. Fructose bisphosphate aldolase, aspartic protease, VHS, glycine-rich RNA-binding protein, and the 26S proteasome non-ATPase regulatory subunit were positive candidate proteins. The bimolecular fluorescence complementation assay revealed the interaction of TaCFBD with partner proteins in the plasma membranes of tobacco cells. Our results suggest that the TaCFBD protein acts as an adaptor between target substrates and the SCF complex and provides substrate specificity to the SCF of ubiquitin ligase complexes.

Corrigendum: 1H NMR-Based Chemometrics to Gain Insights Into the Bran of Radiation-Induced Colored Wheat Mutant.

[This corrects the article DOI: 10.3389/fnut.2021.806744.].

Combining NMR and MS to Describe Pyrrole-2-Carbaldehydes in Wheat Bran of Radiation.

Nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry (MS) are indispensable analytical tools to provide chemical fingerprints in metabolomics studies. The present study evaluated radiation breeding wheat lines for chemical changes by non-targeted NMR-based metabolomics analysis of bran extracts. Multivariate analysis following spectral binning suggested pyrrole-2-carbaldehydes as chemical markers of four mutant lines with distinct NMR fingerprints in a δH range of 9.28-9.40 ppm. Further NMR and MS data analysis, along with chromatographic fractionation and synthetic preparation, aimed at structure identification of marker metabolites and identified five pyrrole-2-carbaldehydes. Quantum-mechanical driven 1H iterative full spin analysis (QM-HiFSA) on synthetic pyrrole-2-carbaldehydes provided a precise description of complex peak patterns. Biological evaluation of pyrrole-2-carbaldehydes was performed with nine synthetic products, and six compounds showed hepatoprotective effects via modulation of reactive oxygen species production. Given that three out of five identified in wheat bran of radiation were described for hepatoprotective activity, the value of radiation mutation to greatly enhance pyrrole-2-carbaldehyde production was supported.

Regulation of Glycosylphosphatidylinositol-Anchored Protein (GPI-AP) Expression by F-Box/LRR-Repeat (FBXL) Protein in Wheat (Triticum aestivum L.).

F-box proteins are substrate recognition components of the Skp1-Cullin-F-box (SCF) complex, which performs many important biological functions including the degradation of numerous proteins via the ubiquitin-26S proteasome system. In this study, we isolated the gene encoding the F-box/LRR-repeat (FBXL) protein from wheat (Triticum aestivum L.) seedlings and validated that the TaFBXL protein is a component of the SCF complex. Yeast two-hybrid assays revealed that TaFBXL interacts with the wheat glycosylphosphatidylinositol-anchored protein (TaGPI-AP). The green fluorescent protein (GFP) fusion protein of TaFBXL was detected in the nucleus and plasma membrane, whereas that of TaGPI-AP was observed in the cytosol and probably also plasma membrane. yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays revealed that TaFBXL specifically interacts with TaGPI-AP in the nucleus and plasma membrane, and TaGPI-AP is targeted by TaFBXL for degradation via the 26S proteasome system. In addition, TaFBXL and TaGPI-AP showed antagonistic expression patterns upon treatment with indole-3-acetic acid (IAA), and the level of TaGPI-AP was higher in tobacco leaves treated with both MG132 (proteasome inhibitor) and IAA than in leaves treated with either MG132 or IAA. Taken together, our data suggest that TaFBXL regulates the TaGPI-AP protein level in response to exogenous auxin application.