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Radish (Raphanus sativus L.) is a vegetable crop with economic value and ecological significance in the genus Radish, family Brassicaceae. In recent years, developed countries have attached great importance to the collection and conservation of radish germplasm resources and their research and utilization, but the lack of population genetic information and molecular markers has hindered the development of the genetic breeding of radish. In this study, we integrated the radish genomic data published in databases for the development of single-nucleotide polymorphism (SNP) markers, and obtained a dataset of 308 high-quality SNPs under strict selection criteria. With the support of Kompetitive Allele-Specific PCR (KASP) technology, we screened a set of 32 candidate core SNP marker sets to analyse the genetic diversity of the collected 356 radish varieties. The results showed that the mean values of polymorphism information content (PIC), minor allele frequency (MAF), gene diversity and heterozygosity of the 32 candidate core SNP markers were 0.32, 0.30, 0.40 and 0.25, respectively. Population structural analysis, principal component analysis and genetic evolutionary tree analysis indicated that the 356 radish materials were best classified into two taxa, and that the two taxa of the material were closely genetically exchanged. Finally, on the basis of 32 candidate core SNP markers we calculated 15 core markers using a computer algorithm to construct a fingerprint map of 356 radish varieties. Furthermore, we constructed a core germplasm population consisting of 71 radish materials using 32 candidate core markers. In this study, we developed SNP markers for radish cultivar identification and genetic diversity analysis, and constructed DNA fingerprints, providing a basis for the identification of radish germplasm resources and molecular marker-assisted breeding as well as genetic research.

Accumulation of anthocyanins in the taproot of radish is an agronomic trait beneficial for human health. Several genetic loci are related to a red skin or flesh color of radish, however, the functional divergence of candidate genes between non-red and red radishes has not been investigated. Here, we report that a novel genetic locus on the R2 chromosome, where RsMYB1.1 is located, is associated with the red color of the skin of radish taproot. A genome-wide association study (GWAS) of 66 non-red-skinned (nR) and 34 red-skinned (R) radish accessions identified three nonsynonymous single nucleotide polymorphisms (SNPs) in the third exon of RsMYB1.1. Although the genotypes of SNP loci differed between the nR and R radishes, no functional difference in the RsMYB1.1 proteins of nR and R radishes in their physical interaction with RsTT8 was detected by yeast-two hybrid assay or in anthocyanin accumulation in tobacco and radish leaves coexpressing RsMYB1.1 and RsTT8. By contrast, insertion-or deletion-based GWAS revealed that one large ATrich low-complexity sequence of 1.3-2 kb was inserted in the promoter region of RsMYB1.1 in the nR radishes (RsMYB1.1nR), whereas the R radishes had no such insertion; this represents a presence/absence variation (PAV). This insertion sequence (RsIS) was radish specific and distributed among the nine chromosomes of Raphanus genomes. Despite the extremely low transcription level of RsMYB1.1nR in the nR radishes, the inactive RsMYB1.1nR promoter could be functionally restored by deletion of the RsIS. The results of a transient expression assay using radish root sections suggested that the RsIS negatively regulates the expression of RsMYB1.1nR, resulting in the downregulation of anthocyanin biosynthesis genes, including RsCHS, RsDFR, and RsANS, in the nR radishes. This work provides the first evidence of the involvement of PAV in an agronomic trait of radish. [ABSTRACT FROM AUTHOR]

Growing microgreens on trays without substrate in a vertical multilayered growing unit offers several advantages over traditional agriculture methods. This study investigated the yield performance and nutritional quality of five selections of radish microgreens grown in sprouting trays, without a substrate using only water, in an indoor multilayer cultivation system using artificial light. Various parameters were measured, including fresh weight, dry matter, chlorophyll, minerals, amino acids, phenolics, flavonoids, anthocyanins, vitamin C, glucosinolates, and antioxidant activity with four different in vitro assays. After ten days, the biomass had increased by 6-10 times, and the dry matter varied from 4.75-7.65%. The highest yield was obtained from ‘Asia red’, while the lowest was from ‘Koregon red’. However, ‘Koregon red’ and ‘Asia red’ had the highest dry matter. ‘Asia red’ was found to have the highest levels of both Chls and vitamin C compared to the other cultivars, while ‘Koregon red’ exhibited the highest levels of total phenolics and flavonoids. Although variations in the levels of individual glucosinolates were observed, there were no significant differences in the total content of glucosinolates among the five cultivars. ‘Asia purple’ had the highest anthocyanin content, while ‘Asia green 2’ had the lowest. The K, Mg, and Na concentrations were significantly highest in ‘Asia green 2’, and the highest Ca was recorded in ‘Asia purple’. Overall, ‘Asia purple’ and ‘Koregon red’ were the best cultivars in terms of nutritional quality among the tested radish microgreens. These cultivars exhibited high levels of dry weight, total phenolics, flavonoids, anthocyanins, essential and total amino acids, and antioxidant activities. Moreover, the implementation of this vertical cultivation method for microgreens, which relies solely on water and seeds known for their tall shoots during the sprouting could hold promise as a sustainable approach. This method can effectively be utilized for cultivar screening and fulfilling the nutritional and functional needs of the population while minimizing the environmental impacts associated with traditional agriculture practices. [ABSTRACT FROM AUTHOR]

Glucosinolates (GSLs) and their degradation products in radish confer plant defense, promote human health, and generate pungent flavor. However, the intact GSLs in radish have not been investigated comprehensively yet. Here, an accurate qualitative and quantitative analyses of 15 intact GSLs from radish, including four major GSLs of glucoraphasatin (GRH), glucoerucin (GER), glucoraphenin (GRE), and 4-methoxyglucobrassicin (4MGBS), were conducted using UHPLC-HRMS/MS in combination with UHPLC-QqQ-MS/MS. Simultaneously, three isomers of hexyl GSL, 3-methylpentyl GSL, and 4-methylpentyl GSL were identified in radish. The highest content of GSLs was up to 232.46 mmol/g DW at the 42 DAG stage in the 'SQY' taproot, with an approximately 184.49-fold increase compared to the lowest content in another sample. That the GSLs content in the taproots of two radishes fluctuated in a similar pattern throughout the five vegetative growth stages according to the metabolic profiling, whereas the GSLs content in the '55' leaf steadily decreased over the same period. Additionally, the proposed biosynthetic pathways of radish-specific GSLs were elucidated in this study. Our findings will provide an abundance of qualitative and quantitative data on intact GSLs, as well as a method for detecting GSLs, thus providing direction for the scientific progress and practical utilization of GSLs in radish. [ABSTRACT FROM AUTHOR]

Microgreens are emerging specialty crops becoming increasingly popular for their rich nutrient profile and variety of colors, flavors, and textures. The growing medium is a significant key factor in microgreen yield, quality, and sustainability. The widespread use of peat-based media raises questions regarding the environmental sustainability of microgreens production, and new substrates that are more sustainable are required. To this purpose, a study was designed with the objective of comparing eight alternative growing media evaluating their physicochemical properties and effect on yield, mineral profile, and nutritional quality of peas and radish microgreens. Tested substrates included a standard peat and perlite mixture (PP), coconut coir (CC), spent mushroom compost (SMC), organic waste compost (CMP), and 50:50 (v:v) mixes of PP and SMC, PP and CMP, CC and SMC, and CC and CMP. The physicochemical properties widely differed among the alternative substrates tested. SMC had high electrical conductivity and salt concentration, which resulted in poor seed germination. Growing media tested significantly influenced the production and nutritional quality of both microgreen species and variations were modulated by the species. With a 39.8% fresh yield increase or a small yield decrease (-14.9%) in radish and peas, respectively, PP+CMP (50:50, v/v) mix provided microgreens of similar or higher nutritional quality than PP, suggesting the potential of substituting at least in part peat with CMP. Using locally available CMP in mix with PP could reduce the microgreens industry reliance on peat while reducing costs and improving the sustainability of the production of microgreens. Further research is needed to evaluate also the potential economic and environmental benefits of using locally available organic materials like CMP as alternative growing media and peat-substitute to produce microgreens. [ABSTRACT FROM AUTHOR]

Glucosinolates (GSLs), precursors of isothiocyanates (ITCs), are present in Brassicaceae plants have been found to have health benefits. Sulforaphane (4-(methylsulfinyl)butyl ITC) is an ITC stored in the formof 4-(methylsulfinyl)butyl GSL (glucoraphanin, 4MSOB) in Brassica vegetables, such as broccoli and kale. Sulforaphane activates Nrf2 expression, a transcription factor responsible for inducing physiological activities such as detoxification in the human body, and it represents a functional component unique to cruciferous vegetables. Raphanobrassica is an inter-generic hybrid between radish and kale, and it contains a high amount of 4MSOB. However, Raphanobrassica contains as much 4-methylsulfinyl-3-butenyl GSL (glucoraphenin, 4MSO3B) as it does 4MSOB. GLUCORAPHASATIN SYNTHASE 1 (GRS1) is an enzyme present in radish that synthesizes 4-methylthio-3-butenyl GSL (glucoraphasatin, 4MT3B), a precursor of 4MSO3B, using 4-(methylthio)butyl GSL (glucoerucin, 4MTB) as a substrate. Since the precursor of 4MSOB is also 4MTB, it was considered that both 4MSOB and 4MSO3B accumulate owing to competition in Raphanobrassica. We hypothesized that owing to the impaired function of GRS1 in Raphanobrassica, it may be possible to breed Raphanobrassica cultivars containing a high 4MSOB content. In this study, we generated Raphanobrassica populations with functional and defective GRS1 and compared the GSL composition in the two populations using high-performance liquid chromatography. The mean 4MSOB content in leaves of the defective-type populations was higher than that in the functionaltype population, and the defective/functional ratio ranged from 2.02 to 2.51-fold, supporting this hypothesis. Furthermore, leaves, flower buds, stems, and roots contained higher amounts of 4MSOB in the defective population than in the functional population. The leaf 4MSOB content of defective Raphanobrassica grown in this study was comparable to that of previously studied vegetables (such as broccoli sprouts) with high 4MSOB content. Raphanobrassica with defective GRS1 represents a new leafy vegetable with high 4MSOB content which exhibits anti-cancerous and anti-inflammatory potentials. [ABSTRACT FROM AUTHOR]

Glucosinolates (GSLs), precursors of isothiocyanates (ITCs), are present in Brassicaceae plants have been found to have health benefits. Sulforaphane (4-(methylsulfinyl)butyl ITC) is an ITC stored in the form of 4-(methylsulfinyl)butyl GSL (glucoraphanin, 4MSOB) in Brassica vegetables, such as broccoli and kale. Sulforaphane activates Nrf2 expression, a transcription factor responsible for inducing physiological activities such as detoxification in the human body, and it represents a functional component unique to cruciferous vegetables. Raphanobrassica is an inter-generic hybrid between radish and kale, and it contains a high amount of 4MSOB. However, Raphanobrassica contains as much 4-methylsulfinyl-3-butenyl GSL (glucoraphenin, 4MSO3B) as it does 4MSOB. GLUCORAPHASATIN SYNTHASE 1 (GRS1) is an enzyme present in radish that synthesizes 4-methylthio-3-butenyl GSL (glucoraphasatin, 4MT3B), a precursor of 4MSO3B, using 4-(methylthio)butyl GSL (glucoerucin, 4MTB) as a substrate. Since the precursor of 4MSOB is also 4MTB, it was considered that both 4MSOB and 4MSO3B accumulate owing to competition in Raphanobrassica. We hypothesized that owing to the impaired function of GRS1 in Raphanobrassica, it may be possible to breed Raphanobrassica cultivars containing a high 4MSOB content. In this study, we generated Raphanobrassica populations with functional and defective GRS1 and compared the GSL composition in the two populations using high-performance liquid chromatography. The mean 4MSOB content in leaves of the defective-type populations was higher than that in the functional-type population, and the defective/functional ratio ranged from 2.02 to 2.51-fold, supporting this hypothesis. Furthermore, leaves, flower buds, stems, and roots contained higher amounts of 4MSOB in the defective population than in the functional population. The leaf 4MSOB content of defective Raphanobrassica grown in this study was comparable to that of previously studied vegetables (such as broccoli sprouts) with high 4MSOB content. Raphanobrassica with defective GRS1 represents a new leafy vegetable with high 4MSOB content which exhibits anti-cancerous and anti-inflammatory potentials.

Radish (Raphanus sativus L.) is an important root vegetable crop belonging to the Brassicaceae family. Anthocyanin rich radish varieties are popular among consumers because of their bright color and high nutritional value. However, the underlying molecular mechanism responsible for skin and flesh induce anthocyanin biosynthesis in transient overexpression, gene silencing and transcriptome sequencing were used to verify its function in radish anthocyanin accumulation, radish remains unclear. Here, we identified a long noncoding RNA LINC15957, overexpression of LINC15957 was significantly increased anthocyanin accumulation in radish leaves, and the expression levels of structural genes related to anthocyanin biosynthesis were also significantly increased. Anthocyanin accumulation and expression levels of anthocyanin biosynthesis genes were significantly reduced in silenced LINC15957 flesh when compared with control. By the transcriptome sequencing of the overexpressed LINC15957 plants and the control, 5,772 differentially expressed genes were identified. A total of 3,849 differentially expressed transcription factors were identified, of which MYB, bHLH, WD40, bZIP, ERF, WRKY and MATE were detected and differentially expressed in the overexpressed LINC15957 plants. KEGG enrichment analysis revealed the genes were significant enriched in tyrosine, L-Phenylalanine, tryptophan, phenylpropanol, and flavonoid biosynthesis. RT-qPCR analysis showed that 8 differentially expressed genes (DEGs) were differentially expressed in LINC15957-overexpressed plants. These results suggested that LINC15957 involved in regulate anthocyanin accumulation and provide abundant data to investigate the genes regulate anthocyanin biosynthesis in radish. [ABSTRACT FROM AUTHOR]

Radish (Raphanus sativus L.) is an economically important vegetable worldwide, but its sustainable production and breeding are highly threatened by blight disease caused by Alternaria brassicae. Melatonin is an important growth regulator that can influence physiological activities in both plants and microbes and stimulate biotic stress resistance in plants. In this study, 0-1500 mM melatonin was exogenously applied to healthy radish seedlings, in vitro incubated A. brassicae, and diseased radish seedlings to determine the effects of melatonin on host, pathogen, and host-pathogen interaction. At sufficient concentrations (0-500 mM), melatonin enhanced growth and immunity of healthy radish seedlings by improving the function of organelles and promoting the biosynthesis of antioxidant enzymes, chitin, organic acid, and defense proteins. Interestingly, melatonin also improved colony growth, development, and virulence of A. brassicae. A strong dosage-dependent effect of melatonin was observed: 50-500 mM promoted host and pathogen vitality and resistance (500 mM was optimal) and 1500 mM inhibited these processes. Significantly less blight was observed on diseased seedlings treated with 500 mM melatonin, indicating that melatonin more strongly enhanced the growth and immunity of radish than it promoted the development and virulence of A. brassicae at this treatment concentration. These effects of MT were mediated by transcriptional changes of key genes as identified by RNA-seq, Dual RNA-seq, and qRT-PCR. The results from this work provide a theoretical basis for the application of melatonin to protect vegetable crops against pathogens. [ABSTRACT FROM AUTHOR]

An ever-increasing population has issued an open challenge to the agricultural sector to provide enough food in a sustainable manner. The upsurge in chemical fertilizers to enhance food production had resulted in environmental problems. The objective of the current study is to assess the utilization of biostimulants for sustainable agricultural production as an alternative to chemical fertilization. For this purpose, two pot experiments were conducted to examine the response of radish against individual and combined applications of biostimulants. In the first experiment, the effects of chemical fertilizer (CK), glycine (G), lysine (L), aspartic acid (A), and vitamin B complex (V) were studied. The results demonstrated that V significantly improved the transpiration rate (81.79%), stomatal conductance (179.17%), fresh weight (478.31%), and moisture content (2.50%). In the second experiment, tested treatments included chemical fertilizer (CK), Isabion® (I), glycine + lysine + aspartic acid (GLA), moringa leaf extract + GLA (M1), 25% NPK + M1 (M2). The doses of biostimulants were 5g L-1 glycine, 1g L-1 lysine, 2g L-1 aspartic acid, and 10ml L-1 moringa leaf extract. The photosynthetic rate improved significantly with GLA (327.01%), M1 (219.60%), and M2 (22.16%), while the transpiration rate was enhanced with GLA (53.14%) and M2 (17.86%) compared to the Ck. In addition, M1 increased the stomatal conductance (54.84%), internal CO2 concentration (0.83%), plant fresh weight (201.81%), and dry weight (101.46%) as compared to CK. This study concludes that biostimulants can effectively contribute to the sustainable cultivation of radish with better growth and yield. [ABSTRACT FROM AUTHOR]

Aquaporins (AQPs) constitute a highly diverse family of channel proteins that transport water and neutral solutes. AQPs play crucial roles in plant development and stress responses. However, the characterization and biological functions of RsAQPs in radish (Raphanus sativus L.) remain elusive. In this study, 61 non-redundant members of AQP-encoding genes were identified from the radish genome database and located on nine chromosomes. Radish AQPs (RsAQPs) were divided into four subfamilies, including 21 plasma membrane intrinsic proteins (PIPs), 19 tonoplast intrinsic proteins (TIPs), 16 NOD-like intrinsic proteins (NIPs), and 5 small basic intrinsic proteins (SIPs), through phylogenetic analysis. All RsAQPs contained highly conserved motifs (motifs 1 and 4) and transmembrane regions, indicating the potential transmembrane transport function of RsAQPs. Tissue- and stage-specific expression patterns of AQP gene analysis based on RNA-seq data revealed that the expression levels of PIPs were generally higher than TIPs, NIPs, and SIPs in radish. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) revealed that seven selected RsPIPs, according to our previous transcriptome data (e.g., RsPIP1-3, 1-6, 2-1, 2-6, 2-10, 2-13, and 2-14), exhibited significant upregulation in roots of salt-tolerant radish genotype. In particular, the transcriptional levels of RsPIP2-6 dramatically increased after 6 h of 150 mM NaCl treatment during the taproot thickening stage. Additionally, overexpression of RsPIP2-6 could enhance salt tolerance by Agrobacterium rhizogenes-mediated transgenic radish hairy roots, which exhibited the mitigatory effects of plant growth reduction, leaf relative water content (RWC) reduction and alleviation of O2 in cells, as shown by nitro blue tetrazolium (NBT) staining, under salt stress. These findings are helpful for deeply dissecting the biological function of RsAQPs on the salt stress response, facilitating practical application and genetic improvement of abiotic stress resistance in radish. [ABSTRACT FROM AUTHOR]

Anthocyanins are water-soluble plant pigments mainly stored in the plant vacuoles. Glutathione S-transferases (GSTs) are a multifunctional enzyme family, which can regulate substance metabolism and biological and abiotic stresses in plants. However, few reports were focused on the involvement of GSTs in anthocyanin sequestration in red skin radish. Here, we identified a glutathione S-transferase gene RsGSTF12 that played roles in anthocyanin sequestration in radish. The bioinformatics analysis revealed that RsGSTF12 belonged to the phi (F) class of glutathione S-transferases and showed a high homology with AtGSTF12, followed by AtGSTF11. The subcellular localization assay showed that RsGSTF12 was located in the endoplasmic reticulum and tonoplast. Temporal and spatial gene expression-specific analyses uncovered a strong correlation of RsGSTF12 with anthocyanin accumulation in radish sprouts. The anthocyanin solubility assay found RsGSTF12 was capable of improving cyanidin water solubility in vitro. Transiently expressing RsGSTF12 in radish cotyledons was able to increase their anthocyanin sequestrations. Furthermore, the functional complementation and overexpression of the Arabidopsis thaliana tt19 mutant and wild type demonstrated that RsGSTF12 might play an indispensable role in anthocyanin accumulation in radish. Taken together, we provide compelling evidence that RsGSTF12 functions critically in how anthocyanins are sequestrated in radish, which may enrich our understanding of the mechanism of anthocyanin sequestration. [ABSTRACT FROM AUTHOR]

Clubroot is a devastating disease that causes substantial yield loss worldwide. However, the inheritance and molecular mechanisms of clubroot resistance during pathogen infection in radish remain largely unclear. In this study, we investigated the inheritance of clubroot resistance in the F2 population derived from crossing clubroot-resistant (CR) and clubroot-susceptible inbred lines "GLX" and "XNQ," respectively. Genetic analysis revealed that a single dominant gene controlled the clubroot resistance of "GLX" with a Mendelian ratio of resistance and susceptibility of nearly 3:1. Bulked segregant analysis combined with whole-genome resequencing (BSA-seq) was performed to detect the target region of RsCr6 on chromosome Rs8. Linkage analysis revealed that the RsCr6 locus was located between two markers, HB321 and HB331, with an interval of approximately 92 kb. Based on the outcomes of transcriptome analysis, in the RsCr6 locus, the R120263140 and R120263070 genes with a possible relation to clubroot resistance were considered candidate genes. In addition, three core breeding materials containing the two reported quantitative trait loci (QTLs) and our novel locus RsCr6 targeting clubroot resistance were obtained using marker-assisted selection (MAS) technology. This study reveals a novel locus responsible for clubroot resistance in radishes. Further analysis of new genes may reveal the molecular mechanisms underlying the clubroot resistance of plants and provide a theoretical basis for radish resistance breeding. [ABSTRACT FROM AUTHOR]

Clubroot is a devastating disease that causes substantial yield loss worldwide. However, the inheritance and molecular mechanisms of clubroot resistance during pathogen infection in radish remain largely unclear. In this study, we investigated the inheritance of clubroot resistance in the F2 population derived from crossing clubroot-resistant (CR) and clubroot-susceptible inbred lines “GLX” and “XNQ,” respectively. Genetic analysis revealed that a single dominant gene controlled the clubroot resistance of “GLX” with a Mendelian ratio of resistance and susceptibility of nearly 3:1. Bulked segregant analysis combined with whole-genome resequencing (BSA-seq) was performed to detect the target region of RsCr6 on chromosome Rs8. Linkage analysis revealed that the RsCr6 locus was located between two markers, HB321 and HB331, with an interval of approximately 92 kb. Based on the outcomes of transcriptome analysis, in the RsCr6 locus, the R120263140 and R120263070 genes with a possible relation to clubroot resistance were considered candidate genes. In addition, three core breeding materials containing the two reported quantitative trait loci (QTLs) and our novel locus RsCr6 targeting clubroot resistance were obtained using marker-assisted selection (MAS) technology. This study reveals a novel locus responsible for clubroot resistance in radishes. Further analysis of new genes may reveal the molecular mechanisms underlying the clubroot resistance of plants and provide a theoretical basis for radish resistance breeding.

The modern agricultural sector faces the challenge of addressing the needs of the fast-growing global population. This process should be both high-yielding and sustainable, without creating risks for the environment and human health. Therefore, natural products are gaining attention in the production of safe and nutritious food. In a systematic effort to develop affordable and effective biostimulants, we examined the impact of botanical extracts on the growth and physiological parameters of radish plants under field conditions. Ultrasound-assisted extraction, mechanical homogenization, and water were used for the production of potential plant-based biostimulants. Foliar applications of the bio-products, developed and used in our study, have led to an increase in the examined parameters (total yield, dry weight, photosynthetic pigments, vitamin C, nitrates, and micro- and macroelements). A decrease in the total phenolic compounds content was also noted, as well as a varied impact on the steam volatile compounds, fatty acids, sterol, and glucosinolates composition. The most beneficial effects on radish, in terms of physiological and biochemical properties, were found in groups treated with extracts based on the common dandelion, valerian, and giant goldenrod. This innovative approach presented in our study could provide a valuable tool for sustainable horticultural production.

Along with being important pigments that determining the flower color in many plants, anthocyanins also perform crucial functions that attract pollinators and reduce abiotic stresses. Purple and white are two different colors of radish petals. In this study, two cDNA libraries constructed with purple and white petal plants were sequenced for transcriptome profiling. Transcriptome results implied that the expression level of the genes participating in the anthocyanin biosynthetic pathway was commonly higher in the purple petals than that in the white petals. In particular, two genes, F3′H and DFR, had a significantly higher expression pattern in the purple petals, suggesting the important roles these genes playing in radish petal coloration. BSA-seq aided-Next Generation Sequencing of two DNA pools revealed that the radish purple petal gene (RsPP) was located on chromosome 7. With additional genotyping of 617 F2 population plants, the RsPP was further confined within a region of 93.23 kb. Transcriptome and Sanger sequencing analysis further helped identify the target gene, Rs392880. Rs392880 is a homologous gene to F3′H, a key gene in the anthocyanin biosynthetic pathway. These results will aid in elucidating the molecular mechanism of plant petal coloration and developing strategies to modify flower color through genetic transformation.

The modern agricultural sector faces the challenge of addressing the needs of the fast-growing global population. This process should be both high-yielding and sustainable, without creating risks for the environment and human health. Therefore, natural products are gaining attention in the production of safe and nutritious food. In a systematic effort to develop affordable and effective biostimulants, we examined the impact of botanical extracts on the growth and physiological parameters of radish plants under field conditions. Ultrasound-assisted extraction, mechanical homogenization, and water were used for the production of potential plant-based biostimulants. Foliar applications of the bio-products, developed and used in our study, have led to an increase in the examined parameters (total yield, dry weight, photosynthetic pigments, vitamin C, nitrates, and micro- and macroelements). A decrease in the total phenolic compounds content was also noted, as well as a varied impact on the steam volatile compounds, fatty acids, sterol, and glucosinolates composition. The most beneficial effects on radish, in terms of physiological and biochemical properties, were found in groups treated with extracts based on the common dandelion, valerian, and giant goldenrod. This innovative approach presented in our study could provide a valuable tool for sustainable horticultural production. [ABSTRACT FROM AUTHOR]

Along with being important pigments that determining the flower color in many plants, anthocyanins also perform crucial functions that attract pollinators and reduce abiotic stresses. Purple and white are two different colors of radish petals. In this study, two cDNA libraries constructed with purple and white petal plants were sequenced for transcriptome profiling. Transcriptome results implied that the expression level of the genes participating in the anthocyanin biosynthetic pathway was commonly higher in the purple petals than that in the white petals. In particular, two genes, F3′H and DFR, had a significantly higher expression pattern in the purple petals, suggesting the important roles these genes playing in radish petal coloration. BSA-seq aided-Next Generation Sequencing of two DNA pools revealed that the radish purple petal gene (RsPP) was located on chromosome 7. With additional genotyping of 617 F2 population plants, the RsPP was further confined within a region of 93.23 kb. Transcriptome and Sanger sequencing analysis further helped identify the target gene, Rs392880. Rs392880 is a homologous gene to F3′H, a key gene in the anthocyanin biosynthetic pathway. These results will aid in elucidating the molecular mechanism of plant petal coloration and developing strategies to modify flower color through genetic transformation. [ABSTRACT FROM AUTHOR]

Radish ( Raphanus sativus L.) is a vegetable crop with economic value and ecological significance in the genus Radish, family Brassicaceae . In recent years, developed countries have attached great importance to the collection and conservation of radish germplasm resources and their research and utilization, but the lack of population genetic information and molecular markers has hindered the development of the genetic breeding of radish. In this study, we integrated the radish genomic data published in databases for the development of single-nucleotide polymorphism (SNP) markers, and obtained a dataset of 308 high-quality SNPs under strict selection criteria. With the support of Kompetitive Allele-Specific PCR (KASP) technology, we screened a set of 32 candidate core SNP marker sets to analyse the genetic diversity of the collected 356 radish varieties. The results showed that the mean values of polymorphism information content (PIC), minor allele frequency (MAF), gene diversity and heterozygosity of the 32 candidate core SNP markers were 0.32, 0.30, 0.40 and 0.25, respectively. Population structural analysis, principal component analysis and genetic evolutionary tree analysis indicated that the 356 radish materials were best classified into two taxa, and that the two taxa of the material were closely genetically exchanged. Finally, on the basis of 32 candidate core SNP markers we calculated 15 core markers using a computer algorithm to construct a fingerprint map of 356 radish varieties. Furthermore, we constructed a core germplasm population consisting of 71 radish materials using 32 candidate core markers. In this study, we developed SNP markers for radish cultivar identification and genetic diversity analysis, and constructed DNA fingerprints, providing a basis for the identification of radish germplasm resources and molecular marker-assisted breeding as well as genetic research., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Xing, Hu, Wang, Li, Wang, Hu, Wei, Yan, Gan, Bao and Wang.)

Cabbage (Brassica oleracea var. capitata) requires a long-term low-temperature exposure for floral induction, causing a delay in the breeding cycle. The objective of this study is to develop a method to induce flowering in cabbage without low-temperature treatment, using a grafting method. We conducted grafting experiments using two flower-induced Chinese kale cultivars (B. oleracea var. alboglabra) and seven radish cultivars/accessions as rootstocks and investigated the flowering response of grafted cabbage scions without low-temperature treatment. “Watanabe-seiko No.1” cabbage, when grafted onto the two Chinese kale cultivars, did not formed flower buds. Flowering was successfully induced in “Watanabe-seiko No.1” by grafting onto three out of the seven tested radish cultivars, and in “Kinkei No.201” and “Red cabbage” by grafting onto one tested radish cultivar. In “Watanabe-seiko No.1,” the earliest flower bud appearance was observed at 29 days after grafting. Seeds were also obtained from the three cabbage cultivars that flowered by grafting. Gene expression analysis of “Watanabe-seiko No.1” cabbage scions which formed flower buds by grafting, revealed high expression of the homolog of the floral integrator, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (BoSOC1), at the time of flower bud appearance. However, in the same leaf samples, we observed low expression of two homologs of florigen, FLOWERING LOCUS T (BoFT.C2 and BoFT.C6). In addition, two homologs of the floral repressor FLOWERING LOCUS C (BoFLC3 and BoFLC4), which are known to be down-regulated before flower bud differentiation in the vernalization pathway, were highly expressed, indicating that grafting onto radish induces cabbage flowering independently of the vernalization pathway. The expression level of the radish FT homolog (RsFT) in “Rat’s tail-G2,” which had highly induced flowering in the grafted cabbage scion, was higher than in the other radish cultivars. However, although “Rat’s tail-CH” effectively induced flowering in the cabbage scion, the expression of RsFT was low in this cultivar. In this study, floral induction of non-vernalized cabbage cannot be explained by the expression levels of RsFT in rootstock plants, alone. The flowering of non-vernalized cabbage would be induced by transmissible agents from rootstocks and not by the expression of cabbage FT, BoFT, from the scion itself.

Accumulation of anthocyanins in the taproot of radish is an agronomic trait beneficial for human health. Several genetic loci are related to a red skin or flesh color of radish, however, the functional divergence of candidate genes between non-red and red radishes has not been investigated. Here, we report that a novel genetic locus on the R2 chromosome, where RsMYB1 . 1 is located, is associated with the red color of the skin of radish taproot. A genome-wide association study (GWAS) of 66 non-red-skinned (nR) and 34 red-skinned (R) radish accessions identified three nonsynonymous single nucleotide polymorphisms (SNPs) in the third exon of RsMYB1 . 1 . Although the genotypes of SNP loci differed between the nR and R radishes, no functional difference in the RsMYB1.1 proteins of nR and R radishes in their physical interaction with RsTT8 was detected by yeast-two hybrid assay or in anthocyanin accumulation in tobacco and radish leaves coexpressing RsMYB1.1 and RsTT8. By contrast, insertion- or deletion-based GWAS revealed that one large AT-rich low-complexity sequence of 1.3-2 kb was inserted in the promoter region of RsMYB1 . 1 in the nR radishes ( RsMYB1 . 1 nR ), whereas the R radishes had no such insertion; this represents a presence/absence variation (PAV). This insertion sequence (RsIS) was radish specific and distributed among the nine chromosomes of Raphanus genomes. Despite the extremely low transcription level of RsMYB1 . 1 nR in the nR radishes, the inactive RsMYB1 . 1 nR promoter could be functionally restored by deletion of the RsIS. The results of a transient expression assay using radish root sections suggested that the RsIS negatively regulates the expression of RsMYB1 . 1 nR , resulting in the downregulation of anthocyanin biosynthesis genes, including RsCHS , RsDFR , and RsANS , in the nR radishes. This work provides the first evidence of the involvement of PAV in an agronomic trait of radish., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Kim, Jang, Huh, Cho, Yim, Jeong, Kim, Yu and Mun.)

Cabbage (Brassica oleracea var. capitata) requires a long-term low-temperature exposure for floral induction, causing a delay in the breeding cycle. The objective of this study is to develop a method to induce flowering in cabbage without low-temperature treatment, using a grafting method. We conducted grafting experiments using two flower-induced Chinese kale cultivars (B. oleracea var. alboglabra) and seven radish cultivars/accessions as rootstocks and investigated the flowering response of grafted cabbage scions without low-temperature treatment. "Watanabe-seiko No.1" cabbage, when grafted onto the two Chinese kale cultivars, did not formed flower buds. Flowering was successfully induced in "Watanabe-seiko No.1" by grafting onto three out of the seven tested radish cultivars, and in "Kinkei No.201" and "Red cabbage" by grafting onto one tested radish cultivar. In "Watanabe-seiko No.1," the earliest flower bud appearance was observed at 29 days after grafting. Seeds were also obtained from the three cabbage cultivars that flowered by grafting. Gene expression analysis of "Watanabe-seiko No.1" cabbage scions which formed flower buds by grafting, revealed high expression of the homolog of the floral integrator, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (BoSOC1), at the time of flower bud appearance. However, in the same leaf samples, we observed low expression of two homologs of florigen, FLOWERING LOCUS T (BoFT.C2 and BoFT.C6). In addition, two homologs of the floral repressor FLOWERING LOCUS C (BoFLC3 and BoFLC4), which are known to be down-regulated before flower bud differentiation in the vernalization pathway, were highly expressed, indicating that grafting onto radish induces cabbage flowering independently of the vernalization pathway. The expression level of the radish FT homolog (RsFT) in "Rat's tail-G2," which had highly induced flowering in the grafted cabbage scion, was higher than in the other radish cultivars. However, although "Rat's tail-CH" effectively induced flowering in the cabbage scion, the expression of RsFT was low in this cultivar. In this study, floral induction of non-vernalized cabbage cannot be explained by the expression levels of RsFT in rootstock plants, alone. The flowering of non-vernalized cabbage would be induced by transmissible agents from rootstocks and not by the expression of cabbage FT, BoFT , from the scion itself. [ABSTRACT FROM AUTHOR]

The MYB-bHLH-WDR (MBW) complex activates anthocyanin biosynthesis through the transcriptional regulation. RsMYB1 has been identified as a key player in anthocyanin biosynthesis in red radish (Raphanus sativus L.), but its partner bHLH transcription factor (TF) remains to be determined. In this study, we isolated a bHLH TF gene from red radish. Phylogenetic analysis indicated that this gene belongs to the TT8 clade of the IIIF subgroup of bHLH TFs, and we thus designated this gene RsTT8. Subcellular localization analysis showed that RsTT8-sGFP was localized to the nuclei of Arabidopsis thaliana protoplasts harboring the RsTT8-sGFP construct. We evaluated anthocyanin biosynthesis and RsTT8 expression levels in three radish varieties (N, C, and D) that display different red phenotypes in the leaves, root flesh, and root skins. The root flesh of the C variety and the leaves and skins of the D variety exhibit intense red pigmentation; in these tissues, RsTT8 expression showed totally positive association with the expression of RsMYB1 TF and of five of eight tested anthocyanin biosynthesis genes (i.e., RsCHS, RsCHI, RsF3H, RsDFR, and RsANS). Heterologous co-expression of both RsTT8 and RsMYB1 in tobacco leaves dramatically increased the expression of endogenous anthocyanin biosynthesis genes and anthocyanin accumulation. Furthermore, a yeast two-hybrid assay showed that RsTT8 interacts with RsMYB1 at the MYB-interacting region (MIR), and a transient transactivation assay indicated that RsTT8 activates the RsCHS and RsDFR promoters when co-expressed with RsMYB1. Complementation of the Arabidopsis tt8-1 mutant, which lacks red pigmentation in the leaves and seeds, with RsTT8 restored red pigmentation, and resulted in high anthocyanin and proanthocyanidin contents in the leaves and seeds, respectively. Together, these results show that RsTT8 functions as a regulatory partner with RsMYB1 during anthocyanin biosynthesis.

Radish (Raphanus sativus) is an important cruciferous root crop with a close relationship to Chinese cabbage (Brassica rapa). RT-qPCR is used extensively to evaluate the expression levels of target genes, and accurate measurement of target gene expression with this method is determined by the valid reference genes used for data nomalization in different experimental conditions. Screening for appropriate reference genes with stable expression based on RT-qPCR data is important for gene expression and functional analysis research in radish and its relatives. However, many researches have thought that almost no single reference gene is widely suitable for all experimental conditions, and few researchers have paid attention to the validation of reference genes in radish gene expression analysis. In the present study, 12 candidate reference genes were selected for analysis. Their expression in 28 samples, including 20 radish samples from different organs and conditions, four Chinese cabbage organs and four organs of their distant hybrid, was assessed by RT-qPCR and then five software tools—ΔCt, geNorm, NormFinder, BestKeeper and RefFinder—were used to compare their expression stability. The results showed that the most suitable reference genes were different in different organs and conditions. GAPDH, DSS1, and UP2 were optimal reference genes for gene expression analysis in all organs and conditions in radish. UPR, GSNOR1, and ACTIN2/7 were the most stable reference genes in different radish organs. UP2 and GAPDH were suitable reference genes for radish pistil development studies. RPII, UBC9, and GAPDH had the most stable expression in radish under various stresses. DSS1, UP2, and TEF2 were the optimal reference genes for Chinese cabbage organs, whereas TUA was optimal for the distant hybrid. UP2, and TEF2 were appropriate reference genes for all of the samples together. The optimal reference genes we identified, UP2, GAPDH, UPR, and GSNOR1 were verified by normalizing the expression patterns of YAB3, RPL, and FUL. These results will provide important information for selecting target reference genes in different research contexts and improve the accuracy and precision of gene expression analysis for radish, Chinese cabbage and their distant hybrid.

To understand the molecular mechanism underlying salt stress response in radish, iTRAQ-based proteomic analysis was conducted to investigate the differences in protein species abundance under different salt treatments. In total, 851, 706, and 685 differential abundance protein species (DAPS) were identified between CK vs. Na100, CK vs. Na200, and Na100 vs. Na200, respectively. Functional annotation analysis revealed that salt stress elicited complex proteomic alterations in radish roots involved in carbohydrate and energy metabolism, protein metabolism, signal transduction, transcription regulation, stress and defense and transport. Additionally, the expression levels of nine genes encoding DAPS were further verified using RT-qPCR. The integrative analysis of transcriptomic and proteomic data in conjunction with miRNAs was further performed to strengthen the understanding of radish response to salinity. The genes responsible for signal transduction, ROS scavenging and transport activities as well as several key miRNAs including miR171, miR395, and miR398 played crucial roles in salt stress response in radish. Based on these findings, a schematic genetic regulatory network of salt stress response was proposed. This study provided valuable insights into the molecular mechanism underlying salt stress response in radish roots and would facilitate developing effective strategies toward genetically engineered salt-tolerant radish and other root vegetable crops.

Growing microgreens on trays without substrate in a vertical multilayered growing unit offers several advantages over traditional agriculture methods. This study investigated the yield performance and nutritional quality of five selections of radish microgreens grown in sprouting trays, without a substrate using only water, in an indoor multilayer cultivation system using artificial light. Various parameters were measured, including fresh weight, dry matter, chlorophyll, minerals, amino acids, phenolics, flavonoids, anthocyanins, vitamin C, glucosinolates, and antioxidant activity with four different in vitro assays. After ten days, the biomass had increased by 6-10 times, and the dry matter varied from 4.75-7.65%. The highest yield was obtained from 'Asia red', while the lowest was from 'Koregon red'. However, 'Koregon red' and 'Asia red' had the highest dry matter. 'Asia red' was found to have the highest levels of both Chls and vitamin C compared to the other cultivars, while 'Koregon red' exhibited the highest levels of total phenolics and flavonoids. Although variations in the levels of individual glucosinolates were observed, there were no significant differences in the total content of glucosinolates among the five cultivars. 'Asia purple' had the highest anthocyanin content, while 'Asia green 2' had the lowest. The K, Mg, and Na concentrations were significantly highest in 'Asia green 2', and the highest Ca was recorded in 'Asia purple'. Overall, 'Asia purple' and 'Koregon red' were the best cultivars in terms of nutritional quality among the tested radish microgreens. These cultivars exhibited high levels of dry weight, total phenolics, flavonoids, anthocyanins, essential and total amino acids, and antioxidant activities. Moreover, the implementation of this vertical cultivation method for microgreens, which relies solely on water and seeds known for their tall shoots during the sprouting could hold promise as a sustainable approach. This method can effectively be utilized for cultivar screening and fulfilling the nutritional and functional needs of the population while minimizing the environmental impacts associated with traditional agriculture practices., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tilahun, Baek, An, Choi, Lee, Hong and Jeong.)

Lead (Pb) is one of the most abundant heavy metal pollutants, which can penetrate the plant through the root and then enter the food chain causing in potential health risks for human beings. Radish is an important root vegetable crop worldwide. To investigate the mechanism underlying plant response to Pb stress in radish, the protein profile changes of radish roots were comprehensively analyzed using iTRAQ (Isobaric Tag for Relative and Absolute Quantification) respectively upon Pb(NO3)2 at 500 mg L-1(Pb500) and Pb(NO3)2 at 1000 mg L-1(Pb1000) exposure. A total of 3, 898 protein species were successfully detected and 2,141 were quantified. Among them, a subset of 721 protein species were differentially accumulated upon at least one Pb treatment, and 135 ones showed significantly abundance changes during both two Pb-stressed conditions. Many critical protein species related to protein translation, processing, and degradation, reactive oxygen species (ROS) scavenging, photosynthesis and respiration and carbon metabolism were successfully identified. Gene Ontology (GO) and pathway enrichment analysis of the 135 differential abundance protein species revealed that the overrepresented GO terms were include in ‘cell wall’, ‘apoplast’, ‘response to metal ion’, ‘vacuole’ and ‘peroxidase activity’, and the critical enriched pathways were involved in ‘citric acid (TCA) cycle and respiratory electron transport’, ‘pyruvate metabolism’, ‘phenylalanine metabolism’, ‘phenylpropanoid biosynthesis’ and ‘carbon metabolism’. Furthermore, the integrative analysis of transcriptomic, miRNA, degradome, metabolomic and proteomic data provided a strengthened understanding of radish response to Pb stress at multiple levels. Under Pb stress, many key enzymes (i.e. ATP citrate lyase, Isocitrate dehydrogenase, fumarate hydratase and malate dehydrogenase) involved in the glycolysis and TCA cycle were severely affected, which ultimately cause alteration of some metabolites including glucose, citrate, and malate. Meanwhile, a series of other defense responses including ascorbate (ASA)–glutathione (GSH) cycle for ROS scavenging and Pb-defense protein species (glutaredoxin, aldose 1-epimerase malate dehydrogenase and thioredoxin), were triggered to cope with Pb-induced injuries. The results would be helpful for further dissecting molecular mechanism underlying plant response to HM stresses, and facilitate effective management of HM contamination in vegetable crops by genetic manipulation.

The MADS-box gene family is an important transcription factor (TF) family that is involved in various aspects of plant growth and development, especially flowering time and floral organogenesis. Although it has been reported in many plant species, the systematic identification and characterization of MADS-box TF family is still limited in radish (Raphanus sativus L.). In the present study, a comprehensive analysis of MADS-box genes was performed, and a total of 144 MADS-box family members were identified from the whole radish genome. Meanwhile, a detailed list of MADS-box genes from other 28 plant species was also investigated. Through the phylogenetic analysis between radish and Arabidopsis thaliana, all the RsMADS genes were classified into two groups including 68 type I (31 Mα, 12 Mβ and 25Mγ) and 76 type II (70 MIKCC and 6 MIKC*). Among them, 41 (28.47%) RsMADS genes were located in nine linkage groups of radish from R1 to R9. Moreover, the homologous MADS-box gene pairs were identified among radish, A. thaliana, Chinese cabbage and rice. Additionally, the expression profiles of RsMADS genes were systematically investigated in different tissues and growth stages. Furthermore, quantitative real-time PCR analysis was employed to validate expression patterns of some crucial RsMADS genes. These results could provide a valuable resource to explore the potential functions of RsMADS genes in radish, and facilitate dissecting MADS-box gene-mediated molecular mechanisms underlying flowering and floral organogenesis in root vegetable crops.

Reports indicate that silver nanoparticles (nAg) are toxic to vegetation, but little is known about their effects in crop plants. This study examines the impacts of nAg on the physiology and nutritional quality of radish (Raphanus sativus) sprouts. Seeds were germinated and grown for five days in nAg suspensions at 0, 125, 250, and 500 mg/L. Seed germination and seedling growth were evaluated with traditional methodologies; the uptake of Ag and nutrients was quantified by inductively coupled plasma-optical emission spectroscopy (ICP-OES) and changes in macromolecules were analyzed by infrared (IR) spectroscopy. None of the nAg concentrations reduced seed germination. However, the water content (% of the total weight) was reduced by 1.62%, 1.65%, and 2.54% with exposure to 125, 250 and 500 mg/L, respectively, compared with the control. At 500 mg/L, the root and shoot lengths were reduced by 47.7% and 40%, with respect to the control. The seedlings exposed to 500 mg/L had 901±150 mg Ag/kg dry wt and significantly less Ca, Mg, B, Cu, Mn, and Zn, compared with the control. The infrared spectroscopy analysis showed changes in the bands corresponding to lipids (3000 – 2800 cm-1), proteins (1550 - 1530), and structural components of plant cells such as lignin, pectin and cellulose. These results suggest that nAg could significantly affect the growth, nutrient content and macromolecule conformation in radish sprouts, with unknown consequences for human health.

Glucosinolates (GSLs) and their degradation products in radish confer plant defense, promote human health, and generate pungent flavor. However, the intact GSLs in radish have not been investigated comprehensively yet. Here, an accurate qualitative and quantitative analyses of 15 intact GSLs from radish, including four major GSLs of glucoraphasatin (GRH), glucoerucin (GER), glucoraphenin (GRE), and 4-methoxyglucobrassicin (4MGBS), were conducted using UHPLC-HRMS/MS in combination with UHPLC-QqQ-MS/MS. Simultaneously, three isomers of hexyl GSL, 3-methylpentyl GSL, and 4-methylpentyl GSL were identified in radish. The highest content of GSLs was up to 232.46 μmol/g DW at the 42 DAG stage in the 'SQY' taproot, with an approximately 184.49-fold increase compared to the lowest content in another sample. That the GSLs content in the taproots of two radishes fluctuated in a similar pattern throughout the five vegetative growth stages according to the metabolic profiling, whereas the GSLs content in the '55' leaf steadily decreased over the same period. Additionally, the proposed biosynthetic pathways of radish-specific GSLs were elucidated in this study. Our findings will provide an abundance of qualitative and quantitative data on intact GSLs, as well as a method for detecting GSLs, thus providing direction for the scientific progress and practical utilization of GSLs in radish., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yan, Huang, Zhang, Cui, Jiao, Peng, Luo, Liu and Qiu.)

Radish (Raphanus sativus L.) is an important worldwide root vegetable crop with high nutrient values and is adversely affected by non-essential heavy metals including chromium (Cr). Little is known about the molecular mechanism underlying Cr stress response in radish. In this study, RNA-Seq technique was employed to identify differentially expressed genes (DEGs) under Cr stress. Based on de novo transcriptome assembly, there were 30,676 unigenes representing 60,881 transcripts isolated from radish root under Cr stress. Differential gene analysis revealed that 2,985 uingenes were significantly differentially expressed between Cr-free (CK) and Cr-treated (Cr600) libraries, among which 1,424 were up-regulated and 1,561 down-regulated. Gene ontology (GO) analysis revealed that these DEGs were mainly involved in primary metabolic process, response to abiotic stimulus, cellular metabolic process and small molecule metabolic process. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that the DEGs were mainly involved in protein processing in endoplasmic reticulum, starch and sucrose metabolism, amino acid metabolism, glutathione metabolism, drug and xenobiotics by cytochrome P450 metabolism. RT-qPCR analysis showed that the expression patterns of 12 randomly selected DEGs were highly accordant with the results from RNA-seq. Furthermore, many candidate genes including signaling protein kinases, transcription factors and metal transporters, chelate compound biosynthesis and antioxidant system, were involved in defense and detoxification mechanisms of Cr stress response regulatory networks. These results would provide novel insight into molecular mechanism underlying plant responsiveness to Cr stress and facilitate further genetic manipulation on Cr uptake and accumulation in radish.

Reports indicate that silver nanoparticles (nAg) are toxic to vegetation, but little is known about their effects in crop plants. This study examines the impacts of nAg on the physiology and nutritional quality of radish (Raphanus sativus) sprouts. Seeds were germinated and grown for 5 days in nAg suspensions at 0, 125, 250, and 500 mg/L. Seed germination and seedling growth were evaluated with traditional methodologies; the uptake of Ag and nutrients was quantified by inductively coupled plasma-optical emission spectroscopy (ICP-OES) and changes in macromolecules were analyzed by infrared (IR) spectroscopy. None of the nAg concentrations reduced seed germination. However, the water content (% of the total weight) was reduced by 1.62, 1.65, and 2.54% with exposure to 125, 250, and 500 mg/L, respectively, compared with the control. At 500 mg/L, the root and shoot lengths were reduced by 47.7 and 40%, with respect to the control. The seedlings exposed to 500 mg/L had 901 ± 150 mg Ag/kg dry wt and significantly less Ca, Mg, B, Cu, Mn, and Zn, compared with the control. The infrared spectroscopy analysis showed changes in the bands corresponding to lipids (3000-2800 cm-1), proteins (1550-1530 cm-1), and structural components of plant cells such as lignin, pectin, and cellulose. These results suggest that nAg could significantly affect the growth, nutrient content and macromolecule conformation in radish sprouts, with unknown consequences for human health. [ABSTRACT FROM AUTHOR]

Radish ( Raphanus sativus L.) is an important root vegetable crop belonging to the Brassicaceae family. Anthocyanin rich radish varieties are popular among consumers because of their bright color and high nutritional value. However, the underlying molecular mechanism responsible for skin and flesh induce anthocyanin biosynthesis in transient overexpression, gene silencing and transcriptome sequencing were used to verify its function in radish anthocyanin accumulation, radish remains unclear. Here, we identified a long noncoding RNA LINC15957 , overexpression of LINC15957 was significantly increased anthocyanin accumulation in radish leaves, and the expression levels of structural genes related to anthocyanin biosynthesis were also significantly increased. Anthocyanin accumulation and expression levels of anthocyanin biosynthesis genes were significantly reduced in silenced LINC15957 flesh when compared with control. By the transcriptome sequencing of the overexpressed LINC15957 plants and the control, 5,772 differentially expressed genes were identified. A total of 3,849 differentially expressed transcription factors were identified, of which MYB, bHLH, WD40, bZIP, ERF, WRKY and MATE were detected and differentially expressed in the overexpressed LINC15957 plants. KEGG enrichment analysis revealed the genes were significant enriched in tyrosine, L-Phenylalanine, tryptophan, phenylpropanol, and flavonoid biosynthesis. RT-qPCR analysis showed that 8 differentially expressed genes (DEGs) were differentially expressed in LINC15957 -overexpressed plants. These results suggested that LINC15957 involved in regulate anthocyanin accumulation and provide abundant data to investigate the genes regulate anthocyanin biosynthesis in radish., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tan, Luo, Lu, Wu, Li, Jin, Peng, Xu, Li and Zhang.)

Radish ( Raphanus sativus L.) is an economically important vegetable worldwide, but its sustainable production and breeding are highly threatened by blight disease caused by Alternaria brassicae . Melatonin is an important growth regulator that can influence physiological activities in both plants and microbes and stimulate biotic stress resistance in plants. In this study, 0-1500 μM melatonin was exogenously applied to healthy radish seedlings, in vitro incubated A. brassicae , and diseased radish seedlings to determine the effects of melatonin on host, pathogen, and host-pathogen interaction. At sufficient concentrations (0-500 μM), melatonin enhanced growth and immunity of healthy radish seedlings by improving the function of organelles and promoting the biosynthesis of antioxidant enzymes, chitin, organic acid, and defense proteins. Interestingly, melatonin also improved colony growth, development, and virulence of A. brassicae . A strong dosage-dependent effect of melatonin was observed: 50-500 μM promoted host and pathogen vitality and resistance (500 μM was optimal) and 1500 μM inhibited these processes. Significantly less blight was observed on diseased seedlings treated with 500 μM melatonin, indicating that melatonin more strongly enhanced the growth and immunity of radish than it promoted the development and virulence of A. brassicae at this treatment concentration. These effects of MT were mediated by transcriptional changes of key genes as identified by RNA-seq, Dual RNA-seq, and qRT-PCR. The results from this work provide a theoretical basis for the application of melatonin to protect vegetable crops against pathogens., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Li, Huang, Xia, Lu, Xu, Liu and Zhang.)

An ever-increasing population has issued an open challenge to the agricultural sector to provide enough food in a sustainable manner. The upsurge in chemical fertilizers to enhance food production had resulted in environmental problems. The objective of the current study is to assess the utilization of biostimulants for sustainable agricultural production as an alternative to chemical fertilization. For this purpose, two pot experiments were conducted to examine the response of radish against individual and combined applications of biostimulants. In the first experiment, the effects of chemical fertilizer (CK), glycine (G), lysine (L), aspartic acid (A), and vitamin B complex (V) were studied. The results demonstrated that V significantly improved the transpiration rate (81.79%), stomatal conductance (179.17%), fresh weight (478.31%), and moisture content (2.50%). In the second experiment, tested treatments included chemical fertilizer (CK), Isabion ® (I), glycine + lysine + aspartic acid (GLA), moringa leaf extract + GLA (M1), 25% NPK + M1 (M2). The doses of biostimulants were 5g L -1 glycine, 1g L -1 lysine, 2g L -1 aspartic acid, and 10 ml L -1 moringa leaf extract. The photosynthetic rate improved significantly with GLA (327.01%), M1 (219.60%), and M2 (22.16%), while the transpiration rate was enhanced with GLA (53.14%) and M2 (17.86%) compared to the Ck. In addition, M1 increased the stomatal conductance (54.84%), internal CO 2 concentration (0.83%), plant fresh weight (201.81%), and dry weight (101.46%) as compared to CK. This study concludes that biostimulants can effectively contribute to the sustainable cultivation of radish with better growth and yield., Competing Interests: Author YG was employed by KOYO Star Agriculture Technology Co., LTD. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Raza, Bashir, Rehim, Ejaz, Raza, Shahzad, Ahmed and Geng.)

Aquaporins (AQPs) constitute a highly diverse family of channel proteins that transport water and neutral solutes. AQPs play crucial roles in plant development and stress responses. However, the characterization and biological functions of RsAQPs in radish ( Raphanus sativus L.) remain elusive. In this study, 61 non-redundant members of AQP-encoding genes were identified from the radish genome database and located on nine chromosomes. Radish AQPs (RsAQPs) were divided into four subfamilies, including 21 plasma membrane intrinsic proteins (PIPs), 19 tonoplast intrinsic proteins (TIPs), 16 NOD-like intrinsic proteins (NIPs), and 5 small basic intrinsic proteins (SIPs), through phylogenetic analysis. All RsAQPs contained highly conserved motifs (motifs 1 and 4) and transmembrane regions, indicating the potential transmembrane transport function of RsAQPs. Tissue- and stage-specific expression patterns of AQP gene analysis based on RNA-seq data revealed that the expression levels of PIPs were generally higher than TIPs , NIPs , and SIPs in radish. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) revealed that seven selected RsPIPs , according to our previous transcriptome data (e.g., RsPIP1-3 , 1-6 , 2-1 , 2-6 , 2-10 , 2-13 , and 2-14 ), exhibited significant upregulation in roots of salt-tolerant radish genotype. In particular, the transcriptional levels of RsPIP2-6 dramatically increased after 6 h of 150 mM NaCl treatment during the taproot thickening stage. Additionally, overexpression of RsPIP2-6 could enhance salt tolerance by Agrobacterium rhizogenes -mediated transgenic radish hairy roots, which exhibited the mitigatory effects of plant growth reduction, leaf relative water content (RWC) reduction and alleviation of O 2- in cells, as shown by nitro blue tetrazolium (NBT) staining, under salt stress. These findings are helpful for deeply dissecting the biological function of RsAQPs on the salt stress response, facilitating practical application and genetic improvement of abiotic stress resistance in radish., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Yi, Sun, Tian, Li, Ni, Ying, Xu, Liu and Wang.)

Anthocyanins are water-soluble plant pigments mainly stored in the plant vacuoles. Glutathione S-transferases (GSTs) are a multifunctional enzyme family, which can regulate substance metabolism and biological and abiotic stresses in plants. However, few reports were focused on the involvement of GSTs in anthocyanin sequestration in red skin radish. Here, we identified a glutathione S-transferase gene RsGSTF12 that played roles in anthocyanin sequestration in radish. The bioinformatics analysis revealed that RsGSTF12 belonged to the phi (F) class of glutathione S-transferases and showed a high homology with AtGSTF12, followed by AtGSTF11. The subcellular localization assay showed that RsGSTF12 was located in the endoplasmic reticulum and tonoplast. Temporal and spatial gene expression-specific analyses uncovered a strong correlation of RsGSTF12 with anthocyanin accumulation in radish sprouts. The anthocyanin solubility assay found RsGSTF12 was capable of improving cyanidin water solubility in vitro . Transiently expressing RsGSTF12 in radish cotyledons was able to increase their anthocyanin sequestrations. Furthermore, the functional complementation and overexpression of the Arabidopsis thaliana tt19 mutant and wild type demonstrated that RsGSTF12 might play an indispensable role in anthocyanin accumulation in radish. Taken together, we provide compelling evidence that RsGSTF12 functions critically in how anthocyanins are sequestrated in radish, which may enrich our understanding of the mechanism of anthocyanin sequestration., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Niu, Bao, Chen, Zhou, Zhang, Zhang, Su and Cui.)

The MYB-bHLH-WDR (MBW) complex activates anthocyanin biosynthesis through the transcriptional regulation. RsMYB1 has been identified as a key player in anthocyanin biosynthesis in red radish ( Raphanus sativus L.), but its partner bHLH transcription factor (TF) remains to be determined. In this study, we isolated a bHLH TF gene from red radish. Phylogenetic analysis indicated that this gene belongs to the TT8 clade of the IIIF subgroup of bHLH TFs, and we thus designated this gene RsTT8 . Subcellular localization analysis showed that RsTT8-sGFP was localized to the nuclei of Arabidopsis thaliana protoplasts harboring the RsTT8-sGFP construct. We evaluated anthocyanin biosynthesis and RsTT8 expression levels in three radish varieties (N, C, and D) that display different red phenotypes in the leaves, root flesh, and root skins. The root flesh of the C variety and the leaves and skins of the D variety exhibit intense red pigmentation; in these tissues, RsTT8 expression showed totally positive association with the expression of RsMYB1 TF and of five of eight tested anthocyanin biosynthesis genes (i.e., RsCHS, RsCHI, RsF3H, RsDFR , and RsANS ). Heterologous co-expression of both RsTT8 and RsMYB1 in tobacco leaves dramatically increased the expression of endogenous anthocyanin biosynthesis genes and anthocyanin accumulation. Furthermore, a yeast two-hybrid assay showed that RsTT8 interacts with RsMYB1 at the MYB-interacting region (MIR), and a transient transactivation assay indicated that RsTT8 activates the RsCHS and RsDFR promoters when co-expressed with RsMYB1. Complementation of the Arabidopsis tt8-1 mutant, which lacks red pigmentation in the leaves and seeds, with RsTT8 restored red pigmentation, and resulted in high anthocyanin and proanthocyanidin contents in the leaves and seeds, respectively. Together, these results show that RsTT8 functions as a regulatory partner with RsMYB1 during anthocyanin biosynthesis.

Radish ( Raphanus sativus ) is an important cruciferous root crop with a close relationship to Chinese cabbage ( Brassica rapa ). RT-qPCR is used extensively to evaluate the expression levels of target genes, and accurate measurement of target gene expression with this method is determined by the valid reference genes used for data nomalization in different experimental conditions. Screening for appropriate reference genes with stable expression based on RT-qPCR data is important for gene expression and functional analysis research in radish and its relatives. However, many researches have thought that almost no single reference gene is widely suitable for all experimental conditions, and few researchers have paid attention to the validation of reference genes in radish gene expression analysis. In the present study, 12 candidate reference genes were selected for analysis. Their expression in 28 samples, including 20 radish samples from different organs and conditions, four Chinese cabbage organs and four organs of their distant hybrid, was assessed by RT-qPCR and then five software tools-ΔCt, geNorm, NormFinder, BestKeeper and RefFinder-were used to compare their expression stability. The results showed that the most suitable reference genes were different in different organs and conditions. GAPDH, DSS1 , and UP2 were optimal reference genes for gene expression analysis in all organs and conditions in radish. UPR, GSNOR1 , and ACTIN2/7 were the most stable reference genes in different radish organs. UP2 and GAPDH were suitable reference genes for radish pistil development studies. RPII, UBC9 , and GAPDH had the most stable expression in radish under various stresses. DSS1, UP2 , and TEF 2 were the optimal reference genes for Chinese cabbage organs, whereas TUA was optimal for the distant hybrid. UP2 , and TEF 2 were appropriate reference genes for all of the samples together. The optimal reference genes we identified, UP2, GAPDH, UPR , and GSNOR1 . These results will provide important information for selecting target reference genes in different research contexts and improve the accuracy and precision of gene expression analysis for radish, Chinese cabbage and their distant hybrid.YAB3, RPL , and FUL . These results will provide important information for selecting target reference genes in different research contexts and improve the accuracy and precision of gene expression analysis for radish, Chinese cabbage and their distant hybrid.

To understand the molecular mechanism underlying salt stress response in radish, iTRAQ-based proteomic analysis was conducted to investigate the differences in protein species abundance under different salt treatments. In total, 851, 706, and 685 differential abundance protein species (DAPS) were identified between CK vs. Na100, CK vs. Na200, and Na100 vs. Na200, respectively. Functional annotation analysis revealed that salt stress elicited complex proteomic alterations in radish roots involved in carbohydrate and energy metabolism, protein metabolism, signal transduction, transcription regulation, stress and defense and transport. Additionally, the expression levels of nine genes encoding DAPS were further verified using RT-qPCR. The integrative analysis of transcriptomic and proteomic data in conjunction with miRNAs was further performed to strengthen the understanding of radish response to salinity. The genes responsible for signal transduction, ROS scavenging and transport activities as well as several key miRNAs including miR171, miR395, and miR398 played crucial roles in salt stress response in radish. Based on these findings, a schematic genetic regulatory network of salt stress response was proposed. This study provided valuable insights into the molecular mechanism underlying salt stress response in radish roots and would facilitate developing effective strategies toward genetically engineered salt-tolerant radish and other root vegetable crops.

Lead (Pb) is one of the most abundant heavy metal (HM) pollutants, which can penetrate the plant through the root and then enter the food chain causing potential health risks for human beings. Radish is an important root vegetable crop worldwide. To investigate the mechanism underlying plant response to Pb stress in radish, the protein profile changes of radish roots respectively upon Pb(NO 3 ) 2 at 500 mg L -1 (Pb500) and 1000 mg L -1 (Pb1000), were comprehensively analyzed using iTRAQ (Isobaric Tag for Relative and Absolute Quantification). A total of 3898 protein species were successfully detected and 2141 were quantified. Among them, a subset of 721 protein species were differentially accumulated upon at least one Pb treatment, and 135 ones showed significantly abundance changes under both two Pb-stressed conditions. Many critical protein species related to protein translation, processing, and degradation, reactive oxygen species (ROS) scavenging, photosynthesis, and respiration and carbon metabolism were successfully identified. Gene Ontology (GO) and pathway enrichment analysis of the 135 differential abundance protein species (DAPS) revealed that the overrepresented GO terms included "cell wall," "apoplast," "response to metal ion," "vacuole," and "peroxidase activity," and the critical enriched pathways were involved in "citric acid (TCA) cycle and respiratory electron transport," "pyruvate metabolism," "phenylalanine metabolism," "phenylpropanoid biosynthesis," and "carbon metabolism." Furthermore, the integrative analysis of transcriptomic, miRNA, degradome, metabolomics and proteomic data provided a strengthened understanding of radish response to Pb stress at multiple levels. Under Pb stress, many key enzymes (i.e., ATP citrate lyase, Isocitrate dehydrogenase, fumarate hydratase and malate dehydrogenase) involved in the glycolysis and TCA cycle were severely affected, which ultimately cause alteration of some metabolites including glucose, citrate and malate. Meanwhile, a series of other defense responses including ascorbate (ASA)-glutathione (GSH) cycle for ROS scavenging and Pb-defense protein species (glutaredoxin, aldose 1-epimerase malate dehydrogenase and thioredoxin), were triggered to cope with Pb-induced injuries. These results would be helpful for further dissecting molecular mechanism underlying plant response to HM stresses, and facilitate effective management of HM contamination in vegetable crops by genetic manipulation.

The MADS-box gene family is an important transcription factor (TF) family that is involved in various aspects of plant growth and development, especially flowering time and floral organogenesis. Although it has been reported in many plant species, the systematic identification and characterization of MADS-box TF family is still limited in radish ( Raphanus sativus L.). In the present study, a comprehensive analysis of MADS-box genes was performed, and a total of 144 MADS-box family members were identified from the whole radish genome. Meanwhile, a detailed list of MADS-box genes from other 28 plant species was also investigated. Through the phylogenetic analysis between radish and Arabidopsis thaliana , all the RsMADS genes were classified into two groups including 68 type I (31 Mα, 12 Mβ and 25Mγ) and 76 type II (70 MIKC C and 6 MIKC ∗ ). Among them, 41 (28.47%) RsMADS genes were located in nine linkage groups of radish from R1 to R9. Moreover, the homologous MADS-box gene pairs were identified among radish, A. thaliana , Chinese cabbage and rice. Additionally, the expression profiles of RsMADS genes were systematically investigated in different tissues and growth stages. Furthermore, quantitative real-time PCR analysis was employed to validate expression patterns of some crucial RsMADS genes. These results could provide a valuable resource to explore the potential functions of RsMADS genes in radish, and facilitate dissecting MADS-box gene-mediated molecular mechanisms underlying flowering and floral organogenesis in root vegetable crops.

Radish (Raphanus sativus L.) is an important worldwide root vegetable crop with high nutrient values and is adversely affected by non-essential heavy metals including chromium (Cr). Little is known about the molecular mechanism underlying Cr stress response in radish. In this study, RNA-Seq technique was employed to identify differentially expressed genes (DEGs) under Cr stress. Based on de novo transcriptome assembly, there were 30,676 unigenes representing 60,881 transcripts isolated from radish root under Cr stress. Differential gene analysis revealed that 2985 uingenes were significantly differentially expressed between Cr-free (CK) and Cr-treated (Cr600) libraries, among which 1424 were up-regulated and 1561 down-regulated. Gene ontology (GO) analysis revealed that these DEGs were mainly involved in primary metabolic process, response to abiotic stimulus, cellular metabolic process and small molecule metabolic process. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that the DEGs were mainly involved in protein processing in endoplasmic reticulum, starch and sucrose metabolism, amino acid metabolism, glutathione metabolism, drug and xenobiotics by cytochrome P450 metabolism. RT-qPCR analysis showed that the expression patterns of 12 randomly selected DEGs were highly accordant with the results from RNA-seq. Furthermore, many candidate genes including signaling protein kinases, transcription factors and metal transporters, chelate compound biosynthesis and antioxidant system, were involved in defense and detoxification mechanisms of Cr stress response regulatory networks. These results would provide novel insight into molecular mechanism underlying plant responsiveness to Cr stress and facilitate further genetic manipulation on Cr uptake and accumulation in radish.