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CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) peptides are a class of small molecules involved in plant growth and development. Although radish (Raphanus sativus) is an important root vegetable crop worldwide, the functions of CLE peptides in its taproot formation remain elusive. Here, a total of 48 RsCLE genes were identified from the radish genome. RNA in situ hybridization showed that RsCLE22a gene was highly expressed in the vascular cambium. Overexpression of RsCLE22a inhibited root growth by impairing stem cell proliferation in Arabidopsis, and radish plants with exogenous supplementation of RsCLE22 peptide (CLE22p) showed a similar phenotype. The vascular cambial activity was increased in RsCLE22a -silenced plants. Transcriptome analysis revealed that CLE22p altered the expression of several genes involved in meristem development and hormone signal transduction in radish. Immunolocalization results showed that CLE22p increased auxin accumulation in vascular cambium. Yeast one-hybrid and dual-luciferase assays showed that the WUSCHEL-RELATED HOMEOBOX 4 (RsWOX4) binds to RsCLE22a promoter and activates its transcription. The expression level of RsWOX4 was related to vascular cambial activity and was regulated by auxin. Furthermore, a RsCLE22a–RsWOX4 module is proposed to regulate taproot vascular cambium activity through an auxin signaling-related pathway in radish. These findings provide novel insights into the regulation of root growth in a horticultural crop. [ABSTRACT FROM AUTHOR]

The green radish (Raphanus sativus L.) contains abundant chlorophyll (Chl). DOF-type transcription factor OBF BINDING PROTEIN (OBP) plays crucial functions in plant growth, development, maturation and responses to various abiotic stresses. However, the metabolism by which OBP transcription factors regulate light-induced Chl metabolism in green radish is not well understood. In this study, six OBP genes were identified from the radish genome, distributed unevenly across five chromosomes. Among these genes, RsOBP2a showed significantly higher expression in the green flesh compared to the white flesh of green radish. Analysis of promoter elements suggested that RsOBPs might be involved in stress responses, particularly in light-related processes. Overexpression of RsOBP2a led to increase Chl levels in cotyledons and adventitious roots of radish, while silencing RsOBP2a expression through TYMV-induced gene silencing accelerated leaf senescence. Further investigations revealed that RsOBP2a was localized in the nucleus and served as a transcriptional repressor. RsOBP2a was induced by light and directly suppressed the expression of STAYGREEN (SGR) and RED CHLOROPHYLL CATABOLITE REDUCTASE (RCCR), thereby delaying senescence in radish. Overall, a novel regulatory model involving RsOBP2a, RsSGR, and RsRCCR was proposed to govern Chl metabolism in response to light, offering insights for the enhancement of green radish germplasm. [ABSTRACT FROM AUTHOR]

Homeodomain‐leucine zipper (HD‐Zip) transcription factors are involved in various biological processes of plant growth, development, and abiotic stress response. However, how they regulate heat stress (HS) response remains largely unclear in plants. In this study, a total of 83 RsHD‐Zip genes were firstly identified from the genome of Raphanus sativus. RNA‐Seq, RT‐qPCR and promoter activity assays revealed that RsHDZ17 from HD‐Zip Class I was highly expressed under heat, salt, and Cd stresses. RsHDZ17 is a nuclear protein with transcriptional activity at the C‐terminus. Ectopic overexpression (OE) of RsHDZ17 in Arabidopsis thaliana enhanced the HS tolerance by improving the survival rate, photosynthesis capacity, and scavenging for reactive oxygen species (ROS). In addition, transient OE of RsHDZ17 in radish cotyledons impeded cell injury and augmented ROS scavenging under HS. Moreover, yeast one‐hybrid, dual‐luciferase assay, and electrophoretic mobility shift assay revealed that RsHDZ17 could bind to the promoter of HSFA1e. Collectively, these pieces of evidence demonstrate that RsHDZ17 could play a positive role in thermotolerance, partially through up‐regulation of the expression of HSFA1e in plants. These results provide novel insights into the role of HD‐Zips in radish and facilitate genetical engineering and development of heat‐tolerant radish in breeding programs. [ABSTRACT FROM AUTHOR]

Melatonin (MT) is a tryptophan-derived natural product that plays a vital role in plant response to abiotic stresses, including heavy metals (HMs). However, it remains elusive how exogenous MT mediates lead (Pb) accumulation and detoxification at the methylation and transcriptional levels in radish. In this study, decreased Pb accumulation and increased antioxidant enzyme activity were detected under MT treatment in radish. Single-base resolution maps of DNA methylation under Pb stress (Pb200) and Pb plus MT treatment (Pb_50MT) were first generated. The genome-wide methylation level was increased under Pb stress, while an overall loss of DNA methylation was observed under MT treatment. The differentially methylated region (DMR)-associated genes between Pb_50MT and Pb200 were uniquely enriched in ion binding terms, including cation binding, iron ion binding, and transition metal ion binding. Hyper-DMRs between Pb200 and Control exhibited a decreasing trend of methylation under Pb_50MT treatment. A few critical upregulated antioxidant genes (e.g., RsAPX2, RsPOD52 and RsGST) exhibited decreased methylation levels under MT treatment, which enabled the radish plants to scavenge lead-induced reactive oxygen species (ROS) and decrease oxidative stress. Notably, several MT-induced HM transporter genes with low methylation (e.g., RsABCF5, RsYSL7 and RsHMT) and transcription factors (e.g., RsWRKY41 and RsMYB2) were involved in reducing Pb accumulation in radish roots. These findings could facilitate comprehensive elucidation of the molecular mechanism underlying MT-mediated Pb accumulation and detoxification in radish and other root vegetable crops. [ABSTRACT FROM AUTHOR]

Background: Taproot is the main edible organ and ultimately determines radish yield and quality. However, the precise molecular mechanism underlying taproot thickening awaits further investigation in radish. Here, RNA-seq was performed to identify critical genes involved in radish taproot thickening from three advanced inbred lines with different root size. Results: A total of 2606 differentially expressed genes (DEGs) were shared between 'NAU-DY' (large acicular) and 'NAU-YB' (medium obovate), which were significantly enriched in 'phenylpropanoid biosynthesis', 'glucosinolate biosynthesis', and 'starch and sucrose metabolism' pathway. Meanwhile, a total of 16 differentially expressed miRNAs (DEMs) were shared between 'NAU-DY' and 'NAU-YH' (small circular), whereas 12 miRNAs exhibited specific differential expression in 'NAU-DY'. Association analysis indicated that miR393a-bHLH77, miR167c-ARF8, and miR5658-APL might be key factors to biological phenomenon of taproot type variation, and a putative regulatory model of taproot thickening and development was proposed. Furthermore, several critical genes including SUS1, EXPB3, and CDC5 were characterized and profiled by RT-qPCR analysis. Conclusion: This integrated study on the transcriptional and post-transcriptional profiles could provide new insights into comprehensive understanding of the molecular regulatory mechanism underlying taproot thickening in root vegetable crops. [ABSTRACT FROM AUTHOR]

Cadmium (Cd) is an environmental pollutant that causes health hazard to living organisms. Melatonin (MT) has emerged as a ubiquitous pleiotropic molecule capable of coordinating heavy metal (HM) stresses in plants. However, it remains unclear how melatonin mediates Cd homeostasis and detoxification at transcriptional and/or post‐transcriptional levels in radish. Herein, the activities of five key antioxidant enzymes were increased, while root and shoot Cd contents were dramatically decreased by melatonin. A combined small RNA and transcriptome sequencing analysis showed that 14 differentially expressed microRNAs (DEMs) and 966 differentially expressed genes (DEGs) were shared between the Cd and Cd + MT conditions. In all, 23 and ten correlated miRNA‐DEG pairs were identified in Con vs. Cd and Con vs. Cd + MT comparisons, respectively. Several DEGs encoding yellow stripe 1‐like (YSL), heavy metal ATPases (HMA), and ATP‐binding cassette (ABC) transporters were involved in Cd transportation and sequestration in radish. Root exposure to Cd2+ induced several specific signaling molecules, which consequently trigger some HM chelators, transporters, and antioxidants to achieve reactive oxygen species (ROS) scavenging and detoxification and eliminate Cd toxicity in radish plants. Notably, transgenic analysis revealed that overexpression of the RsMT1 (Metallothionein 1) gene could enhance Cd tolerance of tobacco plants, indicating that the exogenous melatonin confers Cd tolerance, which might be attributable to melatonin‐mediated upregulation of RsMT1 gene in radish plants. These results could contribute to dissecting the molecular basis governing melatonin‐mediated Cd stress response in plants and pave the way for high‐efficient genetically engineering low‐Cd‐content cultivars in radish breeding programs. [ABSTRACT FROM AUTHOR]

Radish (Raphanus sativus L.) is an economically important and widely cultivated root vegetable crop. The coloration of the green skin and green flesh is an important trait influencing the nutrition and flavor quality in fruit radish. GOLDEN2-LIKEs (GLKs) play critically important roles in plastid development and chlorophyll biosynthesis in plants. However, the molecular mechanism underlying chlorophyll biosynthesis still remain elusive in green fruit radish taproot. Herein, the RsGLK2.1 gene exhibited higher expression level in taproot with a green skin (GS) and green flesh (GF) than that in taproot of the white or red radish genotypes. RsGLK2.1 is a nuclear transcription factor that has intrinsic transcriptional activation activity. Overexpression of RsGLK2.1 increased the total chlorophyll content of 20.68%–45.84% in radish leaves. Knockout of the RsGLK2.1 gene via CRISPR/Cas9 technology resulted in a significant decrease in the chlorophyll content. Overexpression of the RsGLK2.1 gene could restore the phenotype of the glk1glk2 mutant Arabidopsis. RsGLK2.1 was participated in regulating the chlorophyll biosynthesis by directly binding to the promoter of RsHEMA2 and activating its transcription. The interaction of RsNF-YA9a with RsGLK2.1 increased the transcriptional activity of the downstream gene RsHEMA2 under the light condition rather than the dark condition, indicating that both of them regulate the chlorophyll biosynthesis in a light-dependent manner of radish. Overall, these results provided insights into the molecular framework of the RsGLK2.1-RsNF-YA9a module, and could facilitate dissecting the regulatory mechanism underlying chlorophyll biosynthesis in green taproot of radish, and genetic improvement of quality traits in fruit radish breeding programs. • RsGLK2.1 exhibits higher expression level in green taproot than that in white and red radish. • Overexpression of RsGLK2.1 increased the total chlorophyll content in radish leaves. • Knock-down and knock-out of the RsGLK2.1 decreased the chlorophyll content in radish. • The interaction of RsNF-YA9a with RsGLK2.1 positively regulated chlorophyll biosynthesis. [ABSTRACT FROM AUTHOR]

• The identified 65 Aux/IAA genes show an uneven chromosomal distribution in radish. • Six Aux/IAA genes exhibit specifically high expression level in radish root tissues. • RsIAA33 gene might involve in promoting taproot thickening and development in radish. Auxin/indole–3–acetic acid (Aux/IAA) genes encode short lived nuclear proteins that cooperated with auxin or auxin response factor (ARF), which are involved in plant growth and developmental processes. However, it's still ambiguous how the Aux/IAA genes regulate the process governing taproot thickening in radish. Herein, 65 Aux/IAA genes were identified from the radish genome. Gene duplication analysis showed that two pairs of tandem duplication and 17 (27%) segmental duplication events were identified among Aux/IAA family genes in radish. Transcriptomic analysis revealed that most of Aux/IAA genes (52/65) exhibited differential expression pattern in different root tissues, and six root–specific genes were highly expressed in root cortex, cambium, xylem, and root tip in radish. RT–qPCR analysis showed that the expression level of RsIAA33 was the highest at cortex splitting stage (CSS), and early expanding stage (ES). Furthermore, amiRNA–mediated gene silencing of RsIAA33 indicated that it could inhibit the reproductive growth, thus promoting taproot thickening and development. These results would provide valuable information for elucidating the molecular function of Aux/IAA genes involved in taproot thickening in radish. [ABSTRACT FROM AUTHOR]

The CPA (cation proton antiporter) family plays an essential role during plant stress tolerance by regulating ionic and pH homeostasis of the cell. Radish fleshy roots are susceptible to abiotic stress during growth and development, especially salt stress. To date, CPA family genes have not yet been identified in radish and the biological functions remain unclear. In this study, 60 CPA candidate genes in radish were identified on the whole genome level, which were divided into three subfamilies including the Na+/H+ exchanger (NHX), K+ efflux antiporter (KEA), and cation/H+ exchanger (CHX) families. In total, 58 of the 60 RsCPA genes were localized to the nine chromosomes. RNA-seq. data showed that 60 RsCPA genes had various expression levels in the leaves, roots, cortex, cambium, and xylem at different development stages, as well as under different abiotic stresses. RT–qPCR analysis indicated that all nine RsNHXs genes showed up regulated trends after 250 mM NaCl exposure at 3, 6, 12, and 24h. The RsCPA31 (RsNHX1) gene, which might be the most important members of the RsNHX subfamily, exhibited obvious increased expression levels during 24h salt stress treatment. Heterologous over-and inhibited-expression of RsNHX1 in Arabidopsis showed that RsNHX1 had a positive function in salt tolerance. Furthermore, a turnip yellow mosaic virus (TYMV)-induced gene silence (VIGS) system was firstly used to functionally characterize the candidate gene in radish, which showed that plant with the silence of endogenous RsNHX1 was more susceptible to the salt stress. According to our results we provide insights into the complexity of the RsCPA gene family and a valuable resource to explore the potential functions of RsCPA genes in radish. [ABSTRACT FROM AUTHOR]

Radish (Raphanus sativus L.) taproot contains high concentrations of flavonoids, including anthocyanins (ATCs), in red-skinned genotypes. However, little information on the genetic regulation of ATC biosynthesis in radish is available. A genome-wide association study of radish red skin color was conducted using whole-genome sequencing data derived from 179 radish genotypes. The R2R3-MYB transcription factor production of anthocyanin pigment 2 (PAP2) gene was found in the region associated with a leading SNP located on chromosome 2. The amino acid sequence encoded by the RsPAP2 gene was different from those of the other published RsMYB genes responsible for the red skin color of radish. The overexpression of the RsPAP2 gene resulted in ATC accumulation in Arabidopsis and radish, which was accompanied by the upregulation of several ATC-related structural genes. RsPAP2 was found to bind the RsUFGT and RsTT8 promoters, as shown by a dual-luciferase reporter system and a yeast one-hybrid assay. The promoter activities of the RsANS, RsCHI, RsPAL, and RsUFGT genes could be strongly activated by coinfiltration with RsPAP2 and RsTT8. These findings showed the effectiveness of GWAS in identifying candidate genes in radish and demonstrated that RsPAP2 could (either directly or together with its cofactor RsTT8) regulate the transcript levels of ATC-related genes to promote ATC biosynthesis, facilitating the genetic enhancement of ATC contents and other related traits in radish. [ABSTRACT FROM AUTHOR]

• Agrobacterium -mediated in planta transformation system was effectively established in radish. • RsWUSb might play an important role in shoot formation and development. • RsRR18-1-RsWUSb module regulates shoot development through CTK signaling-related pathway. The WUSCHEL-related homeobox (WOX) family genes are involved in cell division and differentiation throughout various stages of plant growth and development. Till now, the function of WOX genes in radish remains unclear. In this study, a total of 23 RsWOX genes were identified from the radish genome. RT-qPCR analysis showed that the expression level of RsWUSb was significantly increased with exogenous cytokinin treatment. Overexpression of RsWUSb in Arabidopsis resulted in apical shoot clustering. Notably, the RsWUSb -overexpressed radish plants were generated by Agrobacterium- mediated in planta transformation approach. Compared with the wide-type (WT) plants, the RsWUSb -OE plants displayed clustered apical shoots, more leaves, and weak apical dominance. Moreover, yeast one-hybrid (Y1H) and dual-luciferase assay (DLA) showed that the RsRR18-1 (RESPONSE REGULATOR 18-1) bound to the promoter of RsWUSb and upregulated its transcription. Furthermore, the expression of RsRR18-1 was significantly activated by exogenous cytokinin, suggesting that cytokinin signaling might regulate the formation and development of radish shoots via RsRR18-1-RsWUSb module. Collectively, these findings indicated that in planta transformation approach is a simple and effective method for generating transgenic plants and gene function validation in radish. [ABSTRACT FROM AUTHOR]

• An efficient transformation system with A. rhizogenes was established in radish. • RsMYB90 promoted anthocyanin accumulation in transgenic hairy roots. • RsMYB90 - RsUFGT module positively regulated the biosynthesis of anthocyanin. Radish (Raphanus sativus L.) is recalcitrant to regeneration and genetic transformation, which has severely limited its gene function verification and germplasm innovation. To overcome the limitation, we have developed a rapid and stable regeneration and transformation of hairy roots in radish to explore the potential function of the critical genes involved in the anthocyanin biosynthesis via the optimization of explant selection, bacteria solution concentration, acetosyrinone (AS) concentration and strain types. The single factor experimental designing method was applied and each treatment was conducted for three independent biological replicates. The hairy roots induction rate was more than 90% when using cotyledon with petiole or rootless seedling as explants, while only 53.33% hypocotyls could induce hairy roots. Phenotypic observation and molecular identification indicated that the highest positive transgenic rate of hairy roots could reach approximately 17.51%, when employed optimal optical density (OD 600) and AS concentration at 1.0 and 300 μM, respectively, as well as the best strain of MSU440. Subsequently, RsMYB90 was found to play a positive role in the anthocyanin accumulation through the established hairy root transgenic system, and a number of anthocyanin-related genes, such as RsUFGT, RsCHS and RsF3H , were up-regulated by RNA-seq and RT-qPCR analysis. In addition, yeast one-hybrid (Y1H) and dual-luciferase assays (DLA) showed that the RsMYB90 could bind to the promoter of RsUFGT to activate its expression. Taken together, the stable A. rhizogenes -mediated transformation system provides an avenue for gene function assay, genetic engineering and study of secondary metabolisms in radish. [ABSTRACT FROM AUTHOR]

Highlights • MeJA and SA have positive effects on GLS accumulation in radish taproot. • Differential expression of genes was observed under MeJA, SA and ABA treatments. • Myrosinase activity and SF content were highly sensitive to SA. • SF is correlated with myrosinase activity and GRA content. Abstract Radish is an important root vegetable crop. The effects of environmental stresses on glucosinolate (GLS) contents were extensively investigated in Brassicaceae species. However, little is known about the accumulation of GLS and sulforaphane (SF) in hormone-treated radish taproot. In this study, the effects of methyl jasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA) on the accumulation of GLS and SF in radish taproot were investigated. The GLS content increased considerably in response to MeJA treatment. Moreover, MeJA also up-regulated the expression level of several transcription factors and GLS biosynthesis related genes. The effects of SA on GLS content were less obvious, while ABA suppressed GLS biosynthesis. Additionally, the SF content increased under treatments with MeJA, SA and ABA. Furthermore, changes in myrosinase activity and glucoraphanin (GRA) content after the application of MeJA, SA and ABA were consistent with the changes in SF contents. The results indicated that the MeJA represents one of the most effective plant growth regulators (PGRs) for increasing the GLS content in radish taproot by up-regulating the expression levels of a few GLS biosynthetic related genes. These findings also implied that SF formation is correlated with myrosinase activity and GRA content in radish taproot. [ABSTRACT FROM AUTHOR]

Radish is an economical cool-season root vegetable crop worldwide. Heat shock protein 70 (HSP70) plays indispensable roles in plant growth, development and abiotic stress responses. Nevertheless, little information is available regarding the identification and functional characterization of HSP70 gene family in radish. Herein, a total of 34 RsHSP70 genes were identified at the radish genome level, among which nine and 25 RsHSP70s were classified into the HSP110/SSE and DnaK subfamilies, respectively. RNA-seq analysis revealed that some RsHSP70 genes had differential expression profile in radish leaf, root, stamen and pistil. A range of RsHSP70 genes exhibited differential expression under several abiotic stresses such as heat, salt and heavy metals. Intriguingly, the expression of four RsHSP70 genes (RsHSP70-7 , RsHSP70-12 , RsHSP70-20 and RsHSP70-22) was dramatically up-regulated under heat stress (HS). RT-qPCR and transient LUC reporter assay indicated that both the expression and promoter activity of RsHSP70-20 was strongly induced by HS. Notably, overexpression of RsHSP70-20 significantly enhanced thermotolerance by decreasing reactive oxygen species and promoting proline accumulation in radish, whereas its knock-down plants exhibited increased thermosensitivity, indicating that RsHSP70-20 positively regulate HS response in radish. These results would provide valuable information to decipher the molecular basis of RsHSP70 -mediated thermotolerance in radish. [Display omitted] • RsHSP70 gene family was systematically characterized at genome-wide level in radish. • Expression and promoter activity of RsHSP70-20 was markedly induced by heat stress. • Transgenic analysis reveals that RsHSP70-20 enhanced thermotolerance of radish plant. [ABSTRACT FROM AUTHOR]

• Totally 42 members of the Histone gene family were firstly identified in radish. • Seven RsCENH3 genes exhibited specifically high expression level in the radish bud. • RsCENH3 gene might be involved in promoting microspore development in radish. Histone, a predominant protein component of chromatin, participates in DNA packaging and transcriptional regulation. However, the available information of Histone gene family is limited in radish. In this study, a total of 42 Histone gene family members were identified from the radish genome. Sequence alignment and phylogenetic analyses classified the Histone family into three groups (H2A, H2B and H3). Motif analysis showed that the functions of some motifs shared by H3 subfamily genes were related to chromosome regulation and cell development activities, such as motif 5 containing Cks1 and PPR region. Analysis of intron/exon structure indicated that RsCENH3 (RsHistone 18) has the characteristics of variant Histone. Furthermore, several motifs, including the LTR, G-box and TC-elements, were found in the promoters of RsHistone genes, which involved in cell development or various abiotic stresses responses. Transcriptome analysis indicated that the RsHistone genes exhibited higher expression level in floral buds than in roots and leaves. Subcellular localization showed that the RsCENH3 was localized on the nucleus, and it was highly expressed in the floral bud of 3.0–4.0 mm in radish. These findings would provide valuable information for characterization and potential utilization of Histone genes, and facilitate the efficient induction of double haploid plants in radish. [ABSTRACT FROM AUTHOR]