Search

Radish is one of the most economical root vegetable crops worldwide. Cold stress dramatically impedes radish taproot formation and development as well as reduces its yield and quality. Although the Cycling Dof Factors (CDFs) play crucial roles in plant growth, development and abiotic stress responses, how CDF TFs mediate the regulatory network of cold stress response remains largely unexplored in radish. Herein, a total of nine RsCDF genes were identified from the radish genome. Among them, the RsCDF3 exhibited obviously up-regulated expression under cold stress, especially at 12 h and 24 h. RsCDF3 was localized to the nucleus and displayed dramatic cold-induced promoter activity in tobacco leaves. Moreover, overexpression of RsCDF3 significantly enhanced cold tolerance of radish plants, whereas its knock-down plants exhibited the opposite phenotype. Interestingly, both in vitro and in vivo assays indicated that the RsCDF3 repressed the transcription of RsRbohA and RsRbohC via directly binding to their promoters, which contributed to maintaining the cellular homeostasis of reactive oxygen species (ROS) production and scavenging in radish. In addition, the RsCDF3 bound to its own promoter to mediate its transcription, thereby forming an autoregulatory feedback loop to cooperatively trigger RsRbohs-dependent cold tolerance. Together, we revealed a novel RsCDF3-RsRbohs module to promote the cold tolerance in radish plants. These findings would facilitate unveiling the molecular mechanism governing RsCDF3-mediated cold stress response in radish., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Radish (Raphanus sativus L.) is one of the most vital root vegetable crops worldwide. Cadmium (Cd), a non-essential and toxic heavy metal, can dramatically restrict radish taproot quality and safety. Although the Peiotrpic Drug Resistance (PDR) genes play crucial roles in heavy metal accumulation and transport in plants, the systematic identification and functional characterization of RsPDRs remain largely unexplored in radish. Herein, a total of 19 RsPDR genes were identified from the radish genome. A few RsPDRs, including RsPDR1, RsPDR8 and RsPDR12, showed significant differential expression under Cd and lead (Pb) stress in the 'NAU-YH' genotype. Interestingly, the plasma membrane-localized RsPDR8 exhibited significantly up-regulated expression and enhanced promoter activity under Cd exposure. Ectopic expression of RsPDR8 conferred Cd tolerance via reducing Cd accumulation in yeast cells. Moreover, the transient transformation of RsPDR8 revealed that it positively regulated Cd tolerance by promoting ROS scavenging and enhancing membrane permeability in radish. In addition, overexpression of RsPDR8 increased root elongation but deceased Cd accumulation compared with the WT plants in Arabidopsis, demonstrating that it could play a positive role in mediating Cd efflux and tolerance in plants. Together, these results would facilitate deciphering the molecular mechanism underlying RsPDR8-mediated Cd tolerance and detoxification in radish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

High-quality radish (Raphanus sativus) genome represents a valuable resource for agronomical trait improvements and understanding genome evolution among Brassicaceae species. However, existing radish genome assembly remains fragmentary, which greatly hampered functional genomics research and genome-assisted breeding. Here, using a NAU-LB radish inbred line, we generated a reference genome of 476.32 Mb with a scaffold N50 of 56.88 Mb by incorporating Illumina, PacBio and BioNano optical mapping techniques. Utilizing Hi-C data, 448.12 Mb (94.08%) of the assembled sequences were anchored to nine radish chromosomes with 40 306 protein-coding genes annotated. In total, 249.14 Mb (52.31%) comprised the repetitive sequences, among which long terminal repeats (LTRs, 30.31%) were the most abundant class. Beyond confirming the whole-genome triplication (WGT) event in R. sativus lineage, we found several tandem arrayed genes were involved in stress response process, which may account for the distinctive phenotype of high disease resistance in R. sativus. By comparing against the existing Xin-li-mei radish genome, a total of 2 108 573 SNPs, 7740 large insertions, 7757 deletions and 84 inversions were identified. Interestingly, a 647-bp insertion in the promoter of RsVRN1 gene can be directly bound by the DOF transcription repressor RsCDF3, resulting into its low promoter activity and late-bolting phenotype of NAU-LB cultivar. Importantly, introgression of this 647-bp insertion allele, RsVRN1 In-536 , into early-bolting genotype could contribute to delayed bolting time, indicating that it is a potential genetic resource for radish late-bolting breeding. Together, this genome resource provides valuable information to facilitate comparative genomic analysis and accelerate genome-guided breeding and improvement in radish., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© 2022 Scandinavian Plant Physiology Society.)

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Radish ( Raphanus sativus L.) is rich in specific glucosinolates (GSLs), which benefit human health and special flavor formation. Although the basic GSLs metabolic pathway in Brassicaceae plants is clear, the regulating mechanism for specific glucosinolates content in radish fleshy taproots is not well understood. In this study, we discovered that there was a significant difference in the GSLs profiles and the content of various GSLs components. Glucoraphasatin (GRH) is the most predominant GSL in radish taproots of different genotypes as assessed by HPLC analysis. Further, we compared the taproot transcriptomes of three radish genotypes with high and low GSLs content by employing RNA-seq. Totally, we identified forty-one differentially expressed genes related to GSLs metabolism. Among them, thirteen genes ( RsBCAT4 , RsIPMDH1 , RsMAM1a , RsMAM1b , RsCYP79F1 , RsGSTF9 , RsGGP1 , RsSUR1 , RsUGT74C1 , RsST5b , RsAPK1 , RsGSL-OH , and RsMYB28 ) were significantly higher co-expressed in the high content genotypes than in low content genotype. Notably, correlation analysis indicated that the expression level of RsMYB28 , as an R2R3 transcription factor directly regulating aliphatic glucosinolate biosynthesis, was positively correlated with the GRH content. Co-expression network showed that RsMYB28 probably positively regulated the expression of the above genes, particularly RsSUR1 , and consequently the synthesis of GRH. Moreover, the molecular mechanism of the accumulation of this 4-carbon (4C) GSL in radish taproots was explored. This study provides new perspectives on the GSLs accumulation mechanism and genetic improvements in radish taproots.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

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.

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 Cd 2+ 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., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

High-density genetic map is a valuable tool for exploring novel genomic information, quantitative trait locus (QTL) mapping and gene discovery of economically agronomic traits in plant species. However, high-resolution genetic map applied to tag QTLs associated with important traits and to investigate genomic features underlying recombination landscape in radish (Raphanus sativus) remains largely unexplored. In this study, an ultra-high-density genetic map with 378 738 SNPs covering 1306.8 cM in nine radish linkage groups (LGs) was developed by a whole-genome sequencing-based approach. A total of 18 QTLs for 11 horticulture traits were detected. The map-based cloning data indicated that the R2R3-MYB transcription factor RsMYB90 was a crucial candidate gene determining the taproot skin colour. Comparative genomics analysis among radish, Brassica rapa and B. oleracea genome revealed several genomic rearrangements existed in the radish genome. The highly uneven distribution of recombination was observed across the nine radish chromosomes. Totally, 504 recombination hot regions (RHRs) were enriched near gene promoters and terminators. The recombination rate in RHRs was positively correlated with the density of SNPs and gene, and GC content, respectively. Functional annotation indicated that genes within RHRs were mainly involved in metabolic process and binding. Three QTLs for three traits were found in the RHRs. The results provide novel insights into the radish genome evolution and recombination landscape, and facilitate the development of effective strategies for molecular breeding by targeting and dissecting important traits in radish., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Basic leucine zipper (bZIP) transcription factors play crucial roles in various abiotic stress responses as well as anthocyanin accumulation. Anthocyanins are most abundant in colorful skin radish, which exhibit strong antioxidant activity that offers benefits for human health. Here, a total of 135 bZIP-encoding genes were identified from radish genome. Synteny analysis showed that 104 radish and 63 Arabidopsis bZIP genes were orthologous. Transcriptome analysis revealed that 10 RsbZIP genes exhibited high-expression levels in radish taproot (RPKM>10). Specifically, RsbZIP010 exhibited down-regulated expression under Cd, Cr and Pb stresses, whereas RsbZIP031 and RsbZIP059 showed significant down-regulation under heat and salt stresses, respectively. RT-qPCR analysis indicated that RsbZIP011 and RsbZIP102 were significantly up-regulated in the tissues of radish with high anthocyanin contents. Furthermore, the promoter sequences of 39 anthocyanin-related genes were found to contain G-box or ACE-box elements that could be recognized by bZIP family members. Taken together, several RsbZIP s might be served as critical regulators in radish taproot under Cd, Cr, Pb, heat and salt stresses. RsbZIP011 and RsbZIP102 were the potential participants in anthocyanin biosynthesis pathway of radish. These results facilitate further investigation on functional characterization of bZIP genes in response to abiotic stress and anthocyanin biosynthesis in radish.

Main conclusion Among 247 RsAP2/ERF identified, the majority of the 21 representatives were preferably expressed under drought and heat while suppressed under heavy metals, indicating their potential roles in abiotic stress responses and tolerance. APETALA2/Ethylene-Responsive factor (AP2/ERF) transcription factor (TF) is one of the largest gene families in plants that play a fundamental role in growth and development as well as biotic and/or abiotic stresses responses. Although AP2/ERFs have been extensively characterized in many plant species, little is known about this family in radish, which is an important root vegetable with various medicinal properties. The available genome provides valuable opportunity to identify and characterize the global information on AP2/ERF TFs in radish. In this study, a total of 247 ERF family genes were identified from the radish genome, and sequence alignment and phylogenetic analyses classified the AP2/ERF superfamily into five groups (AP2, ERF, DREB, RAV and soloist). Motif analysis showed that other than AP2/ERF domains, other conserved regions were selectively distributed among different clades in the phylogenetic tree. Chromosome location analysis showed that tandem duplication may result in the expansion of RsAP2/ERF gene family. The RT-qPCR analysis confirmed that a proportion of AP2/ERF genes were preferably expressed under drought and heat stresses, whereas they were suppressed under the ABA and heavy metal stresses. These results provided valuable information for further evolutionary and functional characterization of RsAP2/ERF genes, and contributed to genetic improvement of stress tolerances in radish and other root vegetable crops., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Abiotic stresses due to climate change pose a great threat to crop production. Heat shock transcription factors (HSFs) are vital regulators that play key roles in protecting plants against various abiotic stresses. Therefore, the identification and characterization of HSFs is imperative to dissect the mechanism responsible for plant stress responses. Although the HSF gene family has been extensively studied in several plant species, its characterization, evolutionary history and expression patterns in the radish (Raphanus sativus L.) remain limited., Results: In this study, 33 RsHSF genes were obtained from the radish genome, which were classified into three main groups based on HSF protein domain structure. Chromosomal localization analysis revealed that 28 of 33 RsHSF genes were located on nine chromosomes, and 10 duplicated RsHSF genes were grouped into eight gene pairs by whole genome duplication (WGD). Moreover, there were 23 or 9 pairs of orthologous HSFs were identified between radish and Arabidopsis or rice, respectively. Comparative analysis revealed a close relationship among radish, Chinese cabbage and Arabidopsis. RNA-seq data showed that eight RsHSF genes including RsHSF-03, were highly expressed in the leaf, root, cortex, cambium and xylem, indicating that these genes might be involved in plant growth and development. Further, quantitative real-time polymerase chain reaction (RT-qPCR) indicated that the expression patterns of 12 RsHSF genes varied upon exposure to different abiotic stresses including heat, salt, and heavy metals. These results indicated that the RsHSFs may be involved in abiotic stress response., Conclusions: These results could provide fundamental insights into the characteristics and evolution of the HSF family and facilitate further dissection of the molecular mechanism responsible for radish abiotic stress responses.

Polyploidy is an important evolutionary factor in most land plant lineages which possess more than two complete sets of chromosomes. Radish (Raphanus sativus L.) is an economically annual/biennial root vegetable crop worldwide. However, the expression patterns of duplicated homologs involved in the autopolyploidization remains unclear. In present study, the autotetraploid radish plants (2n = 4x = 36) were produced with colchicine and exhibited an increase in the size of flowers, leaves, stomata and pollen grains. The differential gene expression (DGE) profiling was performed to investigate the differences in gene expression patterns between diploid and its corresponding autotetraploid by RNA-Sequencing (RNA-Seq). Totally, 483 up-regulated differentially expressed genes (DEGs) and 408 down-regulated DEGs were detected in diploid and autotetraploid radishes, which majorly involved in the pathways of hormones, photosynthesis and stress response. Moreover, the xyloglucan endotransglucosylase/hydrolase (XTH) and pectin methylesterases (PME) family members related to cell enlargement and cell wall construction were found to be enriched in GO enrichment analysis, of which XTH family members enriched in "apoplast" and "cell wall" terms, while PME family members enriched in "cell wall" term. Reverse-transcription quantitative PCR (RT-qPCR) analysis indicated that the expression profile of DEGs were consistent with results from the RNA-Seq analysis. The DEGs involved in cell wall construction and auxin metabolism were predicted to be associated with organs size increase of autotetraploid radishes in the present study. These results could provide valuable information for elucidating the molecular mechanism underlying polyploidization and facilitating further genetic improvements of important traits in radish breeding programs.

Main Conclusion: Differential abundance protein species (DAPS) involved in reducing damage and enhancing thermotolerance in radish were firstly identified. Proteomic analysis and omics association analysis revealed a HS-responsive regulatory network in radish. Heat stress (HS) is a major destructive factor influencing radish production and supply in summer, for radish is a cool season vegetable crop being susceptible to high temperature. In this study, the proteome changes of radish taproots under 40 °C treatment at 0 h (Control), 12 h (Heat12) and 24 h (Heat24) were analyzed using iTRAQ (Isobaric Tag for Relative and Absolute Quantification) approach. In total, 2258 DAPS representing 1542 differentially accumulated uniprotein species which respond to HS were identified. A total of 604, 910 and 744 DAPS was detected in comparison of Control vs. Heat12, Control vs. Heat24, and Heat12 vs. Heat24, respectively. Gene ontology and pathway analysis showed that annexin, ubiquitin-conjugating enzyme, ATP synthase, heat shock protein (HSP) and other stress-related proteins were predominately enriched in signal transduction, stress and defense pathways, photosynthesis and energy metabolic pathways, working cooperatively to reduce stress-induced damage in radish. Based on iTRAQ combined with the transcriptomics analysis, a schematic model of a sequential HS-responsive regulatory network was proposed. The initial sensing of HS occurred at the plasma membrane, and then key components of stress signal transduction triggered heat-responsive genes in the plant protective metabolism to re-establish homeostasis and enhance thermotolerance. These results provide new insights into characteristics of HS-responsive DAPS and facilitate dissecting the molecular mechanisms underlying heat tolerance in radish and other root crops.

Heat stress (HS) causes detrimental effects on plant morphology, physiology, and biochemistry that lead to drastic reduction in plant biomass production and economic yield worldwide. To date, little is known about HS-responsive genes involved in thermotolerance mechanism in radish. In this study, a total of 6600 differentially expressed genes (DEGs) from the control and Heat24 cDNA libraries of radish were isolated by high-throughput sequencing. With Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, some genes including MAPK, DREB, ERF, AP2, GST, Hsf, and Hsp were predominantly assigned in signal transductions, metabolic pathways, and biosynthesis and abiotic stress-responsive pathways. These pathways played significant roles in reducing stress-induced damages and enhancing heat tolerance in radish. Expression patterns of 24 candidate genes were validated by reverse-transcription quantitative PCR (RT-qPCR). Based mainly on the analysis of DEGs combining with the previous miRNAs analysis, the schematic model of HS-responsive regulatory network was proposed. To counter the effects of HS, a rapid response of the plasma membrane leads to the opening of specific calcium channels and cytoskeletal reorganization, after which HS-responsive genes are activated to repair damaged proteins and ultimately facilitate further enhancement of thermotolerance in radish. These results could provide fundamental insight into the regulatory network underlying heat tolerance in radish and facilitate further genetic manipulation of thermotolerance in root vegetable crops.

Key Message: The radish WRKY gene family was genome-widely identified and played critical roles in response to multiple abiotic stresses. The WRKY is among the largest transcription factors (TFs) associated with multiple biological activities for plant survival, including control response mechanisms against abiotic stresses such as heat, salinity, and heavy metals. Radish is an important root vegetable crop and therefore characterization and expression pattern investigation of WRKY transcription factors in radish is imperative. In the present study, 126 putative WRKY genes were retrieved from radish genome database. Protein sequence and annotation scrutiny confirmed that RsWRKY proteins possessed highly conserved domains and zinc finger motif. Based on phylogenetic analysis results, RsWRKYs candidate genes were divided into three groups (Group I, II and III) with the number 31, 74, and 20, respectively. Additionally, gene structure analysis revealed that intron-exon patterns of the WRKY genes are highly conserved in radish. Linkage map analysis indicated that RsWRKY genes were distributed with varying densities over nine linkage groups. Further, RT-qPCR analysis illustrated the significant variation of 36 RsWRKY genes under one or more abiotic stress treatments, implicating that they might be stress-responsive genes. In total, 126 WRKY TFs were identified from the R. sativus genome wherein, 35 of them showed abiotic stress-induced expression patterns. These results provide a genome-wide characterization of RsWRKY TFs and baseline for further functional dissection and molecular evolution investigation, specifically for improving abiotic stress resistances with an ultimate goal of increasing yield and quality of radish.

Cadmium (Cd) is a widespread heavy metal of particular concern with respect to the environment and human health. Although intensive studies have been conducted on Cd-exposed transcriptome profiling, little systematic proteome information is available on the molecular mechanism of Cd stress response in radish. In this study, the radish root proteome under Cd stress was investigated using a quantitative multiplexed proteomics approach. Seedlings were grown in nutrient solution without Cd (control) or with 10 or 50 μM CdCl2 for 12 h (Cd10 and Cd50, respectively). In total, 91 up- and 66 down-regulated proteins were identified in the control vs Cd10 comparison, while 340 up- and 286 down-regulated proteins were identified in the control vs Cd50 comparison. Functional annotation indicated that these differentially expressed proteins (DEPs) were mainly involved in carbohydrate and energy metabolism, stress and defense and signal transduction processes. Correlation analysis showed that 33 DEPs matched with their transcripts, indicating a relatively low correlation between transcript and protein levels under Cd stress. Quantitative real-time PCR evidenced the expression patterns of 12 genes encoding their corresponding DEPs. In particular, several pivotal proteins associated with carbohydrate metabolism, ROS scavenging, cell transport and signal transduction were involved in the coordinated regulatory network of the Cd stress response in radish. Root exposure to Cd2+ activated several key signaling molecules and metal-containing transcription factors, and subsequently some Cd-responsive functional genes were mediated to reduce Cd toxicity and re-establish redox homeostasis in radish. This is a first report on comprehensive proteomic characterization of Cd-exposed root proteomes in radish. These findings could facilitate unraveling of the molecular mechanism underlying the Cd stress response in radish and provide fundamental insights into the development of genetically engineered low-Cd-content radish cultivars., (© The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Radish is an important root vegetable crop with high nutritional, economic, and medicinal value. Lignin is an important secondary metabolite possessing a great effect on plant growth and product quality. To date, lignin biosynthesis-related genes have been identified in some important plant species. However, little information on characterization of critical genes involved in plant lignin biosynthesis is available in radish. In this study, a total of 71,148 transcripts sequences were obtained from radish root, of which 66 assembled unigenes and ten candidate genes were identified to be involved in lignin monolignol biosynthesis. Full-length cDNA sequences of seven randomly selected genes were isolated and sequenced from radish root, and the assembled unigenes covered more than 80% of their corresponding cDNA sequences. Moreover, the lignin content gradually accumulated in leaf during the developmental stages, and it increased from pre-cortex to cortex splitting stage, followed by a decrease at thickening stage and then increased at mature stage in root. RT-qPCR analysis revealed that all these genes except RsF5H exhibited relatively low expression level in root at thickening stage. The expression profiles of Rs4CL5, RsCCoAOMT1, and RsCOMT genes were consistent with the changes of root lignin content, implying that these candidate genes may play important roles in lignin formation in radish root. These findings would provide valuable information for identification of lignin biosynthesis-related genes and facilitate dissection of molecular mechanism underlying lignin biosynthesis in radish and other root vegetable crops.

Background: Alternative splicing (AS) plays important roles in gene expression and proteome diversity. Single nucleotide polymorphism (SNP) and insertion/deletion (InDel) are abundant polymorphisms and co-dominant inheritance markers, which have been widely used in germplasm identification, genetic mapping and marker-assisted selection in plants. So far, however, little information is available on utilization of AS events and development of SNP and InDel markers from transcriptome in radish., Results: In this study, three radish transcriptome datasets were collected and aligned to the reference radish genome. A total of 56,530 AS events were identified from three radish genotypes with intron retention (IR) being the most frequent AS type, which accounted for 59.4% of the total expressed genes in radish. In all, 22,412 SNPs and 9436 InDels were identified with an average frequency of 1 SNP/17.9 kb and 1 InDel/42.5 kb, respectively. A total of 43,680 potential SSRs were identified in 31,604 assembled unigenes with a density of 1 SSR/2.5 kb. The ratio of SNPs with nonsynonymous/synonymous mutations was 1.05:1. Moreover, 35 SNPs and 200 InDels were randomly selected and validated by Sanger sequencing, 83.9% of the SNPs and 70% of the InDels exhibited polymorphism among these three genotypes. In addition, the 15 SNPs and 125 InDels were found to be unevenly distributed on 9 linkage groups. Furthermore, 40 informative InDel markers were successfully used for the genetic diversity analysis on 32 radish accessions., Conclusions: These results would not only provide new insights into transcriptome complexity and AS regulation, but also furnish large amount of molecular marker resources for germplasm identification, genetic mapping and further genetic improvement of radish in breeding programs.

Microsporogenesis is an indispensable period for investigating microspore development and cytoplasmic male sterility (CMS) occurrence. Radish CMS line plays a critical role in elite F1 hybrid seed production and heterosis utilization. However, the molecular mechanisms of microspore development and CMS occurrence have not been thoroughly uncovered in radish. In this study, a comparative analysis of radish floral buds from a CMS line (NAU-WA) and its maintainer (NAU-WB) was conducted using next generation sequencing (NGS) technology. Digital gene expression (DGE) profiling revealed that 3504 genes were significantly differentially expressed between NAU-WA and NAU-WB library, among which 1910 were upregulated and 1594 were downregulated. Gene ontology (GO) analysis showed that these differentially expressed genes (DEGs) were mainly enriched in extracellular region, catalytic activity, and response to stimulus. KEGG enrichment analysis revealed that the DEGs were predominantly associated with flavonoid biosynthesis, glycolysis, and biosynthesis of secondary metabolites. Real-time quantitative PCR analysis showed that the expression profiles of 13 randomly selected DEGs were in high agreement with results from Illumina sequencing. Several candidate genes encoding ATP synthase, auxin response factor (ARF), transcription factors (TFs), chalcone synthase (CHS), and male sterility (MS) were responsible for microsporogenesis. Furthermore, a schematic diagram for functional interaction of DEGs from NAU-WA vs. NAU-WB library in radish plants was proposed. These results could provide new information on the dissection of the molecular mechanisms underlying microspore development and CMS occurrence in radish.

Background: The appropriate timing of bolting and flowering is pivotal for reproductive success in Brassicaceae crops including radish (Raphanus sativus L.). Although several flowering regulatory pathways had been described in some plant species, no study on genetic networks of bolting and flowering regulation was performed in radish. In this study, to generate dataset of radish unigene sequences for large-scale gene discovery and functional pathway identification, a cDNA library from mixed radish leaves at different developmental stages was subjected to high-throughput RNA sequencing (RNA-seq)., Results: A total of 54.64 million clean reads and 111,167 contigs representing 53,642 unigenes were obtained from the radish leaf transcriptome. Among these, 50,385 unigenes were successfully annotated by BLAST searching against the public protein databases. Functional classification and annotation indicated that 42,903 and 15,382 unique sequences were assigned to 55 GO terms and 25 COG categories, respectively. KEGG pathway analysis revealed that 25,973 unigenes were classified into 128 functional pathways, among which 24 candidate genes related to plant circadian rhythm were identified. Moreover, 142 potential bolting and flowering-related genes involved in various flowering pathways were identified. In addition, seven critical bolting and flowering-related genes were isolated and profiled by T-A cloning and RT-qPCR analysis. Finally, a schematic network model of bolting and flowering regulation and pathways was put forward in radish., Conclusions: This study is the first report on systematic identification of bolting and flowering-related genes based on transcriptome sequencing and assembly in radish. These results could provide a foundation for further investigating bolting and flowering regulatory networks in radish, and facilitate dissecting molecular genetic mechanisms underlying bolting and flowering in Brassicaceae vegetable crops.

Embryogenesis is an important component in the life cycle of most plant species. Due to the difficulty in embryo isolation, the global gene expression involved in plant embryogenesis, especially the early events following fertilization are largely unknown in radish. In this study, three cDNA libraries from ovules of radish before and after fertilization were sequenced using the Digital Gene Expression (DGE) tag profiling strategy. A total of 5,777 differentially expressed transcripts were detected based on pairwise comparison in the three libraries (0_DAP, 7_DAP and 15_DAP). Results from Gene Ontology (GO) and pathway enrichment analysis revealed that these differentially expressed genes (DEGs) were implicated in numerous life processes including embryo development and phytohormones biosynthesis. Notably, some genes encoding auxin response factor (ARF ), Leafy cotyledon1 (LEC1) and somatic embryogenesis receptor-like kinase (SERK ) known to be involved in radish embryogenesis were differentially expressed. The expression patterns of 30 genes including LEC1-2, AGL9, LRR, PKL and ARF8-1 were validated by qRT-PCR. Furthermore, the cooperation between miRNA and mRNA may play a pivotal role in the radish embryogenesis process. This is the first report on identification of DEGs profiles related to radish embryogenesis and seed development. These results could facilitate further dissection of the molecular mechanisms underlying embryogenesis and seed development in radish.

Key Message: Transcriptome-based gene expression analysis identifies many critical salt-responsive genes in radish and facilitates further dissecting the molecular mechanism underlying salt stress response. Salt stress severely impacts plant growth and development. Radish, a moderately salt-sensitive vegetable crop, has been studied for decades towards the physiological and biochemical performances under salt stress. However, no systematic study on isolation and identification of genes involved in salt stress response has been performed in radish, and the molecular mechanism governing this process is still indistinct. Here, the RNA-Seq technique was applied to analyze the transcriptomic changes on radish roots treated with salt (200 mM NaCl) for 48 h in comparison with those cultured in normal condition. Totally 8709 differentially expressed genes (DEGs) including 3931 up- and 4778 down-regulated genes were identified. Functional annotation analysis indicated that many genes could be involved in several aspects of salt stress response including stress sensing and signal transduction, osmoregulation, ion homeostasis and ROS scavenging. The association analysis of salt-responsive genes and miRNAs exhibited that 36 miRNA-mRNA pairs had negative correlationship in expression trends. Reverse-transcription quantitative PCR (RT-qPCR) analysis revealed that the expression profiles of DEGs were in line with results from the RNA-Seq analysis. Furthermore, the putative model of DEGs and miRNA-mediated gene regulation was proposed to elucidate how radish sensed and responded to salt stress. This study represents the first comprehensive transcriptome-based gene expression profiling under salt stress in radish. The outcomes of this study could facilitate further dissecting the molecular mechanism underlying salt stress response and provide a valuable platform for further genetic improvement of salt tolerance in radish breeding programs.

The radish (Raphanus sativus L.) is an important root vegetable crop. In this study, the metabolite profiling analysis of radish roots exposed to lead (Pb) and cadmium (Cd) stresses has been performed using gas chromatography-mass spectrometry (GC-MS). The score plots of principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) showed clear discrimination between control and Pb- or Cd-treated samples. The metabolic profiling indicated Pb or Cd stress could cause large metabolite alteration mainly on sugars, amino acids and organic acids. Furthermore, an integrated analysis of the effects of Pb or Cd stress was performed on the levels of metabolites and gene transcripts from our previous transcriptome work in radish roots. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of integration data demonstrated that exposure of radish to Pb stress resulted in profound biochemical changes including carbohydrate metabolism, energy metabolism and glutathione metabolism, while the treatment of Cd stress caused significant variations in energy production, amino acid metabolism and oxidative phosphorylation-related pathways. These results would facilitate further dissection of the mechanisms of heavy metal (HM) accumulation/tolerance in plants and the effective management of HM contamination in vegetable crops by genetic manipulation.

MicroRNAs (miRNAs) play vital regulatory roles in plant growth and development. The phase transition from vegetative growth to flowering is crucial in the life cycle of plants. To date, miRNA-mediated flowering regulatory networks remain largely unexplored in radish. In this study, two small RNA libraries from radish leaves at vegetative and reproductive stages were constructed and sequenced by Solexa sequencing. A total of 94 known miRNAs representing 21 conserved and 13 non-conserved miRNA families, and 44 potential novel miRNAs, were identified from the two libraries. In addition, 42 known and 17 novel miRNAs were significantly differentially expressed and identified as bolting-related miRNAs. RT-qPCR analysis revealed that some miRNAs exhibited tissue- or developmental stage-specific expression patterns. Moreover, 154 target transcripts were identified for 50 bolting-related miRNAs, which were predominately involved in plant development, signal transduction and transcriptional regulation. Based on the characterization of bolting-related miRNAs and their target genes, a putative schematic model of miRNA-mediated bolting and flowering regulatory network was proposed. These results could provide insights into bolting and flowering regulatory networks in radish, and facilitate dissecting the molecular mechanisms underlying bolting and flowering time regulation in vegetable crops.

MicroRNAs (miRNAs) are small noncoding RNAs that play pivotal roles in plant growth, development and stress response. Chromium (Cr) is one of common environmental contaminants possessing potential health hazards to living organisms. To date, little is known about the regulatory roles of miRNAs in response to Cr stress in radish. To systematically identify Cr-responsive miRNAs and their targets in radish, two sRNA libraries derived from Cr-free (CK) and Cr-treated (Cr200) roots were constructed. With Solexa sequencing, 81 known and 72 novel miRNAs were identified, from which 54 known and 16 novel miRNAs were significantly differentially expressed under Cr stress. Several target genes for Cr-responsive miRNAs encode different transcription factor (TF) families, including SPLs, MYBs, ERFs and bZIPs, might regulate corresponding HM-related transcriptional processes in plants. Notably, a few key responsive enzymes or proteins, including HMA, YSL1 and ABC transporter protein were involved in Cr uptake and homeostasis process. Furthermore, the expression patterns of some Cr-responsive miRNAs and their targets were validated by RT-qPCR. This study represents the first characterization of Cr-responsive miRNAs and their targets in radish. The outcomes of this study could provide novel insights into miRNA-mediated regulatory mechanisms underlying plant response to Cr stress in root vegetable crops.

Cadmium (Cd) is a nonessential metallic trace element that poses potential chronic toxicity to living organisms. To date, little is known about the Cd-responsive regulatory network in root vegetable crops including radish. In this study, 31,015 unigenes representing 66,552 assembled unique transcripts were isolated from radish root under Cd stress based on de novo transcriptome assembly. In all, 1496 differentially expressed genes (DEGs) consisted of 3579 transcripts were identified from Cd-free (CK) and Cd-treated (Cd200) libraries. Gene Ontology and pathway enrichment analysis indicated that the up- and down-regulated DEGs were predominately involved in glucosinolate biosynthesis as well as cysteine and methionine-related pathways, respectively. RT-qPCR showed that the expression profiles of DEGs were in consistent with results from RNA-Seq analysis. Several candidate genes encoding phytochelatin synthase (PCS), metallothioneins (MTs), glutathione (GSH), zinc iron permease (ZIPs) and ABC transporter were responsible for Cd uptake, accumulation, translocation and detoxification in radish. The schematic model of DEGs and microRNAs-involved in Cd-responsive regulatory network was proposed. This study represents a first comprehensive transcriptome-based characterization of Cd-responsive DEGs in radish. These results could provide fundamental insight into complex Cd-responsive regulatory networks and facilitate further genetic manipulation of Cd accumulation in root vegetable crops., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Background: Salt stress is one of the most representative abiotic stresses that severely affect plant growth and development. MicroRNAs (miRNAs) are well known for their significant involvement in plant responses to abiotic stresses. Although miRNAs implicated in salt stress response have been widely reported in numerous plant species, their regulatory roles in the adaptive response to salt stress in radish (Raphanus sativus L.), an important root vegetable crop worldwide, remain largely unknown., Results: Solexa sequencing of two sRNA libraries from NaCl-free (CK) and NaCl-treated (Na200) radish roots were performed for systematical identification of salt-responsive miRNAs and their expression profiling in radish. Totally, 136 known miRNAs (representing 43 miRNA families) and 68 potential novel miRNAs (belonging to 51 miRNA families) were identified. Of these miRNAs, 49 known and 22 novel miRNAs were differentially expressed under salt stress. Target prediction and annotation indicated that these miRNAs exerted a role by regulating specific stress-responsive genes, such as squamosa promoter binding-like proteins (SPLs), auxin response factors (ARFs), nuclear transcription factor Y (NF-Y) and superoxide dismutase [Cu-Zn] (CSD1). Further functional analysis suggested that these target genes were mainly implicated in signal perception and transduction, regulation of ion homeostasis, basic metabolic processes, secondary stress responses, as well as modulation of attenuated plant growth and development under salt stress. Additionally, the expression patterns of ten miRNAs and five corresponding target genes were validated by reverse-transcription quantitative PCR (RT-qPCR)., Conclusions: With the sRNA sequencing, salt-responsive miRNAs and their target genes in radish were comprehensively identified. The results provide novel insight into complex miRNA-mediated regulatory network of salt stress response in radish, and facilitate further dissection of molecular mechanism underlying plant adaptive response to salt stress in root vegetable crops.

Background: Radish (Raphanus sativus L.) is an economically important root vegetable crop, and the taproot-thickening process is the most critical period for the final productivity and quality formation. MicroRNAs (miRNAs) are a family of non-coding small RNAs that play an important regulatory function in plant growth and development. However, the characterization of miRNAs and their roles in regulating radish taproot growth and thickening remain largely unexplored. A Solexa high-throughput sequencing technology was used to identify key miRNAs involved in taproot thickening in radish., Results: Three small RNA libraries from 'NAU-YH' taproot collected at pre-cortex splitting stage, cortex splitting stage and expanding stage were constructed. In all, 175 known and 107 potential novel miRNAs were discovered, from which 85 known and 13 novel miRNAs were found to be significantly differentially expressed during taproot thickening. Furthermore, totally 191 target genes were identified for the differentially expressed miRNAs. These target genes were annotated as transcription factors and other functional proteins, which were involved in various biological functions including plant growth and development, metabolism, cell organization and biogenesis, signal sensing and transduction, and plant defense response. RT-qPCR analysis validated miRNA expression patterns for five miRNAs and their corresponding target genes., Conclusions: The small RNA populations of radish taproot at different thickening stages were firstly identified by Solexa sequencing. Totally 98 differentially expressed miRNAs identified from three taproot libraries might play important regulatory roles in taproot thickening. Their targets encoding transcription factors and other functional proteins including NF-YA2, ILR1, bHLH74, XTH16, CEL41 and EXPA9 were involved in radish taproot thickening. These results could provide new insights into the regulatory roles of miRNAs during the taproot thickening and facilitate genetic improvement of taproot in radish.

Background: Radish (Raphanus sativus L.), is an important root vegetable crop worldwide. Glucosinolates in the fleshy taproot significantly affect the flavor and nutritional quality of radish. However, little is known about the molecular mechanisms underlying glucosinolate metabolism in radish taproots. The limited availability of radish genomic information has greatly hindered functional genomic analysis and molecular breeding in radish., Results: In this study, a high-throughput, large-scale RNA sequencing technology was employed to characterize the de novo transcriptome of radish roots at different stages of development. Approximately 66.11 million paired-end reads representing 73,084 unigenes with a N50 length of 1,095 bp, and a total length of 55.73 Mb were obtained. Comparison with the publicly available protein database indicates that a total of 67,305 (about 92.09% of the assembled unigenes) unigenes exhibit similarity (e -value ≤ 1.0e⁻⁵) to known proteins. The functional annotation and classification including Gene Ontology (GO), Clusters of Orthologous Group (COG) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that the main activated genes in radish taproots are predominantly involved in basic physiological and metabolic processes, biosynthesis of secondary metabolite pathways, signal transduction mechanisms and other cellular components and molecular function related terms. The majority of the genes encoding enzymes involved in glucosinolate (GS) metabolism and regulation pathways were identified in the unigene dataset by targeted searches of their annotations. A number of candidate radish genes in the glucosinolate metabolism related pathways were also discovered, from which, eight genes were validated by T-A cloning and sequencing while four were validated by quantitative RT-PCR expression profiling., Conclusions: The ensuing transcriptome dataset provides a comprehensive sequence resource for molecular genetics research in radish. It will serve as an important public information platform to further understanding of the molecular mechanisms involved in biosynthesis and metabolism of the related nutritional and flavor components during taproot formation in radish.

MicroRNAs (miRNAs) are endogenous non-coding small RNAs that play vital regulatory roles in plant growth, development, and environmental stress responses. Cadmium (Cd) is a non-essential heavy metal that is highly toxic to living organisms. To date, a number of conserved and non-conserved miRNAs have been identified to be involved in response to Cd stress in some plant species. However, the miRNA-mediated gene regulatory networks responsive to Cd stress in radish (Raphanus sativus L.) remain largely unexplored. To dissect Cd-responsive miRNAs and their targets systematically at the global level, two small RNA libraries were constructed from Cd-treated and Cd-free roots of radish seedlings. Using Solexa sequencing technology, 93 conserved and 16 non-conserved miRNAs (representing 26 miRNA families) and 28 novel miRNAs (representing 22 miRNA families) were identified. In all, 15 known and eight novel miRNA families were significantly differently regulated under Cd stress. The expression patterns of a set of Cd-responsive miRNAs were validated by quantitative real-time PCR. Based on the radish mRNA transcriptome, 18 and 71 targets for novel and known miRNA families, respectively, were identified by the degradome sequencing approach. Furthermore, a few target transcripts including phytochelatin synthase 1 (PCS1), iron transporter protein, and ABC transporter protein were involved in plant response to Cd stress. This study represents the first transcriptome-based analysis of miRNAs and their targets responsive to Cd stress in radish roots. These findings could provide valuable information for functional characterization of miRNAs and their targets in regulatory networks responsive to Cd stress in radish.

Ascorbate is a primary antioxidant and an essential enzyme cofactor in plants, which has an important effect on the development of plant root system. To investigate the molecular mechanisms of ascorbate accumulation during root development and reveal the key genes of the ascorbate biosynthesis and recycling pathways, the expression of 16 related genes together with ascorbate abundance were analyzed in the flesh and skin of radish (Raphanus sativus L.) fleshy root. The content of ascorbate decreased with root growth in both the flesh and skin. Expression of GDP-d-mannose pyrophosphorylase, GDP-d-mannose-3',5'-epimerase and d-galacturonate reductase were also decreased and correlated with ascorbate levels in the flesh. In the skin, the expression of GDP-d-mannose pyrophosphorylase and l-galactose dehydrogenase was correlated with ascorbate levels. These results suggested that ascorbate accumulation is affected mainly by biosynthesis rather than recycling in radish root, and the l-galactose pathway may be the major biosynthetic route of ascorbate, and moreover, the salvage pathway may also contribute to ascorbate accumulation. The data suggested that GDP-d-mannose pyrophosphorylase could play an important role in the regulation of ascorbate accumulation during radish fleshy taproot development., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Lead (Pb), one of the most toxic heavy metals, can be absorbed and accumulated by plant roots and then enter the food chain resulting in potential health risks for human beings. The radish (Raphanus sativus L.) is an important root vegetable crop with fleshy taproots as the edible parts. Little is known about the mechanism by which radishes respond to Pb stress at the molecular level. In this study, Next Generation Sequencing (NGS)-based RNA-seq technology was employed to characterize the de novo transcriptome of radish roots and identify differentially expressed genes (DEGs) during Pb stress. A total of 68,940 assembled unique transcripts including 33,337 unigenes were obtained from radish root cDNA samples. Based on the assembled de novo transcriptome, 4,614 DEGs were detected between the two libraries of untreated (CK) and Pb-treated (Pb1000) roots. Gene Ontology (GO) and pathway enrichment analysis revealed that upregulated DEGs under Pb stress are predominately involved in defense responses in cell walls and glutathione metabolism-related processes, while downregulated DEGs were mainly involved in carbohydrate metabolism-related pathways. The expression patterns of 22 selected genes were validated by quantitative real-time PCR, and the results were highly accordant with the Solexa analysis. Furthermore, many candidate genes, which were involved in defense and detoxification mechanisms including signaling protein kinases, transcription factors, metal transporters and chelate compound biosynthesis related enzymes, were successfully identified in response to heavy metal Pb. Identification of potential DEGs involved in responses to Pb stress significantly reflected alterations in major biological processes and metabolic pathways. The molecular basis of the response to Pb stress in radishes was comprehensively characterized. Useful information and new insights were provided for investigating the molecular regulation mechanism of heavy metal Pb accumulation and tolerance in root vegetable crops.

MicroRNAs (miRNAs) are endogenous, non-coding, small RNAs that play significant regulatory roles in plant growth, development, and biotic and abiotic stress responses. To date, a great number of conserved and species-specific miRNAs have been identified in many important plant species such as Arabidopsis, rice and poplar. However, little is known about identification of miRNAs and their target genes in radish (Raphanus sativus L.). In the present study, a small RNA library from radish root was constructed and sequenced using the high-throughput Solexa sequencing. Through sequence alignment and secondary structure prediction, a total of 545 conserved miRNA families as well as 15 novel (with their miRNA* strand) and 64 potentially novel miRNAs were identified. Quantitative real-time PCR (qRT-PCR) analysis confirmed that both conserved and novel miRNAs were expressed in radish, and some of them were preferentially expressed in certain tissues. A total of 196 potential target genes were predicted for 42 novel radish miRNAs. Gene ontology (GO) analysis showed that most of the targets were involved in plant growth, development, metabolism and stress responses. This study represents a first large-scale identification and characterization of radish miRNAs and their potential target genes. These results could lead to the further identification of radish miRNAs and enhance our understanding of radish miRNA regulatory mechanisms in diverse biological and metabolic processes., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Real-time quantitative reverse transcription PCR (RT-qPCR) is a rapid and reliable method for gene expression studies. Normalization based on reference genes can increase the reliability of this technique; however, recent studies have shown that almost no single reference gene is universal for all possible experimental conditions. In this study, eight frequently used reference genes were investigated, including Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Actin2/7 (ACT), Tubulin alpha-5 (TUA), Tubulin beta-1 (TUB), 18S ribosomal RNA (18SrRNA), RNA polymerase-II transcription factor (RPII), Elongation factor 1-b (EF-1b) and Translation elongation factor 2 (TEF2). Expression stability of candidate reference genes was examined across 27 radish samples, representing a range of tissue types, cultivars, photoperiodic and vernalization treatments, and developmental stages. The eight genes in these sample pools displayed a wide range of Ct values and were variably expressed. Two statistical software packages, geNorm and NormFinder showed that TEF2, RPII and ACT appeared to be relatively stable and therefore the most suitable for use as reference genes. These results facilitate selection of desirable reference genes for accurate gene expression studies in radish., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Cadmium (Cd) is a widespread soil pollutant and poses a significant threat to human health via the food chain. Large phenotypic variations in Cd concentration of radish roots and shoots have been observed. However, the genetic and molecular mechanisms of Cd accumulation in radish remain to be elucidated. In this study, a genetic linkage map was constructed using an F(2) mapping population derived from a cross between a high Cd-accumulating cultivar NAU-Dysx and a low Cd-accumulating cultivar NAU-Yh. The linkage map consisted of 523 SRAP, RAPD, SSR, ISSR, RAMP, and RGA markers and had a total length of 1,678.2 cM with a mean distance of 3.4 cM between two markers. All mapped markers distributed on nine linkage groups (LGs) having sizes between 134.7 and 236.8 cM. Four quantitative trait loci (QTLs) for root Cd accumulation were mapped on LGs 1, 4, 6, and 9, which accounted for 9.86 to 48.64 % of all phenotypic variance. Two QTLs associated with shoot Cd accumulation were detected on LG1 and 3, which accounted for 17.08 and 29.53 % of phenotypic variance, respectively. A major-effect QTL, qRCd9 (QTL for root Cd accumulation on LG9), was identified on LG 9 flanked by NAUrp011_754 and EM5me6_286 markers with a high LOD value of 23.6, which accounted for 48.64 % of the total phenotypic variance in Cd accumulation of F(2) lines. The results indicated that qRCd9 is a novel QTL responsible for controlling root Cd accumulation in radish, and the identification of specific molecular markers tightly linked to the major QTL could be further applied for marker-assisted selection (MAS) in low-Cd content radish breeding program.