Establishing VIGS and CRISPR/Cas9 techniques to verify RsPDS function in radish

Virus-induced gene silencing(VIGS)and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas)systems are effective technologies for rapid and accurate gene function verification in modern plant biotechnology.However,the investigation of gene silencing and editing in radish remains limited.In this study,a bleaching phenotype was generated through the knockdown of RsPDS using tobacco rattle virus(TRV)-and turnip yellow mosaic virus(TYMV)-mediated gene silencing vectors.The TYMV-mediated gene silencing efficiency was higher than the TRV-based VIGS system in radish.The expression level of RsPDS was significantly inhibited using VIGS in'NAU-067'radish leaves.The rootless seedlings of‘NAU-067'were infected with Agrobacterium rhizogenes using the 2300GN-Ubi-RsPDS-Cas9 vector with two target sequences.Nine adventitious roots were blue with GUs staining,and four of these adventitious roots were edited at target sequence 1 of the RsPDS gene as indicated by Sanger sequencing.Furthermore,albino lines were generated with A.tumefaciens-mediated transformation of radish cotyledons.Five base substitutions and three base deletions occurred at target sequence 2 in Line 1,and three base insertions and three base substitutions occurred at target sequence 1 in Line 2.This study shows that VIGS and CRISPR/Cas9 techniques can be employed to precisely verify the biological functions of genes in radish,which will facilitate the genetic improvement of vital horticultural traits in radish breeding programs.

RsERF40 contributes to cold stress tolerance and cell expansion of taproot in radish (Raphanus sativus L.)

The growth and development of taproots are inhibited by cold stress in radish(Raphanus sativus L.).Ethylene-responsive element binding factors(ERF)are key participators in the cold stress response and growth regulation of plants.However,the function of ERF genes in cold tolerance and root development in radish remains elusive.Here,we showed that the secondary growth of radish taproots was inhibited by cold stress.Comparative transcriptome analysis demonstrated that the RsERF40 gene is an important regulator of the cold stress response and root growth regulation.The cold tolerance of transgenic Arabidopsis plants overexpressing the RsERF40 gene was significantly improved.Overexpressing RsERF40 in the cold-sensitive radish genotype and silencing RsERF40 in the cold-tolerant radish genotype indicated that RsERF40 was beneficial for alleviating oxidative damage under cold stress in radish.Transgenic Arabidopsis seedlings showed an increase in the elongation and radial growth of dark-grown roots.RT-qPCR analysis showed that the expression of the cold-related genes(CORs)RsCOR78 and RsCOR413PM1 and the cell wall strengthening-related genes RsCESA6 and RsEXPB3 was upregulated in transgenic Arabidopsis seedlings.Yeast one-hybrid(Y1H)and dual-luciferase reporter assays(DLA)revealed that RsERF40 directly regulates RsCOR78,RsCOR413PM1,RsCESA6 and RsEXPB3 expression,illustrating that RsERF40 enhances cold tolerance and taproot growth by modulating osmotic adjustment and cell wall mechanical strength in radish.In this study,the RsERF40-regulon was firstly found to be a new cold response pathway independent of the CBF-COR pathway conferring cold stress tolerance with increasing radish taproot growth.These results provided novel insight into the molecular mechanism underlying cold stress response and would facilitate the genetic improvement of cold tolerance in radish and other root vegetable crops.

A genome-wide association study uncovers a critical role of the RsPAP2 gene in red-skinned Raphanus sativus L.

Radish(Raphanus sativus L.)taproot contains high concentrations of flavonoids,including anthocyanins(ATCs),in redskinned 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.

Comparative proteomic analysis provides insight into a complex regulatory network of taproot formation in radish (Raphanus sativus L.)

The fleshy taproot of radish is an important storage organ determining its yield and quality.Taproot thickening is a complex developmental process in radish.However,the molecular mechanisms governing this process remain unclear at the proteome level.In this study,a comparative proteomic analysis was performed to analyze the proteome changes at three developmental stages of taproot thickening using iTRAQ approach.In total,1862 differentially expressed proteins(DEPs)were identified from 6342 high-confidence proteins,among which 256 up-regulated proteins displayed overlapped accumulation in S1(pre-cortex splitting stage)vs.S2(cortex splitting stage)and S1 vs.S3(expanding stage)pairs,whereas 122 up-regulated proteins displayed overlapped accumulation in S1 vs.S3 and S2 vs.S3 pairs.Gene Ontology(GO)and pathway enrichment analysis showed that these DEPs were mainly involved in several processes such as“starch and sucrose metabolism”,“plant hormone signal transduction”,and“biosynthesis of secondary metabolites”.A high concordance existed between iTRAQ and RT-qPCR at the mRNA expression levels.Furthermore,association analysis showed that 187,181,and 96 DEPs were matched with their corresponding differentially expressed genes(DEGs)in S1 vs.S2,S1 vs.S3,and S2 vs.S3 comparison,respectively.Notably,several functional proteins including cell division cycle 5-like protein(CDC5),expansin B1(EXPB1),and xyloglucan endotransglucosylase/hydrolase protein 24(XTH24)were responsible for cell division and expansion during radish taproot thickening process.These results could facilitate a better understanding of the molecular mechanism underlying taproot thickening,and provide valuable information for the identification of critical genes/proteins responsible for taproot thickening in root vegetable crops.