Identification And Characterization Of WRKY,AP2/ERF And NAC Transcription Factors And Their Responses To Abiotic Stresses In Raphanus Sativus L.

Radish(Raphanus sativus L.)is one of the important vegetable crops with an estimated production of about 7 million tons per year,representing approximately 2%of all vegetables.They are mainly important vegetables in Chinese,Japan,Korea and Taiwan.Radish is increasingly popular throughout the world because,among other things,many nutritional and medicinal values.Radish-like any other sessile organisms usually encounter several biotic and abiotic environmental challenges,such as drought,salinity,threshold temperatures(from freezing to scorching),nutrient deficiency and variable light conditions.Additionally,heavy metals(HMs)such as cadmium(Cd),lead(Pb),silver(Ag)and mercury(Hg)have no nutritional functions and are potential stressors in plants.With the onset of global climate changes,abiotic and biotic stresses continuously pose uncertain threats to world’s food security.Growth and development of crops are perpetually suppressed under abiotic and biotic stresses,negatively affecting yield and quality of agricultural produce.Furthermore,the high solubility of toxic metals in water results in uptake by plants,which has deleterious effects on the food chain and human health.To overcome these stresses,higher plants have continuously evolved more complex and diverse mechanisms for their survival under stress conditions.Transcription factors(TFs)including WRKY,AP2/ERF(APETALA2/Ethylene-Responsive factor),and NAC(NAM,no apical meristem;ATAF,Arabidopsis transcription activation factor and CUC,cup-shaped cotyledon)are crucial to the adjustment of gene expression in response to environmental including heat,salinity,and HMs.Integration of bioinformatics studies and expression analysis has provided valuable opportunity to carry out genome-wide characterizations of TF genes in non-model crops such as radish.In radish,genome-wide identification and characterization of essential TFs such as WRKY,AP2/ERF and NAC have not been reported.In this study,bioinformatics and expression analysis were employed to genome-widely characterize these three TFs from the radish genome.These results would provide essential information for exploring potential genes for the development of elite radish cultivars with multiple resistances to abiotic stresses.In the present study,126 putative WRKY genes with 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,gene expression 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,36 of them showed abiotic stress-induced expression patterns.These results provide useful information for further functional dissection and molecular evolution investigation of RsWRKY genes in radish.AP2/ERF TF play a fundamental role in growth and development,as well as in responses to biotic and abiotic stresses.In this study,247 hypothetical ERF family genes were identified from the radish genome.Additionally,sequence alignment and phylogenetic analyses classified AP2/ERF superfamily into five groups(AP2,ERF,DREB,RAV and soloist).According to motif analysis,it was found that other than AP2/ERF domains,other conserved regions were selectively distributed among the different clades in the phylogenetic tree.From genetic mapping analysis,it appears that tandem duplication may have resulted in RsAP2/ERF gene family expansion.Intron/exon structure prediction indicated that a few RsAP2/ERF still conserved the former intron-position patterns being common in the ancestor of monocots and eudicots.The expression analysis confirmed that AP2/ERF genes were preferably expressed under drought and heat and suppressed in response to abscisic acid(ABA),and HM stresses.This study reported the characteristics of the RsAP2/ERF family,which provided valuable details for further evolutionary and functional studies of RsAP2/ERF in R.sativus,and also contribute to genetic improvement of stress tolerances in R.sativus and other root vegetable crops.NAC proteins are important regulators of some biological processes,including cell expansion and differentiation,hormone signaling in addition to both biotic and abiotic stresses adjustments.In this study,172 RsNACs comprising 17 membrane-bound members were categorized from the entire radish genome.In total,98 RsNAC genes were unequally distributed onto the chromosomes.In silico analysis showed expression patterns were tissue-specific for several NAC genes and showed preferential expression in roots and leaves.Furthermore,21 representative genes were selected for expressions of NAC genes gene expression analysis under HMs,salt,heat,drought and ABA stresses,and differential expressions of NAC genes were found under these abiotic stresses.RsNAC023 and RsNAC080 genes positively responded to all stress treatments except ABA,while RsNAC145 responded actively to salt,heat and drought.These results provide novel insight into the diversity and functions of RsNAC genes,which affirm that NACs are critical in plant responses to abiotic stresses,and provide valuable insight on potential genes for further investigation on function characterization of NAC genes under various abiotic stresses.