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.
BackgroundRadish (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.ResultsIn 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 predominately 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-14-836) contains supplementary material, which is available to authorized users.
Radish (Raphanus sativus L.) is one of the most important vegetable crops worldwide. Taproot thickening represents a critical developmental period that determines yield and quality in radish life cycle. To isolate differentially expressed genes (DGEs) involved in radish taproot thickening process and explore the molecular mechanism underlying taproot development, three cDNA libraries from radish taproot collected at pre-cortex splitting stage (L1), cortex splitting stage (L2), and expanding stage (L3) were constructed and sequenced by RNA-Seq technology. More than seven million clean reads were obtained from the three libraries, from which 4,717,617 (L1, 65.35%), 4,809,588 (L2, 68.24%) and 4,973,745 (L3, 69.45%) reads were matched to the radish reference genes, respectively. A total of 85,939 transcripts were generated from three libraries, from which 10,450, 12,325, and 7392 differentially expressed transcripts (DETs) were detected in L1 vs. L2, L1 vs. L3, and L2 vs. L3 comparisons, respectively. Gene Ontology and pathway analysis showed that many DEGs, including EXPA9, Cyclin, CaM, Syntaxin, MADS-box, SAUR, and CalS were involved in cell events, cell wall modification, regulation of plant hormone levels, signal transduction and metabolisms, which may relate to taproot thickening. Furthermore, the integrated analysis of mRNA-miRNA revealed that 43 miRNAs and 92 genes formed 114 miRNA-target mRNA pairs were co-expressed, and three miRNA-target regulatory networks of taproot were constructed from different libraries. Finally, the expression patterns of 16 selected genes were confirmed using RT-qPCR analysis. A hypothetical model of genetic regulatory network associated with taproot thickening in radish was put forward. The taproot formation of radish is mainly attributed to cell differentiation, division and expansion, which are regulated and promoted by certain specific signal transduction pathways and metabolism processes. These results could provide new insights into the complex molecular mechanism underlying taproot thickening and facilitate genetic improvement of taproot in radish.
BackgroundSalt 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.ResultsSolexa 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).ConclusionsWith 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1416-5) contains supplementary material, which is available to authorized users.
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