Identification of anthocyanin biosynthesis related microRNAs in a distinctive Chinese radish (Raphanus sativus L.) by high-throughput sequencing.

Anthocyanins are widely distributed water-soluble phytochemical pigments belonging to the flavonoid group. To date, limited knowledge is available about the regulatory roles of miRNAs in anthocyanin biosynthesis in plants. To identify the miRNAs associated with anthocyanin biosynthesis in radish, five small RNA (sRNA) libraries constructed from 'Xinlimei' radish roots at 11, 21, 44, 56 and 73 days (d) were examined using high-throughput sequencing technology. A total of 102.02 million (M) clean reads were generated, from which 483 known and 1415 novel miRNAs were identified. Combined with target prediction and annotation, 72 differentially expressed miRNAs (52 known and 20 novel miRNAs) were more likely to participate in anthocyanin biosynthesis. Several target genes for these miRNAs encode a few transcription factors, including Myb domain (MYB), basic helix-loop-helix (bHLH), WD40 repeat, squamosa promoter binding protein like (SPL), auxin response factor (ARF), ethylene insensitive 3 (EIN3), WRKY and MADS-box proteins. Furthermore, the expression patterns of some anthocyanin biosynthesis related miRNAs and their corresponding targets were validated by RT-qPCR. Based on the characterization of anthocyanin biosynthesis related miRNAs and their target genes, a putative miRNA-target module regulating anthocyanin biosynthesis was proposed. This study represents the first genome-wide identification of miRNAs associated with anthocyanin biosynthesis in radish, and provides insights into the molecular mechanisms underlying regulation of anthocyanin biosynthesis in radish and other crops.

Sun Y ，Qiu Y ，Duan M ，Wang J ，Zhang X ，Wang H ，Song J ，Li X ... -  《-》

Identification of 'Xinlimei' radish candidate genes associated with anthocyanin biosynthesis based on a transcriptome analysis.

Radish is an economically important vegetable crop belonging to the family Brassicaceae. The high anthocyanin content of the 'Xinlimei' radish roots has been associated with diverse health benefits. However, there is a lack of transcript-level information regarding anthocyanin biosynthesis. In the present study, the 'Xinlimei' radish root transcriptome was analyzed by RNA sequencing at five developmental stages. A total of 222,384,034 clean reads were obtained and 32,253 unigenes were annotated. Expression profiles revealed 10,890 differentially expressed genes (DEGs) among the five analyzed libraries. The DEGs were predominantly involved in KEGG pathways related to the biosynthesis of phenylpropanoids, flavonoids, flavone, and flavonol. The transcriptome data revealed 44 structural and 182 transcription factor genes (TFs) associated with the anthocyanin biosynthetic pathway. Ten structural genes (i.e., 4CL3, CHSB2, CHS1, CHS3, F3H1, F3'H, DFR, DFR1, ANS, and UFGT) and two MYB genes, which were highly and differentially expressed during root development, may be critical for anthocyanin biosynthesis. Additionally, the co-expression of TFs and structural genes was analyzed. Three structural genes (i.e., DFR, ANS, and UFGT) were validated by molecular cloning. The qRT-PCR results indicated that the expression profiles of DEGs were generally consistent with the high-throughput sequencing results. These findings helped identify candidate genes involved in anthocyanin biosynthesis and may be useful for clarifying the molecular mechanism underlying the accumulation of anthocyanins in radish roots.

Sun Y ，Wang J ，Qiu Y ，Liu T ，Song J ，Li X ... -  《-》

Identification of novel and salt-responsive miRNAs to explore miRNA-mediated regulatory network of salt stress response in radish (Raphanus sativus L.).

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. 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). 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.

Sun X ，Xu L ，Wang Y ，Yu R ，Zhu X ，Luo X ，Gong Y ，Wang R ，Limera C ，Zhang K ，Liu L ... -  《BMC GENOMICS》

Transcriptome-wide analysis of chromium-stress responsive microRNAs to explore miRNA-mediated regulatory networks in radish (Raphanus sativus L.).

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.

Liu W ，Xu L ，Wang Y ，Shen H ，Zhu X ，Zhang K ，Chen Y ，Yu R ，Limera C ，Liu L ... -  《Scientific Reports》

Identification of bolting-related microRNAs and their targets reveals complex miRNA-mediated flowering-time regulatory networks in radish (Raphanus sativus L.).

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.

Nie S ，Xu L ，Wang Y ，Huang D ，Muleke EM ，Sun X ，Wang R ，Xie Y ，Gong Y ，Liu L ... -  《Scientific Reports》