Xiaochuan Sun

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

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.

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.

Metabolomic analysis with GC-MS to reveal potential metabolites and biological pathways involved in Pb & Cd stress response of radish roots

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.

De novo transcriptome analysis in radish (Raphanus sativus L.) and identification of critical genes involved in bolting and flowering

BackgroundThe 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).ResultsA 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.ConclusionsThis 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.

Genome-Wide Characterization of the MADS-Box Gene Family in Radish (Raphanus sativus L.) and Assessment of Its Roles in Flowering and Floral Organogenesis

The MADS-box gene family is an important transcription factor (TF) family that is involved in various aspects of plant growth and development, especially flowering time and floral organogenesis. Although it has been reported in many plant species, the systematic identification and characterization of MADS-box TF family is still limited in radish (Raphanus sativus L.). In the present study, a comprehensive analysis of MADS-box genes was performed, and a total of 144 MADS-box family members were identified from the whole radish genome. Meanwhile, a detailed list of MADS-box genes from other 28 plant species was also investigated. Through the phylogenetic analysis between radish and Arabidopsis thaliana, all the RsMADS genes were classified into two groups including 68 type I (31 Mα, 12 Mβ and 25Mγ) and 76 type II (70 MIKCC and 6 MIKC∗). Among them, 41 (28.47%) RsMADS genes were located in nine linkage groups of radish from R1 to R9. Moreover, the homologous MADS-box gene pairs were identified among radish, A. thaliana, Chinese cabbage and rice. Additionally, the expression profiles of RsMADS genes were systematically investigated in different tissues and growth stages. Furthermore, quantitative real-time PCR analysis was employed to validate expression patterns of some crucial RsMADS genes. These results could provide a valuable resource to explore the potential functions of RsMADS genes in radish, and facilitate dissecting MADS-box gene-mediated molecular mechanisms underlying flowering and floral organogenesis in root vegetable crops.

Unraveling the Root Proteome Changes and Its Relationship to Molecular Mechanism Underlying Salt Stress Response in Radish (Raphanus sativus L.)

To understand the molecular mechanism underlying salt stress response in radish, iTRAQ-based proteomic analysis was conducted to investigate the differences in protein species abundance under different salt treatments. In total, 851, 706, and 685 differential abundance protein species (DAPS) were identified between CK vs. Na100, CK vs. Na200, and Na100 vs. Na200, respectively. Functional annotation analysis revealed that salt stress elicited complex proteomic alterations in radish roots involved in carbohydrate and energy metabolism, protein metabolism, signal transduction, transcription regulation, stress and defense and transport. Additionally, the expression levels of nine genes encoding DAPS were further verified using RT-qPCR. The integrative analysis of transcriptomic and proteomic data in conjunction with miRNAs was further performed to strengthen the understanding of radish response to salinity. The genes responsible for signal transduction, ROS scavenging and transport activities as well as several key miRNAs including miR171, miR395, and miR398 played crucial roles in salt stress response in radish. Based on these findings, a schematic genetic regulatory network of salt stress response was proposed. This study provided valuable insights into the molecular mechanism underlying salt stress response in radish roots and would facilitate developing effective strategies toward genetically engineered salt-tolerant radish and other root vegetable crops.

Transcriptome Profiling of Taproot Reveals Complex Regulatory Networks during Taproot Thickening in Radish (Raphanus sativus L.).

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.

Identification of critical genes associated with lignin biosynthesis in radish (Raphanus sativus L.) by de novo transcriptome sequencing

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.