Coordinated Regulation of Anthocyanin Biosynthesis Genes Confers Varied Phenotypic and Spatial-Temporal Anthocyanin Accumulation in Radish (Raphanus sativus L.).

Anthocyanins are natural pigments that have important functions in plant growth and development. Radish taproots are rich in anthocyanins which confer different taproot colors and are potentially beneficial to human health. The crop differentially accumulates anthocyanin during various stages of growth, yet molecular mechanisms underlying this differential anthocyanin accumulation remains unknown. In the present study, transcriptome analysis was used to concisely identify putative genes involved in anthocyanin biosynthesis in radish. Spatial-temporal transcript expressions were then profiled in four color variant radish cultivars. From the total transcript sequences obtained through illumina sequencing, 102 assembled unigenes, and 20 candidate genes were identified to be involved in anthocyanin biosynthesis. Fifteen genomic sequences were isolated and sequenced from radish taproot. The length of these sequences was between 900 and 1,579 bp, and the unigene coverage to all of the corresponding cloned sequences was more than 93%. Gene structure analysis revealed that RsF3'H is intronless and anthocyanin biosynthesis genes (ABGs) bear asymmetrical exons, except RsSAM. Anthocyanin accumulation showed a gradual increase in the leaf of the red radish and the taproot of colored cultivars during development, with a rapid increase at 30 days after sowing (DAS), and the highest content at maturity. Spatial-temporal transcriptional analysis of 14 genes revealed detectable expressions of 12 ABGs in various tissues at different growth levels. The investigation of anthocyanin accumulation and gene expression in four color variant radish cultivars, at different stages of development, indicated that total anthocyanin correlated with transcript levels of ABGs, particularly RsUFGT, RsF3H, RsANS, RsCHS3 and RsF3'H1. Our results suggest that these candidate genes play key roles in phenotypic and spatial-temporal anthocyanin accumulation in radish through coordinated regulation and the major control point in anthocyanin biosynthesis in radish is RsUFGT. The present findings lend invaluable insights into anthocyanin biosynthesis and may facilitate genetic manipulation for enhanced anthocyanin content in radish.

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 Mß 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.

De novo Taproot Transcriptome Sequencing and Analysis of Major Genes Involved in Sucrose Metabolism in Radish (Raphanus sativus L.).

Radish (Raphanus sativus L.) is an important annual or biennial root vegetable crop. The fleshy taproot comprises the main edible portion of the plant with high nutrition and medical value. Molecular biology study of radish begun rather later, and lacks sufficient transcriptomic and genomic data in pubic databases for understanding of the molecular mechanism during the radish taproot formation. To develop a comprehensive overview of the 'NAU-YH' root transcriptome, a cDNA library, prepared from three equally mixed RNA of taproots at different developmental stages including pre-cortex splitting stage, cortex splitting stage, and expanding stage was sequenced using high-throughput Illumina RNA sequencing. From approximately 51 million clean reads, a total of 70,168 unigenes with a total length of 50.28 Mb, an average length of 717 bp and a N50 of 994 bp were obtained. In total, 63,991 (about 91.20% of the assembled unigenes) unigenes were successfully annotated in five public databases including NR, GO, COG, KEGG, and Nt. GO analysis revealed that the majority of these unigenes were predominately involved in basic physiological and metabolic processes, catalytic, binding, and cellular process. In addition, a total of 103 unigenes encoding eight enzymes involved in the sucrose metabolism related pathways were also identified by KEGG pathway analysis. Sucrose synthase (29 unigenes), invertase (17 unigenes), sucrose-phosphate synthase (16 unigenes), fructokinase (17 unigenes), and hexokinase (11 unigenes) ranked top five in these eight key enzymes. From which, two genes (RsSuSy1, RsSPS1) were validated by T-A cloning and sequenced, while the expression of six unigenes were profiled with RT-qPCR analysis. These results would be served as an important public reference platform to identify the related key genes during taproot thickening and facilitate the dissection of molecular mechanisms underlying taproot formation in radish.

Transcriptome-based gene expression profiling identifies differentially expressed genes critical for salt stress response in radish (Raphanus sativus L.).

KEY MESSAGE: Transcriptome-based gene expression analysis identifies many critical salt-responsive genes in radish and facilitates further dissecting the molecular mechanism underlying salt stress response. Salt stress severely impacts plant growth and development. Radish, a moderately salt-sensitive vegetable crop, has been studied for decades towards the physiological and biochemical performances under salt stress. However, no systematic study on isolation and identification of genes involved in salt stress response has been performed in radish, and the molecular mechanism governing this process is still indistinct. Here, the RNA-Seq technique was applied to analyze the transcriptomic changes on radish roots treated with salt (200 mM NaCl) for 48 h in comparison with those cultured in normal condition. Totally 8709 differentially expressed genes (DEGs) including 3931 up- and 4778 down-regulated genes were identified. Functional annotation analysis indicated that many genes could be involved in several aspects of salt stress response including stress sensing and signal transduction, osmoregulation, ion homeostasis and ROS scavenging. The association analysis of salt-responsive genes and miRNAs exhibited that 36 miRNA-mRNA pairs had negative correlationship in expression trends. Reverse-transcription quantitative PCR (RT-qPCR) analysis revealed that the expression profiles of DEGs were in line with results from the RNA-Seq analysis. Furthermore, the putative model of DEGs and miRNA-mediated gene regulation was proposed to elucidate how radish sensed and responded to salt stress. This study represents the first comprehensive transcriptome-based gene expression profiling under salt stress in radish. The outcomes of this study could facilitate further dissecting the molecular mechanism underlying salt stress response and provide a valuable platform for further genetic improvement of salt tolerance in radish breeding programs.

Transcriptome-based gene profiling provides novel insights into the characteristics of radish root response to Cr stress with next-generation sequencing.

Radish (Raphanus sativus L.) is an important worldwide root vegetable crop with high nutrient values and is adversely affected by non-essential heavy metals including chromium (Cr). Little is known about the molecular mechanism underlying Cr stress response in radish. In this study, RNA-Seq technique was employed to identify differentially expressed genes (DEGs) under Cr stress. Based on de novo transcriptome assembly, there were 30,676 unigenes representing 60,881 transcripts isolated from radish root under Cr stress. Differential gene analysis revealed that 2985 uingenes were significantly differentially expressed between Cr-free (CK) and Cr-treated (Cr600) libraries, among which 1424 were up-regulated and 1561 down-regulated. Gene ontology (GO) analysis revealed that these DEGs were mainly involved in primary metabolic process, response to abiotic stimulus, cellular metabolic process and small molecule metabolic process. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis showed that the DEGs were mainly involved in protein processing in endoplasmic reticulum, starch and sucrose metabolism, amino acid metabolism, glutathione metabolism, drug and xenobiotics by cytochrome P450 metabolism. RT-qPCR analysis showed that the expression patterns of 12 randomly selected DEGs were highly accordant with the results from RNA-seq. Furthermore, many candidate genes including signaling protein kinases, transcription factors and metal transporters, chelate compound biosynthesis and antioxidant system, were involved in defense and detoxification mechanisms of Cr stress response regulatory networks. These results would provide novel insight into molecular mechanism underlying plant responsiveness to Cr stress and facilitate further genetic manipulation on Cr uptake and accumulation in radish.