Comparative transcriptome analysis reveals gene network regulating cadmium uptake and translocation in peanut roots under iron deficiency.

BACKGROUND: Iron (Fe) is an essential element for plant growth and development, whereas cadmium (Cd) is non-essential and highly toxic. Previous studies showed that Fe deficiency enhanced Cd uptake and accumulation in peanuts. However, the molecular mechanism underlying the increased Cd accumulation in Fe-deficient peanut plants is poorly understood. RESULTS: We employed a comparative transcriptome analysis approach to identify differentially expressed genes (DEGs) in peanut roots exposed to Fe-sufficient without Cd, Fe-deficient without Cd, Fe-sufficient with Cd and Fe-deficient with Cd. Compared with the control, Fe deficiency induced 465 up-regulated and 211 down-regulated DEGs, whereas the up- and down-regulated DEGs in Cd exposed plants were 329 and 189, respectively. Under Fe-deficient conditions, Cd exposure resulted in 907 up-regulated DEGs and 953 down-regulated DEGs. In the presence of Cd, Fe deficiency induced 1042 up-regulated and 847 down-regulated genes, respectively. Based on our array data, we found that metal transporter genes such as CAX4, COPT1, IRT1, NRAMP5, OPT3, YSL3, VIT3 and VIT4 might be involved in iron homeostasis. Moreover, combined with quantitative real-time PCR, IRT1, NRAMP3, NRAMP5, OPT3, YSL3, ABCC3, ZIP1, and ZIP5 were verified to be responsible for Cd uptake and translocation in peanut plants under iron deficiency. Additionally, a larger amount of ABC transporter genes was induced or suppressed by iron deficiency under Cd exposure, indicating that this family may play important roles in Fe/Cd uptake and transport. CONCLUSIONS: The up-regulated expression of NRAMP5 and IRT1 genes induced by iron deficiency may enhance Cd uptake in peanut roots. The decrease of Cd translocation from roots to shoots may be resulted from the down-regulation of ZIP1, ZIP5 and YSL3 under iron deficiency.

Comparative proteomics analysis of peanut roots reveals differential mechanisms of cadmium detoxification and translocation between two cultivars differing in cadmium accumulation.

BACKGROUND: Peanut is one of the most important oil and protein crops, and it exhibits wide cultivar variations in shoot Cd accumulation ability. However, the mechanism of Cd accumulation in peanut shoots has not been well understood. In this study, the root proteomics of two cultivars differing in seed Cd accumulation, Fenghua 1 (F, low Cd cultivar) and Silihong (S, high Cd cultivar), were investigated under 0 (CK) and 2 µM Cd conditions. RESULTS: A total of 4676 proteins were identified by proteomics screening. Of them, 375, 1762, 1276 and 771 proteins were identified to be differentially expressed proteins (DEPs) for comparison of FCd/FCK, SCd/SCK, FCK/SCK and FCd/SCd, respectively. Silihong is more sensitive to Cd exposure than Fenghua 1 in terms of root proteomics. A total of 30 and 86 DEPs were identified to be related with heavy metal transport and cell wall modification, respectively. The up-regulation of ABCB25, ABCC14, ABCC2, PDR1 and V-ATPases by Cd exposure in Silihong might enhance vacuolar sequestration of Cd and its efflux from symplast to apoplast. The higher Cd accumulation in the root CWs of Silihong might be resulted from its higher capability of CW modification, in which many proteins such as IRX10L, BGLU12-like, BGLU42, EXLB1, XTH30, XTH6, XYL7, PAL3, COMT, CAD1, and CCR1 were involved. CONCLUSIONS: The vacuolar sequestration and efflux of Cd as well as its adsorption in CW might be the principal mechanism of cadmium detoxification in Silihong. The higher capacity of Cd accumulation and translocation of Silihong is an inherent characteristics in which ACA8 and ZIP1 might be involved.

Comparative transcriptome analysis revealed key factors for differential cadmium transport and retention in roots of two contrasting peanut cultivars.

BACKGROUND: Peanut is the world's fourth largest oilseed crop that exhibits wide cultivar variations in cadmium (Cd) accumulation. To establish the mechanisms of Cd distribution and accumulation in peanut plants, eight cDNA libraries from the roots of two contrasting cultivars, Fenghua 1 (low-Cd cultivar) and Silihong (high-Cd cultivar), were constructed and sequenced by RNA-sequencing. The expression patterns of 16 candidate DEGs were validated by RT-qPCR analysis. RESULTS: A total of 75,634 genes including 71,349 known genes and 4484 novel genes were identified in eight cDNA libraries, among which 6798 genes were found to be Cd-responsive DEGs and/or DEGs between these two cultivars. Interestingly, 183 DEGs encoding ion transport related proteins and 260 DEGs encoding cell wall related proteins were identified. Among these DEGs, nine metal transporter genes (PDR1, ABCC4 and ABCC15, IRT1, ZIP1, ZIP11, YSL7, DTX43 and MTP4) and nine cell wall related genes (PEs, PGIPs, GTs, XYT12 CYP450s, LACs, 4CL2, C4H and CASP5) showed higher expression in Fenghua 1 than in Silihong. CONCLUSIONS: Both the metal transporters and cell wall modification might be responsible for the difference in Cd accumulation and translocation between Fenghua 1 and Silihong. These findings would be useful for further functional analysis, and reveal the molecular mechanism responsible for genotype difference in Cd accumulation.

Comparative transcriptome analysis reveals key cadmium transport-related genes in roots of two pak choi (Brassica rapa L. ssp. chinensis) cultivars.

BACKGROUND: Cadmium translocation from roots to shoots is a complex biological process that is controlled by gene regulatory networks. Pak choi exhibits wide cultivar variations in Cd accumulation. However, the molecular mechanism involved in cadmium translocation and accumulation is still unclear. To isolate differentially expressed genes (DEGs) involved in transporter-mediated regulatory mechanisms of Cd translocation in two contrasting pak choi cultivars, Baiyewuyueman (B, high Cd accumulator) and Kuishan'aijiaoheiye (K, low Cd accumulator), eight cDNA libraries from the roots of two cultivars were constructed and sequenced by RNA-sequencing. RESULTS: A total of 244,190 unigenes were obtained. Of them, 6827 DEGs, including BCd10 vs. BCd0 (690), KCd10 vs. KCd0 (2733), KCd0 vs. BCd0 (2919), and KCd10 vs. BCd10 (3455), were identified. Regulatory roles of these DEGs were annotated and clarified through GO and KEEG enrichment analysis. Interestingly, 135 DEGs encoding ion transport (i.e. ZIPs, P1B-type ATPase and MTPs) related proteins were identified. The expression patterns of ten critical genes were validated using RT-qPCR analysis. Furthermore, a putative model of cadmium translocation regulatory network in pak choi was proposed. CONCLUSIONS: High Cd cultivar (Baiyewuyueman) showed higher expression levels in plasma membrane-localized transport genes (i.e., ZIP2, ZIP3, IRT1, HMA2 and HMA4) and tonoplast-localized transport genes (i.e., CAX4, HMA3, MRP7, MTP3 and COPT5) than low Cd cultivar (Kuishan'aijiaoheiye). These genes, therefore, might be involved in root-to-shoot Cd translocation in pak choi.

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

BACKGROUND: 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. RESULTS: 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). CONCLUSIONS: 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.

Transcriptome profiling of root microRNAs reveals novel insights into taproot thickening in radish (Raphanus sativus L.).

BACKGROUND: Radish (Raphanus sativus L.) is an economically important root vegetable crop, and the taproot-thickening process is the most critical period for the final productivity and quality formation. MicroRNAs (miRNAs) are a family of non-coding small RNAs that play an important regulatory function in plant growth and development. However, the characterization of miRNAs and their roles in regulating radish taproot growth and thickening remain largely unexplored. A Solexa high-throughput sequencing technology was used to identify key miRNAs involved in taproot thickening in radish. RESULTS: Three small RNA libraries from 'NAU-YH' taproot collected at pre-cortex splitting stage, cortex splitting stage and expanding stage were constructed. In all, 175 known and 107 potential novel miRNAs were discovered, from which 85 known and 13 novel miRNAs were found to be significantly differentially expressed during taproot thickening. Furthermore, totally 191 target genes were identified for the differentially expressed miRNAs. These target genes were annotated as transcription factors and other functional proteins, which were involved in various biological functions including plant growth and development, metabolism, cell organization and biogenesis, signal sensing and transduction, and plant defense response. RT-qPCR analysis validated miRNA expression patterns for five miRNAs and their corresponding target genes. CONCLUSIONS: The small RNA populations of radish taproot at different thickening stages were firstly identified by Solexa sequencing. Totally 98 differentially expressed miRNAs identified from three taproot libraries might play important regulatory roles in taproot thickening. Their targets encoding transcription factors and other functional proteins including NF-YA2, ILR1, bHLH74, XTH16, CEL41 and EXPA9 were involved in radish taproot thickening. These results could provide new insights into the regulatory roles of miRNAs during the taproot thickening and facilitate genetic improvement of taproot in radish.

De novo transcriptome sequencing of radish (Raphanus sativus L.) and analysis of major genes involved in glucosinolate metabolism.

BACKGROUND: Radish (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. RESULTS: In 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 predominantly 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. CONCLUSIONS: The 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.

Genome-wide identification and characterization of cadmium-responsive microRNAs and their target genes in radish (Raphanus sativus L.) roots.

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.

Identification and differential expression analysis of anthocyanin biosynthetic genes in root-skin color variants of radish (Raphanus sativus L.).

BACKGROUND: Taproot skin color is a major trait for assessing the commercial and nutritional quality of radish, and red-skinned radish is confirmed to improve consumer's interest and health. However, little is known about the molecular mechanisms responsible for controlling the formation of red-skinned radish. OBJECTIVE: This study aimed to identify the differentially expressed anthocyanin biosynthetic genes between red- and white-skinned radishes and understand the molecular regulatory mechanism underlying red-skinned radish formation. METHODS: Based on the published complete genome sequence of radish, the digital gene expression profiles of Yangzhouyuanbai (YB, white-skinned) and Sading (SD, red-skinned) were analyzed using Illumina sequencing. RESULTS: A total of 3666 DEGs were identified in SD compared with YB. Interestingly, 46 genes encoded enzymes related to anthocyanin biosynthesis and 241 genes encoded transcription factors were identified. KEGG pathway analysis showed that the formation of red-skinned radish was mainly controlled by pelargonidin-derived anthocyanin biosynthetic pathway genes. This process included the upregulation of PAL, C4H, 4CL, CHS, CHI, F3H, DFR, LDOX, and UGT enzymes in SD. CHS genes were specifically expressed in SD, and it might be the key point for red pigment accumulation in red-skinned radish. Furthermore, MYB1/2/75, bHLH (TT8), and WD 40 showed higher expression in SD than in YB. Meanwhile, the corresponding low-abundance anthocyanin biosynthesis enzymes and upregulation of MYB4 might be the factors influencing the formation of white-skinned radish. CONCLUSION: These findings provide new insights into the molecular mechanisms and regulatory network of anthocyanin biosynthesis in red-skinned radish.

Variations in root morphology among 18 herbaceous species and their relationship with cadmium accumulation.

This study aims to investigate whether root system morphology is involved in the interspecific variations in Cd accumulation in herbaceous plants. Biomasses, root morphology, and Cd accumulation of 18 herbaceous species were determined under 0, 2, and 10 mg kg-1 Cd conditions. Significant variations were found in biomass production, root system morphology, and Cd accumulation among the 18 species. Cd concentrations in the shoot had negative correlations with the biomass of roots and shoots in the 2 mg kg-1 Cd treatment. Total amounts of Cd in plants showed positive correlations with the biomass of roots and shoots, total root lengths, root surface areas, root volumes, and proportions of the fine roots (diameter <0.2 mm). Percentages of Cd in shoots were positively related to specific root lengths, root surface areas, and plant biomasses but negatively correlated with proportions of roots in the 0.6-0.8-mm diameter class. High-biomass species (rapeseed, Indian mustard, and four-o'clock) have high Cd uptake capacity due to their large root system. Longer and thinner roots might contribute to higher capacity for transferring Cd from roots to shoots, while coarse roots (i.e., diameter of 0.6-0.8 mm) could retain more Cd in the tissues and, consequently, reduce Cd transfer from roots to shoots.

Comparative transcriptomic analysis reveals the roles of ROS scavenging genes in response to cadmium in two pak choi cultivars.

To identify key regulatory genes involved in ROS scavenging in response to cadmium (Cd) exposure in pak choi, eight cDNA libraries from Cd-treated and Cd-free roots of two cultivars, Baiyewuyueman (high Cd accumulator) and Kuishan'aijiaoheiye (low Cd accumulator), were firstly performed by RNA-sequencing. Totally 0.443 billion clean reads and 244,190 unigenes were obtained from eight transcriptome. About 797 and 1167 unigenes encoding ROS related proteins and transcription factors were identified. Of them, 11 and 16 ROS scavenging system related DEGs, and 29 and 15 transcription factors related DEGs were found in Baiyewuyueman and Kuishan'aijiaoheiye, respectively. Ten ROS-scavenging genes (Cu/Zn-SOD, GST1, PODs, TrxR2, PrxR, FER3 and NDPK) showed higher expression levels in Cd-exposed seedings of Baiyewuyueman than those of Kuishan'aijiaoheiye. Four genes (GPX, APX, GRX and GST3) specifically expressed in Cd-free roots of Kuishan'aijiaoheiye. For transcription factors, ERF12/13/22 and WRKY31 was up-regulated by Cd in Baiyewuyueman, while in Kuishan'aijiaoheiye, Cd induced down-regulations of bZIP, NAC and ZFP families. The results indicate that the two cultivars differed in the mechanism of ROS scavenging in response to Cd stress. Fe SOD1, POD A2/44/54/62 and GST1 may be responsible for the difference of Cd tolerance between Baiyewuyueman and Kuishan'aijiaoheiye.

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 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.

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.

De novo sequencing of root transcriptome reveals complex cadmium-responsive regulatory networks in radish (Raphanus sativus L.).

Cadmium (Cd) is a nonessential metallic trace element that poses potential chronic toxicity to living organisms. To date, little is known about the Cd-responsive regulatory network in root vegetable crops including radish. In this study, 31,015 unigenes representing 66,552 assembled unique transcripts were isolated from radish root under Cd stress based on de novo transcriptome assembly. In all, 1496 differentially expressed genes (DEGs) consisted of 3579 transcripts were identified from Cd-free (CK) and Cd-treated (Cd200) libraries. Gene Ontology and pathway enrichment analysis indicated that the up- and down-regulated DEGs were predominately involved in glucosinolate biosynthesis as well as cysteine and methionine-related pathways, respectively. RT-qPCR showed that the expression profiles of DEGs were in consistent with results from RNA-Seq analysis. Several candidate genes encoding phytochelatin synthase (PCS), metallothioneins (MTs), glutathione (GSH), zinc iron permease (ZIPs) and ABC transporter were responsible for Cd uptake, accumulation, translocation and detoxification in radish. The schematic model of DEGs and microRNAs-involved in Cd-responsive regulatory network was proposed. This study represents a first comprehensive transcriptome-based characterization of Cd-responsive DEGs in radish. These results could provide fundamental insight into complex Cd-responsive regulatory networks and facilitate further genetic manipulation of Cd accumulation in root vegetable crops.

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.