Comparative Genomic Analysis of PEBP Genes in Cucurbits Explores the Interactors of Cucumber CsPEBPs Related to Flowering Time.

The family of phosphatidylethanolamine-binding proteins (PEBPs) participates in various plant biological processes, mainly flowering regulation and seed germination. In cucurbit crops, several PEBP genes have been recognized to be responsible for flowering time. However, the investigation of PEBP family members across the genomes of cucurbit species has not been reported, and their conservation and divergence in structure and function remain largely unclear. Herein, PEBP genes were identified from seven cucurbit crops and were used to perform a comparative genomics analysis. The cucurbit PEBP proteins could be classified into MFT, FT, TFL, and PEBP clades, and further, the TFL clade was divided into BFT-like, CEN-like, and TFL1-like subclades. The MFT-like, FT-like, and TFL-like proteins were clearly distinguished by a critical amino acid residue at the 85th position of the Arabidopsis FT protein. In gene expression analysis, CsaPEBP1 was highly expressed in flowers, and its expression levels in females and males were 70.5 and 89.2 times higher, respectively, than those in leaves. CsaPEBP5, CsaPEBP6, and CsaPEBP7 were specifically expressed in male flowers, with expression levels 58.1, 17.3, and 15.7 times higher, respectively, than those of leaves. At least five CsaPEBP genes exhibited the highest expression during the later stages of corolla opening. Through clustering of time-series-based RNA-seq data, several potential transcription factors (TFs) interacting with four CsaPEBPs were identified during cucumber corolla opening. Because of the tandem repeats of binding sites in promoters, NF-YB (Csa4G037610) and GATA (Csa7G64580) TFs appeared to be better able to regulate the CsaPEBP2 and CsaPEBP5 genes, respectively. This study would provide helpful information for further investigating the roles of PEBP genes and their interacting TFs in growth and development processes, such as flowering time regulation in cucurbit crops.

RsERF40 contributes to cold stress tolerance and cell expansion of taproot in radish (Raphanus sativus L.).

The growth and development of taproots are inhibited by cold stress in radish (Raphanus sativus L.). Ethylene-responsive element binding factors (ERF) are key participators in the cold stress response and growth regulation of plants. However, the function of ERF genes in cold tolerance and root development in radish remains elusive. Here, we showed that the secondary growth of radish taproots was inhibited by cold stress. Comparative transcriptome analysis demonstrated that the RsERF40 gene is an important regulator of the cold stress response and root growth regulation. The cold tolerance of transgenic Arabidopsis plants overexpressing the RsERF40 gene was significantly improved. Overexpressing RsERF40 in the cold-sensitive radish genotype and silencing RsERF40 in the cold-tolerant radish genotype indicated that RsERF40 was beneficial for alleviating oxidative damage under cold stress in radish. Transgenic Arabidopsis seedlings showed an increase in the elongation and radial growth of dark-grown roots. RT-qPCR analysis showed that the expression of the cold-related genes (CORs) RsCOR78 and RsCOR413PM1 and the cell wall strengthening-related genes RsCESA6 and RsEXPB3 was upregulated in transgenic Arabidopsis seedlings. Yeast one-hybrid (Y1H) and dual-luciferase reporter assays (DLA) revealed that RsERF40 directly regulates RsCOR78, RsCOR413PM1, RsCESA6 and RsEXPB3 expression, illustrating that RsERF40 enhances cold tolerance and taproot growth by modulating osmotic adjustment and cell wall mechanical strength in radish. In this study, the RsERF40-regulon was firstly found to be a new cold response pathway independent of the CBF-COR pathway conferring cold stress tolerance with increasing radish taproot growth. These results provided novel insight into the molecular mechanism underlying cold stress response and would facilitate the genetic improvement of cold tolerance in radish and other root vegetable crops.

A Comparative Characterization and Expression Profiling Analysis of Fructokinase and Fructokinase-like Genes: Exploring Their Roles in Cucumber Development and Chlorophyll Biosynthesis.

Fructokinase (FRK) and fructokinase-like (FLN), belonging to the phosphofructokinase B type subfamily, share substantial sequence similarity, and are crucial in various plant physiological processes. However, there is limited information regarding what functionally differentiates plant FRKs from FLNs. Here, a total of three CsFRKs and two CsFLNs were identified from the cucumber genome. Their significant difference lay in the structure of their G/AXGD motif, which existed as GAGD in CsFRKs, but as G/ASGD in CsFLNs. Comparative phylogenetic analysis classified CsFRKs and CsFLNs into five sub-branches consistent with their quite different exon/intron organizations. Both transcriptome data and RT-qPCR analyses revealed that CsFRK3 was the most active gene, with the highest expression in the majority of tissues tested. Moreover, the expression levels of two putative plastidic genes, CsFRK1 and CsFLN2, were significantly positively associated with chlorophyll accumulation in the chlorophyll-reduced cucumber mutant. Briefly, both CsFRK and CsFLN genes were involved in the development of sink tissues, especially CsFRK3. CsFRK1 and CsFLN2 were recognized as candidates in the chlorophyll biosynthesis pathway of cucumber. These results would greatly assist in further investigation on functional characterization of FRKs and FLNs, especially in the development and chlorophyll biosynthesis of cucumber.

Genome-wide identification of RsGRAS gene family reveals positive role of RsSHRc gene in chilling stress response in radish (Raphanus sativus L.).

Radish (Raphanus sativus L.) is an important worldwide root vegetable crop. Little information of the GRAS gene family was available in radish. Herein, a total of 51 GRAS family members were firstly identified from radish genome, and unevenly located onto nine radish chromosomes. Expression analysis of RsGRAS genes in taproot displayed that RsSCL15a and RsSHRc were highly expressed in the radish cambium, and its expression level was increased with the taproot thickening. Comparative transcriptome analysis revealed that the expression patterns of RsGRAS genes varied upon exposure to different abiotic stresses including heavy metals, salt and heat. The expression level of six RsGRAS genes including RsSHRc was increased under chilling stress in two radish genotypes with different cold tolerance. Further analysis indicated that RsGRAS genes could respond to cold stress rapidly and the expression of RsSHRc was up-regulated at different development stages (cortex splitting and thickening stages) under long-term cold treatment. Transient expression of RsSHRc gene in radish showed that RsSHRc possessed the reliable function of eliminating reactive oxygen species (ROS), inhibiting the formation of malondialdehyde (MDA) and promoting to accumulate proline under cold stress. Together, these findings provided insights into the function of RsGRAS genes in taproot development and chilling stress response in radish.

Genome-wide characterization of Histone gene family and expression profiling during microspore development in radish (Raphanus sativus L.).

Histone, a predominant protein component of chromatin, participates in DNA packaging and transcriptional regulation. However, the available information of Histone gene family is limited in radish. In this study, a total of 42 Histone gene family members were identified from the radish genome. Sequence alignment and phylogenetic analyses classified the Histone family into three groups (H2A, H2B and H3). Motif analysis showed that the functions of some motifs shared by H3 subfamily genes were related to chromosome regulation and cell development activities, such as motif 5 containing Cks1 and PPR region. Analysis of intron/exon structure indicated that RsCENH3 (RsHistone 18) has the characteristics of variant Histone. Furthermore, several motifs, including the LTR, G-box and TC-elements, were found in the promoters of RsHistone genes, which involved in cell development or various abiotic stresses responses. Transcriptome analysis indicated that the RsHistone genes exhibited higher expression level in floral buds than in roots and leaves. Subcellular localization showed that the RsCENH3 was localized on the nucleus, and it was highly expressed in the floral bud of 3.0-4.0 mm in radish. These findings would provide valuable information for characterization and potential utilization of Histone genes, and facilitate the efficient induction of double haploid plants in radish.

A genome-wide association study uncovers a critical role of the RsPAP2 gene in red-skinned Raphanus sativus L.

Radish (Raphanus sativus L.) taproot contains high concentrations of flavonoids, including anthocyanins (ATCs), in red-skinned genotypes. However, little information on the genetic regulation of ATC biosynthesis in radish is available. A genome-wide association study of radish red skin color was conducted using whole-genome sequencing data derived from 179 radish genotypes. The R2R3-MYB transcription factor production of anthocyanin pigment 2 (PAP2) gene was found in the region associated with a leading SNP located on chromosome 2. The amino acid sequence encoded by the RsPAP2 gene was different from those of the other published RsMYB genes responsible for the red skin color of radish. The overexpression of the RsPAP2 gene resulted in ATC accumulation in Arabidopsis and radish, which was accompanied by the upregulation of several ATC-related structural genes. RsPAP2 was found to bind the RsUFGT and RsTT8 promoters, as shown by a dual-luciferase reporter system and a yeast one-hybrid assay. The promoter activities of the RsANS, RsCHI, RsPAL, and RsUFGT genes could be strongly activated by coinfiltration with RsPAP2 and RsTT8. These findings showed the effectiveness of GWAS in identifying candidate genes in radish and demonstrated that RsPAP2 could (either directly or together with its cofactor RsTT8) regulate the transcript levels of ATC-related genes to promote ATC biosynthesis, facilitating the genetic enhancement of ATC contents and other related traits in radish.

An ultra-high-density genetic map provides insights into genome synteny, recombination landscape and taproot skin colour in radish (Raphanus sativus L.).

High-density genetic map is a valuable tool for exploring novel genomic information, quantitative trait locus (QTL) mapping and gene discovery of economically agronomic traits in plant species. However, high-resolution genetic map applied to tag QTLs associated with important traits and to investigate genomic features underlying recombination landscape in radish (Raphanus sativus) remains largely unexplored. In this study, an ultra-high-density genetic map with 378 738 SNPs covering 1306.8 cM in nine radish linkage groups (LGs) was developed by a whole-genome sequencing-based approach. A total of 18 QTLs for 11 horticulture traits were detected. The map-based cloning data indicated that the R2R3-MYB transcription factor RsMYB90 was a crucial candidate gene determining the taproot skin colour. Comparative genomics analysis among radish, Brassica rapa and B. oleracea genome revealed several genomic rearrangements existed in the radish genome. The highly uneven distribution of recombination was observed across the nine radish chromosomes. Totally, 504 recombination hot regions (RHRs) were enriched near gene promoters and terminators. The recombination rate in RHRs was positively correlated with the density of SNPs and gene, and GC content, respectively. Functional annotation indicated that genes within RHRs were mainly involved in metabolic process and binding. Three QTLs for three traits were found in the RHRs. The results provide novel insights into the radish genome evolution and recombination landscape, and facilitate the development of effective strategies for molecular breeding by targeting and dissecting important traits in radish.

Genome-wide characterization and evolutionary analysis of heat shock transcription factors (HSFs) to reveal their potential role under abiotic stresses in radish (Raphanus sativus L.).

BACKGROUND: Abiotic stresses due to climate change pose a great threat to crop production. Heat shock transcription factors (HSFs) are vital regulators that play key roles in protecting plants against various abiotic stresses. Therefore, the identification and characterization of HSFs is imperative to dissect the mechanism responsible for plant stress responses. Although the HSF gene family has been extensively studied in several plant species, its characterization, evolutionary history and expression patterns in the radish (Raphanus sativus L.) remain limited. RESULTS: In this study, 33 RsHSF genes were obtained from the radish genome, which were classified into three main groups based on HSF protein domain structure. Chromosomal localization analysis revealed that 28 of 33 RsHSF genes were located on nine chromosomes, and 10 duplicated RsHSF genes were grouped into eight gene pairs by whole genome duplication (WGD). Moreover, there were 23 or 9 pairs of orthologous HSFs were identified between radish and Arabidopsis or rice, respectively. Comparative analysis revealed a close relationship among radish, Chinese cabbage and Arabidopsis. RNA-seq data showed that eight RsHSF genes including RsHSF-03, were highly expressed in the leaf, root, cortex, cambium and xylem, indicating that these genes might be involved in plant growth and development. Further, quantitative real-time polymerase chain reaction (RT-qPCR) indicated that the expression patterns of 12 RsHSF genes varied upon exposure to different abiotic stresses including heat, salt, and heavy metals. These results indicated that the RsHSFs may be involved in abiotic stress response. CONCLUSIONS: These results could provide fundamental insights into the characteristics and evolution of the HSF family and facilitate further dissection of the molecular mechanism responsible for radish abiotic stress responses.

Genome- and Transcriptome-Wide Characterization of bZIP Gene Family Identifies Potential Members Involved in Abiotic Stress Response and Anthocyanin Biosynthesis in Radish (Raphanus sativus L.).

Basic leucine zipper (bZIP) transcription factors play crucial roles in various abiotic stress responses as well as anthocyanin accumulation. Anthocyanins are most abundant in colorful skin radish, which exhibit strong antioxidant activity that offers benefits for human health. Here, a total of 135 bZIP-encoding genes were identified from radish genome. Synteny analysis showed that 104 radish and 63 Arabidopsis bZIP genes were orthologous. Transcriptome analysis revealed that 10 RsbZIP genes exhibited high-expression levels in radish taproot (RPKM>10). Specifically, RsbZIP010 exhibited down-regulated expression under Cd, Cr and Pb stresses, whereas RsbZIP031 and RsbZIP059 showed significant down-regulation under heat and salt stresses, respectively. RT-qPCR analysis indicated that RsbZIP011 and RsbZIP102 were significantly up-regulated in the tissues of radish with high anthocyanin contents. Furthermore, the promoter sequences of 39 anthocyanin-related genes were found to contain G-box or ACE-box elements that could be recognized by bZIP family members. Taken together, several RsbZIPs might be served as critical regulators in radish taproot under Cd, Cr, Pb, heat and salt stresses. RsbZIP011 and RsbZIP102 were the potential participants in anthocyanin biosynthesis pathway of radish. These results facilitate further investigation on functional characterization of bZIP genes in response to abiotic stress and anthocyanin biosynthesis in radish.

Transcriptome-based gene expression profiling of diploid radish (Raphanus sativus L.) and the corresponding autotetraploid.

Polyploidy is an important evolutionary factor in most land plant lineages which possess more than two complete sets of chromosomes. Radish (Raphanus sativus L.) is an economically annual/biennial root vegetable crop worldwide. However, the expression patterns of duplicated homologs involved in the autopolyploidization remains unclear. In present study, the autotetraploid radish plants (2n = 4x = 36) were produced with colchicine and exhibited an increase in the size of flowers, leaves, stomata and pollen grains. The differential gene expression (DGE) profiling was performed to investigate the differences in gene expression patterns between diploid and its corresponding autotetraploid by RNA-Sequencing (RNA-Seq). Totally, 483 up-regulated differentially expressed genes (DEGs) and 408 down-regulated DEGs were detected in diploid and autotetraploid radishes, which majorly involved in the pathways of hormones, photosynthesis and stress response. Moreover, the xyloglucan endotransglucosylase/hydrolase (XTH) and pectin methylesterases (PME) family members related to cell enlargement and cell wall construction were found to be enriched in GO enrichment analysis, of which XTH family members enriched in "apoplast" and "cell wall" terms, while PME family members enriched in "cell wall" term. Reverse-transcription quantitative PCR (RT-qPCR) analysis indicated that the expression profile of DEGs were consistent with results from the RNA-Seq analysis. The DEGs involved in cell wall construction and auxin metabolism were predicted to be associated with organs size increase of autotetraploid radishes in the present study. These results could provide valuable information for elucidating the molecular mechanism underlying polyploidization and facilitating further genetic improvements of important traits in radish breeding programs.

Comparative proteomic analysis provides insight into a complex regulatory network of taproot formation in radish (Raphanus sativus L.).

The fleshy taproot of radish is an important storage organ determining its yield and quality. Taproot thickening is a complex developmental process in radish. However, the molecular mechanisms governing this process remain unclear at the proteome level. In this study, a comparative proteomic analysis was performed to analyze the proteome changes at three developmental stages of taproot thickening using iTRAQ approach. In total, 1862 differentially expressed proteins (DEPs) were identified from 6342 high-confidence proteins, among which 256 up-regulated proteins displayed overlapped accumulation in S1 (pre-cortex splitting stage) vs. S2 (cortex splitting stage) and S1 vs. S3 (expanding stage) pairs, whereas 122 up-regulated proteins displayed overlapped accumulation in S1 vs. S3 and S2 vs. S3 pairs. Gene Ontology (GO) and pathway enrichment analysis showed that these DEPs were mainly involved in several processes such as "starch and sucrose metabolism", "plant hormone signal transduction", and "biosynthesis of secondary metabolites". A high concordance existed between iTRAQ and RT-qPCR at the mRNA expression levels. Furthermore, association analysis showed that 187, 181, and 96 DEPs were matched with their corresponding differentially expressed genes (DEGs) in S1 vs. S2, S1 vs. S3, and S2 vs. S3 comparison, respectively. Notably, several functional proteins including cell division cycle 5-like protein (CDC5), expansin B1 (EXPB1), and xyloglucan endotransglucosylase/hydrolase protein 24 (XTH24) were responsible for cell division and expansion during radish taproot thickening process. These results could facilitate a better understanding of the molecular mechanism underlying taproot thickening, and provide valuable information for the identification of critical genes/proteins responsible for taproot thickening in root vegetable crops.

Transcriptomic analysis of Pak Choi under acute ozone exposure revealed regulatory mechanism against ozone stress.

BACKGROUND: Ground-level ozone (O3) is one of the major air pollutants, which cause oxidative injury to plants. The physiological and biochemical mechanisms underlying the responses of plants to O3 stress have been well investigated. However, there are limited reports about the molecular basis of plant responses to O3. In this study, a comparative transcriptomic analysis of Pak Choi (Brassica campestris ssp. chinensis) exposed to different O3 concentrations was conducted for the first time. RESULTS: Seedlings of Pak Choi with five leaves were exposed to non-filtered air (NF, 31 ppb) or elevated O3 (E-O3, 252 ppb) for 2 days (8 h per day, from 9:00-17:00). Compared with plants in the NF, a total of 675 differentially expressed genes (DEGs) were identified in plants under E-O3, including 219 DEGs with decreased expressions and 456 DEGs with increased expressions. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that O3 stress invoked multiple cellular defense pathways to mitigate the impaired cellular integrity and metabolism, including 'glutathione metabolism', 'phenylpropanoid biosynthesis', 'sulfur metabolism', 'glucosinolate biosynthesis', 'cutin, suberine and wax biosynthesis' and others. Transcription factors potentially involved in this cellular regulation were also found, such as AP2-ERF, WRKY, JAZ, MYB etc. Based on the RNA-Seq data and previous studies, a working model was proposed integrating O3 caused reactive oxygen burst, oxidation-reduction regulation, jasmonic acid and downstream functional genes for the regulation of cellular homeostasis after acute O3 stress. CONCLUSION: The present results provide a valuable insight into the molecular responses of Pak Choi to acute O3 stress and the specific DEGs revealed in this study could be used for further functional identification of key allelic genes determining the O3 sensitivity of Pak Choi.

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 WRKY gene family in radish (Raphanus sativus L.) reveals its critical functions under different abiotic stresses.

KEY MESSAGE: The radish WRKY gene family was genome-widely identified and played critical roles in response to multiple abiotic stresses. The WRKY is among the largest transcription factors (TFs) associated with multiple biological activities for plant survival, including control response mechanisms against abiotic stresses such as heat, salinity, and heavy metals. Radish is an important root vegetable crop and therefore characterization and expression pattern investigation of WRKY transcription factors in radish is imperative. In the present study, 126 putative WRKY genes were retrieved from radish genome database. Protein sequence and annotation scrutiny confirmed that RsWRKY proteins possessed highly conserved domains and zinc finger motif. Based on phylogenetic analysis results, RsWRKYs candidate genes were divided into three groups (Group I, II and III) with the number 31, 74, and 20, respectively. Additionally, gene structure analysis revealed that intron-exon patterns of the WRKY genes are highly conserved in radish. Linkage map analysis indicated that RsWRKY genes were distributed with varying densities over nine linkage groups. Further, RT-qPCR analysis illustrated the significant variation of 36 RsWRKY genes under one or more abiotic stress treatments, implicating that they might be stress-responsive genes. In total, 126 WRKY TFs were identified from the R. sativus genome wherein, 35 of them showed abiotic stress-induced expression patterns. These results provide a genome-wide characterization of RsWRKY TFs and baseline for further functional dissection and molecular evolution investigation, specifically for improving abiotic stress resistances with an ultimate goal of increasing yield and quality of radish.

Transcriptomic profiling of two Pak Choi varieties with contrasting anthocyanin contents provides an insight into structural and regulatory genes in anthocyanin biosynthetic pathway.

BACKGROUND: The accumulation of anthocyanin in horticultural crops not only improves their stress tolerances but also their nutritional values. Many key regulatory and structural genes in anthocyanin biosynthesis have been identified in model plants, but limited information is available for non-model plant species featured with colored leaves. In this study, two Pak Choi varieties with green or purple leaves were selected to analyze the anthocyanin biosynthesis through RNA-Seq. RESULTS: A total of 2475 unigenes were differentially expressed between these tested varieties, including 1303 down-regulated and 1172 up-regulated genes in the purple-leafed one. The reliability of the RNA-Seq was further confirmed by using real-time quantitative PCR. Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the differentially expressed genes revealed 'flavonoid biosynthesis' was the only enriched pathway in the purple-leafed variety: In the pathway of phenylpropanoid metabolism, Bra017210, Bra039777, and Bra021637 were expressed at higher levels in the purple-leafed variety;　among the early anthocyanin biosynthetic genes, Bra037747 transcripts were only detected in the purple-leafed variety but not in the green-leafed one; among the late anthocyanin biosynthetic genes, Bra027457, Bra013652, Bra019350, Bra003021, Bra035004, and Bra038445 were all up-regulated in purple-leafed variety; and genes encoding anthocyanin-related transcription factors, such as Bra016164, and genes encoding anthocyanin transportation, such as GST F12, were also identified as up-regulated ones in the purple-leafed variety. CONCLUSIONS: The current result provided a valuable insight into the anthocyanin accumulation in the purple-leafed variety of Pak Choi and a bioinformatic resource for further functional identification of key allelic genes determining the difference of anthocyanin content between Pak Choi varieties.