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Auxin transport is regulated by the AUX1/LAX, PIN, and ABCB gene families, controlling the distribution of auxin and ultimately fruit curving in cucumbers. However, studies on the differential expression of these auxin transporters and their roles in fruit curving are limited. In this study, we identified 36 auxin transporters from cucumber, including CsLAX1–7, CsPIN1–10, and CsABCB1–19. Basic characteristic analysis revealed that all CsLAX proteins were conservative, and a C-terminal NPNTY motif was found in CsPIN1–4/7–10. CsABCB1/5/11/14/17 were categorized as half-size transporters. Phylogenetic analysis revealed a genetic relationship between auxin transporters in Arabidopsis and cucumber. Exogenous auxin treatment on fruits and qPCR analysis indicated that differential expression patterns of auxin transporters control cucumber fruit curving. Co-expression analysis indicated that CsPIN1 and CsLAX2 were substantially negatively correlated, and they displayed opposite expression patterns in curved fruits. A proposed model suggested that CsLAX2 transports extracellular auxin to the convex side of the fruit; however, CsPIN1 inhibits auxin efflux at the same location. This leads to uneven auxin distribution that results in cucumber fruit curving. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Propamocarb is a pesticide widely used to control cucumber downy mildew. The overuse of propamocarb has resulted in residues and phytotoxicity. However, the detoxification and metabolic process of propamocarb have not been documented well. Our previous work showed differences in the propamocarb residues among the different genotypes of cucumber and their regulation by multiple genes. Based on the already reported data on gene expression profiles under propamocarb treatment, we identified the glutathione pathway, including six different genes (Csa4M303130, Csa3M133380, Csa5M409710, Csa7M395820, Csa3M597320, and Csa1M571280), involved in propamocarb detoxification. The qPCR analysis showed that Csa1M571280 (CsGSH2) was most significantly and differentially expressed at 48 h after propamocarb spray in the cucumber varieties Y3F604 (low propamocarb residues) and M729 (high propamocarb residues). In Y3F604, CsGSH2 expression increased from 6 to 48 h after spraying propamocarb, and the expression was positively correlated with propamocarb residues, whereas M729 showed no significant difference in CsGSH2 expression. Therefore, we presumed CsGSH2 as a key gene in managing propamocarb residues. Gene functional analysis showed that propamocarb residues decreased in CsGSH2-overexpressing plants and increased in CsGSH2-antisense plants. Overexpression of CsGSH2 enhanced glutathione (GSH) accumulation and glutathione S-transferase (GST), glutathione reductase (GR), and glutathione peroxidase (GPX) activities, probably for propamocarb detoxification. The activity of antioxidant enzymes (SOD, POD, CAT, and APX) increased to maintain a high antioxidant capacity in CsGSH2-overexpressing plants. The superoxide (O2−), hydrogen peroxide (H2O2), and malondialdehyde (MDA) levels decreased in CsGSH2-overexpressing plants, promoting the antioxidant system composed of ascorbic acid and glutathione (AsA-GSH). Thus, we conclude that CsGSH2 alleviates propamocarb residues and phytotoxicity by enhancing cucumber's antioxidant and glutathione detoxification potential. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

A senescence-induced gene named CsNRT1.7 was cloned from a cucumber cultivar with high capacity for low-N tolerance using a homologous cloning technique based on bioinformatics analysis and cucumber genome information. The full-length cDNA of CsNRT1.7 was 1803 bp with an open reading frame encoding 601 amino acids. Homology analysis showed that CsNRT1.7 had high homology (74%) to the Arabidopsis nitrate transporter gene NRT1.7. Phylogenetic analysis indicated that CsNRT1.7 had a close relationship with NRT1.7 among different nitrate transporters in Arabidopsis. Quantitative RT-PCR and CsNRT1.7pro:GUS reporter analysis showed that CsNRT1.7 was a senescence-induced gene, whose transcription was much stronger in old leaf, followed by mature leaf and young leaf of cucumber plants. CsNRT1.7 could also be induced by low-nitrogen treatment. The transient expression of CsNRT1.7:EGFP in onion epidermal cells indicated that CsNRT1.7 was localized in the plasma membrane. Further functional analysis revealed that 35S:CsNRT1.7/nrt1.7-2 transgenic Arabidopsis plants showed lower nitrate content and larger leaf size than nrt1.7-2 T-DNA insertion Arabidopsis plants. These results provide new clues to further understand the function of CsNRT1.7 regarding senescence-regulated low-nitrogen tolerance in cucumber. [ABSTRACT FROM AUTHOR]

• Totally 42 members of the Histone gene family were firstly identified in radish. • Seven RsCENH3 genes exhibited specifically high expression level in the radish bud. • RsCENH3 gene might be involved in promoting microspore development in radish. 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. [ABSTRACT FROM AUTHOR]

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 ArabidopsisbZIP 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. [ABSTRACT FROM AUTHOR]

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. • The GRAS gene family was comprehensively characterized in radish. • Expression analysis indicates RsSHRc might be involved in radish taproot thickening. • Transient expression analysis revealed RsSHRc gene could enhance cold tolerance of radish plants. [ABSTRACT FROM AUTHOR]

Highlights • MeJA and SA have positive effects on GLS accumulation in radish taproot. • Differential expression of genes was observed under MeJA, SA and ABA treatments. • Myrosinase activity and SF content were highly sensitive to SA. • SF is correlated with myrosinase activity and GRA content. Abstract Radish is an important root vegetable crop. The effects of environmental stresses on glucosinolate (GLS) contents were extensively investigated in Brassicaceae species. However, little is known about the accumulation of GLS and sulforaphane (SF) in hormone-treated radish taproot. In this study, the effects of methyl jasmonate (MeJA), salicylic acid (SA) and abscisic acid (ABA) on the accumulation of GLS and SF in radish taproot were investigated. The GLS content increased considerably in response to MeJA treatment. Moreover, MeJA also up-regulated the expression level of several transcription factors and GLS biosynthesis related genes. The effects of SA on GLS content were less obvious, while ABA suppressed GLS biosynthesis. Additionally, the SF content increased under treatments with MeJA, SA and ABA. Furthermore, changes in myrosinase activity and glucoraphanin (GRA) content after the application of MeJA, SA and ABA were consistent with the changes in SF contents. The results indicated that the MeJA represents one of the most effective plant growth regulators (PGRs) for increasing the GLS content in radish taproot by up-regulating the expression levels of a few GLS biosynthetic related genes. These findings also implied that SF formation is correlated with myrosinase activity and GRA content in radish taproot. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The authors declare that they have no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nanjing Agricultural University.)

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.

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.