Ca2+ deficiency causes irreversible physiological disorders that markedly reduce the yield and quality of many horticultural crops, leading to substantial economic losses. Tip burn in Chinese cabbage (Brassica rapa ssp. pekinensis) is associated with Ca2+ deficiency; however, the underlying molecular mechanisms remain poorly understood. In this study, Ca2+ deficiency suppressed growth and impaired photosynthetic performance in Chinese cabbage seedlings. Under Ca2+ deficiency, cytosolic Ca2+ levels decreased, accompanied by excessive reactive oxygen species (ROS) accumulation in the leaves. Foliar application of dimethylthiourea (DMTU), an ROS scavenger, alleviated Ca2+ deficiency-induced tip burn by reducing ROS accumulation. RNA-seq analysis further revealed significant enrichment of the mitogen-activated protein kinase signaling and plant hormone signal transduction pathways during tip-burn development. Several differentially expressed genes associated with these pathways were identified as responsive to Ca2+ deficiency. Compared with normal Ca2+ and DMTU-rescued conditions, the respiratory burst oxidase homolog D (BrRbohD-2) gene was markedly upregulated under Ca2+ deficiency. Overexpression of BrRbohD-2 increased ROS accumulation and aggravated tip burn in Chinese cabbage seedlings, whereas its suppression reduced ROS levels and alleviated tip burn. These findings provide important insights into the role of BrRbohD-2 and the regulatory mechanisms underlying Ca2+ deficiency-related disorders in Brassicaceae crops.

Self-incompatibility (SI) is a genetic mechanism that promotes out-breeding and maintains genetic diversity in plant species. The inhibition of pollen germination and pollen tube growth is one of the major characteristics of SI response. The involvement of Ca2+ signaling mediated by Ca2+ sensors in the SI response has been demonstrated to affect the germination of incompatible pollen and the normal elongation of pollen tubes. In this study, the calmodulin-like 49 (BrCML49) gene in Chinese cabbage (Brassica rapa L. ssp. pekinensis) was down-regulated expression during the SI process, indicating that BrCML49 may participate in the regulation of SI response. The transient suppression of BrCML49 gene induced the abnormal germination of pollen in vitro, which showed the putative positive roles of BrCML49 in regulating pollen germination. The inhibited germination of self- and cross-pollen in BrCML49-suppressed stigma was also observed, which revealed the activated SI response. After BrCML49 suppression, the increases in Ca2+ level in stigmatic papilla cells were consistent with the increases in ROS/H2O2 contents in the stigma, which may be essential for the occurrence of SI response. Furthermore, heterologous overexpression of BrCML49 in Arabidopsis validated the positive regulation of BrCML49 in pollen germination and pollen tube growth. In addition, BrCML49 overexpressed Arabidopsis exhibited an enhanced tolerance of pollen germination to high Ca2+ condition. These results will provide valuable information for understanding the roles of BrCML49 in regulating pollen germination and pollen tube growth during SI process in Chinese cabbage.

Leaf color is one of the most important phenotypic features in horticultural crops and directly related to the contents of photosynthetic pigments. Most leaf color mutants are determined by the altered chlorophyll or carotenoid, which can be affected by light quality and intensity. Our previous study obtained a Chinese cabbage yellow cotyledon mutant that exhibited obvious yellow phenotypes in the cotyledons and the new leaves. However, the underlying mechanisms in the formation of yellow cotyledons and leaves remain unclear. In this study, the Chinese cabbage yellow cotyledon mutant 19YC-2 exhibited obvious difference in leaf color and abnormal chloroplast ultrastructure compared to the normal green cotyledon line 19GC-2. Remarkably, low-intensity light treatment caused turn-green leaves and a significant decrease in carotenoid content in 19YC-2. RNA-seq analysis revealed that the pathways of photosynthesis antenna proteins and carotenoid biosynthesis were significantly enriched during the process of leaf color changes, and many differentially expressed genes related to the two pathways were identified to respond to different light intensities. Remarkably, BrPDS and BrLCYE genes related to carotenoid biosynthesis showed significantly higher expression in 19YC-2 than that in 19GC-2, which was positively related to the higher carotenoid content in 19YC-2. In addition, several differentially expressed transcription factors were also identified and highly correlated to the changes in carotenoid content, suggesting that they may participate in the regulatory pathway of carotenoid biosynthesis. These findings provide insights into the molecular mechanisms of leaf color changes in yellow cotyledon mutant 19YC-2 of Chinese cabbage.

Calcium (Ca2+) plays essential roles in plant growth and development. Ca2+ deficiency causes a physiological disorder of tip-burn in Brassiceae crops and is involved in the regulation of cellular Ca2+ homeostasis. Although the functions of Ca2+/H+ exchanger antiporters (CAXs) in mediating transmembrane transport of Ca2+ have been extensively characterized in multiple plant species, the potential roles of BrCAX genes remain unclear in Chinese cabbage. In this study, eight genes of the BrCAX family were genome-widely identified in Chinese cabbage. These BrCAX proteins contained conserved Na_Ca_ex domain and belonged to five members of the CAX family. Molecular evolutionary analysis and sequence alignment revealed the evolutionary conservation of BrCAX family genes. Expression profiling demonstrated that eight BrCAX genes exhibited differential expression in different tissues and under heat stress. Furthermore, Ca2+ deficiency treatment induced the typical symptoms of tip-burn in Chinese cabbage seedlings and a significant decrease in total Ca2+ content in both roots and leaves. The expression changes in BrCAX genes were related to the response to Ca2+ deficiency-induced tip-burn of Chinese cabbage. Specially, BrCAX1-1 and BrCAX1-2 genes were highly expressed gene members of the BrCAX family in the leaves and were significantly differentially expressed under Ca2+ deficiency stress. Moreover, overexpression of BrCAX1-1 and BrCAX1-2 genes in yeast and Chinese cabbage cotyledons exhibited a higher Ca2+ tolerance, indicating the Ca2+ transport capacity of BrCAX1-1 and BrCAX1-2. In addition, suppression expression of BrCAX1-1 and BrCAX1-2 genes reduced cytosolic Ca2+ levels in the root tips of Chinese cabbage. These results provide references for functional studies of BrCAX genes and to investigate the regulatory mechanisms underlying Ca2+ deficiency disorder in Brassiceae vegetables.

Self-incompatibility (SI) is a genetic mechanism to promote outcrossing and prevent self-fertilization in plants. Brassicaceae crops have a typical SI response that exhibits the inhibition of incompatible pollen germination and pollen tube growth. Calcium (Ca2+) is a necessary regulator of pollen germination and pollen tube growth. However, the signaling processes by which Ca2+ regulates the SI response are not clear in Chinese cabbage. In this study, exogenous CaCl2 and EGTA spraying was performed to alter the Ca2+ level of the stigma and detect the correlation between Ca2+ and SI response in Chinese cabbage. The decrease in Ca2+ levels in the stigma caused by EGTA treatment could promote pollen germination and break the SI response. Moreover, transmission electron microscopy showed that the accumulation and distribution of cytoplasmic Ca2+ in the stigma were related to the occurrence of the SI response. Additionally, eight BrCaM family genes were identified in Brassica rapa by genome-wide analysis. Expression analysis suggested that the expression of several BrCaM genes was coincident with Ca2+ level alterations and the SI response. These results facilitate understanding the putative roles of BrCaM genes in regulating pollen germination and pollen tube growth and provide valuable references for uncovering the molecular regulatory mechanism of the SI response in Brassicaceae crops.

Although heterosis is commonly used in Chinese cabbage, its molecular basis is poorly understood. In this study, 16Chinese cabbage hybrids were utilized as test subjects to explore the potential molecular mechanism of heterosis. RNA sequencing revealed 5815–10,252 differentially expressed genes (DEGs) (female parent vs. male parent), 1796–5990 DEGs (female parent-vs-hybrid), and 2244–7063 DEGs (male parent vs. hybrid) in 16 cross combinations at the middle stage of heading. Among of them, 72.83–84.20% DEGs conformed to the dominant expression pattern, which is the predominant expression pattern in hybrids. There were 13 pathways in which DEGs were significantly enriched in most cross combinations. Among them, the plant–pathogen interaction (ko04626) and circadian rhythm-plant (ko04712)were significantly enriched by DEGs in strong heterosis hybrids. WGCNA also proved that the two pathways were significantly related to heterosis in Chinese cabbage.

As an important genetic improvement technique in current production practice, heterosis is widely used to enhance the productive traits of hybrid progeny from their parents. Alternative splicing (AS) analysis can be used as a method for exploring the molecular manifestations of heterosis. In our research, 16 hybrids and their parents were utilized to analyze the heterosis performance and AS events. Statistics of plant gross weight (PGW) showed that these hybrids had prominent heterosis, with the mid-parent heterosis values (MPV) ranging from 15.69% to 233.98%. Through pairwise comparison among the female parent, male parent, and hybrid, there were 2980–3205 AS events in each combination, with intron retention being the most common type followed by alternate 3’ splice site, alternative 5’ splice site, skipped exon, and mutually exclusive exon.There were 263–409 differential AS genes (DASGs) between the female parent and the hybrid, and 234–425 DASGs between the male parent and the hybrid in cross combinations. The DASGs were significantly enriched in 33 metabolic pathways in 16 cross combinations, and DASGs of different cross combinations were enriched in different metabolic pathways. Moreover, 76 DASGs in the strong heterosis combinations were identified and significantly enriched in the metabolic pathways related to amino acid metabolism. Further analysis revealed that most of these DASGs in amino acid metabolism were expressed differently in strong heterosis combinations. In addition, the expression levels of BraA06g014310.3C and BraA03g041700.3C in amino acid metabolism significantly correlated with PGW. These results could provide an index for future studies of the genetic and molecular mechanism of heterosis in hybrids.

类钙调蛋白(Calmodulin-like protein,CML)是植物细胞中一类非常重要的钙感受蛋白,且CML蛋白在花粉萌发和花粉管生长中起到重要作用.该研究以大白菜自交不亲和系'91-125'自花授粉后柱头为材料,克隆获得大白菜BrCML49基因cDNA全长序列1046 bp,包括960 bp开放阅读框,编码319个氨基酸,包含2个EF-hand保守结构域.序列分析发现,BrCML49蛋白为稳定的亲水蛋白,没有跨膜区.实时荧光定量(qRT-PCR)表达分析显示,BrCML49基因的表达量在'91-125'的花药中最高,其次是全花和花瓣,在其他组织中表达量较低;BrCML49基因在大白菜自交亲和系'14S23'的全花和柱头中表达量较高,在其他组织中表达量较低.亚细胞定位分析发现BrCML49主要定位于烟草叶片的细胞膜和细胞核中.该研究结果可为进一步研究大白菜BrCML49基因的功能奠定基础,并为阐明大白菜BrCML49基因参与自交不亲和花粉萌发及花粉管生长的信号机制提供理论依据.

Self-incompatibility (SI) is a genetic mechanism in plants to prevent inbreeding, promote outcrossing and thereby increase genetic diversity. Although current studies have well revealed the pathway of SI recognition specificity underlying interaction of female and male determinants in Brassica, the inhibition mechanism of incompatible pollen germination and tube growth is still deficient. In this study, histological analysis showed that the pollen germination and tube growth were obviously suppressed after self-pollination in self-incompatible Chinese cabbage. Furthermore, RNA-seq and gene expression analysis were performed to identify the differentially expressed genes (DEGs) involved in SI response of Chinese cabbage. A total of 59 DEGs belonging to known SI signaling factors and Ca2+-binding proteins were identified in Chinese cabbage after incompatible self-pollination. QRT-PCR analysis found that several genes were significantly differentially expressed during SI response. Chromosomal localization and potential duplicated genes were also predicted. Additionally, spatial temporal expression profiling revealed that 15 DEGs were specifically expressed in the floral organ, some of which exhibited higher expressions in the anther and style. These findings will provide insights into the functional exploration of SI signaling factors in Chinese cabbage, and promote the further understanding of SI signaling networks in Brassica.

To identify genes associated with carotenoid accumulation in petals of Chinese cabbage, the composition and content of carotenoids were analyzed, and comparative transcriptome sequencing was performed between the yellow flower line, 92S105, and the orange flower line, 94C9. High-performance liquid chromatography (HPLC) revealed that petals of 92S105 were high in violaxanthin as well as lutein, whereas petals of 94C9 showed considerable levels of lutein and β-carotene. Transcriptome analysis showed that 3534 and 3833 genes were up- and down-regulated in 94C9, respectively. Among these differentially expressed genes (DEGs), many related to carotenoid accumulation were identified, including 12 carotenoid biosynthesis pathway genes, 4 transcription factor genes, and 1028 specifically expressed genes. β-carotene hydroxylase 1 (BrBCH1), BrBCH2, zeaxanthin epoxidase (BrZEP), and MYB transcription factor gene (BrGAMYB) were down-regulated in petals of 94C9 when compared with petals of 92S105, which caused β-carotene accumulation and may lead to orange petal color in 94C9. Expression levels of 20 DEGs were verified by qPCR and the results were highly consistent with those of transcriptome sequencing. Moreover, Gene Ontology (GO) enrichment analysis revealed that membrane, binding, and metabolic processes were the most significantly enriched GO terms in cellular component, molecular function, and biological process ontologies, respectively. In conclusion, our study analyzed the differences in composition and content of carotenoids between 92S105 and 94C9 and identified potential candidate genes related to carotenoid accumulation in petals, thereby creating a solid foundation for future studies on the mechanism regulating carotenoid accumulation in petals of Chinese cabbage.

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.

Background Calmodulin-like (CML) proteins are a primary family of plant-specific Ca 2+ sensors that specifically bind to Ca 2+ and deliver a Ca 2+ signal. CML proteins have been identified and characterized in many plant species, such as the model plant Arabidopsis and rice. Based on considerable evidence, the roles of CML proteins are crucial in plant growth and development and in the response to various external stimuli. Nevertheless, the characterization and expression profiling of CML genes in Chinese cabbage ( Brassica rapa L. ssp . pekinensis ) remain limited. Results In this study, a genome-wide search and comprehensive analysis were performed, and a total of 79 BrCML genes were identified in Chinese cabbage. Gene structure analysis revealed that these BrCML genes contained two to four conserved EF-hand motifs. Phylogenetic analysis showed that CML homologs between Chinese cabbage and Arabidopsis shared close relationships. The identified BrCML genes were located across ten chromosomes and three different subgenomes of Chinese cabbage. Moreover, 126 pairs of orthologous CML genes were found among Chinese cabbage, Arabidopsis and Brassica oleracea . Expression analysis revealed that the expression of some BrCML genes was tissue-specific and that of some was susceptible to temperature stress. A putative interaction network of BrCML proteins was proposed, which suggested that BrCML2, BrCML6, BrCML15 and BrCML25 were co-expressed and might play roles in flower development and other relevant biological processes of Chinese cabbage. Conclusions The results of this study increased the understanding and characterization of BrCML genes in Chinese cabbage, and will be a rich resource for further studies to investigate BrCML protein function in various developmental processes of Chinese cabbage.

BACKGROUND:The 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).RESULTS:A 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.CONCLUSIONS:This 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.

Both bolting and flowering times influence taproot and seed production in radish. FLOWERING LOCUS C (FLC) plays a key role in plant flowering by functioning as a repressor. Two genomic DNA sequences, a 3 046-bp from an early- and a 2 959-bp from a late-bolting radish line were isolated and named as RsFLC1 and RsFLC2, respectively, for they share approximately 87.03% sequence identity to the FLC cDNA sequences. The genomic DNA sequences, 1 466-bp and 1 744-bp, flanking the 5′-regions of RsFLC1 and RsFLC2, respectively, were characterized. Since both of them harbor the basic promoter elements, the TATA box and CAAT box, they were designated as PRsFLC1 and PRsFLC2. The transcription start site (TSS) was identified at 424 and 336 bp upstream of the start codon in PRsFLC1 and PRsFLC2, respectively. cis-regulatory elements including CGTCA (MeJA-responsive) and ABRE (abscisic acid-responsive) motifs were found in both promoters, while some cis-regulatory elements including TCA element and GARE-motif were present only in PRsFLC1. These sequence differences lead to the diversity of promoter core elements, which could partially result in the difference of bolting and flowering time in radish line NauDY13 (early-bolting) and Naulu127 (late-bolting). Furthermore, to investigate the activity of these promoters, a series of 5′-deletion fragment-GUS fusions were constructed and transformed into tobacco. GUS activity was detected in PRsFLC1-(1 to 4)-GUS-PS1aG-3 and PRsFLC2-(1 to 4)-GUS-PS1aG-3 transgenic tobacco leaf discs, and this activity progressively decreased from PRsFLC-1-GUS-PS1aG-3 to PRsFLC-5-GUS-PS1aG-3. Deletion analysis indicated that the cis-regulatory elements located at −395 bp to +1 bp may be critical for specifying RsFLC gene transcription.

The transition of vegetative growth to bolting and flowering is an important process in the life cycle of plants, which is determined by numerous genes forming an intricate network of bolting and flowering. However, no comprehensive identification and profiling of bolting and flowering-related genes have been carried out in radish. In this study, RNA-Seq technology was applied to analyze the differential gene expressions during the transition from vegetative stage to reproductive stage in radish. A total of 5922 differentially expressed genes (DEGs) including 779 up-regulated and 5143 down-regulated genes were isolated. Functional enrichment analysis suggested that some DEGs were involved in hormone signaling pathways and the transcriptional regulation of bolting and flowering. KEGG-based analysis identified 37 DEGs being involved in phytohormone signaling pathways. Moreover, 95 DEGs related to bolting and flowering were identified and integrated into various flowering pathways. Several critical genes including FT, CO, SOC1, FLC, and LFY were characterized and profiled by RT-qPCR analysis. Correlation analysis indicated that 24 miRNA-DEG pairs were involved in radish bolting and flowering. Finally, a miRNA-DEG-based schematic model of bolting and flowering regulatory network was proposed in radish. These outcomes provided significant insights into genetic control of radish bolting and flowering, and would facilitate unraveling molecular regulatory mechanism underlying bolting and flowering in root vegetable crops.

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.

Transcriptome-based gene expression analysis identifies many critical salt-responsive genes in radish and facilitates further dissecting the molecular mechanism underlying salt stress response.

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.

采用电子克隆与基因组步移策略分离出萝卜RsFPF1基因gDNA和cDNA及启动子序列,并进行表达特征分析及转基因功能验证.序列分析表明,RsFPF1基因长度为330 bp,编码109个氨基酸;蛋白同源分析表明,RsFPF1蛋白与拟南芥及白芥FPF1蛋白间亲缘关系最近.RsFPF1基因5 '上游启动子区序列长度为1 845 bp,采用PLACE和PlantCARE软件分析表明,该启动子序列含有典型调控元件及多个光响应顺式元件.半定量RT-PCR表达分析表明,开花前RsFPF1基因在茎尖表达量最高,开花后在花及花蕾中表达量最高.通过农杆菌介导的遗传转化获得转RsFPF1基因的烟草阳性植株,与野生型相比,转入RsFPF1基因的植株出现花期提前现象.结论:RsFPF1基因能够促进萝卜提早开花,其表达可能受光调控,在调控萝卜抽薹开花及花发生相关基因表达方面发挥着重要作用.