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Wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen) belongs to the Brassica genus of the Cruciferae family, and its leaf curl is a typical feature that distinguishes Wucai from other nonheading cabbage subspecies. Our previous research found that plant hormones were involved in the development of the leaf curl in Wucai. However, the molecular mechanisms and the hormones regulating the formation of leaf curl in Wucai have not yet been reported. This study aimed to understand the molecular functions related to hormone metabolism during the formation of leaf curl in Wucai. A total of 386 differentially expressed genes (DEGs) were identified by transcriptome sequencing of two different morphological parts of the same leaf of Wucai germplasm W7-2, and 50 DEGs were found to be related to plant hormones, which were mainly involved in the auxin signal transduction pathway. Then, we measured the content of endogenous hormones in two different forms of the same leaf of Wucai germplasm W7-2. A total of 17 hormones with differential content were identified, including auxin, cytokinins, jasmonic acids, salicylic acids, and abscisic acid. And we found that treatment with auxin transport inhibitor N-1-naphthylphthalamic acid can affect the leaf curl phenotype of Wucai and pak choi (Brassica rapa L. subsp. Chinensis). These results indicated that plant hormones, especially auxin, are involved in developing the leaf curl of Wucai. Our findings provide a potentially valuable reference for future research on the development of leaf curls., (© 2023 Scandinavian Plant Physiology Society.)

Late embryogenesis abundant (LEA) proteins are important developmental proteins in the response of plants to abiotic stress. In our previous study, BcLEA73 was differentially expressed under low-temperature stress. Herein, we combined bioinformatics analysis, subcellular localization, expression assays, and stress experiments (including salt, drought, and osmotic stress) to identify and analyze the BcLEA gene family. Gene cloning and functional analysis of BcLEA73 were performed in tobacco and Arabidopsis. Based on the sequence homology and the available conservative motif, 82 BrLEA gene family members were identified and were divided into eight subfamilies in the genome-wide database of Chinese cabbage. The analysis showed that the BrLEA73 gene was located on chromosome A09 and belonged to the LEA_6 subfamily. Quantitative real-time PCR analysis indicated that the BcLEA genes were differentially expressed to varying degrees in the roots, stems, leaves, and petioles of Wucai. The overexpressed BcLEA73 transgenic plants exhibited no significant differences in root length and seed germination rates compared to the wild-type (WT) plants under control conditions. Under salt and osmotic stress treatment, the root length and seed germination rates of the BcLEA73-OE strain were significantly greater than those of WT plants. Under salt stress, the total antioxidant capacity (T-AOC) of the BcLEA73-OE lines increased significantly, and the relative conductivity, (REL), hydrogen peroxide (H 2 O 2 ) content, and superoxide anion (O 2 - ) production rate decreased significantly. Under drought treatment, the survival rate of the BcLEA73-OE lines was significantly higher than that of WT plants. These results showed that the BcLEA73 gene of Wucai functions in enhancing the tolerance of plants to salt, drought, and osmotic stress. This study provides a theoretical basis to explore the relevant functions of the BcLEA gene family members of Wucai.

Dehydration responsive element binding protein (DREB) is a significant transcription factor class known to be implicated in abiotic stresses. In this study, we systematically conducted a genome-wide identification and expression analysis of the DREB gene family, including gene structures, evolutionary relationships, chromosome distribution, conserved domains, and expression patterns. A total of 65 DREB family gene members were identified in Chinese cabbage (Brassica rapa L.) and were classified into five subgroups based on phylogenetic analysis. Through analysis of the conserved domains of BrDREB family genes, only one exon existed in the gene structure. Through the analysis of cis-acting elements, these genes were mainly involved in hormone regulation and adversity stress. In order to identify the function of BrDREB2B, overexpressed transgenic Arabidopsis was constructed. After different stress treatments, the germination rate, root growth, survival rate, and various plant physiological indicators were measured. The results showed that transgenic Arabidopsis thaliana plants overexpressing BrDREB2B exhibited enhanced tolerance to salt, heat and drought stresses. Taken together, our results are the first to report the BrDREB2B gene response to drought and heat stresses in Chinese cabbage and provide a basis for further studies to determine the function of BrDREBs in response to abiotic stresses., (© 2022. The Author(s).)

High temperatures have a serious impact on the quality and yield of cold-loving Chinese cabbage, which has evolved to have a unique set of stress mechanisms. To explore the relationship between these mechanisms and the heat-tolerance of Chinese cabbage, the physiological indicators of the heat-tolerant '268' line and heat-sensitive '334' line were measured. Under heat stress, the proline (Pro), soluble sugar (SS), and superoxide dismutase (SOD) indexes of the '268' line increased significantly. When additionally using transcriptome analysis, we found that the identified 3,360 DEGs were abundantly enriched in many metabolic pathways including 'plant hormone signal transduction', 'carbon metabolism', and 'glycolysis/gluconeogenesis'. Dynamic gene expression patterns showed that HKL1 in Cluster 15 may be a key factor in the regulation of sugar homeostasis. The interaction network screened four ABA-related genes in Cluster 15, suggesting that high temperatures lead to changes in hormonal signaling, especially an increase in ABA signaling. Compared with the '334' line, the expressions of Prx50 , Prx52 , Prx54 , SOD1 , and SOD2 in the '268' line were significantly upregulated, and these genes were actively involved in the reactive oxygen species (ROS) scavenging process. In summary, our results revealed the relationship between plant heat tolerance, physiology, and biochemistry and may also provide ideas for the future development of high-quality and heat-tolerant Chinese cabbage germplasm resources., Competing Interests: The authors declare that they have no competing interests., (© 2022 Zhang et al.)

A spontaneous male-sterile mutant ms01 was discovered from the excellent high-generation inbred line 'hx12-6-3' in wucai. Compared with wild-type 'hx12-6-3', ms01 displayed complete male sterility with degenerated stamens and no pollen. In this study, cytological observation revealed that the tapetum of the anthers of ms01 had degraded in advance, and microspore development had stagnated in the mononuclear stage, ultimately resulting in completely aborted pollen. Genetic analysis indicated that the sterility of ms01 was controlled by a single recessive nuclear gene. In the differential proteomic analysis of 'hx12-6-3' and ms01 flower buds using a tandem mass tags-based approach, a comparison of two stages (stage a and stage e) revealed 1272 differentially abundant proteins (DAPs). The abnormal variation of the anther cuticle, pollen coat, and sporopollenin production were effected by lipid metabolism and phenylpropanoid biosynthesis in the mutant ms01. Further analysis elucidated that pollen development was associated with amino acid metabolism, protein synthesis and degradation, carbohydrate metabolism, flavonoid biosynthesis and glutathione metabolism. These results provide novel insights into the molecular mechanism of GMS (genic male sterility) in wucai. SIGNIFICANCE: ms01, as the first indentified spontaneous male-sterile mutant in wucai, plays a significant role in the initial study of GMS (genic male sterility). In our study, the key DAPs related to anther and pollen development were obtained by TMT-based comparative proteomic analysis. We found that the abnormal accumulation of H 2 O 2 might induce premature degradation of the tapetum, causing anther metabolism disorder and pollen abortion. This process involved multiple DAPs and formed a complex regulatory network that generated a series of physiological metabolic alterations, ultimately leading to male sterility. Our results provide a theoretical foundation for further research on the complex anther and pollen development process., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Wucai suffers from low temperature during the growth period, resulting in a decline in yield and poor quality. But the molecular mechanisms of cold tolerance in wucai are still unclear., Results: According to the phenotypes and physiological indexes, we screened out the cold-tolerant genotype "W18" (named CT) and cold-sensitive genotype "Sw-1" (named CS) in six wucai genotypes. We performed transcriptomic analysis using seedling leaves after 24 h of cold treatment. A total of 3536 and 3887 differentially expressed genes (DEGs) were identified between the low temperature (LT) and control (NT) comparative transcriptome in CT and CS, respectively, with 1690 DEGs specific to CT. The gene ontology (GO) analysis showed that the response to cadmium ion (GO:0,046,686), response to jasmonic acid (GO:0,009,753), and response to wounding (GO:0,009,611) were enriched in CT (LT vs NT). The DEGs were enriched in starch and sucrose metabolism and glutathione metabolism in both groups, and α-linolenic acid metabolism was enriched only in CT (LT vs NT). DEGs in these processes, including glutathione S-transferases (GSTs), 13S lipoxygenase (LOX), and jasmonate ZIM-domain (JAZ), as well as transcription factors (TFs), such as the ethylene-responsive transcription factor 53 (ERF53), basic helix-loop-helix 92 (bHLH92), WRKY53, and WRKY54.We hypothesize that these genes play important roles in the response to cold stress in this species., Conclusions: Our data for wucai is consistent with previous studies that suggest starch and sucrose metabolism increased the content of osmotic substances, and the glutathione metabolism pathway enhance the active oxygen scavenging. These two pathways may participated in response to cold stress. In addition, the activation of α-linolenic acid metabolism may promote the synthesis of methyl jasmonate (MeJA), which might also play a role in the cold tolerance of wucai., (© 2022. The Author(s).)

Background: Chlorophyll (Chl) is a vital photosynthetic pigment involved in capturing light energy and energy conversion. In this study, the color conversion of inner-leaves from green to yellow in the new wucai (Brassica campestris L.) cultivar W7-2 was detected under low temperature. The W7-2 displayed a normal green leaf phenotype at the seedling stage, but the inner leaves gradually turned yellow when the temperature was decreased to 10 °C/2 °C (day/night), This study facilitates us to understand the physiological and molecular mechanisms underlying leaf color changes in response to low temperature., Results: A comparative leaf transcriptome analysis of W7-2 under low temperature treatment was performed on three stages (before, during and after leaf color change) with leaves that did not change color under normal temperature at the same period as a control. A total of 67,826 differentially expressed genes (DEGs) were identified. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) analysis revealed that the DEGs were mainly enriched in porphyrin and Chl metabolism, carotenoids metabolism, photosynthesis, and circadian rhythm. In the porphyrin and chlorophyll metabolic pathways, the expression of several genes was reduced [i.e. magnesium chelatase subunit H (CHLH)] under low temperature. Almost all genes [i.e. phytoene synthase (PSY)] in the carotenoids (Car) biosynthesis pathway were downregulated under low temperature. The genes associated with photosynthesis [i.e. photosystem II oxygen-evolving enhancer protein 1 (PsbO)] were also downregulated under LT. Our study also showed that elongated hypocotyl5 (HY5), which participates in circadian rhythm, and the metabolism of Chl and Car, is responsible for the regulation of leaf color change and cold tolerance in W7-2., Conclusions: The color of inner-leaves was changed from green to yellow under low temperature in temperature-sensitive mutant W7-2. Physiological, biochemical and transcriptomic studies showed that HY5 transcription factor and the downstream genes such as CHLH and PSY, which regulate the accumulation of different pigments, are required for the modulation of leaf color change in wucai under low temperature., (© 2021. The Author(s).)

Background: Wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen) is a cold-tolerant plant that is vulnerable to high temperature. This study explored the response mechanism of wucai to low temperature. In this study, wucai seedlings were treated with different temperatures, including low temperature (LT), high temperature (HT), and a control., Results: According to transcriptomics analysis, the number of differentially expressed genes (DEGs) in HT and LT was 10,702 and 7267, respectively, compared with the control. The key genes associated with the physiological response of wucai to the treatments were analyzed. The Kyoto Encyclopedia of Genes and Genomes and Gene Ontology annotations indicated the importance of the photosynthesis and photosynthetic-antenna protein pathways. We found that a high-temperature environment greatly inhibited the expression of important genes in the photosynthetic pathway (BrLhc superfamily members, PsaD, PsaE, PsaD, PsaD, PsbO, PsbP, PsbQ, PsbR, PsbS, PsbW, PsbY, Psb27, and Psb28), whereas low temperature resulted in the expression of certain key genes (BrLhc superfamily members, Psa F, Psa H, Psb S, Psb H, Psb 28). In addition, the wucai seedlings exhibited better photosynthetic performance under low-temperature conditions than high-temperature conditions., Conclusions: Based on the above results, we speculate that upon exposure to low temperature, the plants developed higher cold tolerance by upregulating the expression of genes related to photosynthesis. Conversely, high-temperature stress inhibited the expression of pivotal genes and weakened the self-regulating ability of the plants., (© 2021. The Author(s).)

Background: The discovery of male sterile materials is of great significance for the development of plant fertility research. Wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen) is a variety of non-heading Chinese cabbage. There are few studies on the male sterility of wucai, and the mechanism of male sterility is not clear. In this study, the male sterile mutant MS7-2 and the wild-type fertile plant MF7-2 were studied., Results: Phenotypic characteristics and cytological analysis showed that MS7-2 abortion occurred at the tetrad period. The content of related sugars in the flower buds of MS7-2 was significantly lower than that of MF7-2, and a large amount of reactive oxygen species (ROS) was accumulated. Through transcriptome sequencing of MS7-2 and MF7-2 flower buds at three different developmental stages (a-c), 2865, 3847, and 4981 differentially expressed genes were identified in MS7-2 at the flower bud development stage, stage c, and stage e, respectively, compared with MF7-2. Many of these genes were enriched in carbohydrate metabolism, phenylpropanoid metabolism, and oxidative phosphorylation, and most of them were down-regulated in MS7-2. The down-regulation of genes involved in carbohydrate and secondary metabolite synthesis as well as the accumulation of ROS in MS7-2 led to pollen abortion in MS7-2., Conclusions: This study helps elucidate the mechanism of anther abortion in wucai, providing a basis for further research on the molecular regulatory mechanisms of male sterility and the screening and cloning of key genes in wucai., (© 2021. The Author(s).)

Background: Leaf color mutants are the ideal materials to explore the pathways of chlorophyll (Chl) metabolism, chloroplast development, and photosynthesis system. In this study, a spontaneous yellow-green leaf wucai (Brassica campestris L.) mutant "WY16-13" was identified, which exhibited yellow-green leaf color during its entire growth period. However, current understanding of the molecular mechanism underlying Chl metabolism and chloroplast development of "WY16-13" is limited., Results: Total Chl and carotenoid content in WY16-13 was reduced by 60.92 and 58.82%, respectively, as compared with its wild type parental line W16-13. Electron microscopic investigation revealed fewer chloroplasts per cell and looser stroma lamellae in WY16-13 than in W16-13. A comparative transcriptome profiling was performed using leaves from the yellow-green leaf type (WY16-13) and normal green-leaf type (W16-13). A total of 54.12 million (M) (WY16-13) and 56.17 M (W16-13) reads were generated. A total of 40,578 genes were identified from the mapped libraries. We identified 3882 differentially expressed genes (DEGs) in WY16-13 compared with W16-13 (i.e., 1603 upregulated genes and 2279 downregulated genes). According to the Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, these DEGs are involved in porphyrin and Chl metabolism [i.e., chlorophyllase (CLH), heme oxygenase (HO), chlorophyll (ide) b reductase (NYC), and protochlorophyllide oxidoreductase (POR) genes], carbohydrate metabolism, photosynthesis, and carbon fixation in photosynthetic organisms. Moreover, deficiency in Chl biosynthetic intermediates in WY16-13 revealed that the formation of the yellow-green phenotype was related to the disorder of heme metabolism., Conclusions: Our results provide valuable insights into Chl deficiency in the yellow-green leaf mutant and a bioinformatics resource for further functional identification of key allelic genes responsible for differences in Chl content.

Background: Brassinosteroids (BRs) have a positive effect on many processes during plant growth and development, and in response to various abiotic stressors. Low-temperature (LT) stress constricts the geographic distribution, growth, and development of wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen). However, there is little information on the global gene expression of BRs under LT stress in wucai. In this study, the molecular roles of 24-epibrassinolide (EBR) after exogenously application, were explored by RNA sequencing under LT conditions., Results: According to the Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, photosynthesis was significantly enriched after spraying EBR under LT. The transcripts encoding the photosystem II (PSII) oxygen-evolving enhancer protein, photosystem I (PSI) subunit, light-harvesting chlorophyll protein complexes I and II, and ferredoxin were up-regulated after the application of EBR. Transcripts encoding several key enzymes involved in chlorophyll biosynthesis were also up-regulated, accompanied by significant differences in the contents of 5-aminolevulinic acid (ALA), porphobilinogen (PBG), protoporphyrin IX (Proto IX), Mg-protoporphyrin IX (Mg-proto IX), protochlorophyllide (Pchl), and photosynthetic pigments. Notably, transcriptional and physiological analyses revealed that under LT stress, plant responses to EBR involved a major reorientation of photosynthesis, as well as porphyrin and chlorophyll metabolism., Conclusion: This study explored the role of EBR as an LT stress tolerance mechanism in wucai. At the transcription level, LT tolerance manifests as an enhancement of photosynthesis, and the amelioration of porphyrin and chlorophyll metabolism.

The genotype WS-1, previously identified from novel wucai germplasm, is tolerant to both low-temperature (LT) and high-temperature (HT) stress. However, it is unclear which signal transduction pathway or acclimation mechanisms are involved in the temperature-stress response. In this study, we used the proteomic method of tandem mass tag (TMT) coupled with liquid chromatography-mass spectrometry (LC-MS/MS) to identify 1022 differentially expressed proteins (DEPs) common to WS-1, treated with either LT or HT. Among these 1022 DEPs, 172 were upregulated in response to both LT and HT, 324 were downregulated in response to both LT and HT, and 526 were upregulated in response to one temperature stress and downregulated in response to the other. To illustrate the common regulatory pathway in WS-1, 172 upregulated DEPs were further analyzed. The redox homeostasis, photosynthesis, carbohydrate metabolism, heat-shockprotein, and chaperones and signal transduction pathways were identified to be associated with temperature stress tolerance in wucai. In addition, 35S:BcccrGLU1 overexpressed in Arabidopsis, exhibited higher reduced glutathione (GSH) content and reduced glutathione/oxidized glutathione (GSH/GSSG) ratio and less oxidative damage under temperature stress. This result is consistent with the dynamic regulation of the relevant proteins involved in redox homeostasis. These data demonstrate that maintaining redox homeostasis is an important common regulatory pathway for tolerance to temperature stress in novel wucai germplasm.

Chlorophyll is a vital photosynthetic pigment that plays a key role in plant development, participating in light energy capture and energy conversion. In this study, a novel wucai ( Brassica campestris L.) germplasm with green outer leaves and yellow inner leaves at the adult stage (W7-2) was used to examine chlorophyll metabolism response to cold acclimation. A green leaf wucai genotype without leaf color changes named W7-1 was selected as the control to evaluate the chlorophyll metabolism changes of W7-2. Compared to W7-1, the contents of chlorophyll a (Chl a) and chlorophyll b (Chl b) in W7-2 were significantly reduced at five developmental stages (13, 21, 29, 37, and 45 days after planting (DAP)). An iTRAQ-based quantitative proteomic analysis was carried out at 21 and 29 DAP according to the leaf color changes in both of genotypes. 1409 proteins were identified, while 218 of them displayed differential accumulations between W7-2 and W7-1 during the two developmental stages. The differentially expressed proteins (DEPs) mainly assigned to chlorophyll biosynthesis, photosynthesis, carbohydrate metabolism, ribosome metabolism and posttranslational modification. Among these DEPs, NADPH-protochlorophyllide oxidoreductase (PORB) and Mg-protoporphyrin IX chelatase 1 (CHLI1) were the key enzymes participating in chlorophyll (Chl) biosynthesis, which was down-regulated at 21 DAP and up-regulated at 29 DAP in W7-2 compared with W7-1, respectively. The expression analysis of genes of three subunits of Mg-chelatase ( CHLI1, CHLD, and CHLH), Genomes Uncoupled 4 ( GUN4), and Thioredoxin ( TRX3) associated with chlorophyll metabolism also displayed significant down-regulation in W7-2. In particular, PORB showed significant up-regulation in W7-2, significantly affecting chlorophyll biosynthesis. Additionally, differences in chlorophyll metabolism between W7-2 and W7-1 were in terms of altered photosynthesis, carbohydrate, and energy metabolism. We found that the transcription levels of most photosynthesis proteins showed significantly lower levels, and the genes expression level, associated with carbohydrate and energy metabolism, were lower in W7-2 than in W7-1. Therefore, the present study results help understand the physiological and molecular mechanisms underlying leaf coloring responding to cold acclimation.

Key Message: We developed a novel Agrobacterium-mediated anther transformation for Wucai in planta, and in this procedure, the male germ line was the predominant target. Wucai (Brassica campestris L.), a variant of non-heading Chinese cabbage, is widely cultured in China and only improved by classic breeding methods. Here, a novel and efficient in planta Agrobacterium-mediated anther transformation method is developed based on the optimization of several factors that affect anther transformation. After optimization, transformation with the manual pollination application led to increased transient GUS expression in anthers (reaching 91.59%) and the transformation efficacies in planta (0.59-1.56% for four commercial cultivars). The stable integration and inheritance of the transgenes were further examined by molecular and genetic analyses. Three T 2 transgenic lines presented a segregation ratio of 3:1, which was consistent with the Mendelian feature of a single dominant gene. In addition, the GUS histochemical assay and genetic crossing analysis revealed that the male germ line was the predominant target in this transformation. This optimized transformation system could provide a useful tool for both the improvement of cultivar qualities and investigation of functional genes in Wucai.

Background: Wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen) is a variant of nonheading Chinese cabbage (Brassica campestris L.), which is one of the major vegetables in China. Cytoplasmic male sterility (CMS) has been used for Wucai breeding in recent years. However, the underlying molecular mechanism of Wucai CMS remains unclear. In this study, the phenotypic and cytological features of Wucai CMS were observed by anatomical analysis, and a comparative transcriptome analysis was carried out to identify genes related to male sterility using Illumina RNA sequencing technology (RNA-Seq)., Results: Microscopic observation demonstrated that tapetum development was abnormal in the CMS line, which failed to produce fertile pollen. Bioinformatics analysis detected 4430 differentially expressed genes (DEGs) between the fertile and sterile flower buds. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed to better understand the functions of these DEGs. Among the DEGs, 35 genes (53 DEGS) were implicated in anther and pollen development, and 11 genes were involved in pollen cell wall formation and modification; most of these showed downregulated expression in sterile buds. In addition, several genes related to tapetum development (A6, AMS, MS1, MYB39, and TSM1) and a few genes annotated to flowering (CO, AP3, VIN3, FLC, FT, and AGL) were detected and confirmed by qRT-PCR as being expressed at the meiosis, tetrad, and uninucleate microspore stages, thus implying possible roles in specifying or determining the fate and development of the tapetum, male gametophyte and stamen. Moreover, the top four largest transcription factor families (MYB, bHLH, NAC and WRKY) were analyzed, and most showed reduced expression in sterile buds. These differentially expressed transcription factors might result in abortion of pollen development in Wucai., Conclusion: The present comparative transcriptome analysis suggested that many key genes and transcription factors involved in anther development show reduced gene expression patterns in the CMS line, which might contribute to male sterility in Wucai. This study provides valuable information for a better understanding of CMS molecular mechanisms and functional genome studies in Wucai.

In plants, the accumulation of carotenoids can maintain the balance of the photosystem and improve crop nutritional quality. Therefore, the molecular mechanisms underlying carotenoid synthesis and accumulation should be further explored. In this study, carotenoid accumulation differed significantly among parental Brassica rapa. Genetic analysis was carried out using the golden inner leaf '1900264′ line and the light−yellow inner leaf '1900262′ line, showing that the golden inner leaf phenotype was controlled by a single dominant gene. Using bulked−segregant analysis sequencing, BraA09g007080.3C encoding the ORANGE protein was selected as a candidate gene. Sequence alignment revealed that a 4.67 kb long terminal repeat insertion in the third exon of the BrGOLDEN resulted in three alternatively spliced transcripts. The spatiotemporal expression results indicated that BrGOLDEN might regulate the expression levels of carotenoid−synthesis−related genes. After transforming BrGOLDEN into Arabidopsis thaliana, the seed−derived callus showed that BrGOLDENIns and BrGOLDENDel lines presented a yellow color and the BrGOLDENLdel line presented a transparent phenotype. In addition, using the yeast two−hybrid assay, BrGOLDENIns, BrGOLDENLdel, and Brgoldenwt exhibited strong interactions with BrPSY1, but BrGOLDENDel did not interact with BrPSY1 in the split−ubiquitin membrane system. In the secondary and 3D structure analysis, BrGOLDENDel was shown to have lost the PNFPSFIPFLPPL sequences at the 125 amino acid position, which resulted in the α−helices of BrGOLDENDel being disrupted, restricting the formation of the 3D structure and affecting the functions of the protein. These findings may provide new insights into the regulation of carotenoid synthesis in B. rapa. [ABSTRACT FROM AUTHOR]

• The tapetum was abnormally vacuolated after meiosis in Wucai ogu-CMS, which led to the abortion of microspores. • The TDF1 gene is essential for early tapetal development. • Constitutive expression of BrTDF1 in the Arabidopsis tdf1 mutant transgenic lines could restore its fertility. Ogura cytoplasmic male sterility (ogu -CMS) has been widely used by breeders for vegetables in the genus Brassica ; however, the molecular mechanism of this male sterility remains unclear. The tapetum plays a crucial role in anther development by providing nutrients and secreting callose for pollen development. In this study, we found that the tapetum was abnormally vacuolated after meiosis in Wucai ogu -CMS, which led to the abortion of microspores. The TDF1 gene is essential for early tapetal development. Combined with the characteristics of abortion in ogu -CMS, qRT-PCR analysis showed that the BrTDF1 gene was expressed in all detected tissues from fertile plants, and the transcript levels of this gene in fertile buds were far higher than those in vegetative organs. Compared with the fertile line, BrTDF1 exhibited lower expression in all developmental stages of buds from the sterile line. In addition, the examinations of A6 and EXPB5 genes suggested that the expressions of these genes in ogu -CMS were lower than those in its maintainer line. Further analysis of its function showed that constitutive expression of BrTDF1 in the Arabidopsis tdf1 mutant transgenic lines could restore its fertility. These results indicated that this MYB transcription factor BrTDF1 is required for tapetal development in Wucai. [ABSTRACT FROM AUTHOR]