Systematic Analysis of the BrHAT Gene Family and Physiological Characteristics of Brassica rapa L. Treated with Histone Acetylase and Deacetylase Inhibitors under Low Temperature.

Brassica rapa L. is an important overwintering oilseed crop in Northwest China. Histone acetyltransferases (HATs) play an important role in epigenetic regulation, as well as the regulation of plant growth, development, and responses to abiotic stresses. To clarify the role of histone acetylation in the low-temperature response of B. rapa L., we identified 29 HAT genes in B. rapa L. using bioinformatics tools. We also conducted a comprehensive analysis of the physicochemical properties, gene structure, chromosomal localization, conserved structural domains and motifs, cis-acting regulatory elements, and evolutionary relationships of these genes. Using transcriptome data, we analyzed the expression patterns of BrHAT family members and predicted interactions between proteins; the results indicated that BrHATs play an important role in the low-temperature response of B. rapa L. HAT inhibitor (curcumin; CUR) and histone deacetylase inhibitor (Trichostatin A; TSA) were applied to four B. rapa L. varieties varying in cold resistance under the same low-temperature conditions, and changes in the physiological indexes of these four varieties were analyzed. The inhibitor treatment attenuated the effect of low temperature on seed germination, and curcumin treatment was most effective, indicating that the germination period was primarily regulated by histone acetylase. Both inhibitor treatments increased the activity of protective enzymes and the content of osmoregulatory substances in plants, suggesting that histone acetylation and deacetylation play a significant role in the response of B. rapa L. to low-temperature stress. The qRT-PCR analyses showed that the expression patterns of BrHATs were altered under different inhibitor treatments and low-temperature stress; meanwhile, we found three significantly differentially expressed genes. In sum, the process of histone acetylation is involved in the cold response and the BrHATs gene plays a role in the cold stress response.

Analysis of the Mechanism of Wood Vinegar and Butyrolactone Promoting Rapeseed Growth and Improving Low-Temperature Stress Resistance Based on Transcriptome and Metabolomics.

Rapeseed is an important oil crop in the world. Wood vinegar could increase the yield and abiotic resistance of rapeseed. However, little is known about the underlying mechanisms of wood vinegar or its valid chemical components on rapeseed. In the present study, wood vinegar and butyrolactone (γ-Butyrolactone, one of the main components of wood vinegar) were applied to rapeseed at the seedling stage, and the molecular mechanisms of wood vinegar that affect rapeseed were studied by combining transcriptome and metabolomic analyses. The results show that applying wood vinegar and butyrolactone increases the biomass of rapeseed by increasing the leaf area and the number of pods per plant, and enhances the tolerance of rapeseed under low temperature by reducing membrane lipid oxidation and improving the content of chlorophyll, proline, soluble sugar, and antioxidant enzymes. Compared to the control, 681 and 700 differentially expressed genes were in the transcriptional group treated with wood vinegar and butyrolactone, respectively, and 76 and 90 differentially expressed metabolites were in the metabolic group. The combination of transcriptome and metabolomic analyses revealed the key gene-metabolic networks related to various pathways. Our research shows that after wood vinegar and butyrolactone treatment, the amino acid biosynthesis pathway of rapeseed may be involved in mediating the increase in rapeseed biomass, the proline metabolism pathway of wood vinegar treatment may be involved in mediating rapeseed's resistance to low-temperature stress, and the sphingolipid metabolism pathway of butyrolactone treatment may be involved in mediating rapeseed's resistance to low-temperature stress. It is suggested that the use of wood vinegar or butyrolactone are new approaches to increasing rapeseed yield and low-temperature resistance.

Single and combined effect of polyethylene microplastics (virgin and naturally aged) and cadmium on pakchoi (Brassica rapa subsp. chinensis) under different growth stages.

To protect agro-systems and food security, study on the effect of microplastics and heavy metals on edible plants is of great significance. Existing studies mostly used virgin microplastics to evaluate their effects on plants, effects of naturally aged microplastics and their combined effects with heavy metals are rarely explored. In this study, single and combined effect of polyethylene microplastics (PE, both virgin and naturally aged) and cadmium (Cd) on pakchoi under seedling and mature stages were analyzed from perspectives of growth inhibition, oxidative damage, nutrition content and soil enzyme activities. Results showed that inhibiting effects of naturally aged PE (PEa) on the growth of pakchoi were stronger than virgin PE (PEv), whereas co-contamination of PEa and Cd was less toxic than that of PEv and Cd. The co-contamination of PE and Cd could inhibit pakchoi dry biomass by over 85 %. Both single and combined contamination of PE and Cd promoted soil fluorescein diacetate hydrolase (FDA) activities, which were 1.11 to 2.04 times of that in control group. Soluble sugar contents under co-contamination of PEa and Cd were 14 % to 22 % higher than those in control group. PEa and PEv showed different effects on oxidative damage of pakchoi. Compared with PEv, catalase (CAT) activities were more sensitive with PEa, whereas PEa had lower effect on superoxide dismutase (SOD) activities. The response of pakchoi to PE and Cd changed with growth stage. Chlorophyll contents in pakchoi under seedling stage were generally higher than those under mature stage. For Cd contaminated soils, PE benefited pakchoi growth under seedling stage, i.e. antagonistic effect between Cd and PE but hindered their growth under mature stage, i.e. synergistic effect. The results unraveled here emphasized PE, especially PEa, could trigger negative effects on agro-systems, whereas PE could be beneficial for heavy metal contaminated agro-systems under specific situations.

Melatonin and copper oxide nanoparticles synergistically mitigate clubroot disease and enhance growth dynamics in Brassica rapa.

Clubroot, a devastating soil borne disease affecting 30%∼50% of Brassicaceae crops worldwide, lacks effective control measures. In the present study, we explored the potential of melatonin (MT) and copper oxide nanoparticle (CuO-NPs) in mitigating clubroot severity in the Brassica rapa ssp. pekinensis. Following 18 h priming with MT, CuO-NPs, or both seeds were grown in controlled environment using synthetic potting mix. Inoculated with Plasmodiophora brassicae spores on 5th day, followed by a soil drench phyto-nano treatment with a week interval. Plants were assessed for various health and growth indices including disease, biometrics, photosynthesis, reactive oxygen species (ROS), antioxidant enzyme activity, hormones and genes expression at onset of secondary clubroot infection using established protocols. Statistical analysis employed ANOVA with Fisher's LSD for significance assessment (P < 0.05). Our results revealed that seed priming with both MT (50 µMol/L) and CuO-NPs (200 mg/L), followed by soil drenching significantly reduced clubroot incidence (38%) and disease index (57%), compared to control treatments. This synergistic effect was associated with enhanced plant growth (shoots: 48% and roots: 59%). Plants treated with both MT and CuO-NPs showed robust antioxidant defenses, significantly increased superoxide dismutase (SOD (25/29%)), catalase (CAT (83/55%)), and ascorbate peroxidase (APX (83/46%)) activity in both shoots/roots, respectively, compared to infected control. Notably, salicylic acid and jasmonic acid levels doubled in treated plants, while stress hormone abscisic acid (ABA) decreased by 80% in roots and 21% in shoots. Gene expression analysis corroborated these findings, showing that the combined treatment activated antioxidant defense genes (SOD, APX and CAT) by 1.9-7.2-fold and upregulated hormone signaling genes JAZ1 (7.8-fold), MYC2 (3.9-fold) and SABP2 (36-fold). Conversely, ABA biosynthesis genes (ABA1 and NCED1) were downregulated up to 7.2-fold, while plant resistance genes NPR1, PRB1 and PDF1.2 were dramatically increased by up to 6.3-fold compared to infected plants. Overall, our combined treatment approach significantly reduces clubroot severity in B. rapa via enhanced antioxidant defenses, improved ROS scavenging, coordinated hormonal regulation and increased pathogen response genes. This study offers promising strategy for developing effective control measures against clubroot in susceptible cruciferous crops.

Migration of fluoranthene, phenanthrene, and pyrene in soil environment during the growth of Brassica rapa subsp. chinensis.

The escalating concern surrounding fluoranthene (FLN), phenanthrene (Phe), and pyrene (Pyr), underscores the urgency to investigate their dynamics in the context of agricultural ecosystems. Brassica rapa subsp. chinensis (Bok choy), a globally consumed vegetable, holds particular significance in this scenario. This study explores the migration and transformation of FLN, Phe, and Pyr from soil to Brassica rapa subsp. chinensis during its growth. The germination rates of seeds in these treatments varied, with soil+Bok choy and soil+FLN+Bok choy treatments showing higher rates (77.8 %), while soil+mix+Bok choy exhibited the lowest rate (11.1 %) after 3 days. Analyzing the distribution of FLN, Phe, and Pyr in Brassica rapa subsp. chinensis parts after 30 days revealed a sequence of accumulation in stem> root> leaf. This study provides information on practical implications for regulating the soil-plant migration and transformation of FLN, Phe, and Pyr, offering valuable insights for migration of PAHs pollution in agricultural settings.

Temporal variations in absorption and translocation of heavy metal(loid)s in pak choi (Brassica rapa L.) under open-field and greenhouse cultivation.

Elucidating the absorption and translocation of heavy metal(loid)s by common vegetables across different growth environments and stages is crucial for conducting accurate environmental risk assessments and for associated control. This study investigated temporal variations in the absorption and translocation capacities of pak choi (Brassica rapa L.) for As, Cd, Cr, Cu, Pb, and Zn in polluted soils during the plant growth cycle under greenhouse and open-field cultivation modes. Results showed high root metal(loid) bioconcentration factors and root-to-shoot translocation factors for Cd (0.25 and 1.44, respectively) and Zn (0.26 and 1.01), but low values for As (0.06 and 0.88) and Pb (0.06 and 0.87). The Cd concentration in the aerial edible parts peaked during the early slow growth period, whereas other heavy metal(loid)s peaked during the later stable maturity period. Root bioconcentration and root-to-shoot translocation factors did not significantly differ between cultivation modes. However, greenhouse cultivation exhibited lower average Cd and Zn concentrations in the edible parts and cumulative uptake amounts of most metal(loid)s than open-field cultivation during the typical harvest period spanning days 60 and 90. Short-term transitioning from open-field to greenhouse cultivation may reduce health risks associated with heavy metal(loid) intake via pak choi consumption. These findings facilitate sustainable agricultural practices and food safety management.

Bioregenerative dietary supplementation in space: Brassica rapa var. nipposinica and other Brassica cultivars.

Despite the precise environmental manipulation enabled by controlled environment agriculture (CEA), plant genotype remains a key factor in producing desirable traits. Brassica rapa var. nipposinica (mizuna) is a leading candidate for supplementing deficiencies in the space diet, however, which cultivar of mizuna will respond best to the environment of the international space station (ISS) is unknown. It is also unclear if there are more inter-varietal (mizuna - mustards) or intra-varietal (mizuna - mizuna) differences in response to the ISS environment. Twenty-two cultivars of mustard greens, including 13 cultivars of mizuna, were grown under ISS-like conditions to determine which would provide the greatest yield and highest concentrations of carotenoids, anthocyanins, calcium, potassium, iron, magnesium, ascorbic acid, thiamine, and phylloquinone. The experiment was conducted thrice, and data were analyzed to determine which cultivar is most suited for further optimization of space-based cultivation. It was found that phylloquinone and ß-carotene concentrations did not vary between cultivars, while all other metrics of interest showed some variation. 'Amara' mustard (B. carinata) provided the best overall nutritional profile, despite its low biomass yield of 36.8 g, producing concentrations of 27.85, 0.40, and 0.65 mg·g - 1 of ascorbic acid, thiamine, and lutein, respectively. Of the mizuna cultivars evaluated, open pollinated mibuna provided the best profile, while 'Red Hybrid' mizuna provided a complimentary profile to that of 'Amara', minimally increasing dietary iron while providing beneficial anthocyanins lacking in 'Amara'.

Temperature-dependent jumonji demethylase modulates flowering time by targeting H3K36me2/3 in Brassica rapa.

Global warming has a severe impact on the flowering time and yield of crops. Histone modifications have been well-documented for their roles in enabling plant plasticity in ambient temperature. However, the factor modulating histone modifications and their involvement in habitat adaptation have remained elusive. In this study, through genome-wide pattern analysis and quantitative-trait-locus (QTL) mapping, we reveal that BrJMJ18 is a candidate gene for a QTL regulating thermotolerance in thermotolerant B. rapa subsp. chinensis var. parachinensis (or Caixin, abbreviated to Par). BrJMJ18 encodes an H3K36me2/3 Jumonji demethylase that remodels H3K36 methylation across the genome. We demonstrate that the BrJMJ18 allele from Par (BrJMJ18Par) influences flowering time and plant growth in a temperature-dependent manner via characterizing overexpression and CRISPR/Cas9 mutant plants. We further show that overexpression of BrJMJ18Par can modulate the expression of BrFLC3, one of the five BrFLC orthologs. Furthermore, ChIP-seq and transcriptome data reveal that BrJMJ18Par can regulate chlorophyll biosynthesis under high temperatures. We also demonstrate that three amino acid mutations may account for function differences in BrJMJ18 between subspecies. Based on these findings, we propose a working model in which an H3K36me2/3 demethylase, while not affecting agronomic traits under normal conditions, can enhance resilience under heat stress in Brassica rapa.

Brassica rapa CURLY LEAF is a major H3K27 methyltransferase regulating flowering time.

MAIN CONCLUSION: In Brassica rapa, the epigenetic modifier BraA.CLF orchestrates flowering by modulating H3K27me3 levels at the floral integrator genes FT, SOC1, and SEP3, thereby influencing their expression. CURLY LEAF (CLF) is the catalytic subunit of the plant Polycomb Repressive Complex 2 that mediates the trimethylation of histone H3 lysine 27 (H3K27me3), an epigenetic modification that leads to gene silencing. While the function of CURLY LEAF (CLF) has been extensively studied in Arabidopsis thaliana, its role in Brassica crops is barely known. In this study, we focused on the Brassica rapa homolog of CLF and found that the loss-of-function mutant braA.clf-1 exhibits an accelerated flowering together with pleiotropic phenotypic alterations compared to wild-type plants. In addition, we carried out transcriptomic and H3K27me3 genome-wide analyses to identify the genes regulated by BraA.CLF. Interestingly, we observed that several floral regulatory genes, including the B. rapa homologs of FT, SOC1 and SEP3, show reduced H3K27me3 levels and increased transcript levels compared to wild-type plants, suggesting that they are direct targets of BraA.CLF and key players in regulating flowering time in this crop. In addition, the results obtained will enhance our understanding of the epigenetic mechanisms regulating key developmental traits and will aid to increase crop yield by engineering new Brassica varieties with different flowering time requirements.

Engineering Rapeseed Germination and Root Growth with Mechanical Strength of Polysaccharide Hydrogel.

Plant roots are highly sensitive to physical stress in the soil, with appropriate mechanical impedance promoting root elongation and lateral root growth. However, few studies have quantitatively explored the relationship between the mechanical impedance of the growth medium and the phenotypes of plant roots. In this study, we used a tensile machine equipped with a self-made steel needle mimicking the root tip to measure the force needed to penetrate the hydrogel medium (agar, low acyl gellan gum, and κ-carrageenan), providing insights into the force required for the rapeseed root tip to enter the medium following germination. These findings indicate that root penetration length is inversely associated with the mechanical strength of the growth medium, with variations observed in the root system adaptability across different substrates. Specifically, when the gel puncture resistance of the culture medium without adding MS reached approximately 18.4 mN, root penetration and growth were significantly hindered. With the addition of 1/2 MS medium, the polysaccharide concentration is 1.0 wt %, which is more suitable for cultivating rapeseed. This research not only offers a method for quantifying root phenotypes and medium mechanical impedance but also presents an approach for plant growth regulation and crop breeding.

Genome-Wide Identification, Expression Analysis and Functional Study of DELLA Genes in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

BACKGROUND: DELLA protein is a crucial factor which played pivotal roles in regulating numerous intriguing biological processes in plant development and abiotic stress responses. However, little is known about the function and information of DELLA protein in Chinese cabbage. METHODS: Using 5 DELLA gene sequences in Arabidopsis Thaliana as probes, 5 DELLA genes in Chinese cabbage were identified by Blast search in Chinese cabbage database (Brassica database (BRAD)). The National Center for Biotechnology Information (NCBI), ExPaSy, SWISS-MODEL, DNAMAN, MEGA 11, PlantCARE were used to identify and analyze the DELLA gene family of Chinese cabbage. Gene expression was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). The function of BraA10gRGL3 was verified by overexpression and phenotypic analysis of BraA10gRGL3 and yeast hybrid. RESULTS: In this study, 5 BraDELLAs homologous to Arabidopsis thaliana were identified and cloned based on the Brassica database, namely, BraA02gRGL1, BraA05gRGL2, BraA10gRGL3, BraA06gRGA and BraA09gRGA. All BraDELLAs contain the DELLA, TVHYNP, and GRAS conserved domains. Cis-element analysis revealed that the promoter regions of these 5 DELLA genes all contain light-responsive elements, TCT motif, I-box, G-box, and box 4, which are associated with GA signaling. Transcriptome analysis results proved that the expression of BraA02gRGL1, BraA05gRGL2, and BraA10gRGL3 in Y2 at different growth stages were lower than them in Y7, which is consistent with the phenotype that Y7 exhibited stronger stress tolerance than Y2. It is worth emphasizing that even through the overexpression of BraA10gRGL3-Y7 in Arabidopsis resulted in smaller leaf size and lower fresh weight compared to the wild type (WT) Arabidopsis: Columbia, a stronger response to abiotic stresses was observed in BraA10gRGL3-Y7. It indicated that BraA10gRGL3-Y7 can improve the stress resistance of plants by inhibiting their growth. Moreover, the yeast two-hybrid experiment confirmed that BraA10gRGL3-Y7 can interacted with BraA05gGID1a-Y7, BraA04gGID1b1, BraA09gGID1b3-Y2, and BraA06gGID1c, whereas BraA10gRGL3-Y2 cannot interact with any BraGID1. CONCLUSIONS: Collectively, BraDELLAs play important role in plant development and response to abiotic stress. The differences in amino acid sequences between BraA10gRGL3-Y2 and BraA10gRGL3-Y7 may result in variations in their protein binding sites, thus affecting their interaction with the BraGID1 family proteins. This systematic analysis lays the foundation for further study of the functional characteristics of DELLA genes of Chinese cabbage.

Functional Differentiation of the Duplicated Gene BrrCIPK9 in Turnip (Brassica rapa var. rapa).

Gene duplication is a key biological process in the evolutionary history of plants and an important driving force for the diversification of genomic and genetic systems. Interactions between the calcium sensor calcineurin B-like protein (CBL) and its target, CBL-interacting protein kinase (CIPK), play important roles in the plant's response to various environmental stresses. As a food crop with important economic and research value, turnip (Brassica rapa var. rapa) has been well adapted to the environment of the Tibetan Plateau and become a traditional crop in the region. The BrrCIPK9 gene in turnip has not been characterized. In this study, two duplicated genes, BrrCIPK9.1 and BrrCIPK9.2, were screened from the turnip genome. Based on the phylogenetic analysis, BrrCIPK9.1 and BrrCIPK9.2 were found located in different sub-branches on the phylogenetic tree. Real-time fluorescence quantitative PCR analyses revealed their differential expression levels between the leaves and roots and in response to various stress treatments. The differences in their interactions with BrrCBLs were also revealed by yeast two-hybrid analyses. The results indicate that BrrCIPK9.1 and BrrCIPK9.2 have undergone Asparagine-alanine-phenylalanine (NAF) site divergence during turnip evolution, which has resulted in functional differences between them. Furthermore, BrrCIPK9.1 responded to high-pH (pH 8.5) stress, while BrrCIPK9.2 retained its ancestral function (low K+), thus providing further evidence of their functional divergence. These functional divergence genes facilitate turnip's good adaptation to the extreme environment of the Tibetan Plateau. In summary, the results of this study reveal the characteristics of the duplicated BrrCIPK9 genes and provide a basis for further functional studies of BrrCBLs-BrrCIPKs in turnip.

An adaptive spacing of root-zone hole fertilization to improve production and fertilizer utilization of rapeseed.

BACKGROUND: Root-zone hole fertilization has a positive impact on enhancing crop production and fertilization efficiency. However, a suitable spacing for hole fertilization in rapeseed cultivation is unclear. To explore an adaptive hole spacing for improving rapeseed yield and fertilization efficiency, field experiments were conducted. Four spacings of hole fertilization were designed: 10 (FD10), 20 (FD20), 30 (FD30) and 40 cm (FD40), using no fertilization (F0) and deep-banded placement of fertilizer (DBP) as controls. The burial depth was 10 cm for FD and DBP treatments. RESULTS: Compared to DBP, hole fertilization impacted soil microenvironment, crop growth and yield components, resulting in a significant increase of 28.4% in seed yield and 25.6% in oil yield. Seed yield in FD20 (4345.43 kg ha-1) increased by 4.3%, 9.4% and 15.1% compared to FD10, FD30 and FD40, respectively. Fertilizer partial factor productivity under FD20 was 4.2%, 8.6% and 13.9% greater than FD10, FD30 and FD40, respectively; whereas the increase for agronomic efficiency was 6.0%, 12.7% and 21.0%, and the increase for N recovery efficiency was 39.5%, 52.5% and 62.9%, respectively. CONCLUSION: Fertilization with a hole spacing of 17 cm is a promising practice to maintain high production and fertilization efficiency when cultivating rapeseed. These results provide a theoretical foundation and scientific basis for improving rapeseed productivity and fertilizer utilization. © 2024 Society of Chemical Industry.

Identification of genes related to tipburn resistance in Chinese cabbage and preliminary exploration of its molecular mechanism.

BACKGROUND: Tipburn, also known as leaf tip necrosis, is a severe issue in Chinese cabbage production. One known cause is that plants are unable to provide adequate Ca2+ to rapidly expanding leaves. Bacterial infection is also a contributing factor. Different cultivars have varying degrees of tolerance to tipburn. Two inbred lines of Chinese cabbage were employed as resources in this research. RESULTS: We determined that the inbred line 'J39290' was the tipburn resistant material and the inbred line 'J95822' was the tipburn sensitive material based on the severity of tipburn, and the integrity of cell membrane structure. Ca2+ concentration measurements revealed no significant difference in Ca2+ concentration between the two materials inner leaves. Transcriptome sequencing technology was also used to find the differentially expressed genes (DEGs) of 'J95822' and 'J39290', and there was no significant difference in the previously reported Ca2+ uptake and transport related genes in the two materials. However, it is evident through DEG screening and classification that 23 genes are highly linked to plant-pathogen interactions, and they encode three different types of proteins: CaM/CML, Rboh, and CDPK. These 23 genes mainly function through Ca2+-CaM/CML-CDPK signal pathway based on KEGG pathway analysis, protein interaction prediction, and quantitative real-time PCR (qRT-PCR) of key genes. CONCLUSIONS: By analyzing the Ca2+ concentration in the above two materials, the transcription of previously reported genes related to Ca2+ uptake and transport, the functional annotation and KEGG pathway of DEGs, it was found that Ca2+ deficiency was not the main cause of tipburn in 'J95822', but was probably caused by bacterial infection. This study lays a theoretical foundation for exploring the molecular mechanism of resistance to tipburn in Chinese cabbage, and has important guiding significance for genetics and breeding.

Genome-Wide Identification and Expression Profiling of 2OGD Superfamily Genes from Three Brassica Plants.

The 2-oxoglutarate and Fe(II)-dependent dioxygenase (2OGD) superfamily is the second largest enzyme family in the plant genome, and its members are involved in various oxygenation and hydroxylation reactions. Due to their important biochemical significance in metabolism, a systematic analysis of the plant 2OGD genes family is necessary. Here, we identified 160, 179, and 337 putative 2OGDs from Brassica rapa, Brassica oleracea, and Brassica napus. According to their gene structure, domain, phylogenetic features, function, and previous studies, we also divided 676 2OGDs into three subfamilies: DOXA, DOXB, and DOXC. Additionally, homologous and phylogenetic comparisons of three subfamily genes provided valuable insight into the evolutionary characteristics of the 2OGD genes from Brassica plants. Expression profiles derived from the transcriptome and Genevestigator database exhibited distinct expression patterns of the At2OGD, Br2OGD, and Bo2OGD genes in different developmental stages, tissues, or anatomical parts. Some 2OGD genes showed high expression levels in various tissues, such as callus, seed, silique, and root tissues, while other 2OGD genes were expressed at very low levels in other tissues. Analysis of six Bo2OGD genes in different tissues by qRT-PCR indicated that these genes are involved in the metabolism of gibberellin, which in turn regulates plant growth and development. Our working system analysed 2OGD gene families of three Brassica plants and laid the foundation for further study of their functional characterization.

A single amino acid residue substitution in BraA04g017190.3C, a histone methyltransferase, results in premature bolting in Chinese cabbage (Brassica rapa L. ssp. Pekinensis).

BACKGROUND: Flowering is an important inflection point in the transformation from vegetative to reproductive growth, and premature bolting severely decreases crop yield and quality. RESULTS: In this study, a stable early-bolting mutant, ebm3, was identified in an ethyl methanesulfonate (EMS)-mutagenized population of a Chinese cabbage doubled haploid (DH) line 'FT'. Compared with 'FT', ebm3 showed early bolting under natural cultivation in autumn, and curled leaves. Genetic analysis showed that the early-bolting phenotype was controlled by a single recessive nuclear gene. Modified MutMap sequencing, genotyping analyses and allelism test provide strong evidence that BrEBM3 (BraA04g017190.3 C), encoding the histone methyltransferase CURLY LEAF (CLF), was the strongly candidate gene of the emb3. A C to T base substitution in the 14th exon of BrEBM3 resulted in an amino acid change (S to F) and the early-bolting phenotype of emb3. The mutation occurred in the SET domain (Suppressor of protein-effect variegation 3-9, Enhancer-of-zeste, Trithorax), which catalyzes site- and state-specific lysine methylation in histones. Tissue-specific expression analysis showed that BrEBM3 was highly expressed in the flower and bud. Promoter activity assay confirmed that BrEBM3 promoter was active in inflorescences. Subcellular localization analysis revealed that BrEBM3 localized in the nucleus. Transcriptomic studies supported that BrEBM3 mutation might repress H3K27me3 deposition and activate expression of the AGAMOUS (AG) and AGAMOUS-like (AGL) loci, resulting in early flowering. CONCLUSIONS: Our study revealed that an EMS-induced early-bolting mutant ebm3 in Chinese cabbage was caused by a nonsynonymous mutation in BraA04g017190.3 C, encoding the histone methyltransferase CLF. These results improve our knowledge of the genetic and genomic resources of bolting and flowering, and may be beneficial to the genetic improvement of Chinese cabbage.

Comparative Transcriptome-Based Mining of Senescence-Related MADS, NAC, and WRKY Transcription Factors in the Rapid-Senescence Line DLS-91 of Brassica rapa.

Leaf senescence is a developmental process induced by various molecular and environmental stimuli that may affect crop yield. The dark-induced leaf senescence-91 (DLS-91) plants displayed rapid leaf senescence, dramatically decreased chlorophyll contents, low photochemical efficiencies, and upregulation of the senescence-associated marker gene BrSAG12-1. To understand DLS molecular mechanism, we examined transcriptomic changes in DLS-91 and control line DLS-42 following 0, 1, and 4 days of dark treatment (DDT) stages. We identified 501, 446, and 456 DEGs, of which 16.7%, 17.2%, and 14.4% encoded TFs, in samples from the three stages. qRT-PCR validation of 16 genes, namely, 7 MADS, 6 NAC, and 3 WRKY, suggested that BrAGL8-1, BrAGL15-1, and BrWRKY70-1 contribute to the rapid leaf senescence of DLS-91 before (0 DDT) and after (1 and 4 DDT) dark treatment, whereas BrNAC046-2, BrNAC029-2/BrNAP, and BrNAC092-1/ORE1 TFs may regulate this process at a later stage (4 DDT). In-silico analysis of cis-acting regulatory elements of BrAGL8-1, BrAGL42-1, BrNAC029-2, BrNAC092-1, and BrWRKY70-3 of B. rapa provides insight into the regulation of these genes. Our study has uncovered several AGL-MADS, WRKY, and NAC TFs potentially worthy of further study to understand the underlying mechanism of rapid DLS in DLS-91.

Brassica rapa L. ssp. chinensis Isolated Microspore Culture Protocol.

Culture of isolated microspores is a widely used method to obtain haploid and doubled haploid plants for many crop species. This protocol describes the steps necessary to obtain a large number of microspore derived embryos for pakchoi (Brassica rapa L. ssp. chinensis) and zicaitai (Brassica rapa L. ssp. сhinensis Hanelt var. purpuraria Kitam).

Haploid and Doubled Haploid Plant Production in Brassica rapa L. subsp. pekinensis Via Microspore Culture.

The production of haploid and doubled haploid plants is a biotechnological tool that shortens the breeding process of new cultivars in many species. Doubled haploid plants are homozygous at every locus and they can be utilized as parents to produce F1 hybrids. In this chapter, we describe a protocol for the production of doubled haploid plants in Brassica rapa L. subsp. pekinensis using androgenesis induced by isolated microspore cultures.

Characterization of Brassica rapa metallothionein and phytochelatin synthase genes potentially involved in heavy metal detoxification.

Brassica rapa is an important leafy vegetable that can potentially accumulate high concentrations of cadmium (Cd), posing a risk to human health. The aim of the present study was to identify cadmium detoxifying molecular mechanisms in B. rapa using a functional cloning strategy. A cDNA library constructed from roots of B. rapa plants treated with Cd was transformed into the Cd sensitive yeast mutant strain DTY167 that lacks the yeast cadmium factor (YCF1), and resistant yeast clones were selected on Cd containing media. Two hundred genes potentially conferring cadmium resistance were rescued from the surviving yeast clones and sequenced. Sequencing analysis revealed that genes encoding for metallothionein (MT)1, MT2a, MT2b and MT3, and phytochelatin synthase (PCS)1 and PCS2 accounted for 35.5%, 28.5%, 4%, 11.3%, 18.7% and 2%, respectively of the genes identified. MTs and PCSs expressing DTY167 cells showed resistance to Cd as well as to Zn. PCS1 expressing yeast cells were also more resistant to Pb compared to those expressing MTs or PCS2. RT-PCR results showed that Cd treatment strongly induced the expression levels of MTs in the root and shoot. Furthermore, the different MTs and PCSs exhibited tissue specific expression. The results indicate that MTs and PCS genes potentially play a central role in detoxifying Cd and other toxic metals in B. rapa.