Unveiling molecular mechanisms of pepper resistance to Phytophthora capsici through grafting using iTRAQ-based proteomic analysis.

Phytophthora blight severely threatens global pepper production. Grafting bolsters plant disease resistance, but the underlying molecular mechanisms remain unclear. In this study, we used P. capsici-resistant strain 'ZCM334' and susceptible strain 'Early Calwonder' for grafting. Compared to self-rooted 'Early Calwonder' plants, 'ZCM334' grafts exhibited delayed disease onset, elevated resistance, and reduced leaf cell damage, showcasing the potential of grafting in enhancing pepper resistance to P. capsici. Proteomic analysis via the iTRAQ technology unveiled 478 and 349 differentially expressed proteins (DEPs) in the leaves and roots, respectively, between the grafts and self-rooted plants. These DEPs were linked to metabolism and cellular processes, stimulus responses, and catalytic activity and were significantly enriched in the biosynthesis of secondary metabolites, carbon fixation in photosynthetic organizations, and pyruvate metabolism pathways. Twelve DEPs exhibiting consistent expression trends in both leaves and roots, including seven related to P. capsici resistance, were screened. qRT-PCR analysis confirmed a significant correlation between the protein and transcript levels of DEPs after P. capsici inoculation. This study highlights the molecular mechanisms whereby grafting enhances pepper resistance to Phytophthora blight. Identification of key genes provides a foundation for studying the regulatory network governing the resistance of pepper to P. capsici.

Identification of a new allele of BraA09g066480.3C controlling the wax-less phenotype of Chinese cabbage.

BACKGROUND: Epidermal wax covers the surfaces of terrestrial plants to resist biotic and abiotic stresses. Wax-less flowering Chinese cabbage (Brassica campestris L. ssp. chinesis var. utilis tsen et lee) has the charateristics of lustrous green leaves and flower stalks, which are of high commercial value. RESULTS: To clarify the mechanism of the wax deficiency, the wax-less flowering Chinese cabbage doubled-haploid (DH) line 'CX001' and Chinese cabbage DH line 'FT', obtained from isolated microspore culture, were used in the experiments. Genetic analysis showed that the wax-less phenotype of 'CX001' was controlled by a recessive nuclear gene, named wlm1 (wax-less mutation 1), which was fine-mapped on chromosome A09 by bulked segregant analysis sequencing (BSA-seq) of B.rapa genome V3.0. There was only one gene (BraA09g066480.3C) present in the mapping region. The homologous gene in Arabidopsis thaliana is AT1G02205 (CER1) that encodes an aldehyde decarboxylase in the epidermal wax metabolism pathway. Semi-quantitative reverse transcription PCR and transcriptome analysis indicated that BraA09g066480.3C was expressed in 'FT' but not in 'CX001'. BraA09g066480.3C was lost in the CXA genome to which 'CX001' belonged. CONCLUSION: The work presented herein demonstrated that BraA09g066480.3C was the causal gene for wax-less flowering Chinese cabbage 'CX001'. This study will lay a foundation for further research on the molecular mechanism of epidermal wax synthesis in flowering Chinese cabbage.

Whole transcriptome analysis and construction of a ceRNA regulatory network related to leaf and petiole development in Chinese cabbage (Brassica campestris L. ssp. pekinensis).

BACKGROUND: The growth and development of leaves and petioles have a significant effect on photosynthesis. Understanding the molecular mechanisms underlying leaf and petiole development is necessary for improving photosynthetic efficiency, cultivating varieties with high photosynthetic efficiency, and improving the yield of crops of which the leaves are foodstuffs. This study aimed to identify the mRNAs, long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs) related to leaf and petiole development in Chinese cabbage (Brassica campestris L. ssp. pekinensis). The data were used to construct a competitive endogenous RNA (ceRNA) network to obtain insights into the mechanisms underlying leaf and petiole development. RESULTS: The leaves and petioles of the 'PHL' inbred line of Chinese cabbage were used as research materials for whole transcriptome sequencing. A total of 10,646 differentially expressed (DE) mRNAs, 303 DElncRNAs, 7 DEcircRNAs, and 195 DEmiRNAs were identified between leaves and petioles. Transcription factors and proteins that play important roles in leaf and petiole development were identified, including xyloglucan endotransglucosylase/hydrolase, expansion proteins and their precursors, transcription factors TCP15 and bHLH, lateral organ boundary domain protein, cellulose synthase, MOR1-like protein, and proteins related to plant hormone biosynthesis. A ceRNA regulatory network related to leaf and petiole development was constructed, and 85 pairs of ceRNA relationships were identified, including 71 DEmiRNA-DEmRNA, 12 DEmiRNA-DElncRNA, and 2 DEmiRNA-DEcircRNA pairs. Three LSH genes (BrLSH1, BrLSH2 and BrLSH3) with significant differential expression between leaves and petioles were screened from transcriptome data, and their functions were explored through subcellular localization analysis and transgenic overexpression verification. BrLSH1, BrLSH2 and BrLSH3 were nuclear proteins, and BrLSH2 inhibited the growth and development of Arabidopsis thaliana. CONCLUSIONS: This study identifies mRNAs and non-coding RNAs that may be involved in the development of leaves and petioles in Chinese cabbage, and establishes a ceRNA regulatory network related to development of the leaves and petioles, providing valuable genomic resources for further research on the molecular mechanisms underlying leaf and petiole development in this crop species.

Defect in BrMS1, a PHD-finger transcription factor, induces male sterility in ethyl methane sulfonate-mutagenized Chinese cabbage (Brassica rapa L. ssp. pekinensis).

Male sterility is an ideal character for the female parent in commercial hybrid seed production in Chinese cabbages. We identified three allele male sterile mutants msm2-1/2/3 in progenies of ethyl methane sulfonate mutagenized Chinese cabbage. It was proved that their male sterilities were controlled by a same recessive nuclear gene. Cytological observation showed that the delayed tapetal programmed cell death (PCD) as well as the abnormal pollen exine and intine led to pollen abortion in these mutants. MutMap combined with KASP analyses showed that BraA10g019050.3C, a homologous gene of AtMS1 encoding a PHD-finger transcription factor and regulated pollen development, was the causal gene. A single-nucleotide mutation from G to A occurred at the 2443th base of BrMS1 in msm2-1 which results in premature termination of the PHD-finger protein translation; a single-nucleotide mutation from G to A existed at 1372th base in msm2-2 that makes for frameshift mutation; a single-nucleotide mutation from G to A distributed at 1887th base in msm2-3 which issues in the amino acid changed from Asp to Asn. The three allelic mutations in BrMS1 all led to the male sterile phenotype, which revealed its function in stamen development. Quantitative reverse transcription polymerase chain reaction analysis indicated that BrMS1 specially expressed in the anther at the early stage of pollen development and its expression level was higher in msm2-1/2/3 than that in the wild-type "FT." BrMS1 was located at the nucleus and a length of 12 amino acid residues at the C-terminus had transcriptional activation activity. RNA-seq indicated that the mutation in BrMS1 affected the transcript level of genes related to the tapetum PCD and pollen wall formation, which brought out the pollen abortion. These male sterile mutants we developed provided a novel gene resource for hybrid breeding in Chinese cabbage.

Whole-transcriptome analysis and construction of an anther development-related ceRNA network in Chinese cabbage (Brassica campestris L. ssp. pekinensis).

Anther development is precisely regulated by a complex gene network, which is of great significance to plant breeding. However, the molecular mechanism of anther development in Chinese cabbage is unclear. Here, we identified microRNAs (miRNAs), mRNAs, long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) related to anther development in Chinese cabbage (Brassica campestris L. ssp. pekinensis) to construct competitive endogenous RNA (ceRNA) regulatory networks and provide valuable knowledge on anther development. Using whole-transcriptome sequencing, 9055, 585, 1344, and 165 differentially expressed mRNAs (DEmRNAs), miRNAs (DEmiRNAs), lncRNAs (DElncRNAs), and circRNAs (DEcircRNAs) were identified, respectively, in the anthers of Chinese cabbage compared with those in samples of the vegetative mass of four true leaves. An anther-related ceRNA regulatory network was constructed using miRNA targeting relationships, and 450 pairs of ceRNA relationships, including 97 DEmiRNA-DEmRNA, 281 DEmiRNA-DElncRNA, and 23 DEmiRNA-DEcircRNA interactions, were obtained. We identified important genes and their interactions with lncRNAs, circRNAs, and miRNAs involved in microsporogenesis, tapetum and callose layer development, pollen wall formation, and anther dehiscence. We analyzed the promoter activity of six predominant anther expression genes, which were expressed specifically in the anthers of Arabidopsis thaliana, indicating that they may play an important role in anther development of Chinese cabbage. This study lays the foundation for further research on the molecular mechanisms of anther growth and development in Chinese cabbage.

Proteomic analysis of a plastid gene encoding RPS4 mutant in Chinese cabbage (Brassica campestris L. ssp. pekinensis).

Plastids are important plant cell organelles containing a genome and bacterial-type 70S ribosomes-primarily composed of plastid ribosomal proteins and ribosomal RNAs. In this study, a chlorophyll-deficient mutant (cdm) obtained from double-haploid Chinese cabbage 'FT' was identified as a plastome mutant with an A-to-C base substitution in the plastid gene encoding the ribosomal protein RPS4. To further elucidate the function and regulatory mechanisms of RPS4, a comparative proteomic analysis was conducted between cdm and its wild-type 'FT' plants by isobaric tags and a relative and absolute quantitation (iTRAQ)-based strategy. A total of 6,245 proteins were identified, 540 of which were differentially abundant proteins (DAPs) in the leaves of cdm as compared to those of 'FT'-including 233 upregulated and 307 downregulated proteins. Upregulated DAPs were mainly involved in translation, organonitrogen compound biosynthetic process, ribosomes, and spliceosomes. Meanwhile, downregulated DAPs were mainly involved in photosynthesis, photosynthetic reaction centres, photosynthetic light harvesting, carbon fixation, and chlorophyll binding. These results indicated an important role of RPS4 in the regulation of growth and development of Chinese cabbage, possibly by regulating plastid translation activity by affecting the expression of specific photosynthesis- and cold stress-related proteins. Moreover, a multiple reaction monitoring (MRM) test and quantitative real-time polymerase chain reaction analysis confirmed our iTRAQ results. Quantitative proteomic analysis allowed us to confirm diverse changes in the metabolic pathways between cdm and 'FT' plants. This work provides new insights into the regulation of chlorophyll biosynthesis and photosynthesis in Chinese cabbage.

Whole-transcriptome sequencing reveals a vernalization-related ceRNA regulatory network in chinese cabbage (Brassica campestris L. ssp. pekinensis).

BACKGROUND: The transition from vegetative growth to reproductive growth involves various pathways. Vernalization is a crucial process for floral organ formation and regulation of flowering time that is widely utilized in plant breeding. In this study, we aimed to identify the global landscape of mRNAs, microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) related to vernalization in Chinese cabbage. These data were then used to construct a competitive endogenous RNA (ceRNA) network that provides valuable information to better understand the vernalization response. RESULTS: In this study, seeds sampled from the Chinese cabbage doubled haploid (DH) line 'FT' with or without vernalization treatment were used for whole-transcriptome sequencing. A total of 2702 differentially expressed (DE) mRNAs, 151 DE lncRNAs, 16 DE circRNAs, and 233 DE miRNAs were identified in the vernalization-treated seeds. Various transcription factors, such as WRKY, MYB, NAC, bHLH, MADS-box, zinc finger protein CONSTANS-like gene, and B3 domain protein, and regulatory proteins that play important roles in the vernalization pathway were identified. Additionally, we constructed a vernalization-related ceRNA-miRNA-target gene network and obtained 199 pairs of ceRNA relationships, including 108 DEmiRNA‒DEmRNA, 67 DEmiRNA‒DElncRNA, and 12 DEmiRNA‒DEcircRNA interactions, in Chinese cabbage. Furthermore, several important vernalization-related genes and their interacting lncRNAs, circRNAs, and miRNAs, which are involved in the regulation of flowering time, floral organ formation, bolting, and flowering, were identified. CONCLUSIONS: Our results reveal the potential mRNA and non-coding RNAs involved in vernalization, providing a foundation for further studies on the molecular mechanisms underlying vernalization in Chinese cabbage.

Integrated photocatalysis-adsorption-membrane separation in rotating reactor for synergistic removal of RhB.

A synergistic system of integrated photocatalysis-adsorption-membrane separation in a rotating reactor was designed. The composite membrane was prepared via filtration process under vacuum, and it was composed of graphene oxide (GO) acted as the separation membrane, activated carbon (AC) as the adsorbent and Ag@BiOBr as the photocatalyst, respectively. In this Ag@BiOBr/AC/GO membrane system, rotation of the membrane could avoid the light-shielding effect from organic color pollutants to achieve the complete removal of pollutants. More importantly, the synergistic effect among photocatalysis, adsorption and membrane separation in rotating reactor was significant for the efficient removal of rhodamine B (RhB). In the Ag@BiOBr/AC/GO composite membrane, GO membrane layer could reject the organic molecular by the assistance of AC layer with efficient adsorption capacity, and Ag@BiOBr at outer layer could photodegrade the organics under visible light irradiation. The photocatalysis process could solve the problem of membrane fouling and adsorption could assist GO membrane for stopping the permeation of pollutants. Meanwhile, GO membrane was not only beneficial for catalyst recovery, but also could concentrate the pollutants via the membrane separation to accelerate the photocatalytic degradation. At the same time, both the photocatalysis degradation and membrane separation could promote the adsorption ability of AC. This synergistic system showed the significant potential for the practical application in the future.

Fine mapping of a leaf flattening gene Bralcm through BSR-Seq in Chinese cabbage (Brassica rapa L. ssp. pekinensis).

Leaf flattening influences plant photosynthesis, thereby affecting product yield and quality. Here, we obtained a stably inherited leaf crinkled mutant (lcm), derived from the Chinese cabbage doubled haploid (DH) 'FT' line using EMS mutagenesis combined with isolated microspore culture. The crinkled phenotype was controlled by a single recessive nuclear gene, namely Bralcm, which was preliminarily mapped to chromosome A01 by bulked segregant analysis RNA-seq, and further between markers SSRS-1 and IndelD-20 using 1,575 recessive homozygous individuals in F2 population by a map-based cloning method. The target region physical distance was 126.69 kb, containing 23 genes; the marker SSRMG-4 co-segregated with the crinkled trait. Further, we found SSRMG-4 to be located on BraA01g007510.3C, a homolog of AHA2, which encodes H+-ATPase2, an essential enzyme in plant growth and development. Sequence analysis indicated a C to T transition in exon 7 of BraA01g007510.3C, resulting in a Thr (ACT) to Ile (ATT) amino acid change. Genotyping revealed that the leaf crinkled phenotype fully co-segregated with this SNP within the recombinants. qRT-PCR demonstrated that BraA01g007510.3C expression in lcm mutant leaves was dramatically higher than that in wild-type 'FT'. Thus, BraA01g007510.3C is a strong candidate gene for Bralcm, and AHA2 is possibly associated with leaf flattening in Chinese cabbage.

Investigation of genes associated with petal variations between diploid and autotetraploid in Chinese cabbage (Brassica rapa L. ssp. pekinensis) by RNA-seq and sRNA-seq.

Polyploidy promotes morphological, physiological, and reproductive diversity in plants. The imminent effect of chromosome doubling in plants is the enlargement of organs such as flowers and fruits, which increases the commercial value of crops. Flowering plays a vital role in the growth and development of angiosperms. Here, we prepared an isolated microspore culture of 'FT', a doubled haploid (DH) line of Chinese cabbage (Brassica rapa L. ssp. pekinensis), and obtained diploid and autotetraploid plants with the same genetic background. Compared with diploids, the autotetraploids were characterized by large floral organs, dark petals, delayed flowering, and reduced fertility. The indole-3-acetic acid (IAA) and jasmonic acid (JA) levels in autotetraploid petals were significantly higher and the abscisic acid (ABA) level was significantly lower than those in the diploid petals. The lutein level in autotetraploid petals was nearly two times higher than that in the diploid petals. A comparative transcriptome analysis revealed 14,412 differentially expressed genes (DEGs) between the diploids and autotetraploids, and they were enriched in 117 Gene Ontology terms and 110 Kyoto Encyclopedia of Genes and Genomes pathways. We detected 231 DEGs related to phytohormone signal transduction and 29 DEGs involved in carotenoid biosynthesis. An miRNA-target mRNA analysis showed that 32 DEGs regulated by 16 DEMs were associated with flowering timing (BraA03000336, BraA09004319, and BraA09000515), petal development (BraA05002408, BraA01004006, BraA09004069, and BraA04000966), flower opening (BraA07000350), and pollen development (BraA01000720, BraA09005727, and BraA01000253). This study provides information to help elucidate the molecular mechanisms underlying phenotypic variations induced by autopolyploidy in Chinese cabbage.

iTRAQ-based proteomic analysis of fertile and sterile flower buds from a genetic male sterile line 'AB01' in Chinese cabbage (Brassica campestris L. ssp. pekinensis).

To investigate the molecular basis of multiple-allele-inherited male sterility in Chinese cabbage (Brassica campestris L. ssp. pekinensis), we performed differential proteomic analysis using iTRAQ to identify differentially abundant proteins between fertile and sterile flower buds from the genetic male sterile line 'AB01'. We identified 5932 high-confidence proteins; 1494 were differentially abundant between the two samples, including 749 up- and 745 down-regulated proteins. The up- and down-regulated proteins that could be essential for anther development and male sterility in sterile buds were mainly involved in (1) carbohydrate and energy metabolism (pyruvate dehydrogenase, glycolysis/gluconeogenesis, TCA cycle, starch and sucrose metabolism), (2) pollen wall synthesis and regulation (pectinesterase, polygalacturonase, pectate lyase, beta-galactosidase, glycosyl hydrolase), (3) protein synthesis and degradation (proteasome subunits, ribosome proteins, ABC transporters, RNA transport, protein processing in endoplasmic reticulum), (4) flavonoid biosynthesis, and (5) plant hormone signal transduction. We identified 10 genes/proteins that were both up-regulated and 122 that were both down-regulated in a conjoint analysis. Multiple reaction monitoring and qRT-PCR validation showed that the iTRAQ results were accurate and reliable. These findings will provide valuable information on proteins involved in anther development, and will contribute to the understanding of the molecular mechanism(s) that underlie male sterility in Chinese cabbage. BIOLOGICAL SIGNIFICANCE: Chinese cabbage is an allogamous plant with bisexual flowers that displays significant heterosis. The application of male sterile lines is a very efficient way to produce hybrid seeds, which can generate stronger plants that develop more rapidly and produce higher yield. However, the molecular mechanism(s) underlying multiple-allele-inherited male sterility in Chinese cabbage is unknown. In this study, we used a quantitative proteomic approach (iTRAQ) to identify DAPs between fertile and sterile buds of the GMS line 'AB01'. Subsequently, we also performed conjoined analysis of the iTRAQ results and our previously reported transcriptomics results. The aim of this research was to obtain the key DAPs and to identify the significantly enriched pathways involved in anther development and male sterility. These results may provide new insights into the molecular mechanism(s) underlying multiple-allele-inherited male sterility in Chinese cabbage.

Tsunamis caused by submarine slope failures along western Great Bahama Bank.

Submarine slope failures are a likely cause for tsunami generation along the East Coast of the United States. Among potential source areas for such tsunamis are submarine landslides and margin collapses of Bahamian platforms. Numerical models of past events, which have been identified using high-resolution multibeam bathymetric data, reveal possible tsunami impact on Bimini, the Florida Keys, and northern Cuba. Tsunamis caused by slope failures with terminal landslide velocity of 20 ms-1 will either dissipate while traveling through the Straits of Florida, or generate a maximum wave of 1.5 m at the Florida coast. Modeling a worst-case scenario with a calculated terminal landslide velocity generates a wave of 4.5 m height. The modeled margin collapse in southwestern Great Bahama Bank potentially has a high impact on northern Cuba, with wave heights between 3.3 to 9.5 m depending on the collapse velocity. The short distance and travel time from the source areas to densely populated coastal areas would make the Florida Keys and Miami vulnerable to such low-probability but high-impact events.