Potential Regulatory Networks and Heterosis for Flavonoid and Terpenoid Contents in Pak Choi: Metabolomic and Transcriptome Analyses.

Pak choi exhibits a diverse color range and serves as a rich source of flavonoids and terpenoids. However, the mechanisms underlying the heterosis and coordinated regulation of these compounds-particularly isorhamnetin-remain unclear. This study involved three hybrid combinations and the detection of 528 metabolites from all combinations, including 26 flavonoids and 88 terpenoids, through untargeted metabolomics. Analysis of differential metabolites indicated that the heterosis for the flavonoid and terpenoid contents was parent-dependent, and positive heterosis was observed for isorhamnetin in the two hybrid combinations (SZQ, 002 and HMG, ZMG). Moreover, there was a high transcription level of flavone 3'-O-methyltransferase, which is involved in isorhamnetin biosynthesis. The third group was considered the ideal hybrid combination for investigating the heterosis of flavonoid and terpenoid contents. Transcriptome analysis identified a total of 12,652 DEGs (TPM > 1) in various groups that were used for comparison, and DEGs encoding enzymes involved in various categories, including "carotenoid bio-synthesis" and "anthocyanin biosynthesis", were enriched in the hybrid combination (SZQ, 002). Moreover, the category of anthocyanin biosynthesis also was enriched in the hybrid combination (HMG, ZMG). The flavonoid pathway demonstrated more differential metabolites than the terpenoid pathway did. The WGCNA demonstrated notable positive correlations between the dark-green modules and many flavonoids and terpenoids. Moreover, there were 23 ERF genes in the co-expression network (r ≥ 0.90 and p < 0.05). Thus, ERF genes may play a significant role in regulating flavonoid and terpenoid biosynthesis. These findings enhance our understanding of the heterosis and coordinated regulation of flavonoid and terpenoid biosynthesis in pak choi, offering insights for genomics-based breeding improvements.

BcABF1 Plays a Role in the Feedback Regulation of Abscisic Acid Signaling via the Direct Activation of BcPYL4 Expression in Pakchoi.

Abscisic acid-responsive element-binding factor 1 (ABF1), a key transcription factor in the ABA signal transduction process, regulates the expression of downstream ABA-responsive genes and is involved in modulating plant responses to abiotic stress and developmental processes. However, there is currently limited research on the feedback regulation of ABF1 in ABA signaling. This study delves into the function of BcABF1 in Pakchoi. We observed a marked increase in BcABF1 expression in leaves upon ABA induction. The overexpression of BcABF1 not only spurred Arabidopsis growth but also augmented the levels of endogenous IAA. Furthermore, BcABF1 overexpression in Arabidopsis significantly decreased leaf water loss and enhanced the expression of genes associated with drought tolerance in the ABA pathway. Intriguingly, we found that BcABF1 can directly activate BcPYL4 expression, a critical receptor in the ABA pathway. Similar to BcABF1, the overexpression of BcPYL4 in Arabidopsis also reduces leaf water loss and promotes the expression of drought and other ABA-responsive genes. Finally, our findings suggested a novel feedback regulation mechanism within the ABA signaling pathway, wherein BcABF1 positively amplifies the ABA signal by directly binding to and activating the BcPYL4 promoter.

Chloroplast C-to-U editing, regulated by a PPR protein BoYgl-2, is important for chlorophyll biosynthesis in cabbage.

Leaf color is an important agronomic trait in cabbage (Brassica oleracea L. var. capitata), but the detailed mechanism underlying leaf color formation remains unclear. In this study, we characterized a Brassica oleracea yellow-green leaf 2 (BoYgl-2) mutant 4036Y, which has significantly reduced chlorophyll content and abnormal chloroplasts during early leaf development. Genetic analysis revealed that the yellow-green leaf trait is controlled by a single recessive gene. Map-based cloning revealed that BoYgl-2 encodes a novel nuclear-targeted P-type PPR protein, which is absent in the 4036Y mutant. Functional complementation showed that BoYgl-2 from the normal-green leaf 4036G can rescue the yellow-green leaf phenotype of 4036Y. The C-to-U editing efficiency and expression levels of atpF, rps14, petL and ndhD were significantly reduced in 4036Y than that in 4036G, and significantly increased in BoYgl-2 overexpression lines than that in 4036Y. The expression levels of many plastid- and nuclear-encoded genes associated with chloroplast development in BoYgl-2 mutant were also significantly altered. These results suggest that BoYgl-2 participates in chloroplast C-to-U editing and development, which provides rare insight into the molecular mechanism underlying leaf color formation in cabbage.

In-Depth Characterization of bZIP Genes in the Context of Endoplasmic Reticulum (ER) Stress in Brassica campestris ssp. chinensis.

Numerous studies have been conducted to investigate the genomic characterization of bZIP genes and their involvement in the cellular response to endoplasmic reticulum (ER) stress. These studies have provided valuable insights into the coordinated cellular response to ER stress, which is mediated by bZIP transcription factors (TFs). However, a comprehensive and systematic investigations regarding the role of bZIP genes and their involvement in ER stress response in pak choi is currently lacking in the existing literature. To address this knowledge gap, the current study was initiated to elucidate the genomic characteristics of bZIP genes, gain insight into their expression patterns during ER stress in pak choi, and investigate the protein-to-protein interaction of bZIP genes with the ER chaperone BiP. In total, 112 members of the BcbZIP genes were identified through a comprehensive genome-wide analysis. Based on an analysis of sequence similarity, gene structure, conserved domains, and responsive motifs, the identified BcbZIP genes were categorized into 10 distinct subfamilies through phylogenetic analysis. Chromosomal location and duplication events provided insight into their genomic context and evolutionary history. Divergence analysis estimated their evolutionary history with a predicted divergence time ranging from 0.73 to 80.71 million years ago (MYA). Promoter regions of the BcbZIP genes were discovered to exhibit a wide variety of cis-elements, including light, hormone, and stress-responsive elements. GO enrichment analysis further confirmed their roles in the ER unfolded protein response (UPR), while co-expression network analysis showed a strong relationship of BcbZIP genes with ER-stress-responsive genes. Moreover, gene expression profiles and protein-protein interaction with ER chaperone BiP further confirmed their roles and capacity to respond to ER stress in pak choi.

QTL mapping and candidate gene analysis reveal two major loci regulating green leaf color in non-heading Chinese cabbage.

KEY MESSAGE: Two major-effect QTL GlcA07.1 and GlcA09.1 for green leaf color were fine mapped into 170.25 kb and 191.41 kb intervals on chromosomes A07 and A09, respectively, and were validated by transcriptome analysis. Non-heading Chinese cabbage (NHCC) is a leafy vegetable with a wide range of green colors. Understanding the genetic mechanism behind broad spectrum of green may facilitate the breeding of high-quality NHCC. Here, we used F2 and F7:8 recombination inbred line (RIL) population from a cross between Wutacai (dark-green) and Erqing (lime-green) to undertake the genetic analysis and quantitative trait locus (QTL) mapping in NHCC. The genetic investigation of the F2 population revealed that the variation of green leaf color was controlled by two recessive genes. Six pigments associated with green leaf color, including total chlorophyll, chlorophyll a, chlorophyll b, total carotenoids, lutein, and carotene were quantified and applied for QTL mapping in the RIL population. A total of 7 QTL were detected across the whole genome. Among them, two major-effect QTL were mapped on chromosomes A07 (GlcA07.1) and A09 (GlcA09.1) corresponding to two QTL identified in the F2 population. The QTL GlcA07.1 and GlcA09.1 were further fine mapped into 170.25 kb and 191.41 kb genomic regions, respectively. By comparing gene expression level and gene annotation, BraC07g023810 and BraC07g023970 were proposed as the best candidates for GlcA07.1, while BraC09g052220 and BraC09g052270 were suggested for GlcA09.1. Two InDel molecular markers (GlcA07.1-BcGUN4 and GlcA09.1-BcSG1) associated with BraC07gA023810 and BraC09g052220 were developed and could effectively identify leaf color in natural NHCC accessions, suggesting their potential for marker-assisted leaf color selection in NHCC breeding.

Laser capture microdissection transcriptome (LCM RNA-seq) reveals BcDFR is a key gene in anthocyanin synthesis of non-heading Chinese cabbage.

BACKGROUND: Purple non-heading Chinese cabbage [Brassica campestris (syn. Brassica rapa) ssp. chinensis] has become popular because of its richness in anthocyanin. However, anthocyanin only accumulates in the upper epidermis of leaves. Further studies are needed to investigate the molecular mechanisms underlying the specific accumulation of it. RESULTS: In this study, we used the laser capture frozen section method (LCM) to divide purple (ZBC) and green (LBC) non-heading Chinese cabbage leaves into upper and lower epidermis parts (Pup represents the purple upper epidermis, Plow represents the purple lower epidermis, Gup represents the green upper epidermis, Glow represents the green lower epidermis). Through transcriptome sequencing, we found that the DIHYDROFLAVONOL 4-REDUCTASE-encoding gene BcDFR, is strongly expressed in Pup but hardly in others (Plow, Gup, Glow). Further, a deletion and insertion in the promoter of BcDFR in LBC were found, which may interfere with BcDFR expression. Subsequent analysis of gene structure and conserved structural domains showed that BcDFR is highly conserved in Brassica species. The predicted protein-protein interaction network of BcDFR suggests that it interacts with almost all functional proteins in the anthocyanin biosynthesis pathway. Finally, the results of the tobacco transient expression also demonstrated that BcDFR promotes the synthesis and accumulation of anthocyanin. CONCLUSIONS: BcDFR is specifically highly expressed on the upper epidermis of purple non-heading Chinese cabbage leaves and regulates anthocyanin biosynthesis and accumulation. Our study provides new insights into the functional analysis and transcriptional regulatory network of anthocyanin-related genes in purple non-heading Chinese cabbage.

Investigating the effects of tauroursodeoxycholic acid (TUDCA) in mitigating endoplasmic reticulum stress and cellular responses in Pak choi.

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) within plant cells due to unfavourable conditions leads to ER stress. This activates interconnected pathways involving reactive oxygen species (ROS) and unfolded protein response (UPR), which play vital roles in regulating ER stress. The aim of this study is to investigate the underlying mechanisms of tunicamycin (TM) induced ER stress and explore the potential therapeutic applications of tauroursodeoxycholic acid (TUDCA) in mitigating cellular responses to ER stress in Pak choi (Brassica campestris subsp. chinensis). The study revealed that ER stress in Pak choi leads to detrimental effects on plant morphology, ROS levels, cellular membrane integrity, and the antioxidant defence system. However, treatment with TUDCA in TM-induced ER stressed Pak choi improved morphological indices, pigment contents, ROS accumulation, cellular membrane integrity, and antioxidant defence system restoration. Additionally, TUDCA also modulates the transcription levels of ER stress sensors genes, ER chaperone genes, and ER-associated degradation (ERAD) genes during ER stress in Pak choi. Furthermore, TUDCA has demonstrated its ability to alleviate ER stress, stabilize the UPR, reduce oxidative stress, prevent apoptosis, and positively influence plant growth and development. These results collectively comprehend TUDCA as a promising agent for mitigating ER stress-induced damage in Pak choi plants and provide valuable insights for further research and potential applications in crop protection and stress management.

Integrating Dynamic 3D Chromatin Architecture and Gene Expression Alterations Reveal Heterosis in Brassica rapa.

Heterosis plays a significant role in enhancing variety, boosting yield, and raising economic value in crops, but the molecular mechanism is still unclear. We analyzed the transcriptomes and 3D genomes of a hybrid (F1) and its parents (w30 and 082). The analysis of the expression revealed a total of 485 specially expressed genes (SEGs), 173 differentially expressed genes (DEGs) above the parental expression level, more actively expressed genes, and up-regulated DEGs in the F1. Further study revealed that the DEGs detected in the F1 and its parents were mainly involved in the response to auxin, plant hormone signal transduction, DNA metabolic process, purine metabolism, starch, and sucrose metabolism, which suggested that these biological processes may play a crucial role in the heterosis of Brassica rapa. The analysis of 3D genome data revealed that hybrid F1 plants tend to contain more transcriptionally active A chromatin compartments after hybridization. Supplementaryly, the F1 had a smaller TAD (topologically associated domain) genome length, but the number was the highest, and the expression change in activated TAD was higher than that of repressed TAD. More specific TAD boundaries were detected between the parents and F1. Subsequently, 140 DEGs with genomic structural variants were selected as potential candidate genes. We found two DEGs with consistent expression changes in A/B compartments and TADs. Our findings suggested that genomic structural variants, such as TADs and A/B chromatin compartments, may affect gene expression and contribute to heterosis in Brassica rapa. This study provides further insight into the molecular mechanism of heterosis in Brassica rapa.

Exploring the Regulatory Dynamics of BrFLC-Associated lncRNA in Modulating the Flowering Response of Chinese Cabbage.

Vernalization plays a crucial role in the flowering and yield of Chinese cabbage, a process intricately influenced by long non-coding RNAs (lncRNAs). Our research focused on lncFLC1, lncFLC2a, and lncFLC2b, which emerged as key players in this process. These lncRNAs exhibited an inverse expression pattern to the flowering repressor genes FLOWERING LOCUS C 1 (BrFLC1) and FLOWERING LOCUS C 2 (BrFLC2) during vernalization, suggesting a complex regulatory mechanism. Notably, their expression in the shoot apex and leaves was confirmed through in fluorescent in situ hybridization (FISH). Furthermore, when these lncRNAs were overexpressed in Arabidopsis, a noticeable acceleration in flowering was observed, unveiling functional similarities to Arabidopsis's COLD ASSISTED INTRONIC NONCODING RNA (COOLAIR). This resemblance suggests a potentially conserved regulatory mechanism across species. This study not only enhances our understanding of lncRNAs in flowering regulation, but also opens up new possibilities for their application in agricultural practices.

ETHYLENE RESPONSE FACTOR 070 inhibits flowering in Pak-choi by indirectly impairing BcLEAFY expression.

APETALA2/ethylene-responsive factors respond to ethylene and participate in many biological and physiological processes, such as plant morphogenesis, stress resistance, and hormone signal transduction. Ethylene responsive factor 070 （BcERF070）is important in flowering. However, the underlying molecular mechanisms of BcERF070 in floral transition in response to ethylene signaling have not been fully characterized. Herein, we explored the function of BcERF070 in Pak-choi [Brassica campestris (syn. Brassica rapa) ssp. chinensis]. Ethylene treatment induced BcERF070 expression and delayed flowering in Pak-choi. Silencing of BcERF070 induced flowering in Pak-choi. BcERF070 interacted with major latex protein-like 328 (BcMLP328), which forms a complex with helix-loop-helix protein 30 (BcbHLH30) to enhance the transcriptional activity of BcbHLH30 on LEAFY (BcLFY), ultimately promoting flowering. However, BcERF070 impaired the BcMLP328-BcbHLH30 complex activation of LEAFY (BcLFY), ultimately inhibiting flowering in Pak-choi. BcERF070 directly promoted the expression of the flowering inhibitor gene B-box 29 (BcBBX29) and delayed flowering by reducing FLOWERING LOCUS T (BcFT) expression. These results suggest that BcERF070 mediates ethylene-reduced flowering by impairing the BcMLP328-BcbHLH30 complex activation of BcLFY and by directly promoting the gene expression of the flowering inhibition factor BcBBX29 to repress BcFT expression. The findings contribute to understanding the molecular mechanisms underlying floral transition in response to ethylene in plants.

Regulation of the trade-off between cold stress and growth by glutathione S-transferase phi class 10 (BcGSTF10) in non-heading Chinese cabbage.

Cold stress is a serious threat to global crop production and food security, but plant cold resistance is accompanied by reductions in growth and yield. In this study, we determined that the novel gene BcGSTF10 in non-heading Chinese cabbage [NHCC; Brassica campestris (syn. Brassica rapa) ssp. chinensis] is implicated in resistance to cold stress. Biochemical and genetic analyses demonstrated that BcGSTF10 interacts with BcICE1 to induce C-REPEAT BINDING FACTOR (CBF) genes that enhance freezing tolerance in NHCC and in Arabidopsis. However, BcCBF2 represses BcGSTF10 and the latter promotes growth in NHCC and Arabidopsis. This dual function of BcGSTF10 indicates its pivotal role in balancing cold stress and growth, and this important understanding has the potential to inform the future development of strategies to breed crops that are both climate-resilient and high-yielding.

Dynamic analysis and optimal control of a stochastic investor sentiment contagion model considering sentiments isolation with random parametric perturbations.

Investor sentiment contagion has a profound influence on economic and social development. This paper explores the diverse influences of various investor sentiments in modern society on the economy and society. It also investigates the interference of various uncertain factors on investor sentiments in the modern economy and society. On this basis, the dual-system stochastic SPA2G2R model was constructed, incorporating positive and negative sentiments, as well as a supervision and isolation mechanism. The global existence of positive solutions was established, and sufficient conditions for the disappearance and steady distribution of investor sentiment were calculated. An optimal control strategy for the stochastic model was put forward, with numerical simulation supporting the theoretical analysis results. A comparison with parameter changes in the deterministic model was also conducted. The research reveals a competitive relationship between different investor sentiments. Enhancing societal guidance mechanisms promotes positive investor sentiment contagion. Timely control by the supervisory department effectively curbs the spread of investor sentiment. Additionally, white noise promotes investor sentiment contagion, suggesting effective regulation through control of noise intensity and disturbance parameters.

Construction of an Efficient Genetic Transformation System for Watercress (Nasturtium officinale W. T. Aiton).

Based on the established efficient regeneration system for watercress in our laboratory, we optimized the processes of pretreatment, co-culture, and differentiation culture. Through GFP fluorescence and PCR identification, we successfully obtained transgenic watercress with the DR5 gene, which allowed us to investigate the distribution details of auxin in the growth process of watercress. Our findings provide an effective method for gene function research and lay the foundation for innovative utilization of germplasm resources of watercress.

The Developmental Mechanism of the Root System of Cultivated Terrestrial Watercress.

A well-developed root system is crucial for the rapid growth, asexual reproduction, and adaptation to the drought environments of the watercress. After analyzing the transcriptome of the watercress root system, we found that a high concentration of auxin is key to its adaptation to dry conditions. For the first time, we obtained DR5::EGFP watercress, which revealed the dynamic distribution of auxin in watercress root development under drought conditions. Via the application of naphthylphthalamic acid (NPA), 4-biphenylboronic acid (BBO), ethylene (ETH), abscisic acid (ABA), and other factors, we confirmed that auxin has a significant impact on the root development of watercress. Finally, we verified the role of auxin in root development using 35S::NoYUC8 watercress and showed that the synthesis of auxin in the root system mainly depends on the tryptophan, phenylalanine, and tyrosine amino acids (TAA) synthesis pathway. After the level of auxin increases, the root system of the watercress develops toward adaptation to dry environments. The formation of root aerenchyma disrupts the concentration gradient of auxin and is a key factor in the differentiation of lateral root primordia and H cells in watercress.

BcWRKY22 Activates BcCAT2 to Enhance Catalase (CAT) Activity and Reduce Hydrogen Peroxide (H2O2) Accumulation, Promoting Thermotolerance in Non-Heading Chinese Cabbage (Brassica campestris ssp. chinensis).

WRKY transcription factors (TFs) participate in plant defense mechanisms against biological and abiotic stresses. However, their regulatory role in heat resistance is still unclear in non-heading Chinese cabbage. Here, we identified the WRKY-IIe gene BcWRKY22(BraC09g001080.1), which is activated under high temperatures and plays an active role in regulating thermal stability, through transcriptome analysis. We further discovered that the BcWRKY22 protein is located in the nucleus and demonstrates transactivation activity in both the yeast and plant. Additionally, our studies showed that the transient overexpression of BcWRKY22 in non-heading Chinese cabbage activates the expression of catalase 2 (BcCAT2), enhances CAT enzyme activity, and reduces Hydrogen Peroxide (H2O2) accumulation under heat stress conditions. In addition, compared to its wild-type (WT) counterparts, Arabidopsis thaliana heterologously overexpresses BcWRKY22, improving thermotolerance. When the BcWRKY22 transgenic root was obtained, under heat stress, the accumulation of H2O2 was reduced, while the expression of catalase 2 (BcCAT2) was upregulated, thereby enhancing CAT enzyme activity. Further analysis revealed that BcWRKY22 directly activates the expression of BcCAT2 (BraC08g016240.1) by binding to the W-box element distributed within the promoter region of BcCAT2. Collectively, our findings suggest that BcWRKY22 may serve as a novel regulator of the heat stress response in non-heading Chinese cabbage, actively contributing to the establishment of thermal tolerance by upregulating catalase (CAT) activity and downregulating H2O2 accumulation via BcCAT2 expression.

Integrated Transcriptome and Proteome Analysis Revealed the Regulatory Mechanism of Hypocotyl Elongation in Pakchoi.

Hypocotyl length is a critical determinant for the efficiency of mechanical harvesting in pakchoi production, but the knowledge on the molecular regulation of hypocotyl growth is very limited. Here, we report a spontaneous mutant of pakchoi, lhy7.1, and identified its characteristics. We found that it has an elongated hypocotyl phenotype compared to the wild type caused by the longitudinal growth of hypocotyl cells. Different light quality treatments, transcriptome, and proteomic analyses were performed to reveal the molecular mechanisms of hypocotyl elongation. The data showed that the hypocotyl length of lhy7.1 was significantly longer than that of WT under red, blue, and white lights but there was no significant difference under dark conditions. Furthermore, we used transcriptome and label-free proteome analyses to investigate differences in gene and protein expression levels between lhy7.1 and WT. At the transcript level, 4568 differentially expressed genes (DEGs) were identified, which were mainly enriched in "plant hormone signal transduction", "photosynthesis", "photosynthesis-antenna proteins", and "carbon fixation in photosynthetic organisms" pathways. At the protein level, 1007 differentially expressed proteins (DEPs) were identified and were mainly enriched in photosynthesis-related pathways. The comprehensive transcriptome and proteome analyses revealed a regulatory network of hypocotyl elongation involving plant hormone signal transduction and photosynthesis-related pathways. The findings of this study help elucidate the regulatory mechanisms of hypocotyl elongation in lhy7.1.

Secretory IgA-ETEC F5 Immune Complexes Promote Dendritic Cell Differentiation and Prime T Cell Proliferation in the Mouse Intestine.

Although secretory IgA (SIgA) is the dominant antibody in mucosal secretions, the capacity of the SIgA-antigen complex to prime the activation of dendritic cells (DCs) and T cells in the intestinal epithelium is not well understood. To this end, the SIgA-ETEC F5 immune complexes (ICs) were prepared via Ni-NTA pull-down. After injecting the ICs into the intestines of SPF BALB/c mice, most ICs were observed in the Peyer's patch (PP). We established a microfold (M) cell culture model in vitro for transport experiments and the inhibition test. To evaluate the priming effect of mucosal immunity, we employed the DC2.4 stimulation test, T lymphocyte proliferation assays, and cytokine detection assays. We found that the ICs were taken up via clathrin-dependent endocytosis through M cells. The high expression of costimulatory molecules CD86, CD80, and CD40 indicated that the ICs promoted the differentiation and maturation of DC2.4 cells. The stimulation index (SI) in the complex group was significantly higher than in the control group, suggesting that the ICs stimulated the proliferation of primed T cells. The secretion of some cytokines, namely TNF-α, IFN-γ, IL-2, IL-4, IL-5, and IL-6, in spleen cells from the immunized mice was upregulated. These results indicate that ETEC F5 delivery mediated by SIgA in PPs initiates mucosal immune responses.

BcNAC056 Interacts with BcWRKY1 to Regulate Leaf Senescence in Pak Choi.

Senescence is the final stage of leaf development. For leafy vegetables such as pak choi, leaf senescence is adverse to yield due to the harvest period shortening. However, the regulatory mechanisms of leaf senescence are largely unknown in leafy vegetables. Here, we isolated and characterized a NAC gene, BcNAC056, in pak choi [Brassica campestris (syn. Brassica rapa) ssp. chinensis cv. 49caixin]. BcNAC056-GFP was located in the nucleus at the subcellular level, and BcNAC056 was responsive to leaf senescence and different hormones at the transcriptional level. Heterologous overexpression of BcNAC056 in Arabidopsis promoted leaf senescence, accompanied by the increased expression of senescence-associated genes (SAGs), whereas virus-induced gene silencing-based silencing in pak choi delayed leaf senescence. The following transcriptome analysis showed that heterologous overexpression of BcNAC056 enhanced some AtSAG transcripts in Arabidopsis. Electrophoretic mobility shift assay (EMSA) and dual-luciferase (LUC) reporter assay revealed that BcNAC056 activated SAG12 by directly binding to the promoter. In addition, with the LUC reporter and transient overexpression assays, we proposed that BcNAC056-BcWRKY1 interaction promoted the activation of BcSAG12. Taken together, our findings revealed a new regulatory mechanism of leaf senescence in pak choi.

Ectopic Expression of BcCUC2 Involved in Sculpting the Leaf Margin Serration in Arabidopsis thaliana.

Leaf margin serration is a morphological characteristic in plants. The CUC2 (CUP-SHAPED COTYLEDON 2) gene plays an important role in the outgrowth of leaf teeth and enhances leaf serration via suppression of growth in the sinus. In this study, we isolated the BcCUC2 gene from Pak-choi (Brassica rapa ssp. chinensis), which contains a 1104 bp coding sequence, encoding 367 amino acid residues. Multiple sequence alignment exhibited that the BcCUC2 gene has a typical conserved NAC domain, and phylogenetic relationship analysis showed that the BcCUC2 protein has high identity with Cruciferae plants (Brassica oleracea, Arabidopsis thaliana, and Cardamine hirsuta). The tissue-specific expression analysis displayed that the BcCUC2 gene has relatively high transcript abundance in floral organs. Meanwhile, the expression profile of BcCUC2 was relatively higher in the '082' lines with serrate leaf margins than the '001' lines with smooth leaf margins in young leaves, roots, and hypocotyls. In addition, the transcript level of BcCUC2 was up-regulated by IAA and GA3 treatment, especially at 1-3 h. The subcellular localization assay demonstrated that BcCUC2 was a nuclear-target protein. Furthermore, leaf serration occurred, and the number of the inflorescence stem was increased in the transgenic Arabidopsis thaliana plants' overexpressed BcCUC2 gene. These data illustrated that BcCUC2 is involved in the development of leaf margin serration, lateral branches, and floral organs, contributing to further uncovering and perfecting the regulation mechanism of leaf serration in Pak-choi.

Host Factors Genes BcCLC1 and BcCLC2 Confer Turnip Mosaic Virus Resistance in Non-Heading Chinese Cabbage (Brassica campestris ssp. chinensis).

Clathrin is an evolutionarily highly conserved evolutionary protein consisting of clathrin light chains (CLC) and clathrin heavy chains (CHC), and these form its basic structure. Clathrin is an important host factor in the process of viral infection. In this study, we cloned the BcCLC1 gene and the BcCLC2 gene from the '49CX' variety of non-heading Chinese cabbage (NHCC, Brassica campestris L. ssp. chinensis Makino) and verified their functions. The results showed that BcCLC1 was mainly localized in the cytomembrane and cytoplasm, and only a small amount entered the nucleus. BcCLC2 encoded a protein comprising 265 amino acids that were distributed in the cytomembrane, nucleus, and cytoplasm. A BiFC assay and yeast two-hybrid (Y2H) analysis showed that BcCLCs (BcCLC1 and BcCLC2) could interact with several TuMV proteins. We further investigated the mechanism of BcCLCs in regulating TuMV virus infections in NHCC, and observed that BcCLCs gene silencing inhibited TuMV infections and overexpression of BcCLCs in Arabidopsis promoted TuMV infections in NHCC. Finally, mutants of Arabidopsis homologs of BcCLCs were also screened and subjected to TuMV inoculation tests. In conclusion, we speculate that BcCLCs confer Turnip mosaic virus (TuMV) resistance in NHCC by interacting with TuMV proteins to promote the intracellular transport of the virus.