Leaf Color Classification and Expression Analysis of Photosynthesis-Related Genes in Inbred Lines of Chinese Cabbage Displaying Minor Variations in Dark-Green Leaves.

The leaves of the Chinese cabbage which is most widely consumed come in a wide variety of colors. Leaves that are dark green can promote photosynthesis, effectively improving crop yield, and therefore hold important application and cultivation value. In this study, we selected nine inbred lines of Chinese cabbage displaying slight differences in leaf color, and graded the leaf color using the reflectance spectra. We clarified the differences in gene sequences and the protein structure of ferrochelatase 2 (BrFC2) among the nine inbred lines, and used qRT-PCR to analyze the expression differences of photosynthesis-related genes in inbred lines with minor variations in dark-green leaves. We found expression differences among the inbred lines of Chinese cabbage in photosynthesis-related genes involved in the porphyrin and chlorophyll metabolism, as well as in photosynthesis and photosynthesis-antenna protein pathway. Chlorophyll b content was significantly positively correlated with the expression of PsbQ, LHCA1_1 and LHCB6_1, while chlorophyll a content was significantly negatively correlated with the expression PsbQ, LHCA1_1 and LHCA1_2. Our results provide an empirical basis for the precise identification of candidate genes and a better understanding of the molecular mechanisms responsible for the production of dark-green leaves in Chinese cabbage.

Mutation in a chlorophyll-binding motif of Brassica ferrochelatase enhances both heme and chlorophyll biosynthesis.

The heme branch of tetrapyrrole biosynthesis contributes to the regulation of chlorophyll levels. However, the mechanism underlying the balance between chlorophyll and heme synthesis remains elusive. Here, we identify a dark green leaf mutant, dg, from an ethyl methanesulfonate (EMS)-induced mutant library of Chinese cabbage. The dg phenotype is caused by an amino acid substitution in the conserved chlorophyll a/b-binding motif (CAB) of ferrochelatase 2 (BrFC2). This mutation increases the formation of BrFC2 homodimer to promote heme production. Moreover, wild-type BrFC2 and dBrFC2 interact with protochlorophyllide (Pchlide) oxidoreductase B1 and B2 (BrPORB1 and BrPORB2), and dBrFC2 exhibits higher binding ability to substrate Pchlide, thereby promoting BrPORBs-catalyzed production of chlorophyllide (Chlide), which can be directly converted into chlorophyll. Our results show that dBrFC2 is a gain-of-function mutation contributing to balancing heme and chlorophyll synthesis via a regulatory mechanism in which dBrFC2 promotes BrPORB enzymatic reaction to enhance chlorophyll synthesis.

Construction of a high-density mutant population of Chinese cabbage facilitates the genetic dissection of agronomic traits.

Chinese cabbage (Brassica rapa ssp. pekinensis) is an economically important vegetable crop throughout the world, especially in Asia. High-quality genome sequences are available for Chinese cabbage, but gene functional studies remain challenging. To promote functional genomic studies of Chinese cabbage, we generated an ethyl methane sulfonate (EMS) mutant population of ∼8000 M2 plants using the double haploid inbred line A03 as the parent. The genome of A03 was sequenced and used as a reference for high-throughput functional characterization of gene mutations at the whole-genome level. A total of 300 M2 to M5 EMS mutants were phenotypically screened and then sequenced, revealing 750 629 SNPs and 46 272 InDel mutations that cover 98.27% of all predicted genes in the A03 genome. A forward-genetics approach was successfully used to identify two genes with chloroplast-related functions that are responsible for the yellow leaf mutant trait. A reverse-genetics approach was also used to identify associations between mutations in five genes of the glucosinolate biosynthetic pathway and variations in glucosinolate content of the mutant plants. In addition, we built the Chinese cabbage EMS mutation database (CCEMD, www.bioinformaticslab.cn/EMSmutation/home) to increase the usability of this mutant population resource. In summary, we performed large-scale screening of a heading Chinese cabbage EMS mutant collection at the phenotypic and genotypic levels, which will facilitate gene mining of Chinese cabbage and might also be useful for the study of other Brassica crops.

Internalization and Transport of PEGylated Lipid-Based Mixed Micelles across Caco-2 Cells Mediated by Scavenger Receptor B1.

The aim of this study was to get insight into the internalization and transport of PEGylat-ed mixed micelles loaded by vitamin K, as mediated by Scavenger Receptor B1 (SR-B1) that is abundantly expressed by intestinal epithelium cells as well as by differentiated Caco-2 cells. Inhibition of SR-B1 reduced endocytosis and transport of vitamin-K-loaded 0%, 30% and 50% PEGylated mixed micelles and decreased colocalization of the micelles with SR-B1. Confocal fluorescence microscopy, fluorescence-activated cell sorting (FACS) analysis, and surface plasmon resonance (SPR) were used to study the interaction between the mixed micelles of different compositions (varying vitamin K loading and PEG content) and SR-B1. Interaction of PEGylated micelles was independent of the vitamin K content, indicating that the PEG shell prevented vitamin K exposure at the surface of the micelles and binding with the receptor and that the PEG took over the micelles' ability to bind to the receptor. Molecular docking calculations corroborated the dual binding of both vita-min K and PEG with the binding domain of SR-B1. In conclusion, the improved colloidal stability of PEGylated mixed micelles did not compromise their cellular uptake and transport due to the affinity of PEG for SR-B1. SR-B1 is able to interact with PEGylated nanoparticles and mediates their subsequent internalization and transport.

Development and Application of SSR Markers Related to Genes Involved in Leaf Adaxial-Abaxial Polarity Establishment in Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

In Chinese cabbage (Brassica rapa L. ssp. pekinensis), leaf adaxial-abaxial (ad-ab) polarity is tightly related to leaf incurvature, an essential factor for the formation of leafy heads. Therefore, identification of the genes responsible for leaf ad-ab polarity and studying their genetic variation may clarify the mechanism of leafy head formation. By comparing the sequences of the genes regulating leaf ad-ab polarity development in Arabidopsis thaliana (A. thaliana), 41 candidate genes distributed on 10 chromosomes were found to be responsible for the establishment of ad-ab polarity in Chinese cabbage. Orthologous genes, including 10 single copies, 14 double copies, and one triple copies, were detected in the Chinese cabbage. The gene structure and conserved domain analyses showed that the number of exons of the 41 candidate genes range from one to 25, and that most genes share the conserved motifs 1, 6, and 10. Based on the 41 candidate genes, 341 simple sequence repeats (SSRs) were detected, including five replicated types: single, double, triple, quintuple, and sextuple nucleotide replications. Among these sequence repeat (SSR) loci, 323 loci were used to design 969 specific primers, and 362 primer pairs were selected randomly and evaluated using 12 Chinese cabbage accessions with different heading types. 23 primer pairs resulting with clear, polymorphic bands, combined with other 127 markers, was used to construct a linkage map by using an F2 population containing 214 lines derived from the hybrid of the overlapping heading Chinese cabbage "14Q-141" and the outward curling heading Chinese cabbage "14Q-279." The result showed that the sequences of markers in the genetic linkage map and the physical map was consistent in general. Our study could help to accelerate the breeding process of leafy head quality in Chinese cabbage.

Characterization of Non-heading Mutation in Heading Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

Heading is a key agronomic trait of Chinese cabbage. A non-heading mutant with flat growth of heading leaves (fg-1) was isolated from an EMS-induced mutant population of the heading Chinese cabbage inbred line A03. In fg-1 mutant plants, the heading leaves are flat similar to rosette leaves. The epidermal cells on the adaxial surface of these leaves are significantly smaller, while those on the abaxial surface are much larger than in A03 plants. The segregation of the heading phenotype in the F2 and BC1 population suggests that the mutant trait is controlled by a pair of recessive alleles. Phytohormone analysis at the early heading stage showed significant decreases in IAA, ABA, JA and SA, with increases in methyl IAA and trans-Zeatin levels, suggesting they may coordinate leaf adaxial-abaxial polarity, development and morphology in fg-1. RNA-sequencing analysis at the early heading stage showed a decrease in expression levels of several auxin transport (BrAUX1, BrLAXs, and BrPINs) and responsive genes. Transcript levels of important ABA responsive genes, including BrABF3, were up-regulated in mid-leaf sections suggesting that both auxin and ABA signaling pathways play important roles in regulating leaf heading. In addition, a significant reduction in BrIAMT1 transcripts in fg-1 might contribute to leaf epinastic growth. The expression profiles of 19 genes with known roles in leaf polarity were significantly different in fg-1 leaves compared to wild type, suggesting that these genes might also regulate leaf heading in Chinese cabbage. In conclusion, leaf heading in Chinese cabbage is controlled through a complex network of hormone signaling and abaxial-adaxial patterning pathways. These findings increase our understanding of the molecular basis of head formation in Chinese cabbage.

Influence of PEGylation of Vitamin-K-Loaded Mixed Micelles on the Uptake by and Transport through Caco-2 Cells.

The aim of the study is to investigate the uptake by and transport through Caco-2 cells of two mixed micelle formulations (based on egg phosphatidylcholine and glycocholic acid) of vitamin K, i.e., with and without DSPE-PEG2000. The uptake of vitamin K and fluorescently labeled mixed micelles with and without PEG coating showed similar kinetics and their uptake ratio remained constant over time. Together with the fact that an inhibitor of scavenger receptor B1 (BLT-1) decreased cellular uptake of vitamin K by ∼80% compared to the uptake in the absence of this inhibitor, we conclude that both types of micelles loaded with vitamin K can be taken up intactly by Caco-2 cells via this scavenger receptor. The amount of vitamin K in chylomicrons fraction from Caco-2 cell monolayers further indicates that mixed micelles (with or without PEGylation) are likely packed into chylomicrons after internalization by Caco-2 cells. Uptake of vitamin K from PEGylated mixed micelles increased four- to five-fold at simulated gastrointestinal conditions. In conclusion, PEGylated mixed micelles are stable upon exposure to simulated gastric conditions, and as a result, they do show overall a higher cellular uptake efficiency of vitamin K as compared to mixed micelles without PEG coating.

A Smart Responsive Dual Aptamers-Targeted Bubble-Generating Nanosystem for Cancer Triplex Therapy and Ultrasound Imaging.

The absence of targeted, single treatment methods produces low therapeutic value for treating cancers. To increase the accumulation of drugs in tumors and improve the treatment effectiveness, near-infrared 808 nm photothermal responsive dual aptamers-targeted docetaxel (DTX)-containing nanoparticles is proposed. In this system, DTX and NH4 HCO3 are loaded in thermosensitive liposomes. The surface of liposomes is coated with gold nanoshells and connected with sulfydryl (SH) modified AS1411 and S2.2 aptamers. The nanosystem has good biocompatibility and uniform size (diameter about 200 nm). The drug is rapidly released, reaching a maximum amount (84%) at 4 h under 808 nm laser irradiation. The experiments conducted in vitro and in vivo demonstrate the nanosystem can synergistically inhibit tumor growth by combination of chemotherapy, photothermal therapy, and biological therapy. Dual ligand functionalization significantly increases cellular uptake on breast cancer cell line (MCF-7) cells and achieves ultrasound imaging (USI) at tumor site. The results indicate that this drug delivery system is a promising theranostic agent involving light-thermal response at tumor sites, dual ligand targeted triplex therapy, and USI.

CuS as a gatekeeper of mesoporous upconversion nanoparticles-based drug controlled release system for tumor-targeted multimodal imaging and synergetic chemo-thermotherapy.

In this work, a tumor-targeted multifunctional mesoporous upconversion nanoparticle-based drug controlled release system was developed for UCL/MRI/PAT guided synergetic chemo-thermotherapy. Herein, the core-shell mesoporous upconversion nanoparticles served as drug carrier exhibiting higher upconversion luminescence emission intensity, with CuS as a gatekeeper through a cleavable disulfide bond under the influence of glutathione. CuS could not only prevent drug from early release during the delivery but also improve the delivery system function with the ability of photothermal therapy and photoacoustic tomography. Hyaluronic acid grafted on the surface of mesoporous upconversion nanoparticles could interact with CD44 receptors over-expressed in tumor cells, facilitating the drug delivery system to accumulate in tumor tissues. The synergy between chemotherapy and photothermal therapy was studied in vitro and in vivo, showing powerful anti-tumor effect. In cooperation with the multi-mode imaging, the size, site and morphology of tumor were clearly observed throughout the disease's progression.

A smart upconversion-based light-triggered polymer for synergetic chemo-photodynamic therapy and dual-modal MR/UCL imaging.

We have developed a novel nanocomposite to achieve effective therapy and live surveillance of tumor tissue. In this study, fullerene (C60) with iron oxide (Fe3O4) nanoparticles and upconversion nanophosphors (UCNPs) was loaded into N-succinyl-N'-4-(2-nitrobenzyloxy)-succinyl-chitosan micelles (SNSC) with good biocompatibility. In addition, hydrophobic anticancer drug docetaxel (DTX) was also loaded into the nanocomposites. The experiments conducted in vitro and in vivo demonstrated that C60/Fe3O4-UCNPs@DTX@SNSC can act synergistically to kill tumor cells by releasing chemotherapy drugs at specific target site as well as generating reactive oxygen using 980nm. In addition, it can also be used for non-invasive deep magnetic resonance and upconversion fluorescence dual-mode imaging. The results indicated that this system provided an efficient method to surmount the drawback of UV or visible light-responsive polymeric systems for controlled drug release and generated reactive oxygen in deep tissues and ultimately realized the integration of dual-modal imaging and treatment.

Multimodal imaging-guided, dual-targeted photothermal therapy for cancer.

The ability to selectively destroy cancer cells while sparing normal tissue is highly desirable during cancer therapy. Herein, dual-targeted photothermal therapy was achieved by the integration of upconversion nanoparticles, Fe3O4 nanoparticles (IONPs), Prussian blue nanoparticles (PBNPs) and hyaluronic acid (HA). PBNPs converted near-infrared (NIR) light into heat and HA/Fe3O4 NPs served as dual-targeting moieties. The as-obtained nanocomposites could also be applied as a multimodal probe for upconversion luminescence (UCL) imaging, enhanced T2-weighted magnetic resonance (MR) imaging and photoacoustic tomography (PAT) imaging. This multifunctional nanoparticle (MFNP) system prepared by a layer-by-layer (LBL) assembly method exhibited excellent dispersivity and low toxicity in vitro and in vivo. Furthermore, the research provided effective results for dual-targeted photothermal ablation of cancer with ∼4 fold higher tumor accumulation than that in the absence of HA/magnetic field. The photothermal therapeutic efficacy has been greatly improved in the S180 tumor model. We present a strategy for multimodal imaging-guided, dual-targeted physical cancer therapy and highlight the promise of using multifunctional nanostructures for cancer theranostics.

Multi-functional liposomes showing radiofrequency-triggered release and magnetic resonance imaging for tumor multi-mechanism therapy.

Recently, nanoplatforms with multiple functions, such as tumor-targeting drug carriers, MRI, optical imaging, thermal therapy etc., have become popular in the field of cancer research. The present study reports a novel multi-functional liposome for cancer theranostics. A dual targeted drug delivery with radiofrequency-triggered drug release and imaging based on the magnetic field influence was used advantageously for tumor multi-mechanism therapy. In this system, the surface of fullerene (C60) was decorated with iron oxide nanoparticles, and PEGylation formed a hybrid nanosystem (C60-Fe3O4-PEG2000). Thermosensitive liposomes (dipalmitoylphosphatidylcholine, DPPC) with DSPE-PEG2000-folate wrapped up the hybrid nanosystem and docetaxel (DTX), which were designed to combine features of biological and physical (magnetic) drug targeting for fullerene radiofrequency-triggered drug release. The magnetic liposomes not only served as powerful tumor diagnostic magnetic resonance imaging (MRI) contrast agents, but also as powerful agents for photothermal ablation of tumors. Furthermore, a remarkable thermal therapy combined chemotherapy multi-functional liposome nanoplatform converted radiofrequency energy into thermal energy to release drugs from thermosensitive liposomes, which was also observed during both in vitro and in vivo treatment. The multi-functional liposomes also could selectively kill cancer cells in highly localized regions via their excellent active tumor targeting and magnetic targeted abilities.