Metabolite and Transcriptome Profiles of Proanthocyanidin Biosynthesis in the Development of Litchi Fruit.

The fruit of Litchi chinensis contains high levels of proanthocyanidins (PAs) in the pericarp. These substances can serve as substrates of laccase-mediated rapid pericarp browning after the fruit is harvested. In this study, we found that the major PAs in litchi pericarp were (-)-epicatechin (EC) and several procyanidins (PCs), primarily PC A2, B2, and B1, and the EC and the PC content decreased with the development of the fruit. RNA-seq analysis showed that 43 early and late structure genes related to flavonoid/PA biosynthesis were expressed in the pericarp, including five ANTHOCYANIDIN REDUCTASE (ANR), two LEUCOANTHOCYANIDIN REDUCTASE (LAR), and two ANTHOCYANIDIN SYNTHASE (ANS) genes functioning in the PA biosynthesis branch of the flavonoid pathway. Among these nine PA biosynthesis-related genes, ANR1a, LAR1/2, and ANS1 were highly positively correlated with changes in the EC/PC content, suggesting that they are the key PA biosynthesis-related genes. Several transcription factor (TF) genes, including MYB, bHLH, WRKY, and AP2 family members, were found to be highly correlated with ANR1a, LAR1/2, and ANS1, and their relevant binding elements were detected in the promoters of these target genes, strongly suggesting that these TF genes may play regulatory roles in PA biosynthesis. In summary, this study identified the candidate key structure and regulatory genes in PA biosynthesis in litchi pericarp, which will assist in understanding the accumulation of high levels of browning-related PA substances in the pericarp.

In planta high levels of hydrolysable tannins inhibit peroxidase mediated anthocyanin degradation and maintain abaxially red leaves of Excoecaria Cochinchinensis.

BACKGROUND: Abaxially anthocyanic leaves of deeply-shaded understorey plants play important ecological significance for the environmental adaption. In contrast to the transient pigmentation in other plants, anthocyanins are permanently presented in these abaxially red leaves, however, the mechanism for the pigment maintenance remains unclear. In the present study, we investigated phenolic metabolites that may affect pigment stability and degradation in Excoecaria cochinchinensis (a bush of permanently abaxial-red leaves), via a comparison with Osmanthus fragrans (a bush of transiently red leaves). RESULTS: High levels of galloylated anthocyanins were identified in the Excoecaria but not in the Osmanthus plants. The galloylated anthocyanin showed slightly higher stability than two non-galloylated anthocyanins, while all the 3 pigments were rapidly degraded by peroxidase (POD) in vitro. High levels of hydrolysable tannins [mainly galloylglucoses/ellagitannins (GGs/ETs)] were identified in Excoecaria but none in Osmanthus. GGs/ETs showed inhibition effect on POD, with IC50 ranged from 35.55 to 83.27 µM, correlated to the markedly lower POD activities detected in Excoecaria than in Osmanthus. Strong copigmentation was observed for GGs/ETs and anthocyanins, with more than 30% increase in the red intensity of non-galloylated anthocyanin solutions. In the leaf tissue, the hydrolysable tannins were observed to be co-localized with anthocyanins at the abaxial layer of the Excoecaria leaves, correlated to the low POD activity, more acidity and increased red intensity of the tissue. CONCLUSION: The results suggest that the Excoecaria leaves accumulate a distinct group of phenolic metabolites, mainly GGs/ETs, at the abaxial layer, which prevent anthocyanin degradation and increase the pigment stability, and consequently lead to the permanent maintenance of the red leaves.

BcXyl, a ß-xylosidase Isolated from Brunfelsia Calycina Flowers with Anthocyanin-ß-glycosidase Activity.

Brunfelsia calycina flowers lose anthocyanins rapidly and are therefore well suited for the study of anthocyanin degradation mechanisms, which are unclear in planta. Here, we isolated an anthocyanin-ß-glycosidase from B. calycina petals. The MS/MS (Mass Spectrometry) peptide sequencing showed that the enzyme (72 kDa) was a ß-xylosidase (BcXyl). The enzyme showed high activity to p-Nitrophenyl-ß-d-galactopyranoside (pNPGa) and p-Nitrophenyl-ß-d-xylopyranoside (pNPX), while no activity to p-Nitrophenyl-ß-d-glucopyranoside (pNPG) or p-Nitrophenyl-ß-D-mannopyranoside (pNPM) was seen. The optimum temperature of BcXyl was 40 °C and the optimum pH was 5.0. The enzyme was strongly inhibited by 1 mM D-gluconate and Ag⁺. HPLC (High Performance Liquid Chromatography) analysis showed that BcXyl catalyzed the degradation of an anthocyanin component of B. calycina, and the release of xylose and galactose due to hydrolysis of glycosidic bonds by BcXyl was detected by GC (Gas Chromatography) /MS. A full-length mRNA sequence (2358 bp) of BcXyl (NCBI No. MK411219) was obtained and the deduced protein sequence shared conserved domains with two anthocyanin-ß-glycosidases (Bgln and BadGluc, characterized in fungi). BcXyl, Bgln and BadGluc belong to AB subfamily of Glycoside hydrolase family 3. Similar to BcPrx01, an anthocyanin-degradation-related Peroxidase (POD), BcXyl was dramatically activated at the stage at which the rapid anthocyanin degradation occurred. Taken together, we suggest that BcXyl may be the first anthocyanin-ß-glycosidase identified in higher plants.

A prediction model of sleep disturbances among female nurses by using the BP-ANN.

AIM: To describe sleep disturbances and fatigue among female registered nurses in Beijing and to develop a prediction model for sleep disturbances. BACKGROUND: Chinese nurses are required to work rotating shifts on a weekly basis, which could negatively impact their sleep and well-being. METHOD: A total of 647 registered nurses participated in this study. Self-reported sleep-related data and selected physiological data were collected. Back propagation artificial neural networks was used to develop the prediction model by using the risk management and population health framework. RESULTS: Majority of them reported clinically significant poor sleep (69.4%) and fatigue (75.4%). A total of eight predictors were identified for sleep disturbances, and the top four normalized importance predictors are morning fatigue (100%), body mass index (30.5%), gastrointestinal symptoms (17.6%) and drinking caffeinated beverages at work (17.3%). The cross-entropy error was 206.58, and the model accounted for 77.6% of the variance in sleep disturbances. CONCLUSIONS AND IMPLICATIONS FOR NURSING MANAGEMENT: Female registered nurses in China experience clinically significant sleep disturbances. Morning fatigue severity along with seven significant influencing factors may be used to identify shift nurses who face a higher risk of sleep disturbances. The back propagation artificial neural networks model could be used as the foundation for health promotion interventions for registered nurses.

B Type and Complex A/B Type Epicatechin Trimers Isolated from Litchi pericarp Aqueous Extract Show High Antioxidant and Anticancer Activity.

Litchi (Litchi chinensis Sonn.) fruit is known for its rich source of phenolics. Litchi pericarp contains high levels of epicatechin that may form oligomers of various lengths. Except for several A or B type epicatechin dimers, other soluble oligomers have rarely been identified in the pericarp. Here, bioassay-guided column fractionation was applied to isolate bioactive phenolics from aqueous pericarp extract. A fraction (S3) was obtained by two rounds of Sephadex LH-20 column chromatography, and showed higher antioxidant activity and inhibition on the proliferation of human lung cancer cells (A549) than Litchi anthocyanins. S3 was further separated to isolate fractions P1-P4, which all showed higher antioxidant activity than vitamin C. P3 showed 32.9% inhibition on A549 cells at 30 µg/mL, higher than other fractions and cis-Dichlorodiamineplatinum (DDP, 0.5 µg/mL), but not as high as the combination of the four fractions. Using HPLC-Q-TOF-MS/MS, one B-type and complex A/B type epicatechin trimers were identified in P3; another B-type and two A/B-type trimers were identified in P4. P1 and P2, containing epicatechin and proanthocyanidin B2, respectively, showed no cell inhibition at 30 µg/mL. It is the first time that the two B type trimers of epicatechins (Litchitannin B1 and B2), have been found in Litchi species. The identified proanthocyanidins were detected in the pericarp of the young fruit, and the levels of the compounds decreased as the fruit developed, correlating to the decreasing patterns of the expression of LcLAR and LcANR, two key genes in the catechin biosynthesis pathway.

Characterization of Active Anthocyanin Degradation in the Petals of Rosa chinensis and Brunfelsia calycina Reveals the Effect of Gallated Catechins on Pigment Maintenance.

Anthocyanin degradation decreases ornamental or nutritional values of horticultural products. To investigate factors that may influence colour change in flower development, anthocyanin degradation was compared between the flowers of Brunfelsia calycina and Rosa chinensis, which show rapid and slow degradation, respectively. In-gel activity assays, high performance liquid chromatography (HPLC) analysis of tannins, enzyme kinetics measurement and immune-detection of anthocyanin degradation related-perioxidases (PODs) were carried out for the comparison. Rose petals possessed significantly lower anthocyanin degradation-related POD activities than Brunfelsia petals, which may be related to the high tannin contents. Epicatechin gallate (ECG) and gallocatechin gallate (GCG) were detected in rose as 161.3 ± 12.34 and 273.56 ± 41.23 µg/g FW (Fresh Weight) respectively, while not detected in Brunfelsia. ECG and GCG inhibited the activities of the Brunfelsia POD with half maximal inhibitory concentrations (IC50s) as 21.5 and 29.7 µM respectively, and increased the colour intensities of the anthocyanins. Catechin and epicatechin did not inhibit the POD activity, while serving as POD substrates, with Km (the Michaelis constant) as 0.48 and 1.23 mM. Similar protein levels of the anthocyanin degradation-related 40-kDa PODs were detected in Brunfelsia and rose. In summary, high amount of tannins, particularly ECG and GCG, in red rose petals may inhibit the degradation-related enzymes, leading to the maintenance of anthocyanins in vivo.

An Intracellular Laccase Is Responsible for Epicatechin-Mediated Anthocyanin Degradation in Litchi Fruit Pericarp.

In contrast to the detailed molecular knowledge available on anthocyanin synthesis, little is known about its catabolism in plants. Litchi (Litchi chinensis) fruit lose their attractive red color soon after harvest. The mechanism leading to quick degradation of anthocyanins in the pericarp is not well understood. An anthocyanin degradation enzyme (ADE) was purified to homogeneity by sequential column chromatography, using partially purified anthocyanins from litchi pericarp as a substrate. The purified ADE, of 116 kD by urea SDS-PAGE, was identified as a laccase (ADE/LAC). The full-length complementary DNA encoding ADE/LAC was obtained, and a polyclonal antibody raised against a deduced peptide of the gene recognized the ADE protein. The anthocyanin degradation function of the gene was confirmed by its transient expression in tobacco (Nicotiana benthamiana) leaves. The highest ADE/LAC transcript abundance was in the pericarp in comparison with other tissues, and was about 1,000-fold higher than the polyphenol oxidase gene in the pericarp. Epicatechin was found to be the favorable substrate for the ADE/LAC. The dependence of anthocyanin degradation by the enzyme on the presence of epicatechin suggests an ADE/LAC epicatechin-coupled oxidation model. This model was supported by a dramatic decrease in epicatechin content in the pericarp parallel to anthocyanin degradation. Immunogold labeling transmission electron microscopy suggested that ADE/LAC is located mainly in the vacuole, with essential phenolic substances. ADE/LAC vacuolar localization, high expression levels in the pericarp, and high epicatechin-dependent anthocyanin degradation support its central role in pigment breakdown during pericarp browning.

Floral Response to Heat: A Study of Color and Biochemical Adaptations in Purple Chrysanthemums.

Chrysanthemums are among the world's most popular cut flowers, with their color being a key ornamental feature. The formation of these colors can be influenced by high temperatures. However, the regulatory mechanisms that control the fading of chrysanthemum flower color under high-temperature stress remain unclear. This study investigates the impact of high temperatures on the color and biochemical responses of purple chrysanthemums. Four purple chrysanthemum varieties were exposed to both normal and elevated temperature conditions. High-temperature stress elicited distinct responses among the purple chrysanthemum varieties. 'Zi Feng Che' and 'Chrystal Regal' maintained color stability, whereas 'Zi Hong Tuo Gui' and 'Zi lian' exhibited significant color fading, particularly during early bloom stages. This fading was associated with decreased enzymatic activities, specifically of chalcone isomerase (CHI), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), indicating a critical period of color development under heat stress. Additionally, the color fading of 'Zi Lian' was closely related to the increased activity of the peroxidase (POD) and polyphenol oxidase (PPO). Conversely, a reduction in ß-glucosidase (ßG) activity may contribute significantly to the color steadfastness of 'Zi Feng Che'. The genes Cse_sc027584.1_g010.1 (PPO) and Cse_sc031727.1_g010.1 (POD) might contribute to the degradation of anthocyanins in the petals of 'Zi Hong Tuo Gui' and 'Zi Lian' under high-temperature conditions, while simultaneously maintaining the stability of anthocyanins in 'Zi Feng Che' and 'Chrystal Regal' at the early bloom floral stage. The findings of this research provide new insights into the physiological and biochemical mechanisms by which chrysanthemum flower color responds to high-temperature stress.

Tumor Eradication by Boron Neutron Capture Therapy with 10 B-enriched Hexagonal Boron Nitride Nanoparticles Grafted with Poly(Glycerol).

Boron neutron capture therapy (BNCT) has emerged as a treatment modality with high precision and efficacy of intractable tumors. At the core of effective tumor BNCT are 10 B carriers with facile preparation as well as advantageous pharmacokinetic and therapeutic profiles. Herein, the design and preparation of sub-10 nm 10 B-enriched hexagonal boron nitride nanoparticles grafted with poly(glycerol) (h-10 BN-PG), and their application to cancer treatment by BNCT are reported. By virtue of their small particle size and outstanding stealth property, h-10 BN-PG nanoparticles accumulate efficiently in murine CT26 colon tumors with a high intratumor 10 B concentration of 8.8%ID g-1 or 102.1 µg g-1 at 12 h post-injection. Moreover, h-10 BN-PG nanoparticles penetrate into the inside of the tumor parenchyma and then are taken up by the tumor cells. BNCT comprising a single bolus injection of h-10 BN-PG nanoparticles and subsequent one-time neutron irradiation results in significant shrinkage of subcutaneous CT26 tumors. h-10 BN-PG-mediated BNCT not only causes direct DNA damage to the tumor cells, but also triggers pronounced inflammatory immune response in the tumor tissues, which contributes to long-lasting tumor suppression after the neutron irradiation. Thus, the h-10 BN-PG nanoparticles are promising BNCT agents to eradicate tumor through highly efficient 10 B accumulation.

Activating transcription factor 3 inhibits angiotensin II­induced cardiomyocyte viability and fibrosis by activating the transcription of cysteine­rich angiogenic protein 61.

Depletion of activating transcription factor 3 (ATF3) expression has previously been reported to promote hypertrophy, dysfunction and fibrosis in stress overload­induced hearts; however, the mechanism involved remains poorly understood. In the present study, the mechanism underlying the activation of cysteine­rich angiogenic protein 61 (Cyr61) by ATF3 in hyperproliferative and fibrotic human cardiac fibroblasts (HCFs), induced by angiotensin II (Ang II), was evaluated. The mRNA and protein expression levels of ATF3 and Cyr61 were assessed using reverse transcription­quantitative PCR and western blotting, respectively. The Cell Counting Kit­8 assay was used to assess cell viability. Cell migration was assessed using the wound healing assay and western blotting, whereas the extent of cell fibrosis was evaluated using immunofluorescence staining and western blotting. The binding site of ATF3 to the Cyr61 promoter was predicted using the JASPAR database, and verified using luciferase reporter and chromatin immunoprecipitation assays. The results demonstrated that the mRNA and protein expression levels of ATF3 were significantly upregulated in Ang II­induced HCFs. Overexpression of ATF3 significantly inhibited the Ang II­induced viability, migration and fibrosis of HCFs, whereas ATF3 knockdown mediated significant opposing effects. Mechanistically, ATF3 was demonstrated to transcriptionally activate Cyr61. Cyr61 silencing was subsequently revealed to reverse the effects of ATF3 overexpression on HCFs potentially via regulation of the TGF­ß/Smad signaling pathway. The results of the present study suggested that ATF3 could suppress HCF viability and fibrosis via the TGF­ß/Smad signaling pathway by activating the transcription of Cyr61.

Complete chloroplast genome sequence of a Dutch cultivar of Chrysanthemum, Chrysanthemum morifolium 'Orizaba' (Asteraceae).

The complete plastid genome of Chrysanthemum morifolium 'Orizaba', a cultivar from Holland, was determined and analyzed in this work. It is a circular chromosome and has a length of 151,060 bp. The LSC and SSC of 82,858 bp and 18,294 bp were separated by two IRs of 24,954 bp. The chloroplast genome of C. morifolium 'Orizaba' contains 125 genes, including 83 protein-coding genes, eight ribosomal RNA genes, and 34 transfer RNA genes. Phylogenetic analysis showed that C. morifolium 'Orizaba' clustered together with other Chrysanthemum varieties in the family Asteraceae. The plastome is useful for the elucidation of phylogenetics and evolution in the Asteraceae and Chrysanthemum varieties.

Arabidopsis CHLOROPHYLLASE 1 protects young leaves from long-term photodamage by facilitating FtsH-mediated D1 degradation in photosystem II repair.

The proteolytic degradation of the photodamaged D1 core subunit during the photosystem II (PSII) repair cycle is well understood, but chlorophyll turnover during D1 degradation remains unclear. Here, we report that Arabidopsis thaliana CHLOROPHYLLASE 1 (CLH1) plays important roles in the PSII repair process. The abundance of CLH1 and CLH2 peaks in young leaves and is induced by high-light exposure. Seedlings of clh1 single and clh1-1/2-2 double mutants display increased photoinhibition after long-term high-light exposure, whereas seedlings overexpressing CLH1 have enhanced light tolerance compared with the wild type. CLH1 is localized in the developing chloroplasts of young leaves and associates with the PSII-dismantling complexes RCC1 and RC47, with a preference for the latter upon exposure to high light. Furthermore, degradation of damaged D1 protein is retarded in young clh1-1/2-2 leaves after 18-h high-light exposure but is rescued by the addition of recombinant CLH1 in vitro. Moreover, overexpression of CLH1 in a variegated mutant (var2-2) that lacks thylakoid protease FtsH2, with which CLH1 interacts, suppresses the variegation and restores D1 degradation. A var2-2 clh1-1/2-2 triple mutant shows more severe variegation and seedling death. Taken together, these results establish CLH1 as a long-sought chlorophyll dephytylation enzyme that is involved in PSII repair and functions in long-term adaptation of young leaves to high-light exposure by facilitating FtsH-mediated D1 degradation.

Proteomic Changes in Antioxidant System in Strawberry During Ripening.

To investigate the strawberry antioxidant defense system during fruit ripening, a targeted quantitative proteomic approach using multiple reaction monitoring (MRM) was developed to investigate targeted proteins in the antioxidant enzyme system in strawberry fruit. We investigated 46 proteins and isoforms with 73 identified peptides which may be involved in this antioxidant enzyme system. Among the proteins that changed during ripening, aldo/keto reductase (AKR), superoxide dismutase (SOD) and glutathione transferase (GT) increased significantly, while dehydroascorbate reductase, 2-Cys peroxiredoxin, catalase (CAT), 1-Cys peroxiredoxin and L-ascorbate peroxidase (APX) decreased significantly. These results suggest that fruit ripening of strawberry activates the enzymes of an SOD/glutathione metabolism system. The methodologies used in this study will be useful for systematically characterizing the role of antioxidant enzymes in fruit ripening of other plants.