A key gene, violaxanthin de-epoxidase-like 1, enhances fucoxanthin accumulation in Phaeodactylum tricornutum.

BACKGROUND: Fucoxanthin has been widely investigated owing to its beneficial biological properties, and the model diatom Phaeodactylum tricornutum, possessing fucoxanthin (Fux) chlorophyll proteins as light-harvesting systems, is considered to have the potential to become a commercial cell factory for the pigment production. RESULTS: Here, we compared the pigment contents in 10 different P. tricornutum strains from the globe, and found that strain CCMP631 (Pt6) exhibited the highest Fux content but with a low biomass. Comparison of mRNA levels revealed that higher Fux content in Pt6 was related with the higher expression of gene violaxanthin de-epoxidase-like (VDL) protein 1 (VDL1), which encodes the enzyme catalyzing the tautomerization of violaxanthin to neoxanthin in Fux biosynthesis pathway. Single nucleotide variants of VDL1 gene and allele-specific expression in strains Pt1 (the whole genome sequenced strain CCMP632) and Pt6 were analyzed, and overexpressing of each of the 4 VDL1 alleles, two from Pt1 and two from Pt6, in strain Pt1 leads to an increase in downstream product diadinoxanthin and channels the pigments towards Fux biosynthesis. All the 8 VDL1 overexpression (OE) lines showed significant increases by 8.2 to 41.7% in Fux content without compromising growth, and VDL1 Allele 2 OE lines even exhibited the higher cell density on day 8, with an increase by 24.2-28.7% in two Pt1VDL1-allele 2 OE lines and 7.1-11.1% in two Pt6VDL1-allele 2 OE lines, respectively. CONCLUSIONS: The results reveal VDL1, localized in the plastid stroma, plays a key role in Fux over-accumulation in P. tricornutum. Overexpressing VDL1, especially allele 2, improved both the Fux content and growth rate, which provides a new strategy for the manipulation of Fux production in the future.

Isolation of High-Quality Plastids from the Diatom Phaeodactylum tricornutum.

Phaeodactylum tricornutum, a model pennate diatom, carries a secondary plastid surrounded by four membranes. Its biological function remains mysterious, supposed to combine features of the primary chloroplast and the endomembrane system. Isolation of high-quality plastid from the diatom enables a more conclusive understanding of the special structure and metabolic pathways in the plastid. Due to the direct continuity between the chloroplast endoplasmic reticulum membrane (cERM) and the outer nuclear envelope together with the integration of cERM into the cellular endoplasmic reticulum (ER) system, the plastid isolation is still challenging. In this study, highly purified P. tricornutum plastids with the four-layered membrane are obtained by Percoll density gradient centrifugation. The isolated plastids are unlikely to contain any residue of nuclear and coatomer compartments, and they might contain a relatively small contamination of mitochondrion and ER debris.

Antimildew Effect of Three Phenolic Compounds and the Efficacy of Antimildew Sliced Bamboo Veneer.

The development of the bamboo industry has been hindered by environmental issues caused by the application of bamboo preservatives. Chinese herbal phenolic compounds have been shown to possess broad-spectrum, potent antimildew properties, making them promising candidates for the development of new bamboo mildew inhibitors. In this study, we investigated the antimildew properties of three phenolic compounds, eugenol, carvacrol, and paeonol, against common mildews in bamboo materials using the Oxford cup method and the double-dilution method. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to analyze the antimildew mechanism and its effects on mildew cell morphology. Our results showed that carvacrol exhibited the strongest antimildew activity, with minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values of 1.56 mg/mL and 1.76 mg/mL, respectively, followed by eugenol and paeonol. At a concentration of 25 mg/mL, eugenol and carvacrol had an inhibitory rate of over 50% against various mildews. Different concentrations of the three compounds significantly disrupted the morphology and structural integrity of mildew hyphae, with the extent of damage increasing with concentration and treatment duration. In the sliced bamboo mildew prevention experiment, carvacrol at a concentration of 29.25 mg/mL was found to be highly effective against all tested mildews. Our study provides new insights and a theoretical basis for the development of eco-friendly bamboo mildew inhibitors based on plant phenolic compounds.

Screening of Ionic Liquids against Bamboo Mildew and Its Inhibition Mechanism.

Ionic liquids are a class of organic molten salts that consist entirely of cations and anions. They are characterized by their low vapor pressure, low viscosity, low toxicity, high thermal stability, and strong antifungal potential. In this study, the inhibitory performance of ionic liquid cations against Penicillium citrinum, Trichoderma viride, and Aspergillus niger was investigated, along with the mechanism of cell membrane disruption. The Oxford cup method, SEM, and TEM were employed to examine the extent of damage and the specific site of action of ionic liquids on the mycelium and cell structure of these fungi. The results showed that 1-decyl-3-methylimidazole had a strong inhibitory effect on TV; benzyldimethyldodecylammonium chloride had a weak inhibitory effect on PC, TV, AN, and a mixed culture; while dodecylpyridinium chloride exhibited significant inhibitory effects on PC, TV, AN, and Mix, with more prominent effects observed on AN and Mix, exhibiting MIC values of 5.37 mg/mL, 5.05 mg/mL, 5.10 mg/mL, and 5.23 mg/mL, respectively. The mycelium of the mildews showed drying, partial loss, distortion, and uneven thickness. The cell structure showed separation of the plasma wall. The absorbance of the extracellular fluid of PC and TV reached the maximum after 30 min, while that of AN reached the maximum after 60 min. The pH of the extracellular fluid decreased initially and then increased within 60 min, followed by a continuous decrease. These findings provide important insights for the application of ionic liquid antifungal agents in bamboo, medicine, and food.

CRISPR/Cas9-Mediated Genome Editing via Homologous Recombination in a Centric Diatom Chaetoceros muelleri.

Chaetoceros, the most abundant genus of marine planktonic diatoms, can be used in mariculture. An effective genetic transformation system with a short transformation period was established in Chaetoceros muelleri by electroporation in our previous study. In this study, a sequence-specific clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 vector applicable for C. muelleri was constructed, and the expressions of sgRNA, resistance gene, and Cas9 gene were driven by the endogenous promoters U6, acetyl-CoA acetyltransferase, and fucoxanthin chlorophyll a/c binding protein, respectively, in the vector. Nitrate reductase (NR) and urease (URE) genes were edited in C. muelleri, and the NR knockout and NR/URE double-knockout lines displayed the strict auxotrophic phenotype. In addition, the DNA double-strand break was repaired by homologous recombination when a donor DNA was introduced. CRISPR/Cas9 technology was successfully applied to C. muelleri with an editing efficiency of up to 86%, providing a molecular tool for the study of basic biology in C. muelleri and its synthetic biology applications.

Perylenediimide-Based Hybrid Materials for the Iodoperfluoroalkylation of Alkenes and Oxidative Coupling of Amines: Bay-Substituent-Mediated Photocatalytic Activity.

Inorganic-organic donor-acceptor hybrid compounds are an emerging class of multifunctional crystalline materials with well-defined structures built from semiconductive inorganic and organic components. Perylenediimides (PDIs) are a prominent class of electron-deficient organic dyes, which can undergo consecutive photoinduced electron transfers to generate doublet excited-state radical anions for photoredox-inert chemical bonds. Thus, this is an excellent organic component for building hybrid materials to study the structure-property relationships in organic synthesis. In this context, three molecular structure modified PDI-based hybrid materials, (Me4-PDI)2·SiW12O40 (1), (Me4-Cl4-PDI)2·SiW12O40 (2), and (Me4-Br2-PDI)1.5·HSiW12O40 (3), were studied. By the introduction of different substituent groups at the bay positions, these three hybrid materials were successfully fabricated to investigate the impact of substituent groups on the photocatalytic activity. As expected, all PDI-based hybrid materials easily underwent consecutive photoexcitation to obtain their excited-state radical anions. However, experimental and theoretical analyses showed that these obtained excited-state radical anions displayed unusual bay-substituent-group-dependent photocatalytic conversion activities for the iodoperfluoroalkylation of alkenes and oxidative coupling of amines. Higher conversion yields were obtained for complexes 1 and 3 (bay-unsubstituted and Br-substituted PDI hybrid materials, respectively), and lower conversion was observed for complex 2 (Cl-substituted PDI hybrid material), which is attributed to the excited-state SOMO-1 energies of the PDI radical anions. The structure-property relationship established in this work provides insights for the further exploration of bay-substituted PDI hybrid materials in other small-molecule photocatalytic transformations.

Process and Anti-Mildew Properties of Tea Polyphenol-Modified Citral-Treated Bamboo.

In order to reduce the oxidative degradation of citral, our research group modified citral with the natural antioxidant from tea polyphenols and applied it to bamboo processing to enhance the anti-mold effect of bamboo, but its application to the bamboo treatment process and the anti-mold effect is still not clear. For this reason, in this paper, the tea polyphenol-modified citral anti-mildew treatment of bamboo as well as the anti-mildew properties of bamboo were explored using the orthogonal testing method and a UV-vis spectrophotometer. The results showed that when the concentration of tea polyphenol-modified citral reached 175 mg mL-1 and above, the efficacy of the anti-mildew treated bamboo against common molds reached 100%; the improved anti-mildew treatment process parameters for bamboo were as follows: impregnation pressure 0.6 MPa, impregnation time 150 min, and tea polyphenol-modified citral concentration 200 mg mL-1. Following the tea polyphenol-modified citral anti-mildew treatment of bamboo, not only did it improve the anti-mildew properties of the bamboo materials, but it also added a fresh lemon fragrance without altering the original colour, microstructure, and chemical properties of the bamboo materials.

Antioxidant-Mediated Modification of Citral and Its Control Effect on Mildewy Bamboo.

To reduce the oxidative degradation of citral and improve its antimildew performance, citral was modified with natural antioxidants such as tea polyphenols, ascorbic acid, and theaflavin in the present study. Additionally, the effects of these natural antioxidants on the citral degradation rate and DPPH radical-scavenging rate, as well as the effectiveness of antioxidant-modified citral in the antimildew treatment of bamboo were investigated. Ascorbic acid, theaflavin, and tea polyphenols improved the antioxidant performance of citral to some extent, and the tea polyphenols exhibited the best antioxidant performance. When the amount of tea polyphenols added to citral reached 1.0%, the oxidative degradation of citral was effectively prevented. Compared with citral, tea-polyphenol-modified citral could reduce the efficacy of the bamboo antimildew treatment against all four mildews and the effectiveness of the antimildew treatment reached 100%. Citral modification with antioxidants reduced the amount of citral required in the treatment, thereby reducing the treatment cost for bamboo mildew.

Preparation of Citral Compound and Its Bamboo Antimildew Properties.

To reduce the amount of citral used without reducing the antimildew performance of bamboo, the citral compound preparation process, the distribution of the compound in bamboo, and its antimildew performance were investigated using the Oxford cup method, Fourier-transform infrared spectroscopy, and ultraviolet spectrophotometry. The results revealed that the combination of citral with cinnamaldehyde or thymol may lead to partial chemical reactions, which may change the chemical structure of citral and affect its bacteriostatic properties. The bacteriostatic properties of the citraldehyde thymol compound against common molds of bamboo were considerably superior to those of the citral cinnamaldehyde compound. The limonaldehyde thymol compound showed a low distribution trend outside and vice versa inside in the treated bamboo. The citral thymol compound exhibited good antimildew performance at a concentration of 200 mg/mL. The citral thymol compound could reduce the amount of citral by approximately 67 mg/mL without reducing the antimildew performance of bamboo.

Investigation of the Release Mechanism and Mould Resistance of Citral-Loaded Bamboo Strips.

In the present study, the sustained-release system loading citral was synthesised by using PNIPAm nanohydrogel as a carrier and analysed its drug-release kinetics and mechanism. Four release models, namely zero-order, first-order, Higuchi, and Peppas, were employed to fit the experimental data, and the underlying action mechanism was analysed. The optimised system was applied to treat a bamboo mould, followed by assessment of the mould-proof performance. Our experimental results revealed that the release kinetics equation of the system conformed to the first order; the higher the external temperature, the better the match was. In the release process, PNIPAm demonstrated a good protection and sustained-release effect on citral. Under the pressure of 0.5 MPa, immersion time of 120 min, and the system concentration ratio of 1, the optimal drug-loading parameters were obtained using the slow-release system with the best release parameters. Compared to the other conditions, bamboos treated with pressure impregnation demonstrated a better control effect on bamboo mould, while the control effect on Penicillium citrinum, Trichoderma viride, Aspergillus niger, and mixed mould was 100% after 28 days. Moreover, the structure and colour of bamboo remained unchanged during the entire process of mould control.