Integrated physiological, biochemical, and transcriptomic analyses of Bruguiera gymnorhiza leaves under long-term copper stress: Stomatal size, wax crystals and composition.

Copper (Cu) is a necessary mineral nutrient for plant growth and development and is involved in several morphological, physiological, and biochemical processes; however, high concentrations of Cu can negatively impact these processes. The role of stomata in responding to various biotic and abiotic stimuli has not been studied in Bruguiera gymnorhiza, particularly in terms of their coordinated interactions at the molecular, physiological, and biochemical levels. Moreover, numerous plants employ strategies such as the presence of thick waxy cuticles on their leaf epidermis and the closing of stomata to reduce water loss. Thus, this study investigates the accumulation of Cu in B. gymnorhiza and its effect on leaf morphology and the molecular response under different Cu treatments (0, 200, and 400 mg L⁻¹, Cu0, Cu200, and Cu400, respectively) during a two years stress period. The results show that Cu stress affected accumulation and transport, increased the activities of peroxidase and ascorbate peroxidase, concentrations of soluble sugar, proline, and H2O2, and decreased the activity of catalase and content of malondialdehyde. Also, Cu-induced stress decreased the uptake of phosphorus and nitrogen and inhibited plant photosynthesis, which consequently led to reduced plant growth. Scanning electron microscopy combined with gas chromatography-mass spectrometry showed that B. gymnorhiza leaves had higher wax crystals and compositions under increased Cu stress, which forced the leaf's stomata to be closed. Also, the contents of alkanes, alcohols, primary alcohol levels (C26:0, C28:0, C30:0, and C32:0), n-Alkanes (C29 and C30), and other wax loads were significantly higher, while fatty acid (C12, C16, and C18) was lower in Cu200 and Cu400 compared to Cu0. Furthermore, the transcriptomic analyses revealed 1240 (771 up- and 469 downregulated), 1000 (723 up- and 277 down-regulated), and 1476 (808 up- and 668 downregulated) differentially expressed genes in Cu0 vs Cu200, Cu0 vs Cu400, and Cu200 vs Cu400, respectively. RNA-seq analyses showed that Cu mainly affected eight pathways, including photosynthesis, cutin, suberin, and wax biosynthesis. This study provides a reference for understanding mangrove response to heavy metal stress and developing novel management practices.

Bioremoval of Co(II) by a novel halotolerant microalgae Dunaliella sp. FACHB-558 from saltwater.

Cobalt pollution is harmful to both the aquatic ecosystem and human health. As the primary producer of aquatic ecosystems in hypersaline environments, unicellular planktonic Dunaliella microalgae is considered to be a low-energy and eco-friendly biosorbent that removes excess cobalt and enhances the vitality of coastal and marine ecosystems. In this study, we found that the halotolerant microalga named Dunaliella sp. FACHB-558 could grow under a salinity condition with 0.5-4.5 M NaCl. A phylogenetic analysis based on the rbcL gene revealed that Dunaliella sp. FACHB-558 is a close relative of Dunaliella primolecta TS-3. At lab-scale culture, Dunaliella sp. FACHB-558 exhibited high tolerance to heavy metal stresses, including cobalt, nickel, and cadmium. Treatment with 60 µM cobalt delayed its stationary phase but ultimately led to a higher population density. Furthermore, Dunaliella sp. FACHB-558 has the ability to adsorb the cobalt ions in the aquatic environment, which was evidenced by the decreased amount of cobalt in the culture medium. In addition, the tolerance of Dunaliella sp. FACHB-558 to cobalt stress was correlated with enhanced nitric oxide content and peroxidase activity. The autophagy inhibitor 3-MA enhanced nitric oxide burst, increased peroxidase activity, and accelerated the bioremoval of cobalt, suggesting that the autophagy pathway played a negative role in response to cobalt stress in Dunaliella sp. FACHB-558. In summary, our study identified a novel microalga possessing high cobalt tolerance and provided a promising natural biosorbent for the research and application of heavy metal bioremediation technology.

One-step bioremediation of hypersaline and nutrient-rich food industry process water with a domestic microbial community containing diatom Halamphora coffeaeformis.

Proper treatment of hypersaline and nutrient-rich food industry process water (FIPW) is challenging in conventional wastewater plants. Insufficient treatment leads to serious environmental hazards. However, bioremediation of FIPW with an indigenous microbial community can not only recover nutrients but generate biomass of diverse applications. In this study, monoculture of Halamphora coffeaeformis, together with synthetic bacteria isolated from a local wastewater plant, successfully recovered 91% of NH4+-N, 78% of total nitrogen, 95% of total phosphorus as well as 82% of total organic carbon from medium enriched with 10% FIPW. All identified organic acids and amino acids, except oxalic acid, were completely removed after 14 days treatment. A significantly higher biomass concentration (1.74 g L-1) was achieved after 14 days treatment in the medium with 10% FIPW than that in a nutrient-replete lab medium as control. The harvested biomass could be a potential feedstock for high-value biochemicals and fertilizer production, due to fucoxanthin accumulation (3 mg g-1) and a fantastic performance in P assimilation. Metagenomic analysis revealed that bacteria community in the algal system, dominated by Psychrobacter and Halomonas, also contributed to the biomass accumulation and uptake of nutrients. Transcriptomic analysis further disclosed that multiple pathways, involved in translation, folding, sorting and degradation as well as transport and catabolism, were depressed in H. coffeaeformis grown in FIPW-enriched medium, as compared to the control. Collectively, the proposed one-step strategy in this work offers an opportunity to achieve sustainable wastewater management and a way towards circular economy.

Taurohyocholic acid acts as a potential predictor of the efficacy of tyrosine kinase inhibitors combined with programmed cell death-1 inhibitors in hepatocellular carcinoma.

Background and aims: Tyrosine kinase inhibitors (TKIs) combined with programmed cell death protein-1 (PD-1) have significantly improved survival in patients with unresectable hepatocellular carcinoma (uHCC), but effective biomarkers to predict treatment efficacy are lacking. Peripheral blood bile acids (BAs) are associated with tumor response to therapy, but their roles in HCC remain unclear. Methods: This retrospective study included HCC patients who received first-line TKIs combined with PD-1 inhibitors treatment (combination therapy) in our clinical center from November 2020 to June 2022. The aim of this study was to analyze the changes in plasma BA profiles before and after treatment in both the responding group (Res group) and the non-responding group (Non-Res group). We aimed to explore the potential role of BAs in predicting the response to combination therapy in HCC patients. Results: Fifty-six patients with HCC who underwent combination therapy were included in this study, with 28 designated as responders (Res group) and 28 as non-responders (Non-Res group). There were differences in plasma BA concentrations between the two groups before systemic therapy. Plasma taurohyocholic acid (THCA) levels in the Res group were significantly lower than those in the Non-Res group. Patients with low levels of THCA exhibited superior median progression-free survival (7.6 vs. 4.9 months, p = 0.027) and median overall survival (23.7 vs. 11.6 months, p = 0.006) compared to those of patients with high levels of THCA. Conclusion: Peripheral blood BA metabolism is significantly correlated with combination therapy response and survival in patients with HCC. Our findings emphasize the potential of plasma BAs as biomarkers for predicting combination therapy outcomes and offering novel therapeutic targets for modulating responses to systemic cancer therapy.

Chloroplast gene control: unlocking RNA thermometer mechanisms in photosynthetic systems.

RNA thermometers offer straightforward, protein-independent methods to regulate gene expression at the post-transcriptional level. In this context, Chung and colleagues have discovered a revolutionary RNA thermometer in the chloroplast genome of Chlamydomonas reinhardtii. This will facilitate temperature-driven control of inducible transgene expression for biotechnology applications in plant and algal systems.

Biochemical and multi-omics analyses of response mechanisms of rhizobacteria to long-term copper and salt stress: Effect on soil physicochemical properties and growth of Avicennia marina.

Mangroves are of important economic and environmental value and research suggests that their carbon sequestration and climate change mitigation potential is significantly larger than other forests. However, increasing salinity and heavy metal pollution significantly affect mangrove ecosystem function and productivity. This study investigates the tolerance mechanisms of rhizobacteria in the rhizosphere of Avicennia marina under salinity and copper (Cu) stress during a 4-y stress period. The results exhibited significant differences in antioxidant levels, transcripts, and secondary metabolites. Under salt stress, the differentially expressed metabolites consisted of 30% organic acids, 26.78% nucleotides, 16.67% organic heterocyclic compounds, and 10% organic oxides as opposed to 27.27% organic acids, 24.24% nucleotides, 15.15% organic heterocyclic compounds, and 12.12% phenyl propane and polyketides under Cu stress. This resulted in differential regulation of metabolic pathways, with phenylpropanoid biosynthesis being unique to Cu stress and alanine/aspartate/glutamate metabolism and α-linolenic acid metabolism being unique to salt stress. The regulation of metabolic pathways enhanced antioxidant defenses, nutrient recycling, accumulation of osmoprotectants, stability of plasma membrane, and chelation of Cu, thereby improving the stress tolerance of rhizobacteria and A. marina. Even though the abundance and community structure of rhizobacteria were significantly changed, all the samples were dominated by Proteobacteria, Chloroflexi, Actinobacteriota, and Firmicutes. Since the response mechanisms were unbalanced between treatments, this led to differential growth trends for A. marina. Our study provides valuable inside on variations in diversity and composition of bacterial community structure from mangrove rhizosphere subjected to long-term salt and Cu stress. It also clarifies rhizobacterial adaptive mechanisms to these stresses and how they are important for mitigating abiotic stress and promoting plant growth. Therefore, this study can serve as a reference for future research aimed at developing long-term management practices for mangrove forests.

A vertical-flow constructed wetland-microalgal membrane photobioreactor integrated system for treating high-pollution-load marine aquaculture wastewater: A lab-scale study.

Individual biological water treatment techniques often prove ineffective in removing accumulated high concentrations of nitrogen and phosphorus in the late stages of biofloc aquaculture. To address this issue, we integrated a previously developed autotrophic denitrification and nitrification integrated constructed wetland (ADNI-CW) with a microalgal membrane photobioreactor (MPBR). Under high nitrogen and phosphorus pollution loads in the influent, the standalone ADNI-CW system achieved removal rates of only 24.17 % ± 2.82 % for total nitrogen (TN) and 25.30 % ± 2.59 % for total phosphorus (TP). The optimal conditions for TN and TP degradation and microalgal biomass production in the Chlorella MPBR, determined using response surface methodology, were an inoculum OD680 of 0.394, light intensity of 161.583 µmol/m2/s, and photoperiod of 16.302 h light:7.698 h dark. Under the optimal operating conditions, the integrated ADNI-CW-MPBR system achieved remarkable TN and TP removal rates of 92.63 % ± 2.8 % and 77.46 % ± 8.41 %, respectively, and a substantial microalgal biomass yield of 54.58 ± 6.8 mg/L/day. This accomplishment signifies the successful achievement of efficient nitrogen and phosphorus removal from high-pollution-load marine aquaculture wastewater along with the acquisition of valuable microalgal biomass. A preliminary investigation of the microbial community composition and algal-bacterial interactions in different operational stages of the MPBR system revealed that unclassified_d__Bacteria, Chlorophyta, and Planctomycetes were predominant phyla. The collaborative relationships between bacteria and Chlorella surpassed competition, ensuring highly efficient nitrogen and phosphorus removal in the MPBR system. This study laid the foundation for the green and sustainable development of the aquaculture industry.

A designer synthetic chromosome fragment functions in moss.

Rapid advances in DNA synthesis techniques have enabled the assembly and engineering of viral and microbial genomes, presenting new opportunities for synthetic genomics in multicellular eukaryotic organisms. These organisms, characterized by larger genomes, abundant transposons and extensive epigenetic regulation, pose unique challenges. Here we report the in vivo assembly of chromosomal fragments in the moss Physcomitrium patens, producing phenotypically virtually wild-type lines in which one-third of the coding region of a chromosomal arm is replaced by redesigned, chemically synthesized fragments. By eliminating 55.8% of a 155 kb endogenous chromosomal region, we substantially simplified the genome without discernible phenotypic effects, implying that many transposable elements may minimally impact growth. We also introduced other sequence modifications, such as PCRTag incorporation, gene locus swapping and stop codon substitution. Despite these substantial changes, the complex epigenetic landscape was normally established, albeit with some three-dimensional conformation alterations. The synthesis of a partial multicellular eukaryotic chromosome arm lays the foundation for the synthetic moss genome project (SynMoss) and paves the way for genome synthesis in multicellular organisms.

Extracellular vesicles involved in growth regulation and metabolic modulation in Haematococcus pluvialis.

BACKGROUND: Microalgae-derived extracellular vesicles (EVs), which transfer their cargos to the extracellular environment to affect recipient cells, play important roles in microalgal growth and environmental adaptation. And, they are also considered as sustainable and renewable bioresources of delivery nanocarrier for bioactive molecules and/or artificial drug molecules. However, their molecular composition and functions remain poorly understood. RESULTS: In this study, isolation, characterization, and functional verification of Haematococcus pluvialis-derived EVs (HpEVs) were performed. The results indicated that HpEVs with typical EV morphology and size were secreted by H. pluvialis cells during the whole period of growth and accumulated in the culture medium. Cellular uptake of HpEVs by H. pluvialis was confirmed, and their roles in regulation of growth and various physiological processes of the recipient cells were also characterized. The short-term inhibition of HpEV secretion results in the accumulation of functional cellular components of HpEVs, thereby altering the biological response of these cells at the molecular level. Meanwhile, continuously inhibiting the secretion of HpEVs negatively influenced growth, and fatty acid and astaxanthin accumulation in H. pluvialis. Small RNA high-throughput sequencing was further performed to determine the miRNA cargoes and compelling details in HpEVs in depth. Comparative analysis revealed commonalities and differences in miRNA species and expression levels in three stages of HpEVs. A total of 163 mature miRNAs were identified with a few unique miRNAs reveal the highest expression levels, and miRNA expression profile of the HpEVs exhibited a clear stage-specific pattern. Moreover, a total of 12 differentially expressed miRNAs were identified and their target genes were classified to cell cycle control, lipid transport and metabolism, secondary metabolites biosynthesis and so on. CONCLUSION: It was therefore proposed that cargos of HpEVs, including miRNA constituents, were suggested potential roles in modulate cell physiological state of H. pluvialis. To summarize, this work uncovers the intercellular communication and metabolism regulation functions of HpEVs.

Chlorella pyrenoidosa as a potential bioremediator: Its tolerance and molecular responses to cadmium and lead.

Heavy metal contamination negatively affects plants and animals in water as well as soils. Some microalgae can remove heavy metal contaminants from wastewater. The aim of this study was to screen green microalgae (GM) to identify those that tolerate high concentrations of toxic heavy metals in water as possible candidates for phytoremediation. Analyses of the tolerance, physiological parameters, ultrastructure, and transcriptomes of GM under Cd/Pb treatments were conducted. Compared with the other GM, Chlorella pyrenoidosa showed stronger tolerance to high concentrations of Cd/Pb. The reduced glutathione content and peroxidase activity were higher in C. pyrenoidosa than those in the other GM. Ultrastructural observations showed that, compared with other GM, C. pyrenoidosa had less damage to the cell surface and interior under Cd/Pb toxicity. Transcriptome analyses indicated that the "peroxisome" and "sulfur metabolism" pathways were enriched with differentially expressed genes under Cd/Pb treatments, and that CpSAT, CpSBP, CpKAT2, Cp2HPCL, CpACOX, CpACOX2, and CpACOX4, all of which encode antioxidant enzymes, were up-regulated under Cd/Pb treatments. These results show that C. pyrenoidosa has potential applications in the remediation of polluted water, and indicate that antioxidant enzymes contribute to Cd/Pb detoxification. These findings will be useful for producing algal strains for the purpose of bioremediation in water contamination.

Embracing the era of antimicrobial peptides with marine organisms.

Covering: 2018 to Jun of 2023The efficiency of traditional antibiotics has been undermined by the proliferation of antibiotic-resistant pathogenic microorganisms, necessitating the pursuit of innovative therapeutic agents. Antimicrobial peptides (AMPs), which are part of host defence peptides found ubiquitously in nature, exhibiting a wide range of activity towards bacteria, fungi, and viruses, offer a highly promising candidate solution. The efficacy of AMPs can frequently be augmented via alterations to their amino acid sequences or structural adjustments. Given the vast reservoir of marine life forms and their distinctive ecosystems, marine AMPs stand as a burgeoning focal point in the quest for alternative peptide templates extracted from natural sources. Advances in identification and characterization techniques have accelerated the discoveries of marine AMPs, thereby stimulating AMP customization, optimization, and synthesis research endeavours. This review presents an overview of recent discoveries related to the intriguing qualities of marine AMPs. Emphasis will be placed upon post-translational modifications (PTMs) of marine AMPs and how they may impact functionality and potency. Additionally, this review considers ways in which marine PTM might support larger-scale, heterologous AMP manufacturing initiatives, providing insights into translational applications of these important biomolecules.

Discovering Nature's shield: Metabolomic insights into green zinc oxide nanoparticles Safeguarding Brassica parachinensis L. from cadmium stress.

Heavy metal cadmium (Cd) hinders plants' growth and productivity by causing different morphological and physiological changes. Nanoparticles (NPs) are promising for raising plant yield and reducing Cd toxicity. Nonetheless, the fundamental mechanism of nanoparticle-interfered Cd toxicity in Brassica parachineses L. remains unknown. A novel ZnO nanoparticle (ZnO-NPs) was synthesized using a microalgae strain (Chlorella pyrenoidosa) through a green process and characterized by different standard parameters through TEM, EDX, and XRD. This study examines the effect of different concentrations of ZnO-NPs (50 and 100 mgL-1) in B. parachineses L. under Cd stress through ultra-high-performance liquid chromatography/high-resolution mass spectrometry-based untargeted metabolomics profiling. In the presence of Cd toxicity, foliar spraying with ZnO-NPs raised Cu, Fe, Zn, and Mg levels in the roots and/or leaves, improved seedling development, as demonstrated by increased plant height, root length, and shoot and root fresh weight. Furthermore, the ZnO-NPs significantly enhanced the photosynthetic pigments and changed the antioxidant activities of the Cd-treated plants. Based on a metabolomics analysis, 481 untargeted metabolites were accumulated in leaves under normal and Cd-stressed conditions. These metabolites were highly enriched in producing organic acids, amino acids, glycosides, flavonoids, nucleic acids, and vitamin biosynthesis. Surprisingly, ZnO-NPs restored approximately 60% of Cd stress metabolites to normal leaf levels. Our findings suggest that green synthesized ZnO-NPs can balance ions' absorption, modulate the antioxidant activities, and restore more metabolites associated with plant growth to their normal levels under Cd stress. It can be applied as a plant growth regulator to alleviate heavy metal toxicity and improve crop yield in heavy metal-contaminated regions.

Genome-Wide Identification, Characterization, and Expression Analysis of the Copper-Containing Amine Oxidase Gene Family in Mangrove Kandelia obovata.

Copper-containing amine oxidases (CuAOs) are known to have significant involvement in the process of polyamine catabolism, as well as serving crucial functions in plant development and response to abiotic stress. A genome-wide investigation of the CuAO protein family was previously carried out in sweet orange (Citrus sinensis) and sweet cherry (Prunus avium L.). Six CuAO (KoCuAO1-KoCuAO6) genes were discovered for the first time in the Kandelia obovata (Ko) genome through a genome-wide analysis conducted to better understand the key roles of the CuAO gene family in Kandelia obovata. This study encompassed an investigation into various aspects of gene analysis, including gene characterization and identification, subcellular localization, chromosomal distributions, phylogenetic tree analysis, gene structure analysis, motif analysis, duplication analysis, cis-regulatory element identification, domain and 3D structural variation analysis, as well as expression profiling in leaves under five different treatments of copper (CuCl2). Phylogenetic analysis suggests that these KoCuAOs, like sweet cherry, may be subdivided into three subgroups. Examining the chromosomal location revealed an unequal distribution of the KoCuAO genes across four out of the 18 chromosomes in Kandelia obovata. Six KoCuAO genes have coding regions with 106 and 159 amino acids and exons with 4 and 12 amino acids. Additionally, we discovered that the 2.5 kb upstream promoter region of the KoCuAOs predicted many cis elements linked to phytohormones and stress responses. According to the expression investigations, CuCl2 treatments caused up- and downregulation of all six genes. In conclusion, our work provides a comprehensive overview of the expression pattern and functional variety of the Kandelia obovata CuAO gene family, which will facilitate future functional characterization of each KoCuAO gene.

A Blue Light-Responsive Strong Synthetic Promoter Based on Rational Design in Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii (C. reinhardtii) is a single-cell green alga that can be easily genetically manipulated. With its favorable characteristics of rapid growth, low cost, non-toxicity, and the ability for post-translational protein modification, C. reinhardtii has emerged as an attractive option for the biosynthesis of various valuable products. To enhance the expression level of exogenous genes and overcome the silencing of foreign genes by C. reinhardtii, synthetic promoters such as the chimeric promoter AR have been constructed and evaluated. In this study, a synthetic promoter GA was constructed by hybridizing core fragments from the natural promoters of the acyl carrier protein gene (ACP2) and the glutamate dehydrogenase gene (GDH2). The GA promoter exhibited a significant increase (7 times) in expressing GUS, over the AR promoter as positive control. The GA promoter also displayed a strong responsiveness to blue light (BL), where the GUS expression was doubled compared to the white light (WL) condition. The ability of the GA promoter was further tested in the expression of another exogenous cadA gene, responsible for catalyzing the decarboxylation of lysine to produce cadaverine. The cadaverine yield driven by the GA promoter was increased by 1-2 times under WL and 2-3 times under BL as compared to the AR promoter. This study obtained, for the first time, a blue light-responsive GDH2 minimal fragment in C. reinhardtii, which delivered a doubling effect under BL when used alone or in hybrid. Together with the strong GA synthetic promoter, this study offered useful tools of synthetic biology to the algal biotechnology field.

A chromosome-level genome assembly for the astaxanthin-producing microalga Haematococcus pluvialis.

The green microalga Haematococcus pluvialis can synthesize high amounts of astaxanthin, which is a valuable antioxidant that has been utilized in human health, cosmetics, and aquaculture. To illustrate detailed molecular clues to astaxanthin yield, we performed PacBio HIFI along with Hi-C sequencing to construct an improved chromosome-level haplotypic genome assembly with 32 chromosomes and a genome size of 316.0 Mb. Its scaffold N50 (942.6 kb) and contig N50 (304.8 kb) have been upgraded remarkably from our previous genome draft, and a total of 32,416 protein-coding genes were predicted. We also established a high-evidence phylogenetic tree from seven representative algae species, with the main aim to calculate their divergence times and identify expanded/contracted gene families. We also characterized genome-wide localizations on chromosomes of some important genes such as five BKTs (encoding beta-carotene ketolases) that are putatively involved in astaxanthin production. In summary, we reported the first chromosome-scale map of H. pluvialis, which provides a valuable genetic resource for in-depth biomedical investigations on this momentous green alga and commercial astaxanthin bioproduction.

Effects of different aeration strategies and ammonia-nitrogen loads on nitrification performance and microbial community succession of mangrove constructed wetlands for saline wastewater treatment.

In highly salinized environments, nitrification is the process that limits the rate of nitrogen transformation and removal. Therefore, this study concentrated on the impacts of different aeration strategies and NH4+-N loads on the nitrification performance of mangrove constructed wetlands (CWs), as well as investigating the succession mechanism of ammonia-oxidizing microorganisms (AOMs). The results showed that both the CW with continuous aeration (CA-CW) and intermittent aeration (IA-CW) achieved a nitrification efficiency of more than 98% under an NH4+-N loading of 1.25-4.7 g/(m2·d). However, the total nitrogen removal rates of IA-CW under low and high ammonia-nitrogen loads (LAL, 20.09 ± 4.4% and HAL, 8.77 ± 1.35%, respectively) were higher than those of CA-CW (16.11 ± 4.7% and 3.32 ± 2.3%, respectively), especially under HAL (p < 0.05). Pearson correlation analysis showed that under different operating conditions, the differential secretion of Kandelia candel rhizosphere organic matter had a certain regulatory effect on nitrification and denitrification groups such as Candidatus Nitrocosmicus, Nitrancea, Truepera, Pontibacter, Halomonas, and Sulfurovum in the wetland root layer. The quantitative polymerase chain reaction revealed that the NH4+-N load rate was the primary factor driving the succession of the AOMs, with different aeration strategies exacerbating this process. Overall, this study revealed that the dominant AOMs in mangrove CWs could be significantly altered by regulating the aeration modes and pollution loads to adjust the rhizosphere organic matter in situ, thereby resulting in more efficient nitrification.

Enhancement of ß-carotene content in Chlamydomonas reinhardtii by expressing bacterium-driven lycopene ß-cyclase.

ß-Carotene is one of the economically important carotenoids, having functions as the antioxidant to remove harmful free radicals and as the precursor for vitamin A and other high-valued xanthophyll such as zeaxanthin and astaxanthin. Lycopene cyclase plays an important role in the branching of ß-carotene and α-carotene. Aiming to develop the microalgae with enhanced ß-carotene productivity, the CrtY gene from bacterium Pantoea agglomerans was integrated into Chlamydomonas reinhardtii. The lycopene-producing E. coli harboring CrtY gene produced 1.59 times of ß-carotene than that harboring DsLcyb1 from Dunaliella salina (a microalga with abundant ß-carotene), confirming the superior activity of CrtY on ß-carotene biosynthesis. According to the pigment analysis by HPLC, in microalgal transformants that were confirmed by molecular analysis, the expression of CrtY significantly increased ß-carotene content from 12.48 mg/g to 30.65 mg/g (dry weight), which is about 2.45-fold changes. It is noted that three out of five transformants have statistically significant higher amount of lutein, even though the increment was 20% in maximum. Besides, no growth defect was observed in the transformants. This is the first report of functional expression of prokaryotic gene in eukaryotic microalgae, which will widen the gene pool targeting carotenoids biosynthesis using microalgae as the factory and thereby provide more opportunity for high-valued products engineering in microalgae.

miR397-LACs mediated cadmium stress tolerance in Arabidopsis thaliana.

Cadmium (Cd) is a non-essential heavy metal, assimilated in plant tissue with other nutrients, disturbing the ions' homeostasis in plants. The plant develops different mechanisms to tolerate the hazardous environmental effects of Cd. Recently studies found different miRNAs that are involved in Cd stress. In the current study, miR397 mutant lines were constructed to explore the molecular mechanisms of miR397 underlying Cd tolerance. Compared with the genetically modified line of overexpressed miR397 (artificial miR397, amiR397), the lines of downregulated miR397 (Short Tandem Target Mimic miR397, STTM miR397) showed more substantial Cd tolerance with higher chlorophyll a & b, carotenoid and lignin content. ICP-OES revealed higher cell wall Cd and low total Cd levels in STTM miR397 than in the wild-type and amiR397 plants.Further, the STTM plants produced fewer reactive oxygen species (ROS) and lower activity of antioxidants enzymes (e.g., catalase [CAT], malondialdehyde [MDA]) compared with amiR397 and wild-type plants after stress, indicating that silencing the expression of miR397 can reduce oxidative damage. In addition, the different family transporters' gene expression was much higher in the amiR397 plants than in the wild type and STTM miRNA397. Our results suggest that miR397 plays a role in Cd tolerance in Arabidopsis thaliana. Overexpression of miR397 could decrease Cd tolerance in plants by regulating the expression of LAC 2/4/17, changing the lignin content, which may play an important role in inducing different stress-tolerant mechanisms and protecting the cell from a hazardous condition. This study provides a basis to elucidate the functions of miR397 and the Cd stress tolerance mechanism in Arabidopsis thaliana.

Comparative studies on in vitro antitumor activities and apoptosis-inducing effects of enantiomeric ruthenium(II) complexes.

On the basis of our previous comparative studies on the DNA binding of a pair of ruthenium(II) complex enantiomers, Δ-[Ru(bpy)2PBIP]2+ and Λ-[Ru(bpy)2PBIP]2+ {bpy = 2,2'-bipyridine, PBIP = 2-(4-bromophenyl)imidazo[4,5-f]1,10-phenanthroline}, in this study, their antitumor activities and mechanisms were further investigated comparatively. The cytotoxicity assay demonstrated that both the enantiomers exerted selective antiproliferative effects on cancer cell lines A2780 and PC3. Fluorescence localization experiments suggested that both the enantiomers effectively permeated the nucleus of HeLa cells and co-localized with DNA, resulting in their DNA damage and apoptosis. Flow cytometry experiments showed that the apoptosis was enhanced by increasing the concentration of each enantiomer. Western blotting analyses indicated that both extrinsic and intrinsic apoptosis pathways were activated by the two enantiomers. miRNA microarray analyses displayed that both the enantiomers up- and downregulated multiple miRNAs, some of which were predicted to be associated with carcinogenesis. The above experimental results also showed that the Δ-enantiomer exerted a more potent antitumor activity, a higher efficiency of entering cancer cells and a stronger apoptosis-inducing effect compared with the Λ-enantiomer. Combined with the previously published research results, experimental results from this study implied that the antitumor activity of a metal complex might have originated from the conformation change of DNA in tumor cells caused by the intercalation of the complex, that the antitumor mechanism of a metal complex could be related to its DNA-binding mode, and that the antitumor efficiency of a metal complex could result from its DNA-binding strength.