Direct leaf-peeling method for areca protoplasts: a simple and efficient system for protoplast isolation and transformation in areca palm (Areca catechu).

BACKGROUND: Areca palm (Areca catechu) is a woody perennial plant of both economical and medicinal importance grown in tropical and subtropical climates. Yet, the molecular biology study of areca palm is extremely impeded by its unavailability of a transformation method. An efficient protoplast isolation and transformation system could be highly desirable to overcome this barrier. RESULTS: Here, we described a simple and efficient method for protoplast isolation and transformation from the perennial plant areca palm. A high yield of protoplasts (2.5 × 107 protoplasts per gram of fresh leaf tissues) was obtained from the fresh light green leaflet from the newly-emerged leaf digested overnight in the enzyme solution [2% (w/v) cellulase R10, 0.5% (w/v) macerozyme R10, 0.7 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] by the direct leaf-peeling method. The isolated areca protoplasts maintain viability of 86.6% and have been successfully transformed with a green fluorescent protein (GFP)-tagged plasmid (pGreen0029-GFP, 6.0 kb) via the polyethylene glycol (PEG)-mediated transformation. Moreover, the mannitol concentration (optimal: 0.7 M) was determined as a key factor affecting areca protoplast isolation. We also demonstrated that the optimal density of areca protoplast for efficient transformation was at 1.0-1.5 × 106 cells/ml. With the optimization of transformation parameters, we have achieved a relatively high transformation efficiency of nearly 50%. CONCLUSION: We have established the first efficient protocol for the high-yield isolation and transformation of areca palm protoplasts. This method shall be applied in various biological studies of areca palm, such as gene function analysis, genome editing, protein trafficking and localization and protein-protein interaction. In addition, the protoplast system offers a great genetic transformation approach for the woody perennial plant-areca palm. Moreover, the established platform may be applied in protoplast isolation and transformation for other important species in the palm family, including oil palm and coconut.

The Single Distinct Leader Protease Encoded by Alpinia oxyphylla Mosaic Virus (Genus Macluravirus) Suppresses RNA Silencing Through Interfering with Double-Stranded RNA Synthesis.

The genomic 5'-terminal regions of viruses in the family Potyviridae (potyvirids) encode two types of leader proteases: serine-protease (P1) and cysteine-protease (HCPro), which differ greatly in the arrangement and sequence composition among inter-genus viruses. Most potyvirids have the same tandemly arranged P1 and HCPro, whereas viruses in the genus Macluravirus encode a single distinct leader protease, a truncated version of HCPro with yet-unknown functions. We investigated the RNA silencing suppression (RSS) activity and its underpinning mechanism of the distinct HCPro from alpinia oxyphylla mosaic macluravirus (aHCPro). Sequence analysis revealed that macluraviral HCPros have obvious truncations in the N-terminal and middle regions when aligned to their counterparts in potyviruses (well-characterized viral suppressors of RNA silencing). Nearly all defined elements essential for the RSS activity of potyviral counterparts are not distinguished in macluraviral HCPros. Here, we demonstrated that aHCPro exhibits a similar anti-silencing activity with the potyviral counterpart. However, aHCPro fails to block both the local and systemic spreading of RNA silencing. In line, aHCPro interferes with the dsRNA synthesis, an upstream step in the RNA silencing pathway. Affinity-purification and NanoLC-MS/MS analysis revealed that aHCPro has no association with core components or their potential interactors involving in dsRNA synthesis from the protein layer. Instead, the ectopic expression of aHCPro significantly reduces the transcript abundance of RDR2, RDR6, SGS3, and SDE5. This study represents the first report on the anti-silencing function of Macluravirus-encoded HCPro and the underlying molecular mechanism.

Facilitating Hot Electron Injection from Graphene to Semiconductor by Rectifying Contact for Vis-NIR-Driven H2 O2 Production.

Solar-driven production of hydrogen peroxide (H2 O2 ), as an important industrial chemical oxidant with an extensive range of applications, from oxygen reduction is a sustainable alternative to mainstream anthraquinone oxidation and direct hydrogenation of dioxygen methods. The efficiency of solar to hydrogen peroxide over semiconductor-based photocatalysts is still largely limited by the narrow light absorption to visible light. Here, the authors proposed and demonstrate the proof-of-concept application of light-generated hot electrons in a graphene/semiconductor (exemplified with widely used TiO2 ) dyad to largely extend visible light spectra up to 800 nm for efficient H2 O2 production. The well-designed graphene/semiconductor heterojunction has a rectifying interface with a zero barrier for the hot electron injection, largely boosting excited hot electrons with an average lifetime of ≈0.5 ps into charge carriers with a long fluorescent lifetime (4.0 ns) for subsequent H2 O2 production. The optimized dyadic photocatalyst can provide an H2 O2 yield of 0.67 mm g-1  h-1 under visible light irradiation (λ ≥ 400 nm), which is 20 times of the state-of-the-art noble-metal-free titanium oxide-based photocatalyst, and even achieves an H2 O2 yield of 0.14 mm g-1  h-1 upon photoexcitation by near-infrared-region light (≈800 nm).

A Newly Identified Virus in the Family Potyviridae Encodes Two Leader Cysteine Proteases in Tandem That Evolved Contrasting RNA Silencing Suppression Functions.

Potyviridae is the largest family of plant-infecting RNA viruses and includes many agriculturally and economically important viral pathogens. The viruses in the family, known as potyvirids, possess single-stranded, positive-sense RNA genomes with polyprotein processing as a gene expression strategy. The N-terminal regions of potyvirid polyproteins vary greatly in sequence. Previously, we identified a novel virus species within the family, Areca palm necrotic spindle-spot virus (ANSSV), which was predicted to encode two cysteine proteases, HCPro1 and HCPro2, in tandem at the N-terminal region. Here, we present evidence showing self-cleavage activity of these two proteins and define their cis-cleavage sites. We demonstrate that HCPro2 is a viral suppressor of RNA silencing (VSR), and both the variable N-terminal and conserved C-terminal (protease domain) moieties have antisilencing activity. Intriguingly, the N-terminal region of HCPro1 also has RNA silencing suppression activity, which is, however, suppressed by its C-terminal protease domain, leading to the functional divergence of HCPro1 and HCPro2 in RNA silencing suppression. Moreover, the deletion of HCPro1 or HCPro2 in a newly created infectious clone abolishes viral infection, and the deletion mutants cannot be rescued by addition of corresponding counterparts of a potyvirus. Altogether, these data suggest that the two closely related leader proteases of ANSSV have evolved differential and essential functions to concertedly maintain viral viability.IMPORTANCE The Potyviridae represent the largest group of known plant RNA viruses and account for more than half of the viral crop damage worldwide. The leader proteases of viruses within the family vary greatly in size and arrangement and play key roles during the infection. Here, we experimentally demonstrate the presence of a distinct pattern of leader proteases, HCPro1 and HCPro2 in tandem, in a newly identified member within the family. Moreover, HCPro1 and HCPro2, which are closely related and typically characterized with a short size, have evolved contrasting RNA silencing suppression activity and seem to function in a coordinated manner to maintain viral infectivity. Altogether, the new knowledge fills a missing piece in the evolutionary relationship history of potyvirids and improves our understanding of the diversification of potyvirid genomes.

Use of an Infectious cDNA Clone of Pepper Veinal Mottle Virus to Confirm the Etiology of a Disease in Capsicum chinense.

The pepper cultivar Yellow Lantern, one of the spiciest pepper varieties, is a local germplasm of Capsicum chinense, cultivated exclusively on Hainan Island, China. However, this variety is susceptible to viral diseases that severely affect its production. In this study, we report that pepper veinal mottle virus (PVMV) is associated with foliar chlorosis and rugosity symptoms in Yellow Lantern. To verify this correlation, we constructed a full-length cDNA clone of a PVMV isolate named HNu. The virus progeny derived from the cDNA clone replicated and moved systemically in the pepper, inducing the same symptoms as those induced by PVMV-HNu in Yellow Lantern peppers in the field. The results support that PVMV-HNu is the causal agent of foliar chlorosis and rugosity disease in Yellow Lantern. This knowledge will help in the diagnosis and prevention of disease caused by PVMV. Furthermore, the cDNA clone serves as a reverse genetic tool to study the molecular pathogenesis of PVMV.

Complete genomic sequence of a novel macluravirus, alpinia oxyphylla mosaic virus (AloMV), identified in Alpinia oxyphylla.

A macluravirus, tentatively named alpinia oxyphylla mosaic virus (AloMV), was identified in Alpinia oxyphylla, and its complete genomic sequence determined. The positively single-stranded RNA genome is comprised of 8213 nucleotides excluding the poly (A) tail, and contains one large open reading frame encoding a polyprotein of 2,626 amino acids. Blastp search showed that the polyprotein of AloMV shared 48%~68% aa sequence identities with other reported macluraviruses. Phylogenetic analysis based on the nucleotide sequence of the polyprotein showed that AloMV, together with all other macluraviruses, clustered into the same group most closely related to cardamom mosaic virus, sharing 66.3% nt and 68% aa sequence identities, respectively. These data above suggest that AloMV represents an isolate of a putative new member within the genus Macluravirus.

Three new anthraquinones from the twigs of Cassia fistula and their bioactivities.

Three previously unreported anthraquinones, fistulaquinones A-C (1-3), together with three known ones (4-6) were isolated from the twigs of Cassia fistula. Their structures were determined by means of extensive NMR and MS spectroscopic analyses. All the isolated compounds were tested for their anti-tobacco mosaic virus (anti-TMV) activity, and compound 3 showed significant activity with inhibition rate of 34.5% at 20 µM concentration, even more potent than positive control. Additionally, compounds 1-6 exhibited moderate cytotoxicity with IC50 values ranging from 2.8 to 9.4 µM for some tested human tumor cell lines.

Mariniluteicoccus endophyticus sp. nov., an endophytic actinobacterium isolated from root of Ocimum basilicum.

Two strains of Gram-stain-positive, aerobic, non-motile, irregular coccus-shaped bacteria, designated YIM 2617T and YIM 2617-2, were isolated from the root of Ocimum basilicum. Phylogenetic analysis on the basis of 16S rRNA gene sequence comparison revealed that the two strains were closely related to Mariniluteicoccus flavus YIM M13146T (98.1 and 98.0 % similarities, respectively) and formed a robust clade with M. flavus in the neighbour-joining tree. Optimum growth of the strains was observed at 28-32 °C, at pH 7.0-8.0 and in the presence of 0-2 % (w/v) NaCl. The chemotaxonomic profiles of the strains comprised anteiso-C15 : 0 as the major cellular fatty acid, MK-9(H4) as the predominant menaquinone, alanine, glycine, glutamic acid and ll-diaminopimelic acid as peptidoglycan components, and phosphatidylcholine and diphosphatidylglycerol as the major polar lipids. The G+C contents of the genomic DNA of strains YIM 2617T and YIM 2617-2 were 66.4 and 66.6 mol%, respectively. DNA-DNA hybridizations of the two strains with M. flavus YIM M13146T gave mean relatedness values of 47.6 ± 2.0 and 43.2 ± 1.5 %, respectively, while the relatedness value between them was 92.1 ± 2.2 %. On the basis of the data recorded from the present study, strains YIM 2617T and YIM 2617-2 represent a novel species of the genus Mariniluteicoccus, for which the name Mariniluteicoccus endophyticus sp. nov. is proposed. The type strain is YIM 2617T ( = KCTC 29482T = DSM 28728T = JCM 30097T).

An antialgal compound produced by Streptomyces jiujiangensis JXJ 0074(T).

Previous investigations suggested that Streptomyces jiujiangensis JXJ 0074(T) can secrete antialgal compounds. In this study, an antialgal compound was isolated from the cultured broth of S. jiujiangensis JXJ 0074(T) by using bioassay methods. Based on spectroscopic data, the active compound was identified as 2'-deoxyadenosine, which exhibited a greater antialgal activity against cyanobacteria than its analogues such as adenosine, guanosine, and 2'-deoxyguanosine. The antialgal activity of 2'-deoxyadenosine increased with the content and time. 2'-Deoxyadenosine severely damaged the vegetative cells of cyanobacteria, causing crumpling, collapse, expanding, perforation, breakage of filamentous cyanobacteria, and decrease of the chlorophyll. However, 2'-deoxyadenosine seemed to have less impact on the morphology of heterocysts of filamentous cyanobacteria. The superoxide dismutase (SOD) activity in the treated cells of M. aeruginosa FACHB-905 initially increased with 31.14 ± 2.00% higher than that of the control after 36 h and then decreased quickly. On the same time, there were rapid increases in superoxide anion radical (O2 (-)) and malondialdehyde (MDA) contents with 315.53 ± 12.81 and 84.72 ± 6.15% higher than these of the controls at 60 h, respectively. The intracellular microcystin-LR (MC-LR) content in the treated cells of M. aeruginosa FACHB-905 increased by 36.34 ± 7.35% 1 day later, followed by a rapid decrease with a rate of 90.50 ± 1.08% 8 days later, while the extracellular MC-LR content showed no significant difference with the control. Five days after adding 15 µg/ml of 2'-deoxyadenosine to the culture of M. aeruginosa FACHB-905, there was no 2'-deoxyadenosine detected by HPLC, suggesting that 2'-deoxyadenosine completely degraded. This study provides a new clue to screen natural-based antialgal compounds from nucleoside analogues.

Light-driven Oxidative Demethylation Reaction Catalyzed by a Rieske-type Non-heme Iron Enzyme Stc2.

Rieske-type non-heme iron oxygenases/oxidases catalyze a wide range of transformations. Their applications in bioremediation or biocatalysis face two key barriers: the need of expensive NAD(P)H as a reductant and a proper reductase to mediate the electron transfer from NAD(P)H to the oxygenases. To bypass the need of both the reductase and NAD(P)H, using Rieske-type oxygenase (Stc2) catalyzed oxidative demethylation as the model system, we report Stc2 photocatalysis using eosin Y/sulfite as the photosensitizer/sacrificial reagent pair. In a flow-chemistry setting to separate the photo-reduction half-reaction and oxidation half-reaction, Stc2 photo-biocatalysis outperforms the Stc2-NAD(P)H-reductase (GbcB) system. In addition, in a few other selected Rieske enzymes (NdmA, CntA, and GbcA), and a flavin-dependent enzyme (iodotyrosine deiodinase, IYD), the eosin Y/sodium sulfite photo-reduction pair could also serve as the NAD(P)H-reductase surrogate to support catalysis, which implies the potential applicability of this photo-reduction system to other redox enzymes.

Biological and Molecular Characterization of Two Closely Related Arepaviruses and Their Antagonistic Interaction in Nicotiana benthamiana.

Previously, our group characterized two closely related viruses from Areca catechu, areca palm necrotic ringspot virus (ANRSV) and areca palm necrotic spindle-spot virus (ANSSV). These two viruses share a distinct genomic organization of leader proteases and represent the only two species of the newly established genus Arepavirus of the family Potyviridae. The biological features of the two viruses are largely unknown. In this study, we investigated the pathological properties, functional compatibility of viral elements, and interspecies interactions in the model plant, Nicotiana benthamiana. Using a newly obtained infectious clone of ANRSV, we showed that this virus induces more severe symptoms compared with ANSSV and that this is related to a rapid virus multiplication in planta. A series of hybrid viruses were constructed via the substitution of multiple elements in the ANRSV infectious clone with the counterparts of ANSSV. The replacement of either 5'-UTR-HCPro1-HCPro2 or CI effectively supported replication and systemic infection of ANRSV, whereas individual substitution of P3-7K, 9K-NIa, and NIb-CP-3'-UTR abolished viral infectivity. Finally, we demonstrated that ANRSV confers effective exclusion of ANSSV both in coinfection and super-infection assays. These results advance our understanding of fundamental aspects of these two distinct but closely related arepaviruses.

Single-step Replacement of an Unreactive C-H Bond by a C-S Bond Using Polysulfide as the Direct Sulfur Source in Anaerobic Ergothioneine Biosynthesis.

Ergothioneine, a natural longevity vitamin and antioxidant, is a thiol-histidine derivative. Recently, two types of biosynthetic pathways were reported. In the aerobic ergothioneine biosynthesis, a non-heme iron enzyme incorporates a sulfoxide to an sp2 C-H bond in trimethyl-histidine (hercynine) through oxidation reactions. In contrast, in the anaerobic ergothioneine biosynthetic pathway in a green sulfur bacterium, Chlorobium limicola, a rhodanese domain containing protein (EanB) directly replaces this unreactive hercynine C-H bond with a C-S bond. Herein, we demonstrate that polysulfide (HSSnSR) is the direct sulfur-source in EanB-catalysis. After identifying EanB's substrates, X-ray crystallography of several intermediate states along with mass spectrometry results provide additional mechanistic details for this reaction. Further, quantum mechanics/molecular mechanics (QM/MM) calculations reveal that protonation of Nπ of hercynine by Tyr353 with the assistance of Thr414 is a key activation step for the hercynine sp2 C-H bond in this trans-sulfuration reaction.

Crystal Structure of the Ergothioneine Sulfoxide Synthase from Candidatus Chloracidobacterium thermophilum and Structure-Guided Engineering To Modulate Its Substrate Selectivity.

Ergothioneine is a thiohistidine derivative with potential benefits on many aging-related diseases. The central step of aerobic ergothioneine biosynthesis is the oxidative C-S bond formation reaction catalyzed by mononuclear nonheme iron sulfoxide synthases (EgtB and Egt1). Thus far, only the Mycobacterium thermoresistibile EgtB (EgtB Mth ) crystal structure is available, while the structural information for the more industrially attractive Egt1 enzyme is not. Herein, we reported the crystal structure of the ergothioneine sulfoxide synthase (EgtB Cth ) from Candidatus Chloracidobacterium thermophilum. EgtB Cth has both EgtB- and Egt1-type of activities. Guided by the structural information, we conducted Rosetta Enzyme Design calculations, and we biochemically demonstrated that EgtB Cth can be engineered more toward Egt1-type of activity. This study provides information regarding the factors governing the substrate selectivity in Egt1- and EgtB-catalysis and lays the groundwork for future sulfoxide synthase engineering toward the development of an effective ergothioneine process through a synthetic biology approach.

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis.

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

Application of single nucleotide extension and MALDI-TOF mass spectrometry in proofreading and DNA repair assay.

Proofreading and DNA repair are important factors in maintaining the high fidelity of genetic information during DNA replication. Herein, we designed a non-labeled and non-radio-isotopic simple method to measure proofreading. An oligonucleotide primer is annealed to a template DNA forming a mismatched site and is proofread by Klenow fragment of Escherichia coli DNA polymerase I (pol I) in the presence of all four dideoxyribonucleotide triphosphates. The proofreading excision products and re-synthesis products of single nucleotide extension are subjected to MALDI-TOF mass spectrometry (MS). The proofreading at the mismatched site is identified by the mass change of the primer. We examined proofreading of Klenow fragment with DNAs containing various base mismatches. Single mismatches at the primer terminus can be proofread efficiently. Internal single mismatches can also be proofread at different efficiencies, with the best correction for mismatches located 2-4-nucleotides from the primer terminus. For mismatches located 5-nucleotides from the primer terminus there was partial correction and extension. No significant proofreading was observed for mismatches located 6-9-nucleotides from the primer terminus. We also subjected primers containing 3' penultimate deoxyinosine (dI) lesions, which mimic endonuclease V nicked repair intermediates, to pol I repair assay. The results showed that T-I was a better substrate than G-I and A-I, however C-I was refractory to repair. The high resolution of MS results clearly demonstrated that all the penultimate T-I, G-I and A-I substrates had been excised last 2 dI-containing nucleotides by pol I before adding a correct ddNMP, however, pol I proofreading exonuclease tolerated the penultimate C-I mismatch allowing the primer to be extended by polymerase activity.

Pyrolysis characteristics and kinetics of lignin derived from enzymatic hydrolysis residue of bamboo pretreated with white-rot fungus.

BACKGROUND: The lignocellulose biorefinery based on the sugar platform usually focuses on polysaccharide bioconversion, while lignin is only burned for energy recovery. Pyrolysis can provide a novel route for the efficient utilization of residual lignin obtained from the enzymatic hydrolysis of lignocellulose. The pyrolysis characteristics of residual lignin are usually significantly affected by the pretreatment process because of structural alteration of lignin during pretreatment. In recent years, biological pretreatment using white-rot fungi has attracted extensive attention, but there are only few reports on thermal conversion of lignin derived from enzymatic hydrolysis residue (EHRL) of the bio-pretreated lignocellulose. Therefore, the study investigated the pyrolysis characteristics and kinetics of EHRL obtained from bamboo pretreated with Echinodontium taxodii in order to evaluate the potential of thermal conversion processes of EHRL. RESULTS: Fourier transform infrared spectroscopy spectra showed that EHRL of bamboo treated with E. taxodii had the typical lignin structure, but aromatic skeletal carbon and side chain of lignin were partially altered by the fungus. Thermogravimetric analysis indicated that EHRL pyrolysis at different heating rates could be divided into two depolymerization stages and covered a wide temperature range from 500 to 900 K. The thermal decomposition reaction can be well described by two third-order reactions. The kinetics study indicated that the EHRL of bamboo treated with white-rot fungus had lower apparent activation energies, lower peak temperatures of pyrolysis reaction, and higher char residue than the EHRL of raw bamboo. Pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS) was applied to characterize the fast pyrolysis products of EHRL at 600 ℃. The ratios of guaiacyl-type to syringyl-type derivatives yield (G/S) and guaiacyl-type to p-hydroxy-phenylpropane-type derivatives yield (G/H) for the treated sample were increased by 33.18 and 25.30 % in comparison with the raw bamboo, respectively. CONCLUSIONS: The structural alterations of lignin during pretreatment can decrease the thermal stability of EHRL from the bio-treated bamboo and concentrate the guaiacyl-type derivatives in the fast pyrolysis products. Thus, the pyrolysis can be a promising route for effective utilization of the enzymatic hydrolysis residue from bio-pretreated lignocellulose.

Black N/H-TiO2 Nanoplates with a Flower-Like Hierarchical Architecture for Photocatalytic Hydrogen Evolution.

A facile two-step strategy was used to prepare black of hydrogenated/nitrogen-doped TiO2 nanoplates (NHTA) with a flower-like hierarchical architecture. In situ nitriding and self-assembly was realized by hydrothermal synthesis using tripolycyanamide as a N source and as a structure-directing agent. After thorough characterization, it was found that the hydrogenation treatment did not damage the flower-like architecture but distorted the anatase crystal structure and significantly changed the band structure of NHTA owing to the increased concentration of oxygen vacancies, hydroxyl groups, and Ti3+ cations. Under AM 1.5 illumination, the photocatalytic H2 evolution rate on the black NHTA was approximately 1500 µmol g-1 h-1 , which was much better than the N-doped TiO2 nanoplates (≈690 µmol g-1 h-1 ). This improvement in the hydrogen evolution rate was attributed to a reduced bandgap, enhanced separation of the photogenerated charge carriers, and an increase in the surface-active sites.