Overexpression of a novel cytochrome P450 monooxygenase gene, CYP704B1, from Panax ginseng increase biomass of reproductive tissues in transgenic Arabidopsis.

Cytochrome P450 monooxygenase 704B (CYP704B), a member of the CYP86 clan, was found to be needed in Arabidopsis and rice to biosynthesize precursors of sporopollenin through oxidizing fatty acids. In the present study, we cloned and characterized a CYP704B gene in Panax ginseng, named PgCYP704B1. It shared high sequence identity (98-99%) with CYP704 of Arabidopsis, Theobroma cacao, and Morus notabilis. The phylogenetic comparison of ginseng and higher plants between the members of CYP86 clan revealed that ginseng CYP704 was categorized as a group of CYP704B with dicot plants. The expression of PgCYP704B1 is low in the stem, leaf, and fruit, and high in flower buds, particularly detected in the young gametic cell and tapetum layer of the developing anther. Arabidopsis plants overexpressing PgCYP704B1 improved plant biomass such as plant height, siliques and seed number and size. A cytological observation by transverse and longitudinal semi-thin sections of the siliques cuticles revealed that the cell length increased. Furthermore a chemical analysis showed that PgCYP704B1ox lines increased their cutin monomers contents in the siliques. Our results suggest that PgCYP704B1 has a conserved role during male reproduction for fatty acid biosynthesis and its overexpression increases cutin monomers in siliques that eventually could be used for seed production.

Blocked synthesis of sporopollenin and jasmonic acid leads to pollen wall defects and anther indehiscence in genic male sterile wheat line 4110S at high temperatures.

Environment-sensitive genic male sterility is a valid tool for hybrid production and hybrid breeding, but there are no previous reports of the molecular mechanism of fertility conversion. In this study, RNA-seq, phenotypic and cytological observations, and physiological indexes were applied to analyze thermo-sensitive genic male sterility line 4110S under different temperature conditions to explore the fertility transformation mechanism. In total, 3420 differentially expressed genes (DEGs) were identified comprising 2331 upregulated genes and 1089 downregulated genes. The DEGs were apparently distributed among 54 Gene Ontology functional groups. The phenylpropanoid, long-chain fatty acid, and jasmonic acid (JA) biosynthesis pathways were related to male sterility, where their downregulation blocked the synthesis of sporopollenin and JA. Phenotypic and cytological analyses showed that pollen wall defects and anther indehiscence at high temperatures induced sterility. Moreover, enzyme-linked immunosorbent assay results indicated that the abundance of JA was lower in 4110S under restrictive conditions (high temperature) than permissive conditions (low temperature). A possible regulated network of pathways associated with male sterility was suggested. These results provided insights into the molecular mechanism of fertility conversion in the thermosensitive male sterility system.

Identification of cyp703a3-3 and analysis of regulatory role of CYP703A3 in rice anther cuticle and pollen exine development.

Anther cuticle and pollen exine are two elaborated lipid-soluble barriers protecting pollen grains from environmental and biological stresses. However, less is known about the mechanisms underlying the synthesis of these lipidic polymers. Here, we identified a no-pollen male-sterility mutant cyp703a3-3 from the indica restorer line Zhonghui 8015 (Zh8015) mutant library treated with 60Coγ-ray radiation. Histological analysis indicated that cyp703a3-3 underwent abnormal tapetal cells development, produced few orbicules and secreted less sporopollenin precursors to anther locule, as well as cutin monomers on anther. Genetic analysis revealed that cyp703a3-3 was controlled by a single recessive gene. Map-based cloning was performed to narrow down the mutant gene to a 47.78-kb interval on the chromosome 8 between two markers S15-29 and S15-30. Sequence analysis detected three bases (GAA) deletion in the first exon of LOC_Os08g03682, annotated as CYP703A3 with homologous sequences related to male sterility in Arabidopsis, causing the Asparagine deletion in the mutant site. Moreover, we transformed genomic fragment of CYP703A3 into cyp703a3-3, which male-sterility phenotype was recovered. Both the wild-type and cyp703a3-3 mutant 3D structure of CYP703A3 protein were modeled. Results of qPCR suggested CYP703A3 mainly expressed in anthers with greatest abundance at microspore stage, and genes involved in sporopollenin precursors formation and transportation, such as GAMYB, TDR, CYP704B2, DPW2, OsABCG26 and OsABCG15, were significantly reduced in cyp703a3-3. Collectively, our results further elaborated CYP703A3 plays vital role in anther cuticle and pollen exine development in rice (Oryza sativa L.).

Rice No Pollen 1 (NP1) is required for anther cuticle formation and pollen exine patterning.

Angiosperm male reproductive organs (anthers and pollen grains) have complex and interesting morphological features, but mechanisms that underlie their patterning are poorly understood. Here we report the isolation and characterization of a male sterile mutant of No Pollen 1 (NP1) in rice (Oryza sativa). The np1-4 mutant exhibited smaller anthers with a smooth cuticle surface, abnormal Ubisch bodies, and aborted pollen grains covered with irregular exine. Wild-type exine has two continuous layers; but np1-4 exine showed a discontinuous structure with large granules of varying size. Chemical analysis revealed reduction in most of the cutin monomers in np1-4 anthers, and less cuticular wax. Map-based cloning suggested that NP1 encodes a putative glucose-methanol-choline oxidoreductase; and expression analyses found NP1 preferentially expressed in the tapetal layer from stage 8 to stage 10 of anther development. Additionally, the expression of several genes involved in biosynthesis and in the transport of lipid monomers of sporopollenin and cutin was decreased in np1-4 mutant anthers. Taken together, these observations suggest that NP1 is required for anther cuticle formation, and for patterning of Ubisch bodies and the exine. We propose that products of NP1 are likely important metabolites in the development of Ubisch bodies and pollen exine, necessary for polymerization, assembly, or both.

Pollen wall development: the associated enzymes and metabolic pathways.

Pollen grains are surrounded by a sculpted wall, which protects male gametophytes from various environmental stresses and microbial attacks, and also facilitates pollination. Pollen wall development requires lipid and polysaccharide metabolism, and some key genes and proteins that participate in these processes have recently been identified. Here, we summarise the genes and describe their functions during pollen wall development via several metabolic pathways. A working model involving substances and catalytic enzyme reactions that occur during pollen development is also presented. This model provides information on the complete process of pollen wall development with respect to metabolic pathways.

N-acetylcysteine enhances cystic fibrosis sputum penetration and airway gene transfer by highly compacted DNA nanoparticles.

For effective airway gene therapy of cystic fibrosis (CF), inhaled gene carriers must first penetrate the hyperviscoelastic sputum covering the epithelium. Whether clinically studied gene carriers can penetrate CF sputum remains unknown. Here, we measured the diffusion of a clinically tested nonviral gene carrier, composed of poly-l-lysine conjugated with a 10 kDa polyethylene glycol segment (CK(30)PEG(10k)). We found that CK(30)PEG(10k)/DNA nanoparticles were trapped in CF sputum. To improve gene carrier diffusion across sputum, we tested adjuvant regimens consisting of N-acetylcysteine (NAC), recombinant human DNase (rhDNase) or NAC together with rhDNase. While rhDNase alone did not enhance gene carrier diffusion, NAC and NAC + rhDNase increased average effective diffusivities by 6-fold and 13-fold, respectively, leading to markedly greater fractions of gene carriers that may penetrate sputum layers. We further tested the adjuvant effects of NAC in the airways of mice with Pseudomonas aeruginosa lipopolysaccharide (LPS)-induced mucus hypersecretion. Intranasal dosing of NAC prior to CK(30)PEG(10k)/DNA nanoparticles enhanced gene expression by up to ~12-fold compared to saline control, reaching levels observed in the lungs of mice without LPS challenge. Our findings suggest that a promising synthetic nanoparticle gene carrier may transfer genes substantially more effectively to lungs of CF patients if administered following adjuvant mucolytic therapy with NAC or NAC + rhDNase.

A large-scale genetic screen in Arabidopsis to identify genes involved in pollen exine production.

Exine, the outer plant pollen wall, has elaborate species-specific patterns, provides a protective barrier for male gametophytes, and serves as a mediator of strong and species-specific pollen-stigma adhesion. Exine is made of sporopollenin, a material remarkable for its strength, elasticity, and chemical durability. The chemical nature of sporopollenin, as well as the developmental mechanisms that govern its assembly into diverse patterns in different species, are poorly understood. Here, we describe a simple yet effective genetic screen in Arabidopsis (Arabidopsis thaliana) that was undertaken to advance our understanding of sporopollenin synthesis and exine assembly. This screen led to the recovery of mutants with a variety of defects in exine structure, including multiple mutants with novel phenotypes. Fifty-six mutants were selected for further characterization and are reported here. In 14 cases, we have mapped defects to specific genes, including four with previously demonstrated or suggested roles in exine development (MALE STERILITY2, CYP703A2, ANTHER-SPECIFIC PROTEIN6, TETRAKETIDE α-PYRONE REDUCTASE/DIHYDROFLAVONOL-4-REDUCTASE-LIKE1), and a number of genes that have not been implicated in exine production prior to this screen (among them, fatty acid ω-hydroxylase CYP704B1, putative glycosyl transferases At1g27600 and At1g33430, 4-coumarate-coenzyme A ligase 4CL3, polygalacturonase QUARTET3, novel gene At5g58100, and nucleotide-sugar transporter At5g65000). Our study illustrates that morphological screens of pollen can be extremely fruitful in identifying previously unknown exine genes and lays the foundation for biochemical, developmental, and evolutionary studies of exine production.

Genetic regulation of sporopollenin synthesis and pollen exine development.

Pollen acts as a biological protector of male sperm and is covered by an outer cell wall polymer called the exine, which consists of durable sporopollenin. Despite the astonishingly divergent structure of the exine across taxa, the developmental processes of its formation surprisingly do not vary, which suggests the preservation of a common molecular mechanism. The precise molecular mechanisms underlying pollen exine patterning remain highly elusive, but they appear to be dependent on at least three major developmental processes: primexine formation, callose wall formation, and sporopollenin synthesis. Several lines of evidence suggest that the sporopollenin is built up via catalytic enzyme reactions in the tapetum, and both the primexine and callose wall provide an efficient substructure for sporopollenin deposition. Herein, we review the currently accepted understanding of the molecular regulation of sporopollenin biosynthesis and examine unanswered questions regarding the requirements underpinning proper exine pattern formation, as based on genetic evidence.

Biochemical, mechanical, and spectroscopic analyses of genetically engineered flax fibers producing bioplastic (poly-beta-hydroxybutyrate).

The interest in biofibers has grown in recent years due to their expanding range of applications in fields as diverse as biomedical science and the automotive industry. Their low production costs, biodegradability, physical properties, and perceived eco-friendliness allow for their extensive use as composite components, a role in which they could replace petroleum-based synthetic polymers. We performed biochemical, mechanical, and structural analyses of flax stems and fibers derived from field-grown transgenic flax enriched with PHB (poly-beta-hydroxybutyrate). The analyses of the plant stems revealed an increase in the cellulose content and a decrease in the lignin and pectin contents relative to the control plants. However, the contents of the fibers' major components (cellulose, lignin, pectin) remain unchanged. An FT-IR study confirmed the results of the biochemical analyses of the flax fibers. However, the arrangement of the cellulose polymer in the transgenic fibers differed from that in the control, and a significant increase in the number of hydrogen bonds was detected. The mechanical properties of the transgenic flax stems were significantly improved, reflecting the cellulose content increase. However, the mechanical properties of the fibers did not change in comparison with the control, with the exception of the fibers from transgenic line M13. The generated transgenic flax plants, which produce both components of the flax/PHB composites (i.e., fibers and thermoplastic matrix in the same plant organ) are a source of an attractive and ecologically safe material for industry and medicine.

Ultrastructural characterization of exine development of the transient defective exine 1 mutant suggests the existence of a factor involved in constructing reticulate exine architecture from sporopollenin aggregates.

A male-sterile mutant of Arabidopsis thaliana, in which filament elongation was defective although pollen fertility was normal, was isolated by means of T-DNA tagging. Transmission electron microscopy (TEM) analysis revealed that primexine synthesis and probacula formation, which are thought to be the initial steps of exine formation, were defective, and that globular sporopollenin aggregation was randomly deposited onto the microspore at the early uninucleate microspore stage. Sporopollenin aggregation, which failed to anchor to the microspore plasma membrane, was deposited on the locule wall and in the locule at the uninucleate microspore stage. However, visually normal exine with a basic reticulate structure was observed at the middle uninucleate microspore stage, indicating that the exine formation was restored in the mutant. Thus, the mutant was designated transient defective exine 1 (tde1). These results indicated that tde1 mutation affects the initial process of the exine formation, but does not impair any critical processes. Our results also suggest the existence of a certain factor responsible for exine patterning in A. thaliana. The TDE1 gene was found to be identical to the DE-ETIOLATED 2 gene known to be involved in brassinosteroid (BR) biosynthesis, and the tde1 probacula-defective phenotypes were recovered in the presence of BR application. These results suggest that BRs control the rate or efficiency of initial process of exine pattern formation.

Structural basis for template-independent RNA polymerization.

The 3'-terminal CCA nucleotide sequence (positions 74-76) of transfer RNA is essential for amino acid attachment and interaction with the ribosome during protein synthesis. The CCA sequence is synthesized de novo and/or repaired by a template-independent RNA polymerase, 'CCA-adding enzyme', using CTP and ATP as substrates. Despite structural and biochemical studies, the mechanism by which the CCA-adding enzyme synthesizes the defined sequence without a nucleic acid template remains elusive. Here we present the crystal structure of Aquifex aeolicus CCA-adding enzyme, bound to a primer tRNA lacking the terminal adenosine and an incoming ATP analogue, at 2.8 A resolution. The enzyme enfolds the acceptor T helix of the tRNA molecule. In the catalytic pocket, C75 is adjacent to ATP, and their base moieties are stacked. The complementary pocket for recognizing C74-C75 of tRNA forms a 'protein template' for the penultimate two nucleotides, mimicking the nucleotide template used by template-dependent polymerases. These results are supported by systematic analyses of mutants. Our structure represents the 'pre-insertion' stage of selecting the incoming nucleotide and provides the structural basis for the mechanism underlying template-independent RNA polymerization.

2.1 A crystal structure of human PXR in complex with the St. John's wort compound hyperforin.

The nuclear xenobiotic receptor PXR is activated by a wide variety of clinically used drugs and serves as a master regulator of drug metabolism and excretion gene expression in mammals. St. John's wort is used widely in Europe and the United States to treat depression. This unregulated herbal remedy leads to dangerous drug-drug interactions, however, in patients taking oral contraceptives, antivirals, or immunosuppressants. Such interactions are caused by the activation of the human PXR by hyperforin, the psychoactive agent in St. John's wort. In this study, we show that hyperforin induces the expression of numerous drug metabolism and excretion genes in primary human hepatocytes. We present the 2.1 A crystal structure of hyperforin in complex with the ligand binding domain of human PXR. Hyperforin induces conformational changes in PXR's ligand binding pocket relative to structures of human PXR elucidated previously and increases the size of the pocket by 250 A(3). We find that the mutation of individual aromatic residues within the ligand binding cavity changes PXR's response to particular ligands. Taken together, these results demonstrate that PXR employs structural flexibility to expand the chemical space it samples and that the mutation of specific residues within the ligand binding pocket of PXR tunes the receptor's response to ligands.

Induction of a polyubiquitin gene (ubi1) by potato phytoalexins and heat shock in Gibberella pulicaris.

Gibberella pulicaris, a causal agent of potato dry rot, infects potato tubers via wounds, where it is exposed to the phytoalexins rishitin and lubimin. Incubation of mycelium on agar supplemented with phytoalexins transiently induced the transcription of a polyubiquitin gene consisting of four ubiquitin units arranged head to tail; the fourth unit contains a 54-bp intron and an additional glutamine at the C-terminus of the encoded protein. Southern analysis of the G. pulicaris genome revealed one copy of the isolated polyubiquitin gene and one or two copies of other ubiquitin genes. Increased transcription of the gene was detectable above a threshold of 100 microg/ml of rishitin and at elevated temperatures, which indicates that exposure to phytoalexins causes a stress reaction of hyphal cells similar to that after heat shock.

Polyubiquitin in crustacean striated muscle: increased expression and conjugation during molt-induced claw muscle atrophy.

The claw muscles of decapod crustaceans undergo a molt-induced atrophy to facilitate withdrawal of the claws at ecdysis. Polyubiquitin expression, as well as the levels of ubiquitin conjugates, a ubiquitin-conjugating enzyme involved in the ATP/ubiquitin-dependent proteolytic pathway (crustacean E2(16 kDa) homolog of Drosophila UbcD1), and proteasome, were examined to determine the role of ATP/ubiquitin-dependent proteolysis in the enhanced degradation of myofibrillar proteins during muscle atrophy. A partial-length clone (1.7 kb) of polyubiquitin was isolated from a lobster muscle cDNA library; the 5' end lacked the 5' untranslated region (UTR) and the beginning of the first ubiquitin monomer, while the 3' end contained the terminal ubiquitin monomer and 3' UTR. The deduced amino acid sequence was 100% identical with that from Manduca, Drosophila, and human. In land crab claw muscle, the polyubiquitin mRNA (2.7 kb) increased about 5-fold and ubiquitin-protein conjugates (> 200 kDa) increased about 8-fold during atrophy. In contrast, the level of a ubiquitin-conjugating enzyme (E2(16 kDa)) remained unchanged. The proteasome, which constitutes the catalytic core of the ATP/ubiquitin-dependent proteinase complex, increased about 2-fold during proecdysis, reaching its highest level immediately before ecdysis. These results suggest that the ATP/ubiquitin-dependent proteolytic pathway contributes to the changes in protein metabolism that occur during molt-induced muscle atrophy.

Isolation of a polyubiquitin promoter and its expression in transgenic potato plants.

A polyubiquitin clone (ubi7) was isolated from a potato (Solanum tuberosum) genomic library using a copy-specific probe from a stress-induced ubiquitin cDNA. The genomic clone contained a 569-bp intron immediately 5' to the initiation codon for the first ubiquitin-coding unit. Two chimeric beta-glucuronidase (GUS) fusion transgenes were introduced into potato. The first contained GUS fused to a 1156-bp promoter fragment containing only 5' flanking and 5' untranslated sequences from ubi7. The second transgene contained GUS translationally fused to the carboxy terminus of the first ubiquitin-coding unit and thus included the intron present in the 5' untranslated region of the polyubiquitin gene. Both ubi7-GUS transgenes were activated by wounding in tuber tissue and in leaves by application of exogenous methyl jasmonate. They were also expressed constitutively in the potato tuber peel (outer 1-2 mm). Both transgenes were actively expressed in mature leaves. Exceptionally high levels of expression were observed in senescent leaves. Transgenic clones containing the ubi7 intron and the first ubiquitin-coding unit showed GUS expression levels at least 10 times higher than clones containing GUS fused to the intronless promoter.

Cloning of the polyubiquitin cDNA from the marine sponge Geodia cydonium and its preferential expression during reaggregation of cells.

Ubiquitination of proteins is a critical step in the controlled degradation process of many polypeptides. Here we show that sponges, the simplest multicellular group of eukaryotic organisms, are also equipped with the ubiquitin pathway. The polyubiquitin cDNA was isolated and characterized from the marine sponge Geodia cydonium. The open reading frame contains six ubiquitin moieties, which are lined up head to tail without spacers. A comparison of the predicted amino acid sequence of the six sponge ubiquitin-coding units with those from other organisms revealed a high degree of homology (> 93%). The ubiquitin gene is expressed to almost the same extent in the two main compartments of the sponge, the cortex and the medulla. However, only in the cortex are detectable amounts of the ubiquitin protein synthesized. The ubiquitin protein isolated from the sponge organism was found to initiate protein degradation in the heterologous reticulocyte system in the same manner as bovine ubiquitin. In vitro studies with dissociated sponge cells revealed that the homologous aggregation factor causes (i) a strong increase in the steady-state level of mRNA coding for ubiquitin and (ii) a drastic increase in ubiquitin protein synthesis, while the homologous lectin failed to display that effect in isolated cells. These data suggest that ubiquitin may play a role in sponge morphogenesis.