Reactive oxygen species produced by NADPH oxidase are involved in pollen tube growth

Tip-localized reactive oxygen species (ROS) were detected in growing pollen tubes by chloromethyl dichlorodihydrofluorescein diacetate oxidation, while tip-localized extracellular superoxide production was detected by nitroblue tetrazolium (NBT) reduction. To investigate the origin of the ROS we cloned a fragment of pollen specific tobacco NADPH oxidase (NOX) closely related to a pollen specific NOX from Arabidopsis. Transfection of tobacco pollen tubes with NOX-specific antisense oligodeoxynucleotides (ODNs) resulted in decreased amount of NtNOX mRNA, lower NOX activity and pollen tube growth inhibition. The ROS scavengers and the NOX inhibitor diphenylene iodonium chloride (DPI) inhibited growth and ROS formation in tobacco pollen tube cultures. Exogenous hydrogen peroxide (H2O2) rescued the growth inhibition caused by NOX antisense ODNs. Exogenous CaCl2 increased NBT reduction at the pollen tube tip, suggesting that Ca2+ increases the activity of pollen NOX in vivo. The results show that tip-localized ROS produced by a NOX enzyme is needed to sustain the normal rate of pollen tube growth and that this is likely to be a general mechanism in the control of tip growth of polarized plant cells.     

Marker-free transgenic plants through genetically programmed auto-excision

We present here a vector system to obtain homozygous marker-free transgenic plants without the need of extra handling and within the same time frame as compared to transformation methods in which the marker is not removed. By introducing a germline-specific auto-excision vector containing a cre recombinase gene under the control of a germline-specific promoter, transgenic plants become genetically programmed to lose the marker when its presence is no longer required (i.e. after the initial selection of primary transformants). Using promoters with different germline functionality, two modules of this genetic program were developed. In the first module, the promoter, placed upstream of the cre gene, confers CRE functionality in both the male and the female germline or in the common germline (e.g. floral meristem cells). In the second module, a promoter conferring single germline-specific CRE functionality was introduced upstream of the cre gene. Promoter sequences used in this work are derived from the APETALA1 and SOLO DANCERS genes from Arabidopsis (Arabidopsis thaliana) Columbia-0 conferring common germline and single germline functionality, respectively. Introduction of the genetic program did not reduce transformation efficiency. Marker-free homozygous progeny plants were efficiently obtained, regardless of which promoter was used. In addition, simplification of complex transgene loci was observed.     

NADPH oxidase-dependent reactive oxygen species formation required for root hair growth depends on ROP GTPase

Reactive oxygen species (ROS) production by an NADPH oxidase (NOX) encoded by AtrbohC/RHD2 is required for root hair growth in Arabidopsis thaliana. ROP (RHO of plants) GTPases are also required for normal root hair growth and have been proposed to regulate ROS production in plants. Therefore, the role of ROP GTPase in NOX-dependent ROS formation by root hairs was investigated. Plants overexpressing wild-type ROP2 (ROP2 OX), constitutively active (CA-rop2), or dominant negative (DN-rop2) rop2 mutant proteins were used. Superoxide formation by root hairs was detected by superoxide dismutase-sensitive nitroblue tetrazolium reduction, and ROS production in the root hair differentiation zone was detected by dihydrofluorescein diacetate oxidation. Both probes showed that ROS production was increased in ROP2 OX and CA-rop2 plants, and decreased in DN-rop2 plants, relative to wild-type plants. When CA-rop2 was expressed in the NOX loss-of-function rhd2-1 mutant, ROS formation and root hair growth were impaired, suggesting that RHD2 is required for this ROP2-dependent ROS formation.     

Characterization of fusion determinants points to the involvement of three discrete regions of both E1 and E2 glycoproteins in the membrane fusion process of hepatitis C virus

Infection of eukaryotic cells by enveloped viruses requires the merging of viral and cellular membranes. Highly specific viral surface glycoproteins, named fusion proteins, catalyze this reaction by overcoming inherent energy barriers. Hepatitis C virus (HCV) is an enveloped virus that belongs to the genus Hepacivirus of the family Flaviviridae. Little is known about the molecular events that mediate cell entry and membrane fusion for HCV, although significant progress has been made due to recent developments in infection assays. Here, using infectious HCV pseudoparticles (HCVpp), we investigated the molecular basis of HCV membrane fusion. By searching for classical features of fusion peptides through the alignment of sequences from various HCV genotypes, we identified six regions of HCV E1 and E2 glycoproteins that present such characteristics. We introduced conserved and nonconserved amino acid substitutions in these regions and analyzed the phenotype of HCVpp generated with mutant E1E2 glycoproteins. This was achieved by (i) quantifying the infectivity of the pseudoparticles, (ii) studying the incorporation of E1E2 and their capacity to mediate receptor binding, and (iii) determining their fusion capacity in cell-cell and liposome/HCVpp fusion assays. We propose that at least three of these regions (i.e., at positions 270 to 284, 416 to 430, and 600 to 620) play a role in the membrane fusion process. These regions may contribute to the merging of viral and cellular membranes either by interacting directly with lipid membranes or by assisting the fusion process through their involvement in the conformational changes of the E1E2 complex at low pH.     

Drosophila melanogaster as a model for studying protein-encoding genes that are resident in constitutive heterochromatin

The organization of chromosomes into euchromatin and heterochromatin is one of the most enigmatic aspects of genome evolution. For a long time, heterochromatin was considered to be a genomic wasteland, incompatible with gene expression. However, recent studies--primarily conducted in Drosophila melanogaster--have shown that this peculiar genomic component performs important cellular functions and carries essential genes. New research on the molecular organization, function and evolution of heterochromatin has been facilitated by the sequencing and annotation of heterochromatic DNA. About 450 predicted genes have been identified in the heterochromatin of D. melanogaster, indicating that the number of active genes is higher than had been suggested by genetic analysis. Most of the essential genes are still unknown at the molecular level, and a detailed functional analysis of the predicted genes is difficult owing to the lack of mutant alleles. Far from being a peculiarity of Drosophila, heterochromatic genes have also been found in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Oryza sativa and Arabidopsis thaliana, as well as in humans. The presence of expressed genes in heterochromatin seems paradoxical because they appear to function in an environment that has been considered incompatible with gene expression. In the future, genetic, functional genomic and proteomic analyses will offer powerful approaches with which to explore the functions of heterochromatic genes and to elucidate the mechanisms driving their expression.     

Synthesis and structural characterization of neutral “3 + 2” oxorhenium and oxotechnetium complexes of the 2-mercaptoethyl-N-glycine (SNO)/2,2′-bipyridine (NN) mixed ligand system

Neutral, hexacoordinated “3 + 2” mixed ligand oxorhenium (1) and oxotechnetium (2) complexes of the general formula MO[SNO][NN], where M = Re or ⁹⁹Tc, SNO is 2-mercaptoethyl-N-glycine and NN is 2,2′-bipyridine (bpy), were synthesized by simultaneous action of the tridentate SNO and the bidentate NN ligand on ReOCl₃(PPh₃)₂ or ⁹⁹TcO-gluconate precursors in a 1:1:1 molar ratio. Both complexes were characterized by elemental analysis, IR and NMR spectroscopy. X-ray structure determination of rhenium complex 1 revealed a distorted octahedral coordination geometry where the SNO donor atoms of the tridentate ligand and one bpy nitrogen atom occupy the equatorial positions of the octahedron, whereas the second bpy nitrogen atom and the oxo-group fill the apical positions.     

Two genes in Arabidopsis thaliana encoding GDP-L-galactose phosphorylase are required for ascorbate biosynthesis and seedling viability

Plants synthesize ascorbate from guanosine diphosphate (GDP)-mannose via L-galactose/L-gulose, although uronic acids have also been proposed as precursors. Genes encoding all the enzymes of the GDP-mannose pathway have previously been identified, with the exception of the step that converts GDP-L-galactose to L-galactose 1-P. We show that a GDP-L-galactose phosphorylase, encoded by the Arabidopsis thaliana VTC2 gene, catalyses this step in the ascorbate biosynthetic pathway. Furthermore, a homologue of VTC2, At5g55120, encodes a second GDP-L-galactose phosphorylase with similar properties to VTC2. Two At5g55120 T-DNA insertion mutants (vtc5-1 and vtc5-2) have 80% of the wild-type ascorbate level. Double mutants were produced by crossing the loss-of-function vtc2-1 mutant with each of the two vtc5 alleles. These show growth arrest immediately upon germination and the cotyledons subsequently bleach. Normal growth was restored by supplementation with ascorbate or L-galactose, indicating that both enzymes are necessary for ascorbate generation. vtc2-1 leaves contain more mannose 6-P than wild-type. We conclude that the GDP-mannose pathway is the only significant source of ascorbate in A. thaliana seedlings, and that ascorbate is essential for seedling growth. A. thaliana leaves accumulate more ascorbate after acclimatization to high light intensity. VTC2 expression and GDP-L-galactose phosphorylase activity rapidly increase on transfer to high light, but the activity of other enzymes in the GDP-mannose pathway is little affected. VTC2 and At5g55120 (VTC5) expression also peak in at the beginning of the light cycle and are controlled by the circadian clock. The GDP-L-galactose phosphorylase step may therefore play an important role in controlling ascorbate biosynthesis.