The role of the transit peptide in the routing of precursors toward different chloroplast compartments

The role of the transit peptide in the routing of imported proteins inside the chloroplast was investigated with chimeric proteins in which the transit peptides for the nuclear-encoded ferredoxin and plastocyanin precursors were exchanged. Import and localization experiments with a reconstituted chloroplast system show that the ferredoxin transit peptide directs mature plastocyanin away from its correct location, the thylakoid lumen, to the stroma. With the plastocyanin transit peptide-mature ferredoxin chimera, a processing intermediate is arrested on its way to the lumen. We propose a two domain hypothesis for the plastocyanin transit peptide: the first domain functions in the chloroplast import process, whereas the second is responsible for transport across the thylakoid membrane. Thus, the transit peptide not only targets proteins to the chloroplast, but also is a major determinant in their subsequent localization within the organelle.     

Essential function in chloroplast recognition of the ferredoxin transit peptide processing region

Molecular Genetics and Genomics - Plant ferredoxin is a nuclear-encoded chloroplast protein that is synthesized in the cytoplasm as a transit peptide-containing precursor molecule. To identify...     

Proprotein-processing endoproteases PC6 and furin both activate hemagglutinin of virulent avian influenza viruses.

Among the proprotein-processing subtilisin-related endoproteases, furin has been a leading candidate for the enzyme that activates the hemagglutinin (HA) of virulent avian influenza viruses. In the present study, we examined the cleavage activity of two other recently isolated ubiquitous subtilisin-related proteases, PACE4 and PC6, using wild-type HA of A/turkey/Ireland/1378/83 (H5N8) and a series of its mutant HAs. Vaccinia virus-expressed wild-type HA was not cleaved in human colon adenocarcinoma LoVo cells, which lack active furin. This processing defect was corrected by the expression of furin and PC6 but not of PACE4 and a control wild-type vaccinia virus. PC6 showed a sequence specificity similar to that with the endogenous proteases in cultured cells. When LoVo cells were infected with a virulent avian virus, A/turkey/Ontario/7732/66 (H5N9), only noninfectious virions were produced because of the lack of HA cleavage. However, when the cells were coinfected with vaccinia virus that expressed either furin or PC6, the avian virus underwent multiple cycles of replication, indicating that both furin and PC6 specifically cleave the virulent virus HA at the authentic site. These data suggest that PC6, as well as furin, can activate virulent avian influenza viruses in vivo, implying the presence of multiple HA cleavage enzymes in animals.     

Fructan as a New Carbohydrate Sink in Transgenic Potato Plants

Fructans are polyfructose molecules that function as nonstructural storage carbohydrates in several plant species that are important crops. We have been studying plants for their ability to synthesize and degrade fructans to determine if this ability is advantageous. We have also been analyzing the ability to synthesize fructan in relation to other nonstructural carbohydrate storage forms like starch. To study this, we induced fructan accumulation in normally non-fructan-storing plants and analyzed the metabolic and physiological properties of such plants. The normally non-fructan-storing potato plant was modified by introducing the microbial fructosyltransferase genes so that it could accumulate fructans. Constructs were created so that the fructosyltransferase genes of either Bacillus subtilis (sacB) or Streptococcus mutans (ftf) were fused to the vacuolar targeting sequence of the yeast carboxypeptidase Y (cpy) gene. These constructs were placed under the control of the constitutive cauliflower mosaic virus 35S promoter and introduced into potato tissue. The regenerated potato plants accumulated high molecular mass (>5 [times] 106 D) fructan molecules in which the degree of polymerization of fructose units exceeded 25,000. Fructan accumulation was detected in every plant tissue tested. The fructan content in the transgenic potato plants tested varied between 1 and 30% of dry weight in leaves and 1 and 7% of dry weight in microtubers. Total nonstructural neutral carbohydrate content in leaves of soil-grown plants increased dramatically from 7% in the wild type to 35% in transgenic plants. Our results demonstrated that potato plants can be manipulated to store a foreign carbohydrate by introducing bacterial fructosyltransferase genes. This modification affected photosynthate partitioning in microtubers and leaves and increased nonstructural carbohydrate content in leaves.     

Mannose inhibits Arabidopsis germination via a hexokinase-mediated step

Low concentrations of the glucose (Glc) analog mannose (Man) inhibit germination of Arabidopsis seeds. Man is phosphorylated by hexokinase (HXK), but the absence of germination was not due to ATP or phosphate depletion. The addition of metabolizable sugars reversed the Man-mediated inhibition of germination. Carbohydrate-mediated regulation of gene expression involving a HXK-mediated pathway is known to be activated by Glc, Man, and other monosaccharides. Therefore, we investigated whether Man blocks germination through this system. By testing other Glc analogs, we found that 2-deoxyglucose, which, like Man, is phosphorylated by HXK, also blocked germination; no inhibition was observed with 6-deoxyglucose or 3-O-methylglucose, which are not substrates for HXK. Since these latter two sugars are taken up at a rate similar to that of Man, uptake is unlikely to be involved in the inhibition of germination. Furthermore, we show that mannoheptulose, a specific HXK inhibitor, restores germination of seeds grown in the presence of Man. We conclude that HXK is involved in the Man-mediated repression of germination of Arabidopsis seeds, possibly via energy depletion.     

Dimerization specificity of all 67 B-ZIP motifs in Arabidopsis thaliana: a comparison to Homo sapiens B-ZIP motifs

Basic region-leucine zipper (B-ZIP) proteins are a class of dimeric sequence-specific DNA-binding proteins unique to eukaryotes. We have identified 67 B-ZIP proteins in the Arabidopsis thaliana genome. No A.thaliana B-ZIP domains are homologous with any Homo sapiens B-ZIP domains. Here, we predict the dimerization specificity properties of the 67 B-ZIP proteins in the A.thaliana genome based on three structural properties of the dimeric alpha-helical leucine zipper coiled coil structure: (i) length of the leucine zipper, (ii) placement of asparagine or a charged amino acid in the hydrophobic interface and (iii) presence of interhelical electrostatic interactions. Many A.thaliana B-ZIP leucine zippers are predicted to be eight or more heptads in length, in contrast to the four or five heptads typically found in H.sapiens, a prediction experimentally verified by circular dichroism analysis. Asparagine in the a position of the coiled coil is typically observed in the second heptad in H.sapiens. In A.thaliana, asparagine is abundant in the a position of both the second and fifth heptads. The particular placement of asparagine in the a position helps define 14 families of homodimerizing B-ZIP proteins in A.thaliana, in contrast to the six families found in H.sapiens. The repulsive interhelical electrostatic interactions that are used to specify heterodimerizing B-ZIP proteins in H.sapiens are not present in A.thaliana. Instead, we predict that plant leucine zippers rely on charged amino acids in the a position to drive heterodimerization. It appears that A.thaliana define many families of homodimerizing B-ZIP proteins by having long leucine zippers with asparagine judiciously placed in the a position of different heptads.     

Glucose delays seed germination in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

Here we report that glucose delays germination of Arabidopsis thaliana (L.) Heynh. seeds at concentrations below those known to inhibit early seedling development. This inhibition acts on embryo growth and is independent of hexokinase (HXK) function. Hormones and hormone inhibitors were applied to the germination media and several hormone biosynthesis and signalling mutants were tested on glucose media to investigate a possible role of abscisic acid (ABA), gibberellin and ethylene in the glucose-induced germination delay. Results indicate that the germination inhibition by glucose cannot be antagonized by ethylene or gibberellin and is independent of the HXK1/ABA/ABI4 signalling cascade. These findings suggest that there is a separate regulatory pathway independent of ABI2/ABI4/ABI5. Thus, in a relatively short time frame sugars utilize different signalling cascades to inhibit germination and post-germination growth, underlining the complexity of sugar responses.Abbreviations ABA Abscisic acid - ABI ABA insensitive - ACC 1-Aminocyclopropane-1-carboxylic acid - BR Brassinosteroid - CAB Chlorophyll a/b-binding protein - FUS3 Fusca3 - GA Gibberellin - GA ₃ Gibberellic acid - HXK Hexokinase - LEC1 Leafy cotyledon1 - RBCS Ribulose-1,5-bisphosphate carboxylase small subunit - WT Wild type