Development of a mutant of Trichoderma citrinoviride for enhanced production of cellulases

Considering importance of a microbial strain capable of increased cellulases production and insensitive to catabolite repression for industrial use, we have developed a mutant strain of Trichoderma citrinoviride by multiple exposures to EMS and ethidium bromide. The mutant produced 0.63, 3.12, 8.22 and 1.94 IU ml(-1) FPase, endoglucanase, beta-glucosidase and cellobiase, respectively. These levels were, respectively, 2.14, 2.10, 4.09 and 1.73 fold higher than those in parent strain. Glucose (upto 20 mM) did not repress enzyme production by the mutant under submerged fermentation conditions. In vitro activity assay with partially purified cellulase showed lack of inhibition by glucose. Interestingly, the partially purified endoglucanase and beta-glucosidase were activated by 2.0 fold and 2.6 fold, respectively, by 20 mM and 30 mM ethanol in the assay mixture. Genetic distinction of the mutant was revealed by the presence of two unique amplicans in comparative DNA fingerprinting performed using 20 random primers.     

PTP1B Targets the Endosomal Sorting Machinery: DEPHOSPHORYLATION OF REGULATORY SITES ON THE ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT COMPONENT STAM2*

Dephosphorylation and endocytic down-regulation are distinct processes that together control the signaling output of a variety of receptor tyrosine kinases (RTKs). PTP1B can directly dephosphorylate several RTKs, but it can also promote activation of downstream pathways through largely unknown mechanisms. These positive signaling functions likely contribute to the tumor-promoting effect of PTP1B in mouse cancer models. Here, we have identified STAM2, an endosomal protein involved in sorting activated RTKs for lysosomal degradation, as a substrate of PTP1B. PTP1B interacts with STAM2 at defined phosphotyrosine sites, and knockdown of PTP1B expression augments STAM2 phosphorylation. Intriguingly, manipulating the expression and phosphorylation state of STAM2 did not have a general effect on epidermal growth factor (EGF)-induced EGF receptor trafficking, degradation, or signaling. Instead, phosphorylated STAM2 specifically suppressed Akt activation, and a phosphorylation-deficient STAM2 mutant displayed prolonged localization on endosomes following EGF stimulation. These results reveal a novel link between the dephosphorylation and endocytic machinery and suggest that PTP1B can affect RTK signaling in a previously unrecognized manner.     

Genetic transformation of potato with nptII-gus marker genes enhances foliage consumption by Colorado potato beetle larvae

Little is known about the effect of transgenic plants containing commonly used marker genes, such as aph(3)II (nptII encoding neomycinphosphotransferase) and uidA (gus encoding -glucuronidase) on insect feeding behaviour. We report here, for the first time, that transgenic potato plants containing only nptII and gus marker genes enhance foliage consumption by the Colorado potato beetle (CPB, Leptinotarsa decemlineata S.). Transformation of potato cultivar Désirée was performed with Agrobacterium tumefaciens. Internode explants were inoculated with different strains of bacteria, carrying either nptII-gus or nptII alone. A total of 180 transgenic and untransformed control plants were grown in the greenhouse for the analysis of food consumption by CPB. For each transformed and untransformed line tested, four bioassays were conducted each consisting of 10 second-instar larvae feeding independently on a 2 cm diameter leaf disc for 20 h. Our data show up to 50% increase of mean foliage consumption on plants transformed with the nptII-gus construct, indicating that transgenic plants containing these marker genes can affect the feeding behaviour of the insects. These results were obtained from the primary regenerants (R₀ lines) as well as from tuber-derived plants (R₁ lines). Further tests with transgenic plants containing the nptII marker gene only, showed no significant difference in feeding when compared to untransformed control plants, allowing us to rule out a direct effect of this marker gene on foliage consumption by the insect larvae. It is suggested that gus protein is involved in the increase of foliage consumption by CPB.     

Crk associates with a multimolecular Paxillin/GIT2/beta-PIX complex and promotes Rac-dependent relocalization of Paxillin to focal contacts

We have previously demonstrated that the CrkII and CrkL adapter proteins are required for the spreading of epithelial colonies and the breakdown of adherens junctions in response to hepatocyte growth factor. When overexpressed, CrkII and CrkL promote lamellipodia formation, cell spreading, and the loss of epithelial adherens junctions in the absence of hepatocyte growth factor. The exact mechanism by which Crk proteins elicit these changes is unclear. We show that the overexpression of CrkII or CrkL, but not Src homology 2 or amino-terminal Src homology 3 domain mutant Crk proteins, promotes the relocalization of Paxillin to focal contacts throughout the cell and within lamellipodia in a Rac-dependent manner. In stable cell lines overexpressing CrkII, enhanced lamellipodia formation and cell spreading correlate with an increased association of CrkII with Paxillin, GIT2 (an ARF-GAP) and beta-PIX (a Rac1 exchange factor). Mutants of Paxillin that fail to associate with Crk or GIT2, or do not target to focal adhesions inhibit Crk-dependent cell spreading and lamellipodia formation. We conclude from these studies that the association of Crk with Paxillin is important for the spreading of epithelial colonies, by influencing the recruitment of Paxillin to focal complexes and promoting the enhanced assembly of Paxillin/GIT2/beta-PIX complexes.     

Cellular and subcellular localisation of glutamine synthetase and glutamate dehydrogenase in grapes gives new insights on the regulation of carbon and nitrogen metabolism

The subcellular localisation of glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in grapevine (Vitis vinifera L.) leaves and flowers was investigated using immunogold-labelling experiments. In mature leaf tissue or fully developed flowers, GS was visualised both in the cytosol and in the chloroplasts, a high proportion of the protein being present in the phloem companion cells. GDH was preferentially located in the mitochondria of the phloem companion cells in both leaves and flowers. This observation suggests that, in conjunction with GS, GDH plays a major role in controlling the translocation of organic carbon and nitrogen metabolites in both vegetative and reproductive organs. Significant amounts of GDH protein were also visualised in multivesicular bodies within the flower receptacle. Although the function of such organelles is still unknown, its is possible that the presence of GDH in such cellular structures is important for the recycling of carbon and nitrogen molecules in senescing tissues in which the enzyme is generally induced.     

Differential development of plastids during microspore embryogenesis in barley

Summary In order to understand and limit albino plantlet formation during pollen embryogenesis in barley (Hordeum vulgare L. cv. Igri), plastid feature was followed during pollen embryogenesis under two anther culture conditions and compared to plastid development in the zygotic embryo. The first condition was characterized by cold pretreatment and maltose in the induction medium. Both embryos and calli were then obtained. During pollen embryo development, up to 30% of plastids had abnormal features. Disruptions mainly affected the plastid size, the feature of plastid envelopes, thylakoid and granum organization, as well as starch accumulation. In pollen calli, superficial cells had meristematic features. Up to 50% of plastids exhibited the above mentioned abnormalities. Internal cells were highly vacuolated with amyloplast-like plastids; envelopes had normal features but no internal membrane was detected. Pollen embryo-derived plantlets had a green-to-albino ratio (G/A) being equal to 1.0, whereas calli-derived embryos only formed albino plantlets. The second condition was characterized by mannitol pretreatment and the presence of both maltose and mannitol in the induction medium. No callus was formed but most of microspore-derived structures developed haploid embryos and then the green plantlets (200 plantlets per 100 responding anthers, G/A=9.4). In this case, plastid development in zygotic and pollen embryos were similar and almost no albino plantlets were formed.     

Changes in the cellular and subcellular localization of glutamine synthetase and glutamate dehydrogenase during flag leaf senescence in wheat (Triticum aestivum L.)

In order to improve our understanding of the regulation of nitrogen assimilation and recycling in wheat (Triticum aestivum L.), we studied the localization of plastidic (GS2) and cytosolic (GS1) glutamine synthetase isoenzymes and of glutamate dehydrogenase (GDH) during natural senescence of the flag leaf and in the stem. In mature flag leaves, large amounts of GS1 were detected in the connections between the mestome sheath cells and the vascular cells, suggesting an active transfer of nitrogen organic molecules within the vascular system in the mature flag leaf. Parallel to leaf senescence, an increase of a GS1 polypeptide (GS1b) was detected in the mesophyll cytosol of senescing leaves, while the GS protein content represented by another polypetide (GS1a) in the phloem companion cells remained practically constant in both leaves and stems. Both GDH aminating activity and protein content were strongly induced in senescing flag leaves. The induction occurred both in the mitochondria and in the cytosol of phloem companion cells, suggesting that the shift in GDH cellular compartmentation is important during leaf nitrogen remobilization although the metabolic or sensing role of the enzyme remains to be elucidated. Taken together, our results suggest that in wheat, nitrogen assimilation and recycling are compartmentalized between the mesophyll and the vasculature, and are shifted in different cellular compartments within these two tissues during the transition of sink leaves to source leaves.