Isolation and properties of catechol-cleaving yeasts from coir rets

A suitable method for the selective isolation of catechol-cleaving yeasts from coir rets has been worked out. The yeast strains, all belonging toDebaryomyces hansenii, were found to demand biotin as an essential vitamin. The organism has the ability to grow on catechol, phenol and some related compounds as sole source of carbon. It tolerates 0.4% catechol and 0.26% phenol. Evidence was obtained that the catechol-cleaving enzyme of the isolates is a pyrocatechase. Some properties of the cell-free catechol oxygenase are described.     

First structural evidence of a specific inhibition of phospholipase A2 by alpha-tocopherol (vitamin E) and its implications in inflammation: crystal structure of the complex formed between phospholipase A2 and alpha-tocopherol at 1.8 A resolution

This is the first structural evidence of alpha-tocopherol (alpha-TP) as a possible candidate against inflammation, as it inhibits phospholipase A2 specifically and effectively. The crystal structure of the complex formed between Vipera russelli phospholipase A2 and alpha-tocopherol has been determined and refined to a resolution of 1.8 A. The structure contains two molecules, A and B, of phospholipase A2 in the asymmetric unit, together with one alpha-tocopherol molecule, which is bound specifically to one of them. The phospholipase A2 molecules interact extensively with each other in the crystalline state. The two molecules were found in a stable association in the solution state as well, thus indicating their inherent tendency to remain together as a structural unit, leading to significant functional implications. In the crystal structure, the most important difference between the conformations of two molecules as a result of their association pertains to the orientation of Trp31. It may be noted that Trp31 is located at the mouth of the hydrophobic channel that forms the binding domain of the enzyme. The values of torsion angles (phi, psi, chi(1) and chi(2)) for both the backbone as well as for the side-chain of Trp31 in molecules A and B are -94 degrees, -30 degrees, -66 degrees, 116 degrees and -128 degrees, 170 degrees, -63 degrees, -81 degrees, respectively. The conformation of Trp31 in molecule A is suitable for binding, while that in B hinders the passage of the ligand to the binding site. Consequently, alpha-tocopherol is able to bind to molecule A only, while the binding site of molecule B contains three water molecules. In the complex, the aromatic moiety of alpha-tocopherol is placed in the large space at the active site of the enzyme, while the long hydrophobic channel in the enzyme is filled by hydrocarbon chain of alpha-tocopherol. The critical interactions between the enzyme and alpha-tocopherol are generated between the hydroxyl group of the six-membered ring of alpha-tocopherol and His48 N(delta1) and Asp49 O(delta1) as characteristic hydrogen bonds. The remaining part of alpha-tocopherol interacts extensively with the residues of the hydrophobic channel of the enzyme, giving rise to a number of hydrophobic interactions, resulting in the formation of a stable complex.     

In-vitro selection of Vitis vinifera `Chardonnay' with Elsinoe ampelina culture filtrate is accompanied by fungal resistance and enhanced secretion of chitinase

 Proembryogenic masses of grapevine (Vitis vinifera L.) `Chardonnay' (clone 02Ch) were exposed to the culture filtrate of Elsinoe ampelina (deBary) Shear, the causal agent of anthracnose disease. After four or five cycles of recurrent in-vitro selection with medium containing 40% fungal culture filtrate, putative resistant lines RC 1 and RC 2 respectively, were established. The selected lines inhibited the growth of E. ampelina and Fusarium oxysporium (Schlecht.) (isolated from watermelon) in a dual-culture assay and reduced the growth of mycelium on a conditioned-medium test, thus suggesting the involvement of extracellular compounds in resistance. Sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis of extracellular proteins from spent suspension-culture medium showed enhanced secretion of new proteins by selected lines. A 36-kDa protein was immunodetected by a chitinase antiserum. This chitinase continued to express constitutively in differentiated somatic embryos and also in the intercellular fluids of plants regenerated from the selected lines. Somatic embryos from selected lines grew uninhibitedly in a medium containing 40% fungal culture filtrate, whereas non-selected (control) somatic embryos became necrotic and died within a few days. Plants regenerated from selected lines exhibited resistance to infection by E. ampelina in both greenhouse tests and detached leaf bioassays. Results suggest that embryogenic cells can be selected for resistance following in-vitro selection, resulting in resistant plants. Whether or not resistant cells pre-existed in the original embryogenic culture or were induced by the selection pressure could not be determined. Received: 12 November 1999 / Accepted: 3 December 1999     

Expression of auxin-binding protein1 during plum fruit ontogeny supports the potential role of auxin in initiating and enhancing climacteric ripening

Auxin-binding protein1 (ABP1) is an active element involved in auxin signaling and plays critical roles in auxin-mediated plant development. Here, we report the isolation and characterization of a putative sequence from Prunus salicina L., designated PslABP1. The expected protein exhibits a similar molecular structure to that of well-characterized maize-ABP1; however, PslABP1 displays more sequence polarity in the active-binding site due to substitution of some crucial amino-acid residues predicted to be involved in auxin-binding. Further, PslABP1 expression was assessed throughout fruit ontogeny to determine its role in fruit development. Comparing the expression data with the physiological aspects that characterize fruit-development stages indicates that PslABP1 up-regulation is usually associated with the signature events that are triggered in an auxin-dependent manner such as floral induction, fruit initiation, embryogenesis, and cell division and elongation. However, the diversity in PslABP1 expression profile during the ripening process of early and late plum cultivars seems to be due to the variability of endogenous auxin levels among the two cultivars, which consequently can change the levels of autocatalytic ethylene available for the fruit to co-ordinate ripening. The effect of auxin on stimulating ethylene production and in regulating PslABP1 was investigated. Our data suggest that auxin is involved in the transition of the mature green fruit into the ripening phase and in enhancing the ripening process in both auxin- and ethylene-dependent manners thereafter.     

Responses of embryogenic mango cultures and seedling bioassays to a partially purified phytotoxin produced by a mango leaf isolate of Colletotrichum gloeosporioides penz

Summary Colletotrichum gloeosporioides Penz., the causal agent of mango anthracnose, produces a phytotoxin in vitro. The partially purified phytotoxin, presumably colletotrichin, caused anthracnose-like symptoms on young mango leaves, was toxic to embryogenic suspension cultures of two mango cultivars, ‘Hindi’ and ‘Carabao,’ and inhibited in vitro seed germination of two nonhosts, lettuce and tobacco. There were linear relationships between concentration of the partially purified phytotoxin and mortality of mango embryogenic cultures. Embryogenic cultures grown in the presence of the partially purified phytotoxin showed significantly lower growth rates than the controls. Similarly, embryogenic cultures grown in the presence of 40% (vol/vol) fungal culture filtrate showed significantly lower growth rates than unchallenged controls. Medium containing 40% (vol/vol) Czapek-Dox fungal broth did not reduce growth of embryogenic cultures, indicating the production of phytotoxin in vitro. The results suggest that either fungal culture filtrate or purified phytotoxin can be used as in vitro selection agents to screen for resistance to this fungus.