Luz M. Pérez

Trichoderma aureoviride 7-121, a mutant with enhanced production of lytic enzymes: its potential use in waste cellulose degradation and/or biocontrol

A mutant of the native fungus Trichoderma aureoviride , 7-121, selected for its overproduction of extracellular cellulase and s-glucosidase (cellobiase) was obtained. In shake flask cultures, production of endoglucanase, filter paper activity and cellobiase increased two to four- fold as compared with the wild type strain. The mutant strain is stable and grows rapidly in liquid as well as in solid culture media. Enzyme yields were best when pH was controlled so that it did not fall bellow pH 3.5. Cellobiase production by this mutant is particularly high (approximately 5 U/ml) as compared to other Trichoderma, strains, which makes it a suitable candidate for waste cellulose degradation. In addition, the mutant strain showed enhanced production of fungal cell wall degrading enzymes: chitinases,s-1,3-glucanases and proteases. This improvement in extracellular enzyme production by the mutant T. aureoviride 7-121 suggests that it is a suitable strain to be used in biological control.

Participation of the phosphoinositide metabolism in the hypersensitive response of Citrus limon against Alternaria alternata

Lemon seedlings inoculated with Alternaria alternata develop a hypersensitive response (HR) that includes the induction of Phenylalanine ammonia-lyase (PAL, E. C. 4.3.1.5) and the synthesis of scoparone. The signal transduction pathway involved in the development of this response is unknown. We used several inhibitors of the Phosphoinositide (PI) animal system to study a possible role of Inositol-1,4,5-triphosphate (IP3) in the transduction of the fungal conidia signal in Citrus limon. The HR was only partially inhibited by EGTA, suggesting that not only external but internal calcium as well are necessary for a complete development of the HR. In this plant system, Alternaria alternata induced an early accumulation of the second messenger IP3. When lemon seedlings were watered long term with LiCl, an inhibitor of the phosphoinositide cycle, the IP3 production was reduced, and the LiCl-watered plants could neither induce PAL nor synthesize scoparone in response to fungal conidia. Furthermore, neomycin, a Phospholipase C (PLC, E. C. 3.1.4.3) inhibitor, also inhibited PAL induction and scoparone synthesis in response to A. alternata. These results suggest that IP3 could be involved in the signal transduction pathway for the development of the HR of Citrus limon against A. alternata.

Enhancement of the hydrolysis of geranyl pyrophosphate by bivalent metal ions. A model for enzymic biosynthesis of cyclic monoterpenes

Hydrolysis of geranyl pyrophosphate is catalyzed by salts of Mn2+ and involves C-O bond cleavage. The first order rate constants reach limiting values with [Mn2+] > 10−2 M, and the most reactive species is GPP(Mn2+)2 at the optimum pH of 6.5–7. The products are similar to those from acid hydrolysis except that more cyclic hydrocarbons are formed in the presence of metal ions. Hydrolysis of geranyl phosphate is inhibited, and that of citronnellyl pyrophosphate is weakly catalyzed by Mn2+. Other divalent metal cations catalyze the hydrolysis of geranyl pyrophosphate and the sequence of effectiveness is Cu2+ > Mn2+ > Zn2+ > Co2+ < Mg2+ ~ Ca2+.

Calcium ions promote the response of Citrus limon against fungal elicitors or wounding

Abstract Lemon seedlings treated with 1 μM CaCl 2 increased phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity 1 hr earlier than when treated with fungal elicitors from Alternaria alternata , or when wounded. The calcium dependent response was suppressed by EGTA and Verapamil, and was mimicked by the calcium ionophore A23187.

Oligosaccharides released by pectinase treatment of Citrus limon seedlings are elicitors of the plant response

Abstract Oligosaccharides of different sizes were released from intact Citrus limon seedlings treated with endopolygalacturonase obtained from Alternaria alternata . Also, an increase of phenylalanine ammonia lyase (PAL) activity and the formation of a phytoalexin, were observed. Only mechanically damaged, but not intact seedlings, were able to increase their PAL activity and to synthesize a phytoalexin, in response to isolated oligomers released from plant cell walls or obtained from polygalacturonic acid (PGA). The active oligomers (pectic fragments obtained from cell walls or pectic fragments obtained from PGA) contained between 17 and 23, or 13 to 20 units of galacturonic acid, respectively. Maximal PAL activation was obtained after 20 hr treatment of intact seedlings with endopolygalacturonase and after 7 or 4 hr of treatment of damaged seedlings with endopolygalacturonase or oligosaccharides, respectively. Also, as a result of the increase of PAL activity, the appearance of a phytoalexin was observed, always after 42 hr of PAL activation. Immunoprecipitation with PAL antibodies confirmed that PAL activation was due to an increase in the amount of enzyme.

The expression of extracellular fungal cell wall hydrolytic enzymes in different Trichoderma harzianum isolates correlates with their ability to control Pyrenochaeta lycopersici

Four isolates of Trichoderma harzianum (ThN3, Th11, Th12 and Th16) were selected for their ability to control the in vitro development of the tomato root pathogen Pyrenochaeta lycopersici. Analysis of the mechanisms involved in biocontrol showed that the formation of non-volatile metabolites appears to be one of those involved in biocontrol of P. lycopersici by all T. harzianum isolates tested. Nevertheless, the higher secretion of chitinases, both in number of isoenzymes and activity by the Th11 strain, correlated well with its higher ability to control this agent in laboratory and greenhouse experiments as compared to the other T. harzianum isolates tested. The secretion of beta-1,3-endoglucanases and/or proteases appeared to have less significance than endochitinases in the biological control of P. lycopersici.

Hydrolysis of allylic phosphates by enzymes from the flavedo of Citrus sinensis

Abstract Acid phosphatase activities have been partially purified from an aqueous extract of an acetone powder from orange flavedo. The use of a gel filtration step with an ionic gradient allowed a dissociation of proteins from pigments, thus facilitating purification and stabilization of the enzymes. The enzymes do not require metals for full activity, and they hydrolysed a wide spectrum of phosphorylated substrates. C 10 –C 20 allylic pyrophosphates and monophosphates were hydrolysed sequentially by these ‘prenylphosphatases’. The final product was the corresponding unrearranged prenyl alcohol. This demonstrated the absence of E-Z isomerization and suggested an OP bond cleavage. Prenylphosphatases exhibited a certain degree of chain length specificity. Although the E or Z conformation of the C-2 double bond was not important, its presence was required for full activity. Excess prenylpyrophosphate inhibited the rate of formation of alcohols, most likely through the inhibition of phosphomonoesterase activity. These prenylphosphatases generated the alcoholic components of essential oils from the corresponding pyrophosphates and removed them from the chain lengthening process.

Signal transduction in lemon seedlings in the hypersensitive response against Alternaria alternata: participation of calmodulin, G-protein and protein kinases

The development of an effective hypersensitive response (HR) in any plant system relies, not only in their gene composition and expression, but also on an effective and rapid signal transduction system. Lemon seedlings induce the phenylpropanoid pathway, which results in the de novo biosynthesis of the phytoalexin scoparone, as part of the hypersensitive response against Alternaria alternata. In order to elucidate some of the signaling elements that participate in the development of HR in lemon seedlings, we used several compounds that are known as activators or inhibitors of signal transduction elements in plants or in animal cells. Lemon seedlings treated either with cholera toxin or with phorbol 12-myristate 13-acetate (PMA), in the absence of A. alternata induced phenylalanine ammonia-lyase (PAL, E. C. 4.3.1.5) and the synthesis of scoparone, suggesting the participation of a G-protein and of a serine/threonine kinase, respectively, in signal transduction. The use of trifluoperazine (TFP), W-7, staurosporine, lavendustin A or 2,5-dihydroximethyl cinnamate (DHMC) prevented PAL induction as well as scoparone biosynthesis in response to the fungal inoculation, thus allowing us to infer the participation of Calmodulin (CaM), of serine/threonine and of tyrosine protein kinases (TPK) for signal transduction in Citrus limon in response to A. alternata.

Release of reducing sugars from Citrus seedlings, leaves and fruits. Effect of treatment with pectinase and cellulase from Alternaria and Trichoderma

Abstract Three species of Citrus tree were found to be differentially infected by the fungal association known in Chile as ‘fumagina’. Grapefruit trees showed a 100% presence of fumagina with 34% foliage coverage, whereas lemon and orange trees showed ca 70% presence of the fungal association with only 0.2% foliage coverage. Treatment of Citrus (orange, lemon and grapefruit seedlings and fruits and leaves from adult trees with pectinase obtained from Alternaria chlamydospora, showed that grapefruit tissues were the most sensitive to the action of the enzyme, as determined by the amount of reducing sugars released during the treatment. Cellulase obtained from Trichoderma harzianum had no effect on intact seedlings and released ca 20-fold less reducing sugars from damaged tissues than pectinase. These results correlate well with the natural infection of Citrus by the fungal association fumagina and also supports the role of pectinases in the initiation of plant cell wall degradation.

Prenyltransferases from the flavedo of Citrus sinensis

Abstract A prenyltransferase activity (EC 2.5.1.1) has been partially purified from the flavedo of Citrus sinensis with 30–40-fold purification and 35–60 % yield. The enzyme catalyses the condensation of IPP with DMAPP or GPP. The products are neryl and geranyl pyrophosphate as well as (2 E ,6 E )- and (2 Z ,6 E )-farnesyl pyrophosphate. The two C 15 -products are predominant. The E - and Z -synthetase activities are partially dissociated during the purification procedure, as well as by heat or ageing. Preparations devoid of Z -synthetase were obtained. Mg 2 + is required for full activity. Mn 2 + or Co 2 + can replace Mg 2 + . The ratio of E/Z -products formed is different for each cation. Mg 2 + complexes of allylic substrates or of products protect the enzyme against heat-inactivation and against inactivation by DTNB. The results are interpreted in terms of two or more prenyltransferases stereoselective for the synthesis of E - and Z -products.