Gustavo R. Daleo

Accumulation of cytosolic glyceraldehyde-3-phosphate dehydrogenase RNA under biological stress conditions and elicitor treatments in potato

Plants respond to pathogen infection and environmental stress by regulating the coordinate expression of many stress-related genes. In plants, the expression of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is induced under environmental stress. This work was aimed at investigating whether the expression pattern of cytosolic GAPDH is also modulated upon infection of potato plants (Solanum tuberosum L.) with the late blight fungal agent Phytophthora infestans. Northern blot analysis showed the accumulation of the GAPDH gene transcripts in leaves and stems of inoculated potato plants. When tuber discs were treated with eicosapentaenoic acid (EPA), an elicitor found in P. infestans, GAPDH gene transcripts level increased. This increase was parallel to that of the hydroxymethyl glutharyl coenzyme A reductase (HMGR), an enzyme involved in pathogen defense reactions. Glucans obtained from P. infestans cell wall acts sinergistically with EPA on GAPDH and HMGR gene induction. Salicylic acid, an endogenous signal for inducing systemic acquired resistance, was also effective in stimulating the GAPDH transcript accumulation in potato leaves. These experiments suggest that related multi-component factors, which are part of both primary and secondary metabolism, are probably regulated by similar signal transduction pathways when they are induced under biotic or abiotic stress conditions.

An aspartic protease with antimicrobial activity is induced after infection and wounding in intercellular fluids of potato tubers

Aspartic proteases (APs) one of the main proteinase classes, have different physiological functions in animals, fungi and viruses. In plants, knowledge of the biological roles of APs is less well developed. An AP has been purified from potato tuber and leaves (Guevara et al., 1999, 2001). In this paper, the changes in the level of AP in response to infection by Phytophthora infestans (P. infestans) and wounding were studied in intercellular washing fluids (IWFs) from tuber disks of two potato cultivars differing in their susceptibility to P. infestans. A differential induction was observed between both cultivars: in the resistant cultivar, induction was higher and faster in infected tissues than in wounded ones. In the susceptible cultivar, a lower and later accumulation was observed than in the resistant cultivar. In addition, AP had a direct inhibitory effect on the germination of cysts of P. infestans and conidia of Fusarium solani. The pattern of accumulation and in vitro activity of AP suggest that this enzyme may have a role in the defense response of potato.

Biosynthesis of dolichol phosphate by subcellular fractions from liver.

Since the first report of Behrens and Leloir in 1970 [l] suggesting that dolichyl phosphate glucose could serve as glucosyl donor for the synthesis of glucose-containing proteins, a large amount of work has been done in order to establish the central role of dolichyl phosphate in the biosynthesis of eukaryotic glycoproteins 121. Nevertheless, it is suprising that very little attention has been given to the metabolism of dolichol. This is a generic name for long-chain polyprenols which are composed of an isoprenoid chain of 80 to 110 carbon atoms. Most of the double bonds are cis, and only two isoprene residues near the o end are tram, the cu-isoprene unit being saturated. It is generally accepted that the biosynthesis of dolichyl phosphate follows the standard reaction pattern of head-to-tail condensations of allylic prenyl pyrophosphates with successive isopentenyl pyrophosphates units. These reactions are catalyzed by enzymes usually known as prenyl transferases. A cis prenyl transferase has been partially purified from Lactobacillus plantarum, giving undecaprenyl pyrophosphate as final product [ 31. In vivo synthesis of dolichol has been achieved in rat liver from [4 S-3H]-mevalonate [4]. Most of the

Synthesis of dolichol phosphate by a cell-free extract from pea.

Dolichols are long-chain polyprenols found in eucaryotes made up of 16-22 isoprene residues linked head to tail. The molecule has cis and frans doublebonds and the a-isoprene unit is saturated. Usually polyprenols are isolated as mixtures differing from each other only in one or two isoprene residues [ 1,2]. The phosphorylated polyprenols appear as biologically active carriers of sugars in the biosynthesis of bacterial wall polysaccharides and eucaryotic glycoproteins [ 1,3] . Bacterial undecaprenol has been synthesized in vitro with preparations of Micrococcus lysodeikticus [4] , Salmonella newington [S] and Luctobacillus plantarum [6,7] . The cis-undecaprenyl pyrophosphate synthetase from L. plantarum has recently been partially purified and the reaction requirements were studied [8]. Little is known about the biosynthesis of polyprenols in higher organisms. Incorporation of mevalonate into betulaprenol by woody tissue of Betula verrucosa [9] and into dolichol by rat liver [lo] has been achieved in vivo. We report here the first evidence for the synthesis of dolichol phosphate by a cell-free preparation from pea epicotyls and the ability of this compound to accept glucose from UDP-glucose.

Effect of glucans from different races of Phytophthora infestans on defense reactions in potato tuber

It has been proposed that susceptibility of potato to Phytophthora infestans would be a consequence of suppression and /or delaying of defense reactions by a soluble glucan which is released by compatible races of the fungus. In this report, the reaction of potato tuber slices (Solanum tuberosum cv. Huinkul) infected with either race I (1,4,7,8,10,11) or C (1,4,10,11) of Phytophthora infestans was studied. Race C grew better on slices than race I. Glucans from both races were isolated and their effect on the accumulation of phytoalexins and glucanases in tuber slices was studied. The glucans from the less virulent race (I) did not affect the accumulation of phytoalexins and glucanases in tuber slices infected or elicited with eicosapentaenoic acid, whereas the glucans from race C produced 70% inhibition of phytoalexin accumulation and reduced by 50% the induction of glucanase activities. Purified glucanases from potato degraded the glucans from race C but not from race I. The results reported here show that, at least on this cultivar, glucans from both races affected defense responses in a different manner, which could reflect structural differences between these glucans.

Production of phytoalexins, glycoalkaloids and phenolics in leaves and tubers of potato cultivars with different degrees of field resistance after infection withPhytophthora infestans

SummaryThe kinetics of accumulation of phytoalexins, glycoalkaloids and phenolics was studied in two potato cultivars differing in their degrees of field resistance when infected withPhytophthora infestans. Tuber slices and leaves of cvs Pampeana INTA (high degree of field resistance, free of R genes) and Bintje (susceptible) were infected with race C (complex race 1, 3, 5, 7, 11) ofPhytophthora infestans. Phytoalexins and phenolics accumulated in tuber and leaf tissues which had been inoculated. The levels of these compounds in the susceptible cv. Bintje were relatively low and similar to those found before inoculation. Leaves of cv. Pampeana INTA had a very high glycoalkaloid content, suggesting that glycoalkaloids may play a role in protection of leaves against the fungus. However, we could find no correlation between resistance and glycoalkaloid content of tubers. Our results suggest a major role of phytoalexins, phenolics and glycoalkaloids in the complex mechanisms of field resistance.

Defence reactions in two potato cultivars following infection with two races ofPhytophthora infestans

SummaryThe kinetics of accumulation of chitinases, glucanases and phytoalexins were studied in two potato cultivars, differing in their degrees of vertical and horizontal resistance, when infected with two races ofPhytophthora infestans. Tuber disks of cvs Kennebec (susceptible, low horizontal resistance) and Huinkul (tolerant, high horizontal resistance) were infected with either race “0” (avirulent) or “C” (complex, race 1.3.5.7.11) ofPhytophthora infestans. Extracts of tuber tissue (0–7 days) from cv. Kennebec infected with race “0” showed a strong increase in phytoalexin production and in chitinase and glucanase activities when compared with those infected with race “C”. These results indicate that race “C” is able to block defensive reactions. No significant differences were observed in cv. Huinkul infected with both races. The contribution of these reactions to horizontal resistance is unknown, and our results would not support a conclusive role for them in the interaction.

The presence of phospholipids and diglyceride kinase activity in microtubules from different tissues

Abstract Microtubular preparations obtained by different procedures from chick embryonic muscles and brains and from HeLa cells have associated, in addition to the protein kinase which can phosphorylate tubulin, an enzymatic activity which has been identified as that of a diglyceride kinase. They have also consistently associated various phospholipids, lecithin being the principal one. The phosphatidic acid formation is greatly stimulated by exogenous diglycerides or pretreatment with phospholipase C, and it is not present in partially purified preparations of cytoplasmic protein kinases of chick muscle. The significance of these findings is discussed.

Steryl glucoside biosynthesis in the alga Prototheca zopfii

Abstract A particulate enzyme fraction from the Chlorophyta Prototheca zopfii catalysed the transfer of glucose-[U- 14 C]from UDP-Glc-[U- 14 C] to endogenous sterol acceptors and the esterification of steryl glucosides with fatty acids from an endogenous acyl donor. Glucose was the only sugar present, and it appeared to have the β-configuration. In the acylated derivatives the glucose-acyl linkage appeared in the C-6 position of glucose, as indicated by periodate oxidation. UDP-Glc:sterol glucosyltransferase was solubilized with detergent and purified 34-fold. The solubilized enzyme showed no specificity for the sterol but a high affinity for the sugar nucleotide UDP-Glc. Time-course incorporation into steryl glucoside (SG) and the acylderivative (ASG) indicated that SG was the precursor of ASG and that phosphatidyl ethanolamine stimulated the formation of the latter compound, presumably acting as acyl donor. A high sterol glucosylating activity was found in the Golgirich fraction. All this evidence indicates that steryl glucosides and their acylated derivatives were synthesized by algae. The early assumption that these compounds were not present in algae must be revised.

Isolation and characterization of a Solanum tuberosum subtilisin-like protein with caspase-3 activity (StSBTc-3)

Plant proteases with caspase-like enzymatic activity have been widely studied during the last decade. Previously, we have reported the presence and induction of caspase-3 like activity in the apoplast of potato leaves during Solanum tuberosum- Phytophthora infestans interaction. In this work we have purified and identified a potato extracellular protease with caspase-3 like enzymatic activity from potato leaves infected with P. infestans. Results obtained from the size exclusion chromatography show that the isolated protease is a monomeric enzyme with an estimated molecular weight of 70 kDa approximately. Purified protease was analyzed by MALDI-TOF MS, showing a 100% of sequence identity with the deduced amino acid sequence of a putative subtilisin-like protease from S. tuberosum (Solgenomics protein ID: PGSC0003DMP400018521). For this reason the isolated protease was named as StSBTc-3. This report constitutes the first evidence of isolation and identification of a plant subtilisin-like protease with caspase-3 like enzymatic activity. In order to elucidate the possible function of StSBTc-3 during plant pathogen interaction, we demonstrate that like animal caspase-3, StSBTc-3 is able to produce in vitro cytoplasm shrinkage in plant cells and to induce plant cell death. This result suggest that, StSBTc-3 could exert a caspase executer function during potato- P. infestans interaction, resulting in the restriction of the pathogen spread during plant-pathogen interaction.