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Systemic Acquired Resistance in Conifers

#G-983


Mechanism of Induction of Systemic Acquired Resistance in Conifers by Saponin-Bound 3- Hydroxy-3-Methylglutaric Acid

Tech Area / Field

  • AGR-PPR/Plant Protection/Agriculture
  • BIO-CHM/Biochemistry/Biotechnology

Status
3 Approved without Funding

Registration date
10.02.2003

Leading Institute
Durmishidze Institute of Biochemistry and Biotechnology, Georgia, Tbilisi

Collaborators

  • Michigan Technological University / School of Forest Resources and Environmental Science, USA, MI, Houghton\nNational Institute of Agrobiological Sciences, Japan, Tsukuba\nIstituto di Chimica Biomolecolare/Consiglio Nazionale delle Ricerche (ICB-CNR), Italy, Napoli\nToyama Prefectural University, Japan, Toyama\nKyoto University / Division of Applied Life Sciences, Japan, Kyoto\nNihon University / College of Science & Technology, Japan, Tokyo\nKinki University / School of Biology-oriented Science and Technology, Japan, Wakayama\nMax-Planck-Institute for Chemical Ecology, Germany, Jena\nCNRS / Institut de Biologie Moleculaire de Plantes, France, Strasbourg\nForestry and Forest Products Research Institute, Japan, Tsukuba\nNorth Carolina State University, USA, NC, Raleigh

Project summary

Conifers in Georgia are of inestimable value as aesthetic, environmental, and economic resources. Each year untold numbers of trees throughout the state are damaged or killed by destructive insects and diseases. If values could be accurately placed on trees destroyed, annual statewide losses would easily reach into the tens of millions of dollars. The global application of chemicals in forestry and agriculture of Georgia resulted in environmental pollution and negative ecological consequences. Environmental pollution, causes many diseases characterized with increasing epidemiological parameters nowadays in Georgia.

Main objective of our project is to work out the methods of isolation of natural bioregulators (saponins - acilated with 3-hydroxy-3-methylglutaric acid) from plant materials capable to induce systemic acquired resistance in conifers against bark beetles and its associated pathogenic fungi and investigation of molecular mechanism of their action. Scientific team, leading by Dr.K.Gurielidze has more than 25 years research expertise and experience in the study of biosynthesis, metabolism, chemical structure and biological activity of plant secondary compounds (steroid glycosides, terpenoids and flavonoids). The investigations carried out by this team showed, that these compounds practically are ideal bioregulators as preparations for constructing biocomposites.

A new point of the suggested project is exploring acquired defense mechanisms in conifers against bark beetles and its associated pathogenic fungi, induced by the action of saponin-bound-3-hydroxy-3-methylglutaric acid. Low toxicity for environment, high bio-activity and proper economics determine an efficiency of new preparations.

Scientific significance of the project. An, interdisciplinary, research project aimed at understanding the molecular mechanism of induction of systemic acquired resistance in Caucasian fir (Abies normanniana) as a model system induced by the action of saponins, against bark beetles and its associated pathogenic fungi and study the structure-activity relationship of natural saponins.

Practical significance of project will be isolation and wide using ecologically harmless cheap preparations of plant origin to protect conifers. Bioregulators prepared on the base of plant saponins will possess high biological effect which consists of increased conifer resistance to pathogens and pests. Our study aims to show that saponins cause in Caucasian fir (Abies normanniana) to naturally produce increased levels of terpenes, which are toxic to fungi and insects. Conifer diseases will be significantly decreased. Application of chemicals: pesticides and fungicides, will be appreciable reduced.

The role of foreign collaborators will be expressed as follows:


· Information exchange in the course of project implementation;
· Consultations on the methods used in the study and help with analysis and presentation of results;
· Provide comments to the technical reports (quarterly, annual final, etc.), submitted by project participants to the ISTC;
· Provide necessary consultations and help on this project and help with preparation of this proposal;
· Joint discussion of obtained results at working seminars;
· Joint series of exchange visits and attendance of international meetings.

Equipments that will be used by Georgian team in collaboration with Prof. J.Gershenzon, Max-Planck Institute,include: 500-, 300- and 200-MHz NMR instruments, GC-MS ion trap instrumentation, an HP1100 HPLC system, an HP6890 GC-FID system.

To achieve goals of the project it is necessary to solve the following main tasks:


1. Isolation, purification and determination of chemical structures of steroid saponins from leaves and cell culture of: Allium erubescens., Dioscorea caucasica, Dioscorea deltoidea. Isolation of radioactive saponins
2. Study of In vivo conversion of saponin-bound-3-hydroxy-3-methylglutaric acid in cultured Abies normanniana Cells
3. Study of saponin-bound -3-hydroxy-3-methylglutaric acid -induced terpenoid synthesis in monoclonal trees Abies normanniana
4. Study of regulation of terpenoid synthase gene expression induced by saponin-bound-3-hydroxy-3-methylglutaric acid in monoclonal trees and cultured Abies normanniana Cells.

Isolation and purification of saponins The Plant material will be successively extracted by percolation with CHCl3 and 95% EtOH and will be fractionated on a silica gel column eluted with a increase gradient of CHCl3-MeOH-H2O (35:5:2 → 35:20:2). HPLC Buchi column, Lichroprep RP-8 using appropriate MeOH-H2O mixts. TLC silica gel and HPTLC silica gel for nano TLC; solvents for saponins: EtOAc-MeOH-H2O (20:5:2) and (200:27:20). Study of chemical structures of saponins will be carried out using: IR: KBr disc. 1H and 13C NMR spectra, total acid hydrolysis; partial acid hydrolysis; enzymatic hydrolysis, permetilation, Kiliani hydrolysis of permethylates, periodic acid oxidation of methylated sugars, Smith degradation of saponins, acetate of saponins, saponification.

Isolation and purification of radioactive saponins. Radioactive [14C]saponin-bound[14C]-3-hydroxy-3-methylglutaric acid will be isolated and purified as mentioned above from leaves and cell culture of Allium erubescens., Dioscorea caucasica, Dioscorea deltoidea after introducing [2-14С]acetate. In molecules of [14C]saponin-bound[14C]-3-hydroxy-3-methylglutaric acid received by this method, ~2/3 radioactivity is localized in 3-hydroxy-3-methylglutaric acid part and the rest in steroid part of the molecule. For control experiments will be exploited the same [14C]saponins, from which preliminary will be separated labeled 3-hydroxy-3-methylglutaric acid via saponification. Radio purity of saponins will be tested by the TCL method. Radioactivity of saponins-precursors and the products of labeled saponins transformation will be estimated by scintillation counter SL-400 (“Intertechnique” France).

Cell-tissue cultures of Abies normanniana will be grown in Gamborg medium (Gamborg et al. 1968), supplemented with 2,4-dichlorophenoxyacetic acid, kinetin, and sucrose. Radioactive saponins will be introduced into the nutrient medium cell culture of Abies normanniana. Three experiments will be carried out from in a monoclonal stand of Abies normanniana planted by the Georgian Mountain Forest Research Institute.

Saponin and MeJA (for comparison) Treatment. To test dose-dependent effects and time course experiments will be done with saplings, sprayed with solutions of [14C]saponin-bound[14C]-3-hydroxy-3-methylglutaric acid and MeJA. In controls were used the same [14C]saponins with preliminarily separated labeled 3-hydroxy-3-methylglutaric acid.

Monoterpene synthase assay will be carried out using the method suggested by Gijzen et al., 1991, Sesquiterpene synthase assay - by Steele et al., 1998, Diterpene synthase assay - by LaFever et al., 1994. Analysis of monoterpenes, sesquiterpenes and diterpenes will be carried out using the method described by Gershenzon et al., 1993. The syntheses of [1-3H]geranyl diphosphate will be carried out - by Croteau et al., 1994, [1-3H]farnesyl diphosphate - by Dehal and Croteau, 1988, and [1-3H]geranylgeranyl diphosphate by – La Fever et al., 1994. All other reagents will be purchased from Aldrich and Sigma unless otherwise specified. RNA extraction and purification will be carried out using a published protocol (Lewinsohn et al., 1994). Class-directed probes for transcripts encoding either monoterpene, sesquiterpene, or diterpene synthases will be designed based on comparison of the corresponding cDNA sequences for these enzymes (Bohlmann et al., 1997) (Steele et al., 1998), (LaFever et al., 1994). The monoterpene synthase probe will be labeled using a random-priming protocol (Sambrook et al., 1989). The sesquiterpene synthase and diterpene synthase probes used in RNA-blot hybridization will be labeled via PCR according to the manufacturer's (Gibco-BRL) protocol. A cDNA insert encoding soybean (Glycine max) ubiquitin will labeled using random hexamers and will be employed for monitoring constitutive gene expression. Plasmid DNA slot-blot analyses will be conducted to assess the class selectivity of the probes (Bohlmann et al., 1997). RNA Blotting and Analysis:Total RNA will be separated under denaturing conditions (Ausubel et al., 1991). The resulting labeled blots will be exposed to an imaging screen (Bio-Rad) and analyzed with a molecular imaging system (GS-525 system and Molecular Analyst software, version 2.1, Bio-Rad). Following analysis, each of the three separate blots (for monoterpene, sesquiterpene, and diterpene synthases) will be stripped and reprobed with the soybean ubiquitin cDNA as a constitutively expressed gene marker to correct for loading and blotting variations between samples.

The project will give a possibility to the Georgian scientists previously involved in military research to redirect towards civilian activities. The project together with other members of this project will facilitate to the upcoming integration of Georgian scientists in the international scientific society. Realization of the project will promote the application of elaborated methods by governmental ecological organizations, as well as by private companies not only in Georgia, but in other countries too.


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The International Science and Technology Center (ISTC) is an intergovernmental organization connecting scientists from Kazakhstan, Armenia, Tajikistan, Kyrgyzstan, and Georgia with their peers and research organizations in the EU, Japan, Republic of Korea, Norway and the United States.

 

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