Project Objectives

The main objectives of the present research are:
1. The establishment of different in vitro cultures of selected chemotypes of T. caespititius;
2. The isolation of putative TPSs sequences from different T. caespititius chemotypes, eventually responsible for variations in the essential oils composition;
3. The construction of a cDNA library from T. caespititius tissues with active glandular hairs. This library should be used to obtain and clone the full-length TPSs alleles;
4. Compare the TPSs alleles obtained, with other TPSs genes of Lamiaceae plants reported in databases;
5. Understand how the expression of TPSs alleles is regulated by different stimuli, using the in vitro cultures of T. caespititius chemotypes established in (1.);
6. The analysis of the chemical composition of the essential oils of the plants in vitro and the detection of putative changes resulting from the different stimuli applied;
7. Perform controlled crosses using thyme plants belonging to different chemotypes;
8. Progeny analysis to understand the genetics of the chemotype inheritance;
9. Karyotype analysis of the different T. caespititius chemotypes and the respective progeny from controlled crosses.

Abstract

Research in medicinal and aromatic plants has developed tremendously in the last four decades. In this regard the family Lamiaceae, to which Thymus belongs, is extremely important comprising many species which are used as aromatic herbs for flavoring foods due to their essential oil composition. Terpenoids are a structurally diverse group of plant natural products serving a variety of different functions in basic and specialized metabolism, some operating as direct or indirect defence agents.
Most of the terpenoid volatiles detected in Thymus oils are monoterpenes, usually making up more than 90% of the oils. In Thymus oils, about 270 terpenes have been identified, although some occur in very limited amounts. The phenolic compounds, thymol and carvacrol, are of high importance because of their activity as antioxidants and antimicrobial agents; in the plant kingdom their production is quite restricted and the genus Thymus is one of the few which produces these compounds. Volatile identification and terpene composition of a Portuguese Thymus species, T. caespititius, has been performed using populations and now the existence of four distinct chemotypes is well established in this species, namely thymol, carvacrol, α-terpineol and sabinene, with mixed chemotypes, thymol/sabinene, thymol/carvacrol and thymol/sabinene/carvacrol, occurring as well in populations. A genetic approach using random markers has revealed that different chemotypes occur in closely-related plants either genetic and geographically, and inversely the same chemotype can occur in individuals which are genetically very distant from one another. This observation suggest that, as proposed for basil, most lineages of thyme might have the genetic potential to synthesize most, if not all, the compounds that have been found collectively in this species [4]. However, the putative mechanisms behind such chemical diversity are not clear.
Genetic background and environmental conditions influence the yield and composition of the volatiles produced by thyme plants. Genetic control of the chemical characteristics has been proven and the monoterpenes produced by the plant are controlled by a series of loci with epistatic relationships between them. Biochemically, terpenes are derived from common precursors and synthesized by terpene synthases (TPSs), which are a family of enzymes, further divided into seven subfamilies, TPSa through TPSg. TPSs making different products may be more similar to each other than TPSs making the same products in different species, suggesting that convergent evolution is common in this family of proteins.
In what concerns genomic organization of plant terpenoids synthase genes, it has been nicely described by Trapp and Croteau, where these TPS genes are divided in three classes, which have a different exon and intron numbers. Several papers have been published since then and a quick search through the literature, including Genbank reveals several sequences deposited for Lamiaceae and other plant families.
Terpene synthases have also been studied in some detail by biochemists, and several papers by Croteau have been published. In Thymus, however, the literature is scarce.
Taken all this together, we propose an integrated approach to address the rationale for chemotypes. Molecular tools will be directed towards terpene synthases and controlled crosses will be performed between plants with distinct chemotypes. Progenies will be analysed to elucidate about chemotype inheritance and variation. A third approach will involve the use of in vitro cultures, including T. caespititius hairy roots obtained from different chemotypes, which will provide a system to study regulation of the expression of TPS alleles, following different stimuli.
The members from “Centro de Biotecnologia Vegetal” have more than 20 years experience on volatile analysis, hairy root cultures and micropropagation. More recently we have been involved in genetic characterization of aromatic plants using molecular markers. From collaboration with Plant Genetic Engineering Lab (ITQB), the team will be able to address the study of TPS genes. The joint
participation of “Escola Superior Agrária de Coimbra” will guarantee field experience and know-how in breeding, to allow the maintenance of the T. caespititius genotype collection and the controlled crosses.
The molecular strategy involves the construction of a cDNA library and isolation of TPS sequences from different T. caespititius chemotypes aiming to identify alleles putatively responsible for variations in the volatile composition. The in vitro cultures provide a system to study gene expression which, together with controlled crosses and progeny analysis, will provide an overall view on monoterpene synthesis control and trait inheritance in thyme.