Table of Contents:
Hydroxycinnamic esters and amides e.g. hydroxycinnamoylquinate, hydroxycinnamoyl-shikimate, hydroxycinnamoyltyramine and rosmarinate are quite abundant in the plant kingdom. Plants can use these compounds as defense strategies against herbivores, pathogens or UV-irradiation. Rosmarinic acid synthase (RAS, 4-coumaroyl-CoA:hydroxyl-phenyllactic acid hydroxycinnamoyltransferase) is an important enzyme in the formation of rosmarinic acid (RA), the main phenolic compound in Coleus blumei. For structural analyses of RAS, the RAS-cDNA was heterologously expressed in Escherichia coli. The protein was mostly found as insoluble inclusion bodies. After solubilization and refolding of CbRAS, the protein was insufficiently active. So the expression was performed with SoluBL21TM E. coli and after purification by gel permeation chromatography, very active and pure protein could be used for crystallization. Unfortunately no crystals were growing.
RAS, HST (hydroxycinnamoyl-CoA:shikimate hydroxycinnamoyltransferase), which is important for the formation of monolignols, and HQT (hydroxycinnamoyl-CoA:quinate hydroxyl¬cinnamoyltransferase), a compound essential for the formation of chlorogenic acid (CA), are important members of the hydroxycinnamoyltransferases (HCTs). The cloning and characterization of these HCTs present in Glechoma hederacea was an objective of this study. Glechoma hederacea L. (Lamiaceae) is a perennial plant, which is distributed widely in Europe, Asia and America and an ideal candidate, because it produces RA, CA and caffeic acid. By several attempts a partial sequence putatively encoding a HSCQT was isolated from Glechoma hederacea. Additionally, the presence of caffeic acid, CA, RA and the transcript abundances of RAS, HST and an unknown HCT in flowers, leaves, stems and roots of naturally grown G. hederacea were determined. The RAS, HST and HCT genes were expressed in all organs apart from roots. Flowers accumulated astonishingly 12.5% RA in their dry mass and leaves, stems and roots around 1%.
In another part of this project the accumulation of caffeic acid, CA and RA and the determination of RAS and phenylalanine ammonia-lyase (PAL) activities in a suspension culture of Glechoma hederacea was studied. Growth, medium and secondary metabolism parameters were assessed during a cultivation period of 14 days. The maximum PAL activity was observed on day 5, while the maximum of the RAS activity was accomplished on day 8. The main caffeic acid derivative definitely was RA, which reached 25.9% of the dry mass on day 7. This is among the highest level of a secondary metabolite in plant cells ever observed.
The activity and substrate acceptance of already constructed chimeric proteins consisting of RAS and HST halves (CbRAS/HST, CbHST/RAS) were determined. The assumption is that hydroxycinnamoyltransferases are formed by two almost equally sized domains I and II. The chimeras were designed by exchanging the two main domains of RAS and HST, which is important for the formation of monolignols. RAS and HST activity assays revealed low RAS-activities in both chimeric proteins while HST activity could not be detected.
Lemon balm (Melissa officinalis) is a commonly used medicinal plant, which belongs to the family Lamiaceae (subfamily Nepetoideae). The pharmaceutical properties are e.g. sedative, carminative, spasmolytic, antibacterial and antiviral mostly due to the content of essential oil (citral, citronellal) and phenolic acid esters. The influence of low ozone concentrations on primary and secondary metabolism of lemon balm was studied. Melissa plants were exposed to low ozone (O3) dosages (80 ppb for 5 h), because high background levels of O3 are considered to be as harmful as episodic O3 peaks. Samples were taken 0, 3, 5, 12 and 24 h from beginning of exposure (FBE). The influence on different ecophysiological, biochemical and structural parameters were analyzed. At the end of the ozone exposure, no visible foliar symptoms were detectable, but at microscopic level a small number of dead cells were found. Nevertheless several photosynthetic reactions were significantly affected.
Several enzymes are responsible for the biosynthesis of RA, among them PAL, 4-coumarate:coenzyme A ligase (4CL), tyrosine aminotransferase (TAT) and RAS. The transcript levels of these genes have been investigated by quantitative RT-PCR in lemon balm. All analyzed genes were quickly up-regulated at 3 h of O3 exposure, but at 24 h from beginning of exposure (FBE) only RAS and PAL were up-regulated. The specific activity of RAS was closely correlated with a decrease in RA content, while the specific activity of PAL increased at 12 h FBE to 163% in comparison to control levels.
In a second treatment with 80 ppb ozone for 5 h the antioxidant effects of Melissa officinalis were assessed. Sampling took place 0, 3, 5, 12, 24 and 48 h FBE. A significant increase of ascorbate, dehydroascorbate, total ascorbate 48 h FBE and a biphasic redox status was measured. Strong scavenging capacities, measured with the DPPH assay, were correlating with a high content of total phenols and carotenoids. Furthermore the hydrogen peroxide and proline contents also increased significantly 48 h FBE. The catalase activtity showed a biphasic trend with an increase 5 h FBE and a decrease 48 h FBE.
In conclusion these experiments provide insight into the effect of abiotic stress caused by ozone on the pharmaceutically important plant Melissa officinalis.