Biochemical and Molecular Investigations of Arabidopsis thaliana Transformed with Genes of Rosmarinic Acid Biosynthesis
Rosmarinic acid (RA) is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid (DHPL). This compound is present in various higher plants and some lower plants. However, the occurrence is not consistent since not all members in each level of plant taxa where RA has been shown to be present conta...
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|Summary:||Rosmarinic acid (RA) is an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid (DHPL). This compound is present in various higher plants and some lower plants. However, the occurrence is not consistent since not all members in each level of plant taxa where RA has been shown to be present contain RA. Recent investigations confirm that a number of enzymes involved in the formation of RA are present in all higher plant species and also active in other metabolic pathways. In Arabidopsis thaliana (Brassicaceae), the presence of certain RA biosynthesis genes has been described, but one or more RA specific genes are missing and thus the plant is unable to synthesise RA. Therefore, introducing the cDNA of CbRAS into Arabidopsis might prove the role of enzymes such as HPR2 (a photorespiratory enzyme) and cytochrome P450 monooxygenases from the CYP98A family (enzymes in the formation of monolignols) in providing the substrates and finishing the formation of RA or RA-like esters. The transformed Arabidopsis thaliana was obtained via Agrobacterium tumefaciens-mediated transformation and has been successfully established as cell suspension cultures. The transformed cells were R-lines (plants transformed with the vector containing cDNA of CbRAS) and L-lines (control plants transformed with the empty vector). Molecular characterisation showed that the T-DNA parts, the RAS and CaMV 35S promoter sequences, have been stably integrated in the plant genome. However, the presence of the KanR gene (a non-T-DNA part) in all established cell suspension cultures revealed a phenomenon which is called the integration of the non-T-DNA binary vector backbone sequences. This has been reported in several plant transformation events. The optimum growth of cell suspension cultures including the cell biomass and protein contents was observed in the first week of the cultivation period. Overexpression of the RAS gene slightly reduced the cell growth but did not impair the cells. In addition, strong expression of the RAS gene did not increase the protein content. All the RA biosynthetic enzymes were found to be highly active during the logarithmic growth phase. Enzymes such as TAT, C4H and 4CL reached their maximum activity on day 4, while PAL and RAS on day 6. Overexpression of RAS gene had no influence on the activity of the enzymes in the upstream pathway of RA. Their activities were found to be cell line-specific. The strong expression of the RAS gene did not make all ras-transformed lines having RAS activity. Some of them had exhibited the activity, but later on, the activity was lost. Gene expression analysis did not show any post-transcriptional gene silencing (PTGS) event. Therefore, a complex regulation at the post-translational level might be involved. The transcript level of the RAS gene varied among ras-transformed lines which might be caused by the number and site(s) of T-DNA integration events or cellular responses of the cell lines. The expression pattern of other RA-biosynthetic genes cannot be clearly distinguished between ras-transformed lines, control lines and the wild type. In general, gene transcripts fluctuated and were line-specific. The attempt to overexpress two genes, CbHPPR and CbRAS, in Arabidopsis was achieved by generating hairy roots from ras-transformed lines with the help of Agrobacterium rhizogenes carrying the CbHPPR gene. Only one single hairy root line could be obtained containing both genes. However, the transformed roots exhibited neither RAS activity nor detectable levels of ras transcript; this might be caused by PTGS events. Also, the transformed roots did not show any significant change of the HPPR activity although high expression of the HPPR gene was clearly observed. Accordingly, the attempt to increase the formation of RAS-ester products by double expression of hppr and ras remains unsuccessful. Analysis of metabolite contents by LC-MS showed that ras-transformed cells probably accumulated RA and Caf-pHPL, although the yields were extremely low. Thus, the presence of RAS in Arabidopsis thaliana demonstrates the dual role of HP(P)R in primary and secondary metabolism, by providing glycerate (in photorespiration) and pHPL (for RA biosynthesis), respectively. It also leads us into new perspectives regarding the role of CYP98As in Arabidopsis, the biosynthetic pathway of RA, and the role of vacuoles for RA accumulation. This, however, should be answered in future experiments.|