A possible approach for altering alkaloid biosynthesis in plants is the expression of genes encoding key enzymes of a pathway such as lysine decarboxylase (ldc) in transgenic plants. Two strategies were followed here: one focused on expression of the gene in the cytoplasm, the other on subsequent targeting of the protein to the chloroplasts. The ldcgene from Hafnia alvei was therefore (a) placed under the control of the 1' promoter of the bidirectional Tr promoter from Agrobacterium tumefaciens Ti-plasmid, and (b) cloned behind the rbcS promoter from potato fused to the coding region of the rbcS transit peptide. Both ldc constructs, introduced into Nicotiana tabacum with the aid of A. tumefaciens, were integrated into the plant genome and transcribed as shown by Southern and northern hybridization. However, LDC activity was only detectable in plants expressing mRNA under the control of the rbcS promoter directing the LDC fusion protein into chloroplasts with the aid of the transit peptide domain. In plants expressing the processed bacterial enzyme cadaverine levels increased from nearly zero to 0.3-1% of dry mass.

1. A highly purified sporopollenin fraction from Corylus avellana pollen was obtained using a gentle method employing hydrolyzing enzymes (pronase, lipase, cellulase, amylase, cellulysin) and an exhaustive extraction using different solvents. 2. The sporopollenin fractions were degraded by potash-fusion and nitrobenzene oxidation and the low molecular decomposition products were analyzed by TLC and HPLC. The investigation centered solely on the proof of phenolic compounds. 3. The degradation by potash-fusion yielded p-hydroxybenzoic acid as a main component, while the degradation by nitrobenzene oxidation (NBO) resulted in the formation of p-hydroxybenzaldehyde as the main component. In addition phenolic components such as p-coumaric acid, ferulic acid, vanillin and vanillic acid were formed to different degrees by using NBO as degradation procedure. 4. A comparison of the products formed following degradation of Corylus sporopollenin and “synthetic sporopollenin” shows, that phenolic compounds, if they indeed occurred as degradation products of “synthetic sporopollenin”, are generated only in extremely small quantities. It appears that, in contrast to several reports in the literature, phenols are integral constituents of natural sporopollenin. This view is supported by unpublished tracer experiments.

The gene of a bacterial lysine decarboxylase (ldc) fused to a rbcS transit peptide coding sequence (tp), and under the control of the CaMV 35S promoter, was expressed in hairy root cultures of Nicotiana tabacum. The fusion of the ldc to the targeting signal sequence improved the performance of the bacterial gene in the plant cells in many respects. Nearly all transgenic hairy root cultures harbouring the 35S-tp-ldc gene contained distinctly higher lysine decarboxylase activity (from 1.5 to 30 pkat LDC per mg protein) than those which had been transformed with constructs in which the gene had been directly cloned behind the CaMV 35S promoter. The higher enzyme activity led to the accumulation of up to 0.7% cadaverine on a dry mass basis. In addition, part of the cadaverine pool was used for increased biosynthesis of anabasine, an alkaloid which was hardly detectable in control cultures. The best line contained anabasine levels of 0.5% dry mass, which could be further be enhanced by feeding of lysine.

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