To return to Chapter 12 of Volume 7 ‘‘Secondary Plants Products’’ in the series The Biochemistry of Plants (Waller and Dermer, 1981) authored by George Waller and Otis Dermer, the authors concluded at the time that the important characteristics of alkaloid biosynthetic enzymes should be catalytic properties, regulation, intracellular localization, and tissue distribution. The authors were arguably not satisfied with the progress that had been made in these areas up until 1979. Since that time, astonishingly much progress has been made in our understanding the nature of the enzymes that synthesize alkaloids in plants. With the advent of the application of molecular genetic methods to the field, we have sophisticated tools at our disposal to study the regulation of enzyme biosynthesis as well as the cellular and subcellular localization. We still purify enzymes with ever more refined instrumentation, but we can also identify biosynthetic enzymes with genetic approaches. The first reports of successful crystallization of enzymes of alkaloid biosynthesis, a requisite to X-ray crystallographic structure determination, have now appeared (Ma et al., 2004a,b). Notably, the first alkaloid biosynthetic enzyme for which a crystal structure has been determined is also the first one for which a cDNA was isolated, strictosidine synthase from R. serpentina (Ma et al., 2006). A topic not touched upon at all by Waller and Dermer was the possibility to alter alkaloid metabolism in plants and plant tissue and cell cultures. In 2006, we are just beginning to metabolically engineer alkaloid metabolism in plants and in in vitro culture. Multicellular compartmentation of alkaloid pathways must be considered if meaningful metabolic engineering experiments are to be designed. We will need to use promoters that drive transgene expression in the correct cell types. Regulation of these pathways at the gene and enzyme level is complex, and there is still much to be learned about metabolite levels and pathway interconnections as we systematically overexpress and suppress gene transcription. Today, pathway engineering in plants remains highly variable. When we perturb cellular physiology, metabolite homeostasis and intra- and intercellular partitioning can be affected in unpredictable ways. Another aspect that needs attention is the development of efficient transformation and regeneration protocols for alkaloid-producing plants that do not belong to the Solanaceae. All told, there is still much to be achieved.
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