Yiji Xia

Plants produce a wide array of natural products, many of which are likely to be useful bioactive structures. Unfortunately, these complex natural products usually occur at very low abundance and with restricted tissue distribution, thereby hindering their evaluation. Here, we report a novel approach for enhancing the accumulation of natural products based on activation tagging by Agrobacterium-mediated transformation with a T-DNA that carries cauliflower mosaic virus 35S enhancer sequences at its right border. Among approximately 5000 Arabidopsis activation-tagged lines, we found a plant that exhibited intense purple pigmentation in many vegetative organs throughout development. This upregulation of pigmentation reflected a dominant mutation that resulted in massive activation of phenylpropanoid biosynthetic genes and enhanced accumulation of lignin, hydroxycinnamic acid esters, and flavonoids, including various anthocyanins that were responsible for the purple color. These phenotypes, caused by insertion of the viral enhancer sequences adjacent to an MYB transcription factor gene, indicate that activation tagging can overcome the stringent genetic controls regulating the accumulation of specific natural products during plant development. Our findings suggest a functional genomics approach to the biotechnological evaluation of phytochemical biodiversity through the generation of massively enriched tissue sources for drug screening and for isolating underlying regulatory and biosynthetic genes.

Activation tagging using T-DNA vectors that contain multimerized transcriptional enhancers from the cauliflower mosaic virus (CaMV) 35S gene has been applied to Arabidopsis plants. New activation-tagging vectors that confer resistance to the antibiotic kanamycin or the herbicide glufosinate have been used to generate several tens of thousands of transformed plants. From these, over 30 dominant mutants with various phenotypes have been isolated. Analysis of a subset of mutants has shown that overexpressed genes are almost always found immediately adjacent to the inserted CaMV 35S enhancers, at distances ranging from 380 bp to 3.6 kb. In at least one case, the CaMV 35S enhancers led primarily to an enhancement of the endogenous expression pattern rather than to constitutive ectopic expression, suggesting that the CaMV 35S enhancers used here act differently than the complete CaMV 35S promoter. This has important implications for the spectrum of genes that will be discovered by this method.

A 470-kb segment from the long arm of chromosome 3 of Zea mays (inbred LH82), encompassing the a1-sh2 interval, was cloned as a yeast artificial chromosome. Comparison of the sizes of the restriction fragments generated from the cloned DNA fragment and from the DNA isolated from the maize inbred line LH82 established the colinearity of the a1-sh2 interval in these DNAs. By utilizing a chromosome fragmentation technique, a yeast artificial chromosome encompassing the a1-sh2 interval was separately fragmented at the a1 and sh2 loci. Comparison of the sizes of these fragmentation products established the physical distance between the a1 and sh2 loci to be 140 kb. Furthermore, these fragmentation experiments established the physical orientation of the a1 and sh2 genes relative to the maize centromere. The molecular cloning of the contiguous region between the a1 and sh2 loci made it possible to define the relationship between physical and genetic distances over a relatively large segment of the maize genome. In this interval, the relationship between physical and genetic distances is 1560 kb/centimorgan, which compares with 1460 kb/centimorgan for the entire maize genome, and 217 kb/centimorgan for a 1-kb segment within the a1 locus. Therefore, these findings are consistent with the hypothesis that genes per se are preferred sites for meiotic recombination rather than the hypothesis that genes reside in large recombinationally active segments of the genome.