Richard A. Jorgensen. Associate Professor, Department of Plant Sciences. Ph.D., University of Wisconsin, Madison. RNA silencing and chromatin-based mechanisms of gene regulation.

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Richard A. Jorgensen. Associate Professor, Ph.D., University of Wisconsin, Madison. RNA silencing and chromatin-based mechanisms of gene regulation. e-mail: raj@ag.arizona.edu

My principal research interests involve epigenetic mechanisms of gene regulation in plants. This is fundamental research that has application to problems in the control of gene expression in the genetic engineering of crop plants, as well as being important in plant development and physiology. My approach exploits reporter genes and reverse genetics to monitor and manipulate patterns of gene expression. There are two major areas of investigation of gene regulation in my lab: 1) RNA silencing and 2) functional genomics of chromatin remodelling genes.

RNA silencing. My interests in RNA silencing center on the phenomenon of cosuppression, which we discovered in 1990. Cosuppression is a surprising, but common outcome of experiments that are designed to overexpress an endogenous plant gene product by the introduction of a transgene in which the endogene's coding sequence has been fused to a strong promoter. Instead of overexpression, often the expression of both the transgene and the homologous endogene is “cosuppressed” by a post-transcriptional mechanism that remains to be solved.

Flower pigmentation genes are especially useful tools for studying cosuppression because they confer a phenotype that is visible, dispensable, and cell-autonomous. Cosuppression of Chalcone synthase (Chs) genes in petunia produces a diverse set of flower color patterns that range from simple to complex, ordered to disordered, and stable to metastable. Chs transgene dosage experiments and promoter modification studies suggest that a slight difference in transcription distinguishes the cosuppressed state of white flowers from the coexpressed state of purple flowers. Thus, Chs cosuppression appears to be threshold-dependent, i.e., it is an extremely nonlinear response to high initial levels of transgene expression. It is also interesting that cosuppression can be transmitted between cells and throughout the plant via plasmodesmata and the phloem, respectively, probably via an RNA signal molecule.

Petunia plants exhibiting Chs cosuppression frequently undergo epigenetic changes that produce novel flower color patterns. These patterns are often heritable through the germ-line, though they are also reversible at high frequency. These epigenetic events are promoted by the presence of extra copies of the Chs transgene, suggesting that they are due to interactions between transgenes. Recently, we have shown that duplication of only the Chs coding sequences is sufficient to induce novel patterns of cosuppression. The fact that these patterns are based on petal veins and that they depend on duplication of only transcribed sequences and not the transgene promoter suggests that the duplication produces an RNA signal that transmits the cosuppression state through the phloem.

Global control of gene expression: functional genomics of chromatin genes. My interests in chromatin remodelling are being explored through a large multi-investigator, multi-university genome project which involves the generation of mutations in genes that control gene expression at the level of chromatin. The overall goal of the project is to identify and functionally analyze most, if not all, of the several hundred genes in Arabidopsis and maize (corn) that contribute to chromatin-level control of gene expression. Chromatin is the proteinaceous material that together with DNA comprises chromosomes. A key requirement for the expression of genes in chromosomes is that chromatin be remodelled (i.e., “opened”) in such a way that transcriptional activator proteins and RNA polymerases can have access to the DNA, permitting the assembly of a transcription complex which then transcribes the gene into messenger RNA.

The approach exploits conserved chromatin genes identified in the human, yeast, worm, and fly genome projects and uses bioinformatics to identify similar genes in the complete Arabidopsis genome sequence. Certain tests of chromatin gene function require dominant mutations, so dominant negative mutations will be made for each target chromatin gene. Most importantly, all mutations will be characterized to determine their effects on genetic transmission, plant growth and development, and a comprehensive battery of biochemical and epigenetic tests. These tests include histone acetylation, DNA methylation, the processes of epimutation and paramutation, reactivation of silenced transgenes and transposons, the efficiency of Agrobacterium T-DNA integration, and nucleolar dominance. Also, fusions of chromatin gene products to the GAL4 DNA binding domain will be tested for effects on a reporter transgene possessing a GAL4 DNA binding site to determine the ability of candidate genes to reverse or promote the formation of repressive chromatin. These lines will be valuable for isolation of additional mutations that suppress activity of chromatin genes. This “forward” genetic approach will be important to identify the many interesting regulatory components in chromatin that are not highly conserved or are plant specific.

This project will result in the generation and classification of a large set of useful mutations that will facilitate investigations of gene regulation in plants, leading to deeper understanding of the complex mechanisms by which plants control the expression of their genes. Equally important, a chromatin database and web site will be created that will facilitate communication among scientists and dissemination of information on chromatin level control in plants and other organisms. Further information can be obtained at our web site, The Plant Chromatin Database: http://Ag.Arizona.Edu/chromatin/chromatin.html.

Jorgensen, R.A., R.G. Atkinson, R.L.S. Forster, and W.J. Lucas. 1998. An RNA-based information superhighway in plants. Science 279:1486-1487.

Que, Q., and R.A. Jorgensen. 1998. Homology-based control of gene expression patterns in transgenic petunia flowers. Developmental Genetics 22:100-109.

Que, Q., H.-Y. Wang, and R.A. Jorgensen. 1998. Distinct patterns of pigment suppression are produced by allelic sense and antisense chalcone synthase transgenes in petunia flowers. Plant Journal 13: 401-409.

Que, Q, H-Y Wang, JJ English, RA Jorgensen. 1997. The frequency and degree of cosuppression by sense chalcone synthase transgenes are dependent on transgene promoter strength and are reduced by premature nonsense codons in the transgene coding sequence. Plant Cell 9:1357-1368.

Jorgensen, RA, PD Cluster, J English, Q Que, CA Napoli. 1996. Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences. Plant Molecular Biology, 31:957-973.

Jorgensen, RA. 1995. Cosuppression, flower color patterns, and metastable gene expression states. Science 268: 686-691.

Jorgensen, R. 1994. Developmental significance of epigenetic impositions on the plant genome: a paragenetic function for chromosomes. Developmental Genetics 15: 523-532.

Jorgensen, R. 1993. The germinal inheritance of epigenetic information in plants. Phil Trans Roy Soc Lond B 339:173-181.

Jorgensen, R. 1992. Silencing of plant genes by homologous transgenes. AgBiotech News Info. 4: 265N-273N.

Dooner, HK, TP Robbins, RA Jorgensen. 1991. Genetic and developmental control of anthocyanin biosynthesis. Annual Review of Genetics. 25:173-199.

Napoli, C., C. Lemieux, and R. Jorgensen. 1990. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co‑suppression of expression of homologous genes in trans. Plant Cell 2:279‑289.