Arabidopsis thaliana, Meet Microarray Technology

Courtesy of Steve Kay Data derived from the Science Watch/Hot Papers database and the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. P. Schenk et al., "Coordinated plant defense responses in Arabidopsis revealed by microarray analysis," Proc Natl Acad Sci, 97:11655-60, 2000. (Cited in 118 papers) S. Harmer et al., "Orchestrated transcription of key pathways in Arabidopsis by the circadian clock," Science, 290:2110-3, 2000. (Cited in 112 papers) Even before researchers had sequenced the genome of Arabidopsis thaliana, the small flowering relative of broccoli and a favorite model organism,1 others in the budding field of microarray analysis were attempting to glean its secrets. This issue's Hot Papers represent two early and successful attempts at connecting Arabidopsis gene expression to Arabidopsis biology, using only a fraction of its genome. Peer Schenk and his colleagues studied responses to pathogens or defense-stimulating molecules in the leaf, and ended up uncovering unexpected complexities in the plant's defense responses.2 Steve Kay's team analyzed the genetic underpinnings of the plant's circadian rhythms, and learned a thing or two about how the phenylpropanoid gene expresses itself just before sunrise.3 "[People] had been developing Arabidopsis microarrays for a number of years, and these were some of the first papers that published high-quality data with real biological conclusions," says Chris Town, an associate investigator at The Institute for Genomic Research. COMPLICATED RELATIONSHIPS Schenk and colleagues analyzed the gene expression profile of Arabidopsis leaves after exposing them to the fungus Alternaria brassicicola, or one of three signaling molecules: salicylic acid (SA), methyl jasmonate (MJ), or ethylene. The results surprised them in more ways than one. The plant's defense mechanisms turned out to be more coordinated than they had expected, with 169 genes regulated by more than one treatment. Previous attempts to group defense genes into distinct signaling pathways "may have been a simplistic view," says Schenk, a senior research officer at the Cooperative Research Centre for Tropical Plant Protection at the University of Queensland, Brisbane, Australia. The new results counter previous ideas about the SA and MJ pathways. When applied to a plant, the two chemicals prompt unique defense responses; some studies based on the expression patterns of individual genes had suggested that the two antagonize one another.4,5 But in the microarray analysis, both chemicals upregulated about one-fourth of the genes induced by SA or MJ; the results point to a more complicated relationship. Since this paper, Schenk says, "we have also carried out cluster analysis that showed coordinated expression for some genes and antagonistic expression for others."6 Of the genes whose expression levels changed significantly, at least 25% are believed to play a role in signal recognition and transduction. That is key, because pinpointing the key regulatory genes "would give valuable insights into how plant defense is activated and controlled," says Schenk. And that could lead to the design of new resistance strategies that could be applied to transgenic crops. "Upregulation of all plant defense responses comes at a cost for the plant ... and as a result, yield losses are experienced. If we knew which gene specifically regulates the activation of plant defenses [e.g., the MJ pathway], we would have a tool for making plants resistant against certain [kinds of] pathogens only. As a result yield losses may not occur," says Schenk. Schenk reasons that the paper received so much attention because "to our knowledge, this was the first large-scale microarray analysis of plant defense responses. The notion that complex regulatory interactions underlie the defense response is relatively new." The paper also serves as a platform for other researchers. All in all, the team identified 705 genes that had a change in expression in response to plant defense-related treatments. Using the available data sets, says Schenk, "researchers can search ... for their genes of interest." And search they have. Work in the field has continued apace since this paper was published. One group identified a potential key regulatory gene for plant defense responses.7 Another team subjected about half of the entire Arabidopsis transcriptome (the DNA that gets transcribed to RNA) to various plant defense treatments and bacterial pathogens.8 A recent review considered the timing of plant defenses.9 Courtesy of Steve Kay SLATHER ON THE SUNSCREEN Kay's interest, meanwhile, gravitated towards the plant's circadian rhythms. He wanted to get a better idea of the key genetic regulators and systems underlying the plant's daily responses to light and darkness. To get there, the team used microarrays to determine the mRNA levels of 8,000 genes, about one-third of the plant's total genome, at four-hour intervals throughout the day. Six percent of the genes showed varied mRNA levels in step with the circadian rhythm. The researchers, in search of patterns, analyzed the expression levels. "We were hoping we might be able to rationalize some of the biology, and we got a lot of help because clocks have been fairly well-described in plants," says Kay, a cell biologist at Scripps Research Institute in San Diego. "It wasn't a surprise to find photosynthesis genes peaking during the day, but a lot of the biology did surprise us." One eye-opener involved the expression of the phenylpropanoid gene just before dawn. The pathway churns out a variety of molecules that play a part in structural support, defense and signaling. "It turns out to make a great deal of sense," explains Kay. "One of the products [of the pathway] is pigments that screen from UV damage. We always quipped that this was the plant putting on its sunscreen each day." The results also supplemented and expanded previous observations. Before, researchers had subjected plants to cold stress and identified genes that ramped up in response. "But nobody had studied the genes under natural daylight conditions. It was a surprise to find that genes thought as being cold-induced were actually really there to protect the plant from a daily cold shock it gets at night," Kay says. The researchers also got a surprise when the analysis turned up a highly conserved promoter sequence critical to circadian expression patterns. All but one of the 31 genes it accompanied peaked in the evening. "I think what you're seeing here is [that] this element represents a major way of controlling evening or morning-specific expression, but there still must be other elements that control the other phases. I think what we found was one of the easiest ones to find," Kay says. Since then, further studies in mice and flies have turned up similar promoter elements, though these differ significantly from the promoters found in Arabidopsis.10,11 The team also identified genes under circadian control that induce flowering. That led to a recent paper showing that a specific gene uses the 24-hour clock to measure the time of the season.12 "I think [the paper] captured people's imagination [regarding] how pervasive circadian rhythms are," Kay says. They're important in humans, too. "Body temperature, blood pressure, cognitive function, the way we metabolize cholesterol. They are all hugely clock-regulated. So is cell division, and it has an important implication in [the timing of] cancer treatment." The paper also laid groundwork for identifying gene clusters that have a common controlling promoter element. "The evening element is a striking example of that. It may have been one of the first," says TIGR's Town. The work by Schenk's team was also seminal, according to John McDowell, assistant professor at Virginia Tech. The work shot down earlier ideas of relatively simple pathways switched on and off by specific kinds of pathogens. "Its biggest impact was that it provided a dramatic illustration of the regulatory complexity within the defensive response network." Jim Kling (jkling@nasw.org) is a freelance writer in Washington, DC. References 1. The Arabidopsis Genome Initiative, "Analysis of the genome sequence of the flowering plant Arabidopsis thaliana," Nature, 408:796-815, 2000. 2. P. Schenk et al., "Coordinated plant defense responses in Arabidopsis revealed by microarray analysis," Proc Natl Acad Sci, 97:11655-60, 2000. (Cited in 118 papers) 3. S. Harmer et al., "Orchestrated transcription of key pathways in Arabidopsis by the circadian clock," Science, 290:2110-3, 2000. (Cited in 112 papers) 4. S.H. Doares et al., "Salicylic acid inhibits synthesis of proteinase inhibitors in tomato leaves induced by systemin and jasmonic acid," Plant Physiol, 108:1741-6, 1995. 5. B. Thomma et al., "Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens," Proc Natl Acad Sci, 95:15107-11, 1998. 6. K. Kazan et al., "DNA microarrays: New tools in the analysis of plant defense responses," Molec Plant Pathol, 2:177-85, 2001. 7. A. Maldonado et al., "A putative lipid transfer protein involved in systemic resistance signalling in Arabidopsis," Nature, 419:399-403, 2002. 8. K. Maleck et al., "The transcriptome of Arabidopsis thaliana during systemic acquired resistance," Nat Genet, 26:403-9, 2000. 9. U. Conrath et al., "Priming in plant-pathogen interactions," Trends Plant Sci, 7:210-16, 2002. 10. M. Ceriani et al., "Genome-wide expression analysis in Drosophila reveals genes controlling circadian behavior," J Neurosci, 22:9305-19, 2002. 11. S. Panda et al., "Coordinated transcription of key pathways in the mouse by the circadian clock," Cell, 109:307-20, 2002. 12. M. Yanovsky et al., "Molecular basis of seasonal time measurement in Arabidopsis," Nature, 419:308-12, 2002.

Arabidopsis thaliana, Meet Microarray Technology

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