Advances in understanding cis regulation of the plant gene with an emphasis on comparative genomics.

The plant gene model remains largely an extrapolation from animals, with the cis functional unit, the gene, cast as a dynamic looping structure. Molecular genetics with model plants continues to make advances; highlighted here are quantitative-occupancy results from the Arabidopsis thaliana (Arabidopsis) Phytochrome-Interacting bHLH transcription Factors (PIF) quartet. Compared to this complex snapshot, results from chromatin occupancy and other Encyclopedia of DNA Elements (ENCODE)-like approaches increase our transcription factor-motif cognate library, but regulation cannot by itself be inferred from binding. Complementary published Arabidopsis conserved noncoding sequence lists are compared, evaluated, merged, and released. Comparative genomic approaches have identified a cis modifier of a gene's expression-hypothetically, a transposon-based 'rheostat'-that works in all cells, times and places.

Temporal global expression data reveal known and novel salicylate-impacted processes and regulators mediating powdery mildew growth and reproduction on Arabidopsis.

Salicylic acid (SA) is a critical mediator of plant innate immunity. It plays an important role in limiting the growth and reproduction of the virulent powdery mildew (PM) Golovinomyces orontii on Arabidopsis (Arabidopsis thaliana). To investigate this later phase of the PM interaction and the role played by SA, we performed replicated global expression profiling for wild-type and SA biosynthetic mutant isochorismate synthase1 (ics1) Arabidopsis from 0 to 7 d after infection. We found that ICS1-impacted genes constitute 3.8% of profiled genes, with known molecular markers of Arabidopsis defense ranked very highly by the multivariate empirical Bayes statistic (T(2) statistic). Functional analyses of T(2)-selected genes identified statistically significant PM-impacted processes, including photosynthesis, cell wall modification, and alkaloid metabolism, that are ICS1 independent. ICS1-impacted processes include redox, vacuolar transport/secretion, and signaling. Our data also support a role for ICS1 (SA) in iron and calcium homeostasis and identify components of SA cross talk with other phytohormones. Through our analysis, 39 novel PM-impacted transcriptional regulators were identified. Insertion mutants in one of these regulators, PUX2 (for plant ubiquitin regulatory X domain-containing protein 2), results in significantly reduced reproduction of the PM in a cell death-independent manner. Although little is known about PUX2, PUX1 acts as a negative regulator of Arabidopsis CDC48, an essential AAA-ATPase chaperone that mediates diverse cellular activities, including homotypic fusion of endoplasmic reticulum and Golgi membranes, endoplasmic reticulum-associated protein degradation, cell cycle progression, and apoptosis. Future work will elucidate the functional role of the novel regulator PUX2 in PM resistance.

A novel, two-component system for cell lethality and its use in engineering nuclear male-sterility in plants.

Ablation of cells by the controlled expression of a lethal gene can be used to engineer plant traits such as male sterility and disease resistance. However, it may not be possible to achieve sufficient specificity of expression to prevent secondary effects in non-targeted tissues. In this paper we demonstrate that the extracellular ribonuclease, barnase, can be engineered into two complementary fragments, allowing overlapping promoter specificity to be used to enhance targeting specificity. Using a transient system, we first show that barnase can be split into two inactive peptide fragments, that when co-expressed can complement each other to reconstitute barnase activity. When a luciferase reporter gene was introduced into plant cells along with genes encoding both partial barnase peptides, a substantial reduction in luciferase activity was seen. Cytotoxicity of the reconstituted barnase was demonstrated by crossing together parents constitutively expressing each of the barnase fragments, then assaying their progeny for the presence of both partial barnase genes. None of over 300 tomato seeds planted resulted in a viable progeny that inherited both transgenes. When expression of the partial barnase genes was instead targeted to the tapetum, male sterility resulted. All 13 tomato progeny that inherited both transgenes were male sterile, whereas the three progeny inheriting only the N-terminal barnase gene were male fertile. Finally, we describe how male sterility generated by this type of two-component system can be used in hybrid seed production.