With the completion of the Arabidopsis genome sequencing project, the next major challenge is the large-scale determination of gene function. As a model organism for agricultural biotechnology, Arabidopsis presents the opportunity to provide key insights into the way that gene function can affect commercial crop production. In an attempt to aid in the rapid discovery of gene function, we have established a high throughput phenotypic analysis process based on a series of defined growth stages that serve both as developmental landmarks and as triggers for the collection of morphological data. The data collection process has been divided into two complementary platforms to ensure the capture of detailed data describing Arabidopsis growth and development over the entire life of the plant. The first platform characterizes early seedling growth on vertical plates for a period of 2 weeks. The second platform consists of an extensive set of measurements from plants grown on soil for a period of ∼2 months. When combined with parallel processes for metabolic and gene expression profiling, these platforms constitute a core technology in the high throughput determination of gene function. We present here analyses of the development of wild-type Columbia (Col-0) plants and selected mutants to illustrate a framework methodology that can be used to identify and interpret phenotypic differences in plants resulting from genetic variation and/or environmental stress.

Summary: Previous studies suggest that salicylic acid (SA) plays an important role in influencing plant resistance to ozone (O₃). To further define the role of SA in O₃‐induced responses, we compared the responses of two Arabidopsis genotypes that accumulate different amounts of SA in response to O₃ and a SA‐deficient transgenic Col‐0 line expressing salicylate hydroxylase (NahG). The differences observed in O₃‐induced changes in SA levels, the accumulation of active oxygen species, defense gene expression, and the kinetics and severity of lesion formation indicate that SA influences O₃ tolerance via two distinct mechanisms. Detailed analyses indicated that features associated with a hypersensitive response (HR) were significantly greater in O₃‐exposed Cvi‐0 than in Col‐0, and that NahG plants failed to exhibit these HR‐like responses. Furthermore, O₃‐induced antioxidant defenses, including the redox state of glutathione, were greatly reduced in NahG plants compared to Col‐0 and Cvi‐0. This suggests that O₃‐induced cell death in NahG plants is due to the loss of SA‐mediated potentiation of antioxidant defenses, while O₃‐induced cell death in Cvi‐0 is due to activation of a HR. This hypothesis is supported by the observation that inhibition of NADPH‐oxidases reduced O₃‐induced H₂O₂ levels and the O₃‐induced cell death in Cvi‐0, while no major changes were observed in NahG plants. We conclude that although SA is required to maintain the cellular redox state and potentiate defense responses in O₃ exposed plants, high levels of SA also potentiate activation of an oxidative burst and a cell death pathway that results in apparent O₃ sensitivity.

Recent studies suggest that cross-talk between salicylic acid (SA)–, jasmonic acid (JA)–, and ethylene-dependent signaling pathways regulates plant responses to both abiotic and biotic stress factors. Earlier studies demonstrated that ozone (O₃) exposure activates a hypersensitive response (HR)–like cell death pathway in the Arabidopsis ecotype Cvi-0. We now have confirmed the role of SA and JA signaling in influencing O₃-induced cell death. Expression of salicylate hydroxylase (NahG) in Cvi-0 reduced O₃-induced cell death. Methyl jasmonate (Me-JA) pretreatment of Cvi-0 decreased O₃-induced H₂O₂ content and SA concentrations and completely abolished O₃-induced cell death. Cvi-0 synthesized as much JA as did Col-0 in response to O₃ exposure but exhibited much less sensitivity to exogenous Me-JA. Analyses of the responses to O₃ of the JA-signaling mutants jar1 and fad3/7/8 also demonstrated an antagonistic relationship between JA- and SA-signaling pathways in controlling the magnitude of O₃-induced HR-like cell death.

Plant cells respond to low concentrations of auxin by cell expansion, and at a slightly higher concentration, these cells divide. Previous work revealed that null mutants of the α-subunit of a putative heterotrimeric G protein (GPA1) have reduced cell division. Here, we show that this prototypical G protein complex acts mechanistically by controlling auxin sensitivity toward cell division. Loss-of-function G protein mutants have altered auxin-mediated cell division throughout development, especially during the auxin-induced formation of lateral and adventitious root primordia. Ectopic expression of the wild-type Gα-subunit phenocopies the Gβ mutants (auxin hypersensitivity), probably by sequestering the Gβγ-subunits, whereas overexpression of Gβ reduces auxin sensitivity and a constitutively active (Q222L) mutant Gα behaves like the wild type. These data are consistent with a model in which Gβγ acts as a negative regulator of auxin-induced cell division. Accordingly, basal repression of approximately one-third of the identified auxin-regulated genes (47 of 150 upregulated genes among 8300 quantitated) is lost in the Gβ transcript-null mutant. Included among these are genes that encode proteins proposed to control cell division in root primordia formation as well as several novel genes. These results suggest that although auxin-regulated cell division is not coupled directly by a G protein, the Gβ-subunit attenuates this auxin pathway upstream of the control of mRNA steady state levels.

Exposure of Arabidopsis thaliana to ozone results in the expression of a number of defense-related genes that are also induced during a hypersensitive response. A potential common link between the activation of defense gene expression during a hypersensitive response and by ozone treatment is the production of active oxygen species and the accumulation of hydrogen peroxide. Here we report that salicylic acid accumulation, which can be induced by hydrogen peroxide and is required for the expression of both a hypersensitive response and systemic acquired resistance, is also required for the induction of some, but not all, ozone-induced mRNAs examined. In addition, we show that ozone exposure triggers induced resistance of A. thaliana to infection with virulent phytopathogenic Pseudomonas syringae strains. Infection of transgenic plants expressing salicylate hydroxylase, which prevents the accumulation of salicylic acid, or npr1 mutant plants, which are defective in the expression of systemic acquired resistance at a step downstream of salicylic acid, demonstrated that the signaling pathway activated during ozone-induced resistance overlaps with the systemic acquired resistance activation pathway and is salicylic acid dependent. Interestingly, plants expressing salicylate hydroxylase exhibited increased sensitivity to ozone exposure. These results demonstrate that ozone activates at least two distinct signaling pathways, including a salicylic acid dependent pathway previously shown to be associated with the activation of pathogen defense reactions, and that this latter pathway also induces a protective response to ozone.

Changes in gene expression induced by toxic levels of Al were characterized to investigate the nature of Al stress. A cDNA library was constructed from Arabidopsis thaliana seedlings treated with Al for 2 h. We identified five cDNA clones that showed a transient induction of their mRNA levels, four cDNA clones that showed a longer induction period, and two down-regulated genes. Expression of the four long-term-induced genes remained at elevated levels for at least 48 h. The genes encoded peroxidase, glutathione-S-transferase, blue copper-binding protein, and a protein homologous to the reticuline:oxygen oxidoreductase enzyme. Three of these genes are known to be induced by oxidative stresses and the fourth is induced by pathogen treatment. Another oxidative stress gene, superoxide dismutase, and a gene for Bowman-Birk protease inhibitor were also induced by Al in A. thaliana. These results suggested that Al treatment of Arabidopsis induces oxidative stress. In confirmation of this hypothesis, three of four genes induced by Al stress in A. thaliana were also shown to be induced by ozone. Our results demonstrate that oxidative stress is an important component of the plant's reaction to toxic levels of Al.