Keith Davis

Growth Stage–Based Phenotypic Analysis of Arabidopsis: A Model for High Throughput Functional Genomics in Plants

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.

Jasmonic Acid Signaling Modulates Ozone-Induced Hypersensitive Cell Death

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.

Induction of Arabidopsis defense genes by virulent and avirulent Pseudomonas syringae strains and by a cloned avirulence gene.

We developed a model system to study the signal transduction pathways leading to the activation of Arabidopsis thaliana genes involved in the defense against pathogen attack. Here we describe the identification and characterization of virulent and avirulent Pseudomonas syringae strains that elicit disease or resistance symptoms when infiltrated into Arabidopsis leaves. The virulent and avirulent strains were characterized by determining growth of the pathogen in Arabidopsis leaves and by measuring accumulation of mRNA corresponding to Arabidopsis phenylalanine ammonia-lyase (PAL), beta-1,3-glucanase (BG), and chalcone synthase (CHS) genes in infected leaves. The virulent strain, P. syringae pv maculicola ES4326, multiplied 10(5)-fold in Arabidopsis leaves and strongly elicited BG1, BG2, and BG3 mRNA accumulation but had only a modest effect on PAL mRNA accumulation. In contrast, the avirulent strain, P. syringae pv tomato MM1065, multiplied less than 10-fold in leaves and had only a minimal effect on BG1, BG2, and BG3 mRNA accumulation, but it induced PAL mRNA accumulation. No accumulation of CHS mRNA was found with either ES4326 or MM1065. We also describe the cloning of a putative avirulence (avr) gene from the avirulent strain MM1065 that caused the virulent strain ES4326 to grow less well in leaves and to strongly elicit PAL but not BG1 and BG3 mRNA accumulation. These results suggest that the Arabidopsis PAL and BG genes may be activated by distinct signal transduction pathways and show that differences in plant gene induction by virulent and avirulent strains can be attributed to a cloned presumptive avr gene.

An Arabidopsis thaliana Lipoxygenase Gene Can Be Induced by Pathogens, Abscisic Acid, and Methyl Jasmonate

We isolated and characterized a 2.8-kb, full-length, Arabidopsis thaliana cDNA clone encoding a lipoxygenase. DNA sequence analysis showed that the deduced amino acid sequence of the Arabidopsis protein is 72 to 78% similar to that of legume seed lipoxygenases. DNA blot analysis indicated that Arabidopsis contains a single gene, LOX1, with appreciable homology to the cDNA clone. RNA blot analysis showed that the LOX1 gene is expressed in Arabidopsis leaves, roots, inflorescences, and young seedlings. LOX1 expression levels were highest in roots and young seedlings. In mature plants, LOX1 mRNA levels increased upon treatment with the stress-related hormones abscisic acid and methyl jasmonate and remained high for at least 96 h. Expression of the LOX1 gene was examined following infiltration of leaves with virulent (Psm ES4326) and avirulent (Pst MM1065) strains of Pseudomonas syringae. LOX1 mRNA levels were induced approximately 6-fold by both virulent and avirulent strains; however, the response to avirulent strains was much more rapid. Infiltration of leaves with Pst MM1065 resulted in maximal induction within 12 h, whereas maximal induction by Psm ES4326 did not occur until 48 h. When a cloned avr gene, avrRpt2, was transferred to Psm ES4326, LOX1 mRNA accumulated in a pattern similar to that observed for the avirulent strain Pst MM1065.

The β-Subunit of the Arabidopsis G Protein Negatively Regulates Auxin-Induced Cell Division and Affects Multiple Developmental Processes

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.

The physiology of ozone induced cell death.

Ozone (O3) is a toxic air pollutant that is, somewhat paradoxically, both beneficial and harmful to life on earth. While stratospheric O3 shields biologically harm ful UY radiation from reaching the earths surface, tropospheric 03 is toxic to biological organisms. The discovery of the phytotoxicity of O3 during the mid 1950s (Richards et al. 1958) prompted widespread studies on the effects of O3 on plant growth and development. The effect of 03 on biological organisms is attributed to its ability to spontaneously dismutate or react with cellular constituents to generate excess active oxygen species (AOS; Rao et al. 2000a). Several excellent reviews that discuss the source and the chemical reactions of 03 for mation, and the physiological effects of 03 on flora are available, and will not be discussed here (Darrall 1989; Heath and Taylor 1997; Pell et al. 1997). Instead, recent advances on the complex signal transduction pathways of 03-induced cell death will be reviewed.

Real-world assessment of therapy changes, suboptimal treatment and associated costs in patients with ulcerative colitis or Crohn's disease

Treatments for Crohns disease (CD) and ulcerative colitis (UC) are not uniformly effective, thus necessitating dose changes, switching, and augmentation and carry adverse event risk, often requiring discontinuation, which reduces treatment benefits.

Arabidopsis Thaliana as a Model System for Studying Plant- Pathogen Interactions

Although plants are continuously exposed to a variety of potentially pathogenic microorganisms, successful infections are rare. Plants utilize a diverse array of defense mechanisms to prevent microbial infections. Some of these defense strategies involve constitutively expressed physical and chemical barriers that provide a first line of defense against potential pathogens (Mansfield 1983). Other defense mechanisms are specifically induced upon attempted infection. These induced defense responses include the synthesis of polyphenolic lignins and hydroxyproline-rich glycoproteins which are incorporated into plant cell walls, causing the walls to be more resistant to microbial invasion? the synthesis of the hydrolases (β-1,3- glucanase and chitinase, which may inhibit fungi by degrading their cell walls; and the synthesis and accumulation of antimicrobial compounds called phytoalexins (Bell 1981, Collinge and Slusarenko 1987, Darvill and Albersheim 1984, Hahlbrock and Scheel 1987).

Treatment Patterns, Statin Intolerance, and Subsequent Cardiovascular Events Among Japanese Patients With High Cardiovascular Risk Initiating Statin Therapy

BACKGROUNDnThis study examined treatment patterns, possible statin intolerance, and incidence of cardiovascular events (CVEs) in 2 cohorts of patients with high cardiovascular risk (i.e., patients with atherosclerotic cardiovascular disease [ASCVD] and patients with diabetes mellitus).Methodsu2004andu2004Results:A retrospective cohort study examined adults initiating either a statin or ezetimibe from 1 January 2006 to 31 May 2014 in the Japan Medical Data Center database. The first observed statin or ezetimibe prescription defined the index date. Patients had ≥12 months of pre- and post-index date plan enrollment. Two high-risk cohorts, the ASCVD cohort and diabetes cohort, were created based on diagnoses observed during the 12 months pre-index date. Treatment patterns, possible statin intolerance, and incidence of CVEs were reported. In the ASCVD cohort (n=5,302), 32.9% discontinued therapy, 7.7% switched to a non-index statin or non-statin lipid-lowering therapy, and 11.2% augmented index therapy in the 12 months post-index date; only 0.3% were using high-intensity statins and 10% had possible statin intolerance. Also, 8.1% had any new CVE during the follow-up period. Treatment patterns and incidence of CVEs among the diabetes cohort were similar to those of the ASCVD cohort.nnnCONCLUSIONSnHigh cardiovascular risk Japanese patients had frequent treatment modifications, although use of high-intensity statin doses was rare. These patterns may indicate that alternative therapies for lipid lowering are needed.

Recent Trends in Costs, Length of Stay, and Mortality Associated with Infant Pertussis Hospitalizations in the United States:

ISPOR 19th Annual International Meeting May 31-June 4, 2014 Montreal, QC, Canada Table 1a. Characteristics of Inpatient Admissions for Infant Pertussis in the United States, 2000-2005 200