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The plant hormone cytokinin is responsible for regulation of a diverse set of biological and developmental phenomena in plants such as leaf senescence, organ formation, nutrient distribution, abiotic stress response, and others. The core components of the cytokinin signaling pathway have been identified and characterized in Arabidopsis thaliana and other plant species, but questions remain about whether biological factors exist that may participate in or regulate the pathway. In this work, I present the results of a sensitized genetic screen for enhancers of ahp (eah) mutants which are hyposensitive to exogenously supplied cytokinin signals. The eah screen identified several new alleles of ahk4, one of the cytokinin receptors in Arabidopsis, as well as implicating the light-signaling transcription factor HY5. As part of the screen, the “mutagenomics” strategy for analyzing uncharacterized mutants in a screen was developed. Mutagenomics involves high-throughput resequencing of a small number of mutant plants with the goal of detecting protein-altering single nucleotide polymorphisms (SNPs). The SNPs detected across the experiment are then assessed to determine if any genes were mutated more frequently than would be expected by chance alone. A second aspect of mutagenomics involves resequencing plant lines of common descent, referred to as siblings, to determine which mutations are homozygous in all related plants. The causative mutation should be contained in the list of shared homozygous mutations. The mutagenomics strategies have the potential to accelerate the pace of genetic screens. I also present a primer design tool, indCAPS, developed to aid the search for novel CRISPR/Cas9-derived mutant alleles. The indCAPS platform is intended to be used for Cleaved Amplified Polymorphic Sequence (CAPS) or derived CAPS (dCAPS) restriction-digest genotyping assays. Tools exist for designing dCAPS primers but they assume the researcher is genotyping a SNP allele. CRISPR/Cas9-derived alleles are typically insertion/deletion (indel) events that result in a frameshift of a protein, and the previously developed primer design tools often fail when given sequences with indels. The indCAPS platform was developed with the ability to distinguish indel alleles as well as SNP alleles.

For self-pollinating plants to reproduce, male and female organ development must be coordinated as flowers mature. The Arabidopsis transcription factors AUXIN RESPONSE FACTOR 6 (ARF6) and ARF8 regulate this complex process by promoting petal expansion, stamen filament elongation, anther dehiscence, and gynoecium maturation, thereby ensuring that pollen released from the anthers is deposited on the stigma of a receptive gynoecium. ARF6 and ARF8 induce jasmonate production, which in turn triggers expression of MYB21 and MYB24, encoding R2R3 MYB transcription factors that promote petal and stamen growth. To understand the dynamics of this flower maturation regulatory network, we have characterized morphological, chemical, and global gene expression phenotypes of arf, myb, and jasmonate pathway mutant flowers. We found that MYB21 and MYB24 promoted not only petal and stamen development but also gynoecium growth. As well as regulating reproductive competence, both the ARF and MYB factors promoted nectary development or function and volatile sesquiterpene production, which may attract insect pollinators and/or repel pathogens. Mutants lacking jasmonate synthesis or response had decreased MYB21 expression and stamen and petal growth at the stage when flowers normally open, but had increased MYB21 expression in petals of older flowers, resulting in renewed and persistent petal expansion at later stages. Both auxin response and jasmonate synthesis promoted positive feedbacks that may ensure rapid petal and stamen growth as flowers open. MYB21 also fed back negatively on expression of jasmonate biosynthesis pathway genes to decrease flower jasmonate level, which correlated with termination of growth after flowers have opened. These dynamic feedbacks may promote timely, coordinated, and transient growth of flower organs. U.S. NSFNational Science Foundation (NSF) [IBN-0344257, IOS-0744874]; Max Planck SocietyMax Planck Society This work was supported by U.S. NSF grants IBN-0344257 and IOS-0744874, and by funds of the Max Planck Society. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Dermal exposure to 1,6-hexamethylene diisocyanate (HDI) and its oligomers HDI biuret and HDI isocyanurate has been implicated in occupation asthma in automotive spray-painters. Dermal exposure assessment for these compounds has been hampered due to low sample concentrations for these compounds. A sample concentration process, where samples are dried under a stream of nitrogen gas and resuspended in a smaller volume of liquid, was developed and tested in order to improve sensitivity and specificity for sample quantification and exposure assessment. The method was evaluated by comparing tape-strips samples spiked with derivatized standard or clearcoat solutions before and after concentration. In addition, the method was evaluated using 412 dermal tape-strip samples collected from automotive refinishing workers occupationally exposed to diisocyanates. Concentrations of the urea derivatives of HDI, biuret, and isocyanurate were determined using LC-MS analysis via selective ion monitoring. The results showed that both the dry-down process and skin exposure produced significant variation in the spiked sample concentrations. In the worker samples, the dry-down process increased the number of samples with detectable levels of isocyanurate from 125 to 302 while the number of samples for biuret increased from 8 to 70. Interference from unknown compounds impaired analysis of HDI after drying. In summary, the dry-down procedure has a potential to increase the sensitivity and specificity of isocyanurate in the tape-strip samples collected from the exposed workers. However, the potential biases introduced in the sample processing and the increased analytical cost indicate that this method is not suitable in its current state for adaptation to the processing of samples collected from exposed workers.