\u3ci\u3eArabidopsis thaliana GH3.9\u3c/i\u3e influences primary root growth

Auxins regulate a complex signal transduction network to direct plant development. Auxin-responsive genes fit into three major classes: the so-called auxin/indole- 3-acetic acid (Aux/IAA), the GH3, and the small auxin-up RNA (SAUR) gene families. The 20-member Arabidopsis thaliana GH3 gene family has been subdivided into three groups. In vitro studies have shown that most Group II members function as IAA–amido synthetases to conjugate amino acids to the plant hormone auxin. Here we report the role of a previously uncharacterized GH3 gene family member, GH3.9, in root growth. Unlike most other Group II family members, GH3.9 expression was repressed by low concentrations of exogenous IAA in seedlings. Transgenic plants harboring a GH3.9 promoter::reporter gene construct indicate that GH3.9 is expressed in the root-hypocotyl junction, leaves and the shoot apical meristem of young seedlings, in mature embryos, and in the root vascular tissue. Expression was also observed in lateral root tips when seedlings were treated with exogenous IAA. Inverse PCR was used to identify an activation tagged T-DNA insertion in chromosome 2 near the 5′UTR region of At2g47750 (GH3.9). Plants homozygous for the T-DNA insertion (gh3.9-1 mutants) had reduced GH3.9 expression, no obvious effects on apical dominance or leaf morphology, greater primary root length, and increased sensitivity to indole- 3-acetic acid (IAA)-mediated root growth inhibition. Additional T-DNA insertion alleles and transgenic plants with reduced GH3.9 transcript levels due to RNA-interference (RNAi) also showed these same phenotypes. Our results provide new information on the function of GH3.9 in roots where it is likely to control auxin activity through amino acid conjugation

Role of a Transcriptional Regulator in Programmed Cell Death and Plant Development

The long-term goal of this research is to understand the role(s) and molecular mechanisms of programmed cell death (PCD) in the controlling plant growth, development and responses to biotic and abiotic stress. We developed a genetic selection scheme to identify A. thaliana FB1-resistant (fbr) mutants as a way to find genes involved in PCD (Stone et al., 2000; Stone et al., 2005; Khan and Stone, 2008). The disrupted gene in fbr6 (AtSPL14) responsible for the FB1-insensitivity and plant architecture phenotypes encodes a plant-specific SBP DNA-binding domain transcriptional regulator (Stone et al., 2005; Liang et al., 2008). This research plan is designed to fill gaps in the knowledge about the role of SPL14 in plant growth and development. The work is being guided by three objectives aimed at determining the pathways in which SPL14 functions to modulate PCD and/or plant development: (1) determine how SPL14 functions in plant development, (2) identify target genes that are directly regulated by SPL14, and (3) identify SPL14 modifications and interacting proteins. We made significant progress during the funding period. Briefly, some major accomplishments are highlighted below: (1) To identify potential AtSPL14 target genes, we identified a consensus DNA binding site for the AtSPL14 SBP DNA-binding domain using systematic evolution of ligands by exponential selection (SELEX) and site-directed mutagenesis (Liang et al., 2008). This consensus binding site was used to analyze Affymetrix microarray gene expression data obtained from wild-type and fbr6 mutant plants to find possible AtSPL14-regulated genes. These candidate AtSPL14-regulated genes are providing new information on the molecular mechanisms linking plant PCD and plant development through modulation of the 26S proteasome. (2) Transgenic plants expressing epitope-tagged versions of AtSPL14 are being used to confirm the AtSPL14 targets (by ChIP-PCR) and further dissect the molecular interactions (Nazarenus, Liang and Stone, in preparation) (3) Double mutants generated between fbr6 and various accelerated cell death (acd) mutants indicate that sphingolipid metabolism is influenced by AtSPL14 and sphingolipidomics profiling supports this conclusion (Lin, Markham and Stone, in preparation). (4) A new set of phenotypes have been uncovered in the original fbr6-1 mutant, including a short-root phenotype related to auxin signaling and altered photosynthetic parameters related to stomatal density and conductance (Lin and Stone, in preparation; Lin, Madhavan and Stone, in preparation). Additional AtSPL14-related mutants and transgenic plants have been generated to effectively dissect the functions of AtSPL14, including a dominant negative fbr6-2 allele and transgenic plants overexpressing FBR6/AtSPL14 that display an accelerated cell death (acd) phenotype

A family-based probabilistic method for capturing de novo mutations from high-throughput short-read sequencing data

Recent advances in high-throughput DNA sequencing technologies and associated statistical analyses have enabled in-depth analysis of whole-genome sequences. As this technology is applied to a growing number of individual human genomes, entire families are now being sequenced. Information contained within the pedigree of a sequenced family can be leveraged when inferring the donors' genotypes. The presence of a de novo mutation within the pedigree is indicated by a violation of Mendelian inheritance laws. Here, we present a method for probabilistically inferring genotypes across a pedigree using high-throughput sequencing data and producing the posterior probability of de novo mutation at each genomic site examined. This framework can be used to disentangle the effects of germline and somatic mutational processes and to simultaneously estimate the effect of sequencing error and the initial genetic variation in the population from which the founders of the pedigree arise. This approach is examined in detail through simulations and areas for method improvement are noted. By applying this method to data from members of a well-defined nuclear family with accurate pedigree information, the stage is set to make the most direct estimates of the human mutation rate to date

ORM Expression Alters Sphingolipid Homeostasis and Differentially Affects Ceramide Synthase Activity

Sphingolipid synthesis is tightly regulated in eukaryotes. This regulation in plants ensures sufficient sphingolipids to support growth while limiting the accumulation of sphingolipid metabolites that induce programmed cell death. Serine palmitoyltransferase (SPT) catalyzes the first step in sphingolipid biosynthesis and is considered the primary sphingolipid homeostatic regulatory point. In this report, Arabidopsis (Arabidopsis thaliana) putative SPT regulatory proteins, orosomucoidlike proteins AtORM1 and AtORM2, were found to interact physically with Arabidopsis SPT and to suppress SPT activity when coexpressed with Arabidopsis SPT subunits long-chain base1 (LCB1) and LCB2 and the small subunit of SPT in a yeast (Saccharomyces cerevisiae) SPT-deficient mutant. Consistent with a role in SPT suppression, AtORM1 and AtORM2 overexpression lines displayed increased resistance to the programmed cell death-inducing mycotoxin fumonisin B1, with an accompanying reduced accumulation of LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Conversely, RNA interference (RNAi) suppression lines of AtORM1 and AtORM2 displayed increased sensitivity to fumonisin B1 and an accompanying strong increase in LCBs and C16 fatty acid-containing ceramides relative to wild-type plants. Overexpression lines also were found to have reduced activity of the class I ceramide synthase that uses C16 fatty acid acyl-coenzyme A and dihydroxy LCB substrates but increased activity of class II ceramide synthases that use very-long-chain fatty acyl-coenzyme A and trihydroxy LCB substrates. RNAi suppression lines, in contrast, displayed increased class I ceramide synthase activity but reduced class II ceramide synthase activity. These findings indicate that ORM mediation of SPT activity differentially regulates functionally distinct ceramide synthase activities as part of a broader sphingolipid homeostatic regulatory network

A population genetic approach to mapping neurological disorder genes using deep resequencing

Deep resequencing of functional regions in human genomes is key to identifying potentially causal rare variants for complex disorders. Here, we present the results from a large-sample resequencing (n  =  285 patients) study of candidate genes coupled with population genetics and statistical methods to identify rare variants associated with Autism Spectrum Disorder and Schizophrenia. Three genes, MAP1A, GRIN2B, and CACNA1F, were consistently identified by different methods as having significant excess of rare missense mutations in either one or both disease cohorts. In a broader context, we also found that the overall site frequency spectrum of variation in these cases is best explained by population models of both selection and complex demography rather than neutral models or models accounting for complex demography alone. Mutations in the three disease-associated genes explained much of the difference in the overall site frequency spectrum among the cases versus controls. This study demonstrates that genes associated with complex disorders can be mapped using resequencing and analytical methods with sample sizes far smaller than those required by genome-wide association studies. Additionally, our findings support the hypothesis that rare mutations account for a proportion of the phenotypic variance of these complex disorders

Evaluating the relationship between interannual variations in the Antarctic ozone hole and Southern Hemisphere surface climate in chemistry-climate models

Studies have recently reported statistically significant relationships between observed year-to-year spring Antarctic ozone variability and the Southern Hemisphere Annular Mode and surface temperatures in spring-summer. This study investigates whether current chemistry-climate models (CCMs) can capture these relationships, in particular, the connection between November total column ozone (TCO) and Australian summer surface temperatures, where years with anomalously high TCO over the Antarctic polar cap tend to be followed by warmer summers. The interannual ozone-temperature teleconnection is examined over the historical period in the observations and simulations from the Whole Atmosphere Community Climate Model (WACCM) and nine other models participating in the Chemistry-Climate Model Initiative (CCMI). There is a systematic difference between the WACCM experiments forced with prescribed observed sea surface temperatures (SSTs) and those with an interactive ocean. Strong correlations between TCO and Australian temperatures are only obtained for the uncoupled experiment, suggesting that the SSTs could be important for driving both variations in Australian temperatures and the ozone hole, with no causal link between the two. Other CCMI models also tend to capture this relationship with more fidelity when driven by observed SSTs, though additional research and targeted modelling experiments are required to determine causality and further explore the role of model biases and observational uncertainty. The results indicate that CCMs can reproduce the relationship between spring ozone and summer Australian climate reported in observational studies, suggesting that incorporating ozone variability could improve seasonal predictions, however more work is required to understand the difference between the coupled and uncoupled simulations

Evaluating the relationship between interannual variations in the Antarctic ozone hole and Southern Hemisphere surface climate in chemistry-climate models

Studies have recently reported statistically significant relationships between observed year-to-year spring Antarctic ozone variability and the Southern Hemisphere Annular Mode and surface temperatures in spring-summer. This study investigates whether current chemistry-climate models (CCMs) can capture these relationships, in particular, the connection between November total column ozone (TCO) and Australian summer surface temperatures, where years with anomalously high TCO over the Antarctic polar cap tend to be followed by warmer summers. The interannual ozone-temperature teleconnection is examined over the historical period in the observations and simulations from the Whole Atmosphere Community Climate Model (WACCM) and nine other models participating in the Chemistry-Climate Model Initiative (CCMI). There is a systematic difference between the WACCM experiments forced with prescribed observed sea surface temperatures (SSTs) and those with an interactive ocean. Strong correlations between TCO and Australian temperatures are only obtained for the uncoupled experiment, suggesting that the SSTs could be important for driving both variations in Australian temperatures and the ozone hole, with no causal link between the two. Other CCMI models also tend to capture this relationship with more fidelity when driven by observed SSTs, though additional research and targeted modelling experiments are required to determine causality and further explore the role of model biases and observational uncertainty. The results indicate that CCMs can reproduce the relationship between spring ozone and summer Australian climate reported in observational studies, suggesting that incorporating ozone variability could improve seasonal predictions, however more work is required to understand the difference between the coupled and uncoupled simulations